Basic and Clinical Endocrinology 7th International student edition Edition
Introduction to Endocrinology
John D. Baxter MD
Ralff C.J. Ribeiro MD, PhD
Paul Webb PhD
THE ENDOCRINE SYSTEM
The endocrine and nervous systems are the major controllers of the flow of information between different cells and tissues (Figure 1-1). The term “endocrine” denotes internal secretion of biologically active substances—in contrast to “exocrine,” which denotes secretion outside the body, eg, through sweat glands or ducts that lead into the gastrointestinal tract. The endocrine system uses internal secretion of hormones into the circulation to convey information to target cells that express cognate receptors. This system of internal hormone secretion is subject to complex regulatory mechanisms that govern receptor activity and hormone synthesis, release, transport, metabolism, and delivery to the interior of the target cells. The endocrine system also has complex relationships with the nervous and immune systems and exerts widespread effects upon development, growth, and metabolism. This chapter provides a broad overview of the field of endocrinology, including basic science facts and principles that are important for the diagnosis of endocrine disorders and the management of patients.
Figure 1-1. Actions of hormones and neurotransmitters. Endocrine and neurotransmitter cells synthesize hormones and release them by specialized secretory pathways of diffusion. The hormones act on the producer cell (autocrine) or on neighboring target cells, including neurotransmitter cells, without entering the circulation (juxtacrine and paracrine). They may go to the target cell through the circulation (hormonal). Neurotransmitter cells release neurotransmitters from nerve terminals. The same neurotransmitters can be released to act as hormones through the synaptic junctions or directly by the cell. (H, hormone; R, receptor; N, neurotransmitter.)
HORMONES: ENDOCRINE, PARACRINE, & AUTOCRINE ACTIONS
The endocrine system uses hormones to convey information between different tissues (Figure 1-1). Hormones are released by endocrine glands and transported through the bloodstream to tissues where they bind to specific receptor molecules and regulate target tissue function (Chapter 3). Some hormones (eg insulin, growth hormone, prolactin, leptin, catecholamines) bind cell surface receptors. Other hormones (eg, steroids, thyroid hormone) bind to intracellular receptors that act in the nucleus. Receptors have bifunctional properties of both recognition (ie, ability to distinguish the hormone from all other molecules to which they are exposed) and signal activation. The hormone acts as an allosteric effector that alters receptor conformation, and this conformational alteration transmits the binding information into postreceptor events that influence cellular function.
In addition to this traditional view, hormones can also act locally by binding to receptors that are expressed by cells that are close to the site of release. When hormones act on neighboring non-hormone-producing cells, the action is called “paracrine,” as illustrated by actions of sex steroids in the ovary, angiotensin II in the kidney, and platelet-derived growth factor in the vascular wall. As a variant of this mechanism, peptide hormones can remain in the membrane of one cell and interact with a receptor on a juxtaposed cell. This is seen, for example, with hematopoietic growth factors and is termed “juxtacrine”regulation. When hormone is released and acts on receptors located on the same cell, the action is referred to as “autocrine.”Autocrine actions may be important in promoting unregulated growth of cancer cells. Hormones can also act inside the cell without being released, ie, an “intracrine” effect. For example, insulin can inhibit its own release from pancreatic islet B cells and somatostatin can inhibit its own release from pancreatic D cells (Chapter 17).
Figure 1-2. Precursors of hormones. Shown are representations of the sources of the major hormones, with examples of different hormones that reflect each chemical type.
CHEMICAL COMPOSITION OF HORMONES
Hormones derive from the major classes of biologic molecules (Figure 1-2 and Figure 1-3; see also Chapter 2). Thus, hormones can be proteins (including glycoproteins),
peptides or peptide derivatives, amino acid analogs, or lipids. Polypeptide hormones are direct translation products of specific mRNAs, cleavage products of larger precursor proteins, or modified peptides. Catecholamines and thyroid hormones are amino acid derivatives. Steroid hormones and vitamin D are derived from cholesterol. Retinoids are derived from carotenoids in the diet that are modified by the body. Eicosanoids are derived from fatty acids.
Figure 1-3. Relations between the source, class, and actions of various molecules involved in the endocrine system. These molecules are or become hormones, eicosanoids, oncogene products, and vitamins. Normal genes encode proteins that are regulatory proteins, neurotransmitters, hormones, and paracrine or autocrine factors (or polypeptides from which these are derived). Normal genes also encode enzymes which generate amino acid analogs that can be neurotransmitters, hormones, and autocrine or paracrine factors; steroids that can be hormones or autocrine or paracrine factors; or eicosanoids that can be autocrine or paracrine factors. Oncogenes encode proteins that can be regulatory proteins, hormones, or autocrine or paracrine factors. Vitamin D can be obtained from the diet or synthesized by the body. It can act as a hormone or as an autocrine or paracrine factor.
New hormones are still being discovered. Classically, the field of endocrinology has progressed from discovery of hormones that mediate physiologic effects to identification of receptors. With the advent of molecular biology and large-scale genomic sequencing, it has become possible to identify a receptor based on sequence homology before the hormone is identified. This approach shifts endocrinology into reverse and gives rise to a search for new hormones and their signaling pathways. For example, many orphan nuclear receptors (with no known ligands) were identified on the basis of sequence similarities with other nuclear receptors, and new hormones that interact with many of these orphan receptors were subsequently identified. Similarly, genes encoding proteins with homology to known cell surface receptors have been cloned in recent years. This suggests that a number of ligands or hormones that act upon cell surface receptors are yet to be discovered. Urotensin II, a vasoactive somatostatin-like peptide, was shown to bind tightly to a previously orphaned G protein-coupled receptor, GPR14, in cardiovascular tissues.
RELATIONSHIPS BETWEEN HORMONES & OTHER SIGNALING MOLECULES
Endocrine hormones are part of a large complement of small intercellular signaling molecules. The following sections review parallels and overlaps between the endocrine system and other signaling systems.
NEUROTRANSMITTERS & HORMONES
Traditionally, the endocrine system is distinguished from the nervous system by the fact that endocrine signals are released systemically whereas the nervous system
is directly connected to target tissues through neurons (Figure 1-1). Hormones are widely distributed, and reliance is placed on the receptor to distinguish the hormone from other molecules and then to generate responses in specific cells (Figure 1-1). By contrast, the neurotransmitter is synthesized in the cell body of the neuron and travels down the axon, where it is stored in synaptic vesicles, released upon depolarization, and binds specific receptors on the postsynaptic neuron. Here, specificity of response is dependent on targeted local release of signaling molecules.
While there are differences between the endocrine and nervous systems, there are also similarities. Neurotransmitter actions involve ligand-receptor interactions that resemble those of the endocrine system. Indeed, the same molecule can be both a neurotransmitter and a hormone (Figure 1-1). Catecholamines are neurotransmitters when released by nerve terminals and hormones when released by the adrenal medulla. Furthermore, catecholamines utilize the same types of adrenergic receptors and the same postreceptor intracellular signaling pathways in the central nervous system and the peripheral tissues.
Other molecules behave as hormones and neurotransmitters. Thyrotropin-releasing hormone (TRH) is a hormone when it is produced by the hypothalamus, but it has diverse neurotransmitter actions in the central nervous system. Dopamine, corticotropin-releasing hormone (CRH), calcitonin gene-related hormone (CGRH), somatostatin, gonadotropin-releasing hormone (GnRH), vasoactive intestinal peptide (VIP), gastrin, secretin, cholecystokinin, and steroids (neurosteroids) and their receptors are also found in various parts of the brain.
There is also overlap between the function of endocrine glands and the nervous system. For example, the hypothalamus contains specialized neurons that secrete hormones into the circulation. This is reviewed below in the section on neuroendocrinology and is discussed also inChapter 5.
VITAMINS & HORMONES
Vitamins (Figure 1-3) are essential substances required in small quantities from the diet. They are utilized by the body as cofactors and regulators of cellular functions. Although this definition is acceptable, the body produces molecules that have been described as “vitamins,” and vitamins have actions that resemble hormones. For example, vitamin D is produced in individuals who are exposed to sunlight, and supplementation is required only when there is inadequate exposure to sunlight (Chapter 8). Furthermore, the active product of vitamin D is a derivative of the ingested vitamin. Vitamins can also act by mechanisms that resemble hormones. For example, vitamin D and the retinoids (retinoic acid, 9-cis-retinoic acid, and others) act through nuclear receptors belonging to the same family as the steroids and thyroid hormone (see Chapter 3).
ONCOGENES & HORMONES
Oncogenes are mutated versions of normal genes that promote cancer (see Chapter 21 and Figure 1-3). Oncogenes were originally identified in oncogenic viruses that captured genes from their host's genome. The chicken erythroleukemia virus contains two cooperating oncogenes that are homologs of hormone receptors: v-erbA (viral erbA), which is similar to the thyroid hormone receptor (sometimes designated as c-erbA, cellular erbA); and v-erbB, which is similar to the epidermal growth factor (EGF) receptor (c-erbB). Oncogenes were subsequently identified in the genomes of cancer cells.
In many cases, oncogenes are analogs of the genes encoding hormones, hormone receptors, or factors that are downstream of the hormone receptor. Typically, the oncogene codes for a mutated version of its normal cellular counterpart which is not subject to the usual regulatory controls. Thus, v-erbA does not bind thyroid hormone and acts constitutively as a transcriptional repressor. The mechanisms of action of oncogene products are described in Chapter 3 and Chapter 21.
THE IMMUNE SYSTEM & HORMONES
Interrelationships between the endocrine and immune systems are illustrated in Figure 1-4. Many immune signaling events resemble events of endocrine signaling. Thus, antigen recognition and the mechanisms that inhibit
the recognition of self-antigens involve ligand-receptor interactions similar to those used by hormones (see Chapter 3). Moreover, immunologically competent cells release peptide signaling molecules that resemble endocrine hormones in their actions. These include cytokines (eg, interleukins, interferons, tumor necrosis factor [TNF], and plasminogen activator) that bind to receptors on target cells and stimulate growth, mediate cytotoxicity, or suppress antibody production by B cells, and lymphokines that attract macrophages and neutrophils to an area of infection. In some cases, immune cells produce peptides that have been traditionally considered as hormones (eg, corticotropin [ACTH], prolactin [PRL], and gonadotropin-releasing hormone [GnRH]). The roles of these immune cell-produced classic hormones are not yet clear, though GnRH appears to play a role in lymphocyte homing.
Figure 1-4. Interrelations between the endocrine and immune systems. The plus or minus signs indicate that the influences can be stimulatory or inhibitory.
There is extensive cross-talk between the endocrine and immune systems. Substances released by immune cells can affect the function of the endocrine system. For example, TNF can influence the release and metabolism of thyroid hormones (see Chapter 7). Moreover, endocrine hormones regulate the actions of the immune system. Finally, autoimmune-induced disorders of the endocrine glands comprise a major component of endocrine practice. These include autoimmune destruction of glands, as is seen in type 1 diabetes mellitus (Chapter 17) and the most common form of Addison's disease (Chapter 9); and autoimmune stimulation, as is seen in the most common form of hyperthyroidism (Chapter 7).
EICOSANOIDS: PROSTAGLANDINS & RELATED COMPOUNDS
Eicosanoids (including prostaglandins, prostacyclins, leukotrienes, and thromboxanes; Figure 1-2 and Figure 1-3) are derived from polyunsaturated fatty acids with 18-, 20-, or 22-carbon skeletons. Arachidonic acid (cis-5,8,11,14-eicosatetraenoic acid) is the most abundant eicosanoid precursor in humans. Eicosanoids are produced by most cells, released with little storage, cleared rapidly from the circulation, and thought to act in a paracrine or autocrine fashion. They have mechanisms of action similar to those of hormones and act through both cell surface receptors and nuclear receptors.
There is cross-talk between eicosanoids and endocrine systems. Eicosanoids regulate hormone release and actions. For example, prostaglandin E (PGE) inhibits growth hormone (GH) and prolactin (PRL) release from the pituitary. Eicosanoid synthesis is also frequently stimulated by hormones, and in these instances eicosanoids act as downstream mediators of hormone action.
MECHANISMS OF HORMONE ACTION
The mechanisms of hormone action are discussed in greater detail in Chapter 3.
Hormones bind specifically to hormone receptors with high affinity and promote allosteric changes within the receptor molecule that translate the signal into biologic activities. The receptors can be expressed on the cell surface or within the cell. Whereas traditionally it was thought that the unliganded receptors are inactive—and only become activated upon hormone binding—there are now many examples (eg, with some nuclear receptors) of unliganded receptors that are active in the absence of hormone, and ligand binding reverses this effect and promotes a different activity of the receptor. The various receptor types are discussed separately in following paragraphs and in more detail in Chapter 3.
Cell Surface Receptors
Cell surface receptors have ligand recognition domains that are exposed on the outer surface of the cell membrane, one or more membrane-spanning domains, and a ligand-regulated cytoplasmic effector domain. This organization allows the cell to sense extracellular events and to pass this information to the intracellular environment.
The cell surface receptors can be divided into four types.
- (1) Seven-transmembrane domain receptors—also known as G-protein coupled receptors—mediate actions of catecholamines, prostaglandins, ACTH, glucagon, parathyroid hormone (PTH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and others (Chapter 3). They contain a surface-exposed amino terminal domain followed by seven transmembrane domains that span the lipid bilayer and a hydrophilic carboxyl terminal domain that lies in the cytoplasm. These receptors are coupled to the guanylyl nucleotide binding “G proteins.” Binding of ligand to the receptor activates G proteins, which in turn act on effectors such as adenylyl cyclase and phospholipase C and in that way initiate production of second messengers with resultant influences on cell organization, enzymatic activities, and transcription.
- (2) Receptors with intrinsic ligand-regulated enzymatic activities mediate the actions of growth factors, atrial natriuretic peptide (ANP), and TGFβ. Each contains
an amino terminal surface exposed ligand-binding domain, a single membrane-spanning domain, and a carboxyl terminal catalytic domain. Growth factor receptors, including those for insulin and EGF, possess tyrosine kinase activity. Ligand binding results in dimerization, activation of tyrosine kinase, and autophosphorylation. These events lead to recruitment of additional factors that trigger second messenger cascades such as the MAP kinase phosphorylation pathway and activation of the pI3-kinase-protein kinase B-Akt system. The ANP receptors are monomers with ligand-regulated guanylyl cyclase activity, which generates the second messenger cGMP. The TGFβ receptor forms heterodimers upon ligand binding and contains ligand-dependent serine-threonine kinase activity.
- (3) Cytokine receptors are part of a large class of receptors that also mediate the actions of growth hormone (GH) and leptin. Like growth factor receptors, this class contains a surface-exposed amino terminal domain that binds ligand, a single membrane-spanning domain, and a carboxyl terminal effector domain. They also function as dimers. However, cytokine receptors do not possess intrinsic enzymatic activity. Instead, liganded receptors associate with cytoplasmic tyrosine kinases (such as the Janus kinases; JAKs) that mediate downstream signaling events such as activation of associated transcription factors (signal transducers and activators of transcription; STATs) and cross-talk with kinase cascades.
- (4) Ligand-regulated transporters can bind ligands such as acetylcholine and respond by opening the channel for ion flow. In this case, the ion flux acts as the second messenger.
Other cell surface molecules resemble “receptors” but transport hormones into cells for degradation. Examples include the type C (clearance) ANP receptor and those for low-density lipoprotein (LDL), mannose 6-phosphate, and transferrin. Each of these proteins shares a short cytoplasmic domain with no known signal transduction function. Since most of these receptors are engaged in internalization by endocytosis and degradation of ligands, they are often called “transporters” rather than receptors.
Nuclear receptors mediate actions of steroid hormones, vitamin D, thyroid hormones, retinoids, fatty acids, bile acids, eicosanoids, xenobiotics, and other molecules. As described above, new nuclear receptor ligands are still being identified and new ligand specificities are being uncovered. Recent examples are the discoveries that the receptor HNF-4A binds tightly to 14-chain and 18-chain fatty acids and that the vitamin D receptor binds bile acids.
Nuclear receptors control gene expression by binding to either DNA response elements in the promoters of target genes or to other transcription factors. They then recruit large corepressor and coactivator complexes that modulate gene expression by modifying chromatin or contacting the basal transcription machinery. The DNA elements are specific sequences, usually a repeated hexanucleotide separated by a variable number of nucleotides aligned either as a direct repeat, a palindrome, or a reverse palindrome. These elements are termed hormone response elements (HREs). Nuclear receptor-protein interactions modulate the activities of heterologous DNA-bound transcription factors such as AP1, SP1, and NFκB.
Nuclear receptors have similar structures and functions. Each is composed of three domains that can act somewhat independently. The amino terminal domain is the most variable and mediates effects on transcription. The DNA-binding domain is well conserved and mediates HRE recognition and dimerization and contributes to modulation of heterologous transcription factor activity. The carboxyl terminal domain is also well conserved and mediates ligand binding, dimerization, and effects on transcription. The genomic sequence of several organisms is now available, and this permits an estimate of the numbers of nuclear receptor genes based on sequence homology. The human genome contains 48 distinct nuclear receptor genes all of which were previously identified by alternative approaches.
While nuclear receptors exhibit similar organization, there are subclasses that differ in details of their actions. Unliganded steroid receptors form inactive cytoplasmic complexes with heat shock proteins. Ligand binding promotes dissociation from the heat shock protein complex and formation of active receptor homodimers that translocate to the nucleus where they recruit coactivators. By contrast, unliganded thyroid hormone, retinoid, vitamin D, and peroxisomal proliferator-activated receptors bind tightly to chromatin, usually as heterodimers with the retinoid X receptor. Here, ligand promotes dissociation of corepressors and subsequent recruitment of coactivators with a resulting shift from repression to activation of gene expression at positively regulated genes. Other nuclear receptors exhibit variations on the standard structural organization. Steroidogenic factor-1, for example, contains a large hinge domain between the DNA-binding and putative ligand-binding functions, and this region has second messenger-regulated transcriptional activation properties. SHP (short heterodimer partner) consists of an isolated ligand-binding domain that represses the activity of other nuclear receptors and may work in the same way as a cofactor. Other nuclear receptors function as monomers.
Given that there are many transcription factors, it is surprising that there is only a single nuclear hormone receptor family. The aryl hydrocarbon receptor is a widely distributed transcription factor that binds xenobiotics, including man-made chemicals such as dioxin. This receptor is a member of a family of proteins that differ from nuclear receptors but also bind DNA. Although no endogenous ligand is known to bind these receptors, it is possible that natural ligands for the aryl hydrocarbon receptor and other nonclassic nuclear receptors will be identified. There are also complex interrelations between membrane receptors, nuclear receptors, and their ligands. Nuclear receptor ligands can exert rapid effects on the cell membrane. Some of these ligands interact with specialized membrane receptors. Thus, progesterone can antagonize the action of oxytocin by direct and highly selective binding to G protein-coupled membrane oxytocin receptors. However, in other cases, classic nuclear receptors (including estrogen, androgen, and progesterone receptors) interact with proteins at the inner surface of the cell membrane such as the tyrosine kinase Src and the p85 subunit of pI3-kinase, and initiate second messenger cascades. The estrogen receptor (ER) may also be able to interact with the cell membrane directly. In either case, the fact that a single ligand can influence events within the nucleus and at the membrane raises the possibility that these diverse events could exert concerted effects upon cellular function. Finally, nuclear receptors can be activated by second messenger signaling systems in the absence of the ligand. For example, the progesterone receptor can be activated by dopamine through phosphorylation.
HORMONE EFFECTS ON RECEPTOR ACTIVITY
The traditional view of hormone receptor action is that the receptor is inactive in the absence of hormone and that hormone activates the receptor. In this sense, the receptor is often described as an “on-off” switch. However, this simple model does not always account for the full spectrum of hormone effects on receptor activity. As mentioned above, some nuclear receptors are active in the absence of ligand. Thyroid hormone and retinoic acid receptors bind to DNA in the absence of ligand and actively repress transcription of nearby genes. Ligand promotes release of corepressors and recruitment of coactivators and, consequently, promotes simultaneous relief of inhibition and further activation of transcription. The unliganded thyroid hormone receptor can also activate negatively regulated genes, including that of TSH. Here, ligand reverses the activation that is obtained with the unliganded receptor and suppresses transcription below basal levels. Thus, thyroid hormone and retinoic acids are more properly described as altering the spectrum of receptor activities rather than turning the receptor on or off in the traditional sense. Other receptors can be active in the absence of ligand and either inactive or less active in the presence of ligand. Examples from the nuclear receptor family include constitutive androstane receptor (CAR) and the estrogen-related receptors (ERRs). Among cell surface receptors, the melanocortin receptors (MCRs) may be either stimulated by agonists or inhibited by natural antagonists.
Even if a hormone does promote a true transition from an inactive receptor state to an active state, it need not always elicit maximal agonist responses. Different peptides that bind to the growth factor receptors can show qualitatively different effects on downstream signaling events. ER activity and cofactor binding is dependent on the DNA sequence of the hormone response element or upon whether receptor acts through classic DNA binding sites or through heterologous transcription factors, such as AP1 (see sections on partial agonistand mixed agonist-antagonist activity, below).
CLASSES OF HORMONE ACTION
Hormones and hormone analogs can be classified using two criteria (Figure 1-5). The first utilizes the receptor through which ligand acts. The second utilizes the activity the ligand elicits (agonist, antagonist, etc). Thus, a compound that produces estrogenic effects on breast through ER is an ER agonist. Conversely, a compound that binds to the ER and blocks binding of estrogens but does not allow the receptor to adopt a functionally active state is said to be an ER antagonist.
CLASSIFICATION OF HORMONE ACTION BY RECEPTOR TYPE
Traditionally, hormones were classified according to effects. Glucocorticoids were named for carbohydrate-regulating activities, mineralocorticoids for salt-regulating activities, and pituitary hormones for various tropisms. This nomenclature can pose problems. The effects of a particular hormone that are recognized first might represent a subset of its primary effects. For example, glucocorticoids, named for their glucose-regulating activities, also have widespread anti-inflammatory effects and can influence abdominal fat deposition. Furthermore, several different hormones exert the same
effects through the same receptor (Figure 1-5 and Figure 1-6). For example, both the “glucocorticoid” cortisol and the“mineralocorticoid” aldosterone regulate mineral metabolism by interactions with mineralocorticoid receptors. Two hormones can also have the same effect through interactions with different receptors. Both glucocorticoids and insulin promote glycogen deposition. Finally, the same hormone can act through more than one receptor with different physiologic consequences. Prostaglandins and progesterone can act through distinct cell surface and nuclear receptors.
Figure 1-5. Classification of actions of ligands that interact with hormone receptors. Shown are examples of different types of ligands with classification of the type of ligand and the receptors through which they interact. (GRE, glucocorticoid response element; AP1, activating protein 1; MRE, mineralocorticoid response element; PRE, progesterone response element; ERE, estrogen response element; ARE, androgen response element.)
Other hormones were named for the endocrine gland (eg, parathyroid hormone). This classification can also be confusing since most glands produce multiple hormones and some hormones are made in more than one site. For example, progesterone is made in both the placenta and the ovaries.
Figure 1-6. Possible pathways of transmission of hormonal signals. Each hormone can work through one or more receptors; each hormone-receptor complex can work through one or more mediator proteins; and each mediating protein or enzyme activated by hormone-receptor complexes can affect one or more effector functions.
One way to avoid confusion is by utilizing the receptor for classifying hormone action (Figure 1-5). This method of classification was used by pharmacologists even before receptors were proved to exist. For example, the classification of catecholamine actions through the α- and β-adrenergic receptors is familiar to clinicians and scientists. This receptor-based method of classification has the advantage that it acknowledges the spectrum of receptor-mediated actions while preserving the historical name. By this system, most actions of cortisol are mediated through glucocorticoid receptors, but cortisol actions through mineralocorticoid receptors are termed a mineralocorticoid action of cortisol.
CLASSIFICATION OF HORMONE ACTION BY LIGAND TYPE
Classification of ligands as agonists, partial agonists-partial antagonists, antagonists, or inactive compounds has been widely utilized. Classification of mixed agonist-antagonist compounds and inverse agonists accounts for new information about mechanisms of action of various ligands whose properties do not fit the other categories. The effects of these compounds vary
in different tissues as well as with respect to the factors that interact with hormone-responsive genes. Thus, the same compound in one tissue or context can act differently in another context.
Inactive compounds are those that do not bind to receptors and have neither agonist nor antagonist activity.
An agonist binds to a receptor and transforms binding into a response. Most naturally produced ligands are agonists. However, synthetic hormone analogs may have more potent activity than the natural hormone. Examples include synthetic glucocorticoids such as prednisone, dexamethasone, and triamcinolone that are used to suppress inflammatory and immunologic responses (Chapter 9). It should be noted that some agonists can exert so-called inverse agonist effects—ie, they inhibit activity of active unliganded receptors.
An antagonist binds a receptor but does not transform binding into a response. The antagonist usually competes for agonist binding and thereby prevents agonist actions, defining the term antagonist. The body produces nuclear receptor antagonists, but these usually circulate at levels too low to be effective. For example, progesterone can act as a mineralocorticoid or glucocorticoid receptor antagonist but interacts with each of these receptors with low affinity. Normal progesterone concentrations are too low for the steroid to occupy substantial numbers of either receptor. However, synthetic nuclear receptor hormone antagonists are clinically useful. Examples include the antiestrogens tamoxifen and raloxifene and the antiprogestin and antiglucocorticoid mifepristone (RU 486). In contrast, there are examples of natural high-affinity antagonists for cell surface receptors. The melanocortin 4 receptor (MC4R), which is expressed in hypothalamus and regulates feeding behavior, binds α-MSH, a natural agonist, and agouti-related peptide, a natural antagonist. The balance of α-MSH and agouti-related peptide is affected by hormones such as leptin and factors related to feeding and energy storage, such as fasting behavior. This balance, in turn, dictates the direction of MC4R activity.
Most hormone antagonists compete with the agonist for the hormone binding site and are referred to as competitive antagonists. However, it should be possible to identify other types of antagonists. Noncompetitive antagonists of hormone response could prevent the allosteric alteration that signals ligand binding or could block interactions with downstream cofactors that mediate the signal. For example, efforts are being made to develop drugs that mimic the peptide interactions between nuclear receptors and their downstream coactivators.
Partial Agonist-Partial Antagonists
Partial agonists or partial antagonists bind to receptors and yield a response that is less than that of a full agonist at saturating ligand concentrations. A partial agonist will block binding of a full agonist and suppress receptor activity to the level induced by the partial agonist alone, thereby justifying its “partial antagonist” designation. Some of these compounds are naturally produced, although—as in the case of antagonists—the occupancy of receptors has not been shown to be important in normal circumstances. However, many plant estrogens (phytoestrogens), such as genistein, may behave as partial ER agonists; it has been speculated that this property is related to the apparent protective effect of these compounds on breast cancer incidence.
These compounds act in different ways through the same receptor type depending on the context (which cells, which promoter, etc). As an example, the estrogen “antagonists” tamoxifen and raloxifene act mostly as antagonists in breast but have estrogen agonist actions in bone and uterus. This property has been exploited clinically, since the effects on both breast and bone are useful.
Ligands with Reverse Pharmacology
This classification has been used to describe ligands that exert agonist effects which are completely distinct from those of the native ligand. For example, when estradiol binds to ERβ there is little or no effect at genes with AP1 sites, whereas tamoxifen and raloxifene show potent stimulatory effects at these sites.
MECHANISMS OF LIGAND ACTION
The mechanisms of agonist and antagonist hormone action are addressed in Chapter 3. Although there are exceptions, as discussed earlier, unliganded receptors are commonly in an inactive conformational state. When agonists bind to these receptors, they induce conformational changes that transduce postbinding information into responses. These changes result in alterations of the complement of receptor associated proteins, alterations in receptor enzymatic activity, or
modifications of the receptor (such as phosphorylation and ubiquitination).
REGULATION OF HORMONE RESPONSIVENESS
Hormone response depends on the presence of a response system and the availability of hormone. A common mechanism for ensuring specificity of hormone receptor action is to restrict receptor expression. However, even in the absence of effects that restrict receptor expression to particular contexts, hormone responsiveness of a particular tissue or cell type to a particular hormone is not fixed (Figure 1-7). As discussed in a later section on disorders of the endocrine system, alteration of responsiveness to hormones is a major factor in disease. The best-known example is type 2 diabetes mellitus, where insulin resistance plays a prominent role. The factors that regulate overall hormone responsiveness are reviewed in the next sections.
HORMONE RESPONSES CAN BE SELF-LIMITING
Continuous hormone stimulation does not always result in a continuous response. Instead, hormone responses are often self-limiting. Chronic stimulation of cells with peptide hormones can decrease the amounts of receptor that are expressed on the cell surface. Immediate postreceptor signaling events can also exert feedback inhibition of hormone response by decreasing receptor activity. For example, phosphorylation of the β-adrenergic receptor can foster interactions with a protein (arrestin) that locks the receptor in an unresponsive state (Chapter 3). While acute cell surface signaling events are rarely regulated at the transcriptional level, hormone stimulation usually results in induction of genes that mediate the response to the original signal (feed forward) and genes that limit the original signal (feed back). The duration of membrane signaling events can have important functional consequences for cell fate. Acute stimulation of MAP kinase cascades in nerve cells can enhance cellular proliferation, whereas chronic stimulation inhibits cellular proliferation and promotes differentiation.
Figure 1-7. Regulation of hormone responsiveness by homologous hormone-receptor complexes can occur at multiple loci. Shown are feedback loops that regulate responsiveness through effects on the receptor, effector, or response limbs in any of the elements of the response network. The plus or minus signs indicate that the influences can be stimulatory or inhibitory.
Nuclear receptor responses can also be self-limiting. Ligands often exert transient effects on gene transcription that peak a few hours after the initial signal and then diminish. In some cases, this may be due to ligand-induced degradation of the receptor. Alternatively, enzymatic activities in the fully assembled nuclear receptor coactivator complex can inhibit the receptor-coactivator interaction.
Finally, far downstream effects of hormone action can feed back and influence the response to the original hormone. One example is the effect of insulin on blood sugar levels, which in turn influence insulin action. Hormone response can also be influenced by counterregulatory hormones. For example, in stress, the rise in glucocorticoids mitigates the deleterious effects of other hormones (Chapter 9). Occasionally, these effects can feed back to enhance the primary hormone response.
DIFFERENT SIGNALS INFLUENCE HORMONE RESPONSIVENESS
Hormone response networks are subject to multiple regulatory inputs, including endocrine, paracrine, autocrine, and juxtacrine signals; central nervous system inputs; and cell-cell contacts. Different hormones and other signals can interact in different ways (Figure 1-6). In some cases, different hormones bind the same receptor. Thus, insulin and the insulin-like growth factors (IGFs) both bind the IGF receptor, and glucocorticoids and mineralocorticoids bind to each other's receptors. More commonly, different signals interact downstream or upstream of the receptor. In general, these interactions can be classified into four groups:
- (1) Cell surface signals stimulate common or interacting second-messenger systems that modulate activities
of the homologous receptor or other receptors or downstream signal transduction systems or alter expression of genes that encode cell surface receptors or their signal transduction proteins.
- (2) Nuclear receptors modulate each others' expression or activities by direct interactions (such as heterodimerization) or by interactions through common cofactors (such as shared coactivators or corepressors). Alternatively, nuclear receptor activities can converge at the level of the same response element, gene, or heterologous transcription factor complexes.
- (3) Cell surface receptors activate second-messenger cascades that lead to phosphorylation of the nuclear receptors themselves, their coactivators and corepressors, or transcription factors that cooperate with the nuclear receptors.
- (4) Nuclear receptors regulate expression of genes that encode cell surface receptors or components of their downstream signaling pathways.
Together, these schemes for cross-talk allow for wide scope in combinatorial interactions between signaling systems. Combinatorial interactions between signaling systems represents a critical component of endocrine control and can be synergistic, additive, or antagonistic. Figure 1-8 illustrates a hypothetical synergistic hormone response between different receptors. The response to individual hormones is small, but the combination produces a response that is greater than additive. Combinatorial interactions could modulate behavior of an entire response network or particular hormone responses. For example, signal transduction events that lead to phosphorylation of nuclear receptors can potentiate nuclear receptor activity in a wide variety of contexts. However, signal transduction events that lead to phosphorylation of a transcription factor which cooperates with nuclear receptors might only selectively amplify hormone responses in the context of a limited number of promoters.
Figure 1-8. Schematic representation of a synergistic hormone response. Note that in this case neither hormone has a major effect alone.
MODULATION OF HORMONE LEVELS
Hormone response is regulated by hormone concentration. This in turn is governed by hormone production, efficiency of delivery, and metabolism. In many cases, hormones and their actions have short half-lives. Thus, response can be rapidly initiated or terminated by modulating hormone concentration. The following sections review the processes that determine hormone concentration, delivery, and intracellular levels.
Details of synthesis of individual hormones are provided in Chapter 2 and in the individual chapters devoted to them. Protein hormone production often does not require special machinery. Thus, growth hormone, prolactin, and PTH are produced similarly to other secreted proteins. However, some peptide hormones (eg, insulin, ACTH, CRH, and glucagon) are produced by cleavage of a larger protein. In these cases, peptide production may be dependent on specific proteases. Other hormones with unique structures are generated by specialized enzymes. Thyroid hormone is produced by iodination and coupling of tyrosine residues contained in a large protein, thyroglobulin, whereas catecholamine production involves modifications of phenylalanine. Steroid hormones are produced from cholesterol in a series of reactions that involve cleavage of the cholesterol side chain residues to yield pregnenolone, followed by a variety of modifications that include hydroxylations, more cleavage reactions, and modification of the ring structures.
REGULATION OF HORMONE PRODUCTION
Hormone production can be regulated at the level of transcription, at the level of posttranscriptional mechanisms that modulate messenger RNA levels or translation efficiency, and at the level of release. Transcriptional regulation can be seen, for example, at polypeptide hormone genes or enzymes involved in steroid synthesis. Control of hormone release can be
seen in cases in which secretagogues stimulate the release of stored peptide hormones. Finally, hormone production can be augmented by the effects of tropic hormones or growth factors on endocrine cells, which increases the number and size of cells that are actively producing the hormone.
There are a number of patterns of regulation of hormone release (Figure 1-9). Many hormones are linked to the hypothalamic-pituitary axis (discussed in the section on neuroendocrinology, below). Here, hormones that are produced in the hypothalamus and pituitary (ACTH, TSH, etc) enhance production of hormones in peripheral glands (cortisol, thyroid hormone, etc). Production of hypothalamic and pituitary hormones is subject to negative regulation by peripheral hormones in classic feedback loops. Other systems are more freestanding. Parathyroid hormone (PTH) increases plasma Ca2+ concentration, and this exerts a dominant feedback inhibition on the release of PTH by binding Ca2+ sensor receptors in the membrane of PTH-producing cells (Chapter 8). Insulin production leads to decreased glucose levels, and this effect leads to cessation of the stimulus to release more insulin. Hormone release can be triggered by inputs from the nervous system. Hormone production is regulated by all types of regulatory molecules, including tropic hormones and counterregulatory hormones (discussed below), traditional growth factors, eicosanoids, and ions.
Figure 1-9. The two major types of control of endocrine gland function. I: The hypothalamic-pituitary-target gland systems involve central nervous system regulation of releasing hormones from the hypothalamus that stimulate the pituitary to release tropic hormones which act on peripheral glands to release hormones. These hormones can be regulated by other factors. The hormones from the peripheral glands exert feedback control on the hypothalamus and pituitary. II: Free-standing endocrine glands (eg, parathyroid and islet cells) release hormones that stimulate a target tissue to produce an effect (eg, a rise in serum calcium or a fall in blood sugar) which in turn modifies the function of the gland.
HORMONE TRANSPORT IN THE CIRCULATION
Most peptide hormones circulate at low concentrations and are not bound to other proteins. Exceptions include growth hormone, which binds to a protein identical to the hormone-binding portion of the growth hormone receptor; IGF-I and IGF-II, which bind to a variety of IGF-binding proteins (Chapter 6); and vasopressin and oxytocin, which are bound to neurophysins (Chapter 5). By contrast, circulating steroids, thyroid hormones, and vitamin D are bound to plasma proteins (Figure 1-10). The major plasma binding proteins are CBG, which binds cortisol and progesterone (Chapter 9); SHBG, which binds testosterone and estradiol (Chapter 12 and Chapter 13); thyroid hormone-binding globulin (TBG) (Chapter 7); and vitamin D-binding protein (Chapter 8). Hormone binding to plasma proteins occurs through noncovalent interactions and increases the half-life of the hormone in the circulation.
The free hormone is that which is not bound by plasma proteins. This fraction is available for receptor binding, dictates feedback inhibition of hormone release, is that which is cleared from the circulation, and correlates best with clinical states of hormone excess and deficiency. Thus, for some clinical tests (discussed later) the best measurement is the free hormone.
Generally, transport proteins bind most soluble circulating hormone, such that the free hormone is a small portion of the total. In general, the binding capacity of transport proteins barely exceeds the normal concentrations of the hormone in plasma. Thus, modest decreases in transport protein levels or modest elevations of hormone concentrations can lead to large increases in free active hormone. Thus, whereas the presence of low or high levels of hormone transport proteins generally does not itself lead to clinical abnormalities, these changes can influence interpretation of laboratory tests. One likely role of transport proteins is to facilitate even delivery of hormones across target tissues (Figure 1-10).
Thus, a free hormone might be completely sequestered in the proximal portions of the liver as the blood flows through the tissue. By contrast, if the hormone were bound to transport proteins, free hormone would be sequestered in proximal portions of the liver, and additional hormone would be released from the bound fraction as the blood moves distally, making the hormone available for these regions. Differences in the uniformity of hormone delivery probably explain why deletion of the plasma vitamin D-binding protein affects vitamin D action.
Figure 1-10. Role of plasma binding in delivery of hormones to peripheral tissues. Shown are examples with a hormone that is bound (solid circles) to a plasma protein (large circles) and a hormone that is not bound (open circles). With the bound hormone, only the free fraction is available for tissue uptake. As the free fraction is taken up, additional hormone dissociates from the plasma-binding protein as the blood moves to more distal portions of the tissue and becomes available for tissue uptake. In contrast, all of the hormone that does not bind to plasma proteins is available for uptake by the proximal part of the tissue.
TRANSPORT OF HORMONES ACROSS THE MEMBRANE
Nuclear receptor ligands are hydrophobic and are often presumed to enter and exit the cells by traversing the lipid bilayer. However, in some cases influx and efflux varies in different tissues and cell types, as has been demonstrated for thyroid hormones, pointing toward active import and export mechanisms. Specific differences in transport in target cells may regulate hormone concentration and action.
METABOLISM & ELIMINATION OF HORMONES
Metabolism can degrade the hormones and hormone precursors to inactive forms or lead to the generation of more active products (Figure 1-11). These processes affect the plasma levels of the hormone and can have regulatory roles.
Peptide hormones have short half-lives in the circulation (a few minutes), as seen with ACTH, insulin, glucagon, PTH, and the releasing hormones. The glycosylated glycoprotein hormones are more stable; CG has a half-life of several hours. The major mechanism for hormone degradation is through binding to cell surface receptors and nonreceptor hormone-binding sites, with subsequent uptake (internalization) and degradation. An important source for these enzymes is the lysosome, which fuses with endocytotic vesicles to expose its enzymes and its acid environment to the internalized hormone-receptor complex.
Steroid & Thyroid Hormones & Vitamin D
The hydrophobic steroid hormones and the D vitamins are filtered and reabsorbed by the kidney. For instance, 1% of the cortisol that is produced daily appears in the urine (Chapter 9). These compounds are ordinarily metabolized to water-soluble, inactive forms that are more effectively eliminated. Metabolic inactivation generally involves reduction and conjugation to glucuronide and sulfate groups.
Intracellular degradation of ligands provides a means both for eliminating the hormones and for exerting tissue-selective control of hormone levels. For example, cortisol would completely occupy the mineralocorticoid receptor in the kidney if it were not inactivated by 11β-hydroxysteroid dehydrogenase. This process allows aldosterone, which is not affected by the enzyme, to act as the major mineralocorticoid in this tissue. Thyroid hormones are degraded to inactive forms by at least three different deiodinases whose levels vary in different tissues (see Chapter 7). Deaminations and decarboxylations of the alanine side chains as well as conjugations with glucuronic acid and sulfate groups are also involved in degrading thyroid hormones.
Metabolism of prohormones to active forms also plays an important role. Testosterone is reduced to dihydrotestosterone in target tissues. 3,5,3′,5′-Tetraiodo-L-thyronine (thyroxine; T4) is the major form of thyroid
hormone released from the gland, but T4 becomes deiodinated to the more active 3,5,3′-triiodo-L-thyronine (triiodothyronine; T3) in peripheral tissues. The major active form of vitamin D is generated by sequential hydroxylation reactions that occur in the liver and kidney.
Figure 1-11. Hormone metabolism. Shown are various pathways along which hormones are metabolized and the effects on those pathways with production of additional precursors; with production of more active hormones for local or systemic actions; with selective degradation of hormones to prevent local actions; and with degradation to inactive forms that are eliminated.
Finally, hormones can be produced locally in target tissues. Thus, testosterone can be produced from androstenedione and dehydroepiandrosterone, and estradiol can be produced from testosterone. These mechanisms can provide for high local concentrations of hormones, thereby providing additional control of the hormone response.
Catecholamines & Eicosanoids
Metabolism of catecholamines is discussed in Chapter 11. These compounds have a very short half-life of about 2 minutes. Catecholamines are degraded by two principal routes: catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO). Measurement of some of the metabolites—normetanephrine, metanephrine, and vanillylmandelic acid (VMA)—can be useful in evaluating possible catecholamine overproduction. Prostaglandins are also rapidly metabolized—within seconds—by widely distributed enzymes, particularly through oxidation of the 15-hydroxyl group that renders the prostaglandin molecule inactive.
RECOMBINANT DNA, GENOMICS, PROTEOMICS, & ENDOCRINOLOGY
The cloning of endocrine system genes has had an enormous impact on endocrinology. This impact will increase with the information provided from sequencing of the human genome. Some contributions of recombinant DNA technology are listed in Table 1-1.
Recombinant DNA makes possible the production of therapeutic agents such as GH and insulin and of diagnostic materials such as polypeptide hormones. More importantly, it has provided information about the function of the endocrine system in health and disease. Elucidation of mechanisms that regulate gene expression has provided insights into mechanisms of hormone action (see Chapter 3) and regulation of the entire system, as detailed in subsequent chapters of this book.
Table 1-1. Impact on endocrinology of molecular biology, recombinant DNA technology, and sequencing of the human genome.
Recent research has emphasized larger-scale approaches to understanding of basic biological phenomena. These are loosely termed genomics and proteomics for their emphasis upon the genome and its expression pattern (genomics) and protein expression and function (proteomics), respectively. These approaches capture larger overall pictures of the behavior of cellular signaling pathways rather than focusing on particular genes or proteins.
SEQUENCING OF THE HUMAN GENOME
Completion of the human genome sequence will have an enormous impact on endocrinology. It is now clear that there are about 30,000 genes in humans and that further diversity is generated by alternative RNA processing or alternative promoter usage. The data tell us conclusively, for example, that there are 48 nuclear receptors in humans (discussed earlier) as opposed to previous estimates, which ranged up to 600. Readily available sequence data facilitate studies of gene regulation or of sequences that become altered in disease. Comparison of sequence data from different species will provide clues about altered regulation of endocrine signaling pathways in evolution.
GENOMICS & PATTERNS OF TRANSCRIPTION
In addition to understanding the structure of the genome, genomics can be applied to larger-scale understanding of genome transcription. Most studies use so-called microarray technologies in which small glass or silicon slides are imprinted with cDNAs or oligonucleotides corresponding to a few selected (less than 100) or many (over 20,000) genes. The microarrays are probed with labeled cDNA corresponding to RNA from a given cell line or tissue by conventional DNA hybridization approaches. The information can be used to identify individual genes—or gene families—that may be essential for particular endocrine responses and will help elucidate the functional linkages between different genes that might identify new targets for drug discovery. The technology can also be used to understand the way a particular signal influences the overall patterns of expression of related clusters of genes (cell cycle, metabolic pathways, stress response, etc), which can be of great predictive value. For example, malignancy and hormone responsiveness of breast tumors can be typed according to the overall expression profile of their genes. Coexpression of ER and the cell surface receptor HER2/neu, coupled with amplification of the gene for the coactivator protein AIB1, is predictive of tamoxifen resistance in breast tumors. Other patterns of gene expression in breast tumors may suggest whether more or less aggressive therapies are appropriate. It is likely that analysis of transcription patterns in different tumors will become part of standard clinical diagnosis for tumor biopsies in the future and dictate treatment strategies.
Ultimately, understanding of biologic responses will require a complete understanding of patterns of protein expression, their interconnections, and their activity. To some extent, protein expression patterns can be inferred from the pattern of transcription. However, gene expression studies do not assess the role of factors that govern translation rates or protein stability. Moreover, gene expression studies do not elucidate the functional connections between proteins or alterations in protein activity (which can be influenced by subcellular localization, secondary modifications, etc). For example, regulation of enzymatic activity, protein localization,
and stabilization are particularly important in rapid response to peptide hormone signals.
Proteomics covers large-scale approaches that capture the expression patterns and activity states of proteins in a given context. Large-scale yeast two hybrid assays can be used to obtain descriptions of potential protein-protein interactions between many different gene products. Large scale mass spectroscopy of cellular protein preparations can identify the proteins that are expressed in a given context. When this technique is coupled with fractionation of proteins in subcellular compartments—or fractionation according to the biochemical properties of proteins or their particular protein-protein interactions—it is possible to begin to gain insight into the ways in which protein expression and activity bring about changes in endocrine signaling pathways. For example, it may be possible to observe accumulation, down-regulation, or nuclear translocation of subsets of proteins in response to a particular signal. Targeted analysis of proteins in signaling pathways (eg, banks of phosphoprotein-specific antibodies for molecules participating in the insulin or ERK signaling pathways) can also be applied to detect the alterations in activity of subsets of response pathways in given contexts. Like functional genomics, the results of these approaches can identify individual proteins that may be important for a particular endocrine signal or may give an overall picture of the pattern of protein activity alteration in a given endocrine signal.
MOLECULAR BASIS OF GENETIC ENDOCRINE DISEASE
Many endocrine diseases have a strong genetic component. This applies to rare inherited syndromes that are often the result of a single monogenic mutation, such as defects in steroid biosynthesis (Chapter 9, Chapter 12, Chapter 13, Chapter 14, and Chapter 15); and mutations in hormone genes, such as GH (Chapter 6). Analysis of inherited monogenic diseases has been greatly facilitated by linkage maps of every human chromosome and by human DNA sequence data, which helps track a particular disease-causing allele through affected families. More common diseases also have genetic components, usually thought to result from the interplay of common variant proteins (polygenic disease). This applies to hypertension (Chapter 10) and type 2 diabetes mellitus (Chapter 17). The genetic basis of polygenic diseases is often less clear than that of monogenic diseases. This is because particular genetic variants may be associated with—but neither necessary nor sufficient for—a disease state. Accordingly, large studies of affected populations are required to achieve sufficient statistical power to elucidate the contributions of individual genes. Future linkage studies of polygenic diseases may be facilitated in two ways: first, by the realization that much human genetic variation is actually relatively common; and second, by our understanding that long stretches of human DNA exist in so-called haplotype blocks. These long stretches of chromosomal DNA are areas in which very little recombination occurs. Relatively few common haplotypes may exist within each block, and understanding how haplotype blocks are inherited within populations may help to highlight the areas of the genome that contribute to the risk of particular diseases.
The mechanisms underlying genetic disease are discussed in the chapters that follow. Components of the endocrine system can be affected by a wide range of genetic defects. Single nucleotide substitutions alter the coding sequence or create a translational frame shift or premature stop codon. Examples can be seen with loss of mutated polypeptide hormones such as insulin or growth hormone; transcription factors such as Pit-1, which regulates GH expression; or hormone receptors, as in the testicular feminization syndrome (Chapter 14). Point mutations can also lead to gain of function; a point mutation in the mineralocorticoid receptor increases its binding to progesterone and leads to a rare syndrome of pregnancy-associated hypertension. Simple mutations can also affect noncoding sequences that play a role in gene regulation. Larger deletions of all or parts of genes usually result in a complete loss of function, as with deletions of polypeptide genes such as growth hormone (Chapter 6), hormone receptors, or enzymes involved in hormone synthesis. The latter can be observed in congenital adrenal hyperplasia (Chapter 9, Chapter 13, Chapter 14, and Chapter 15). Amplifications of genes, particularly in cancer, can lead to increased gene activity. There can also be insertions or rearrangements between genes. The latter may lead to profound alterations in cell function. For example, rearrangement of growth factor genes can place them under control of a different gene promoter in some malignancies (Chapter 21). It is also observed in a form of glucocorticoid-remediable hypertension where the enzyme that promotes production of aldosterone is placed under the regulatory control of another gene that is regulated by ACTH (Chapter 10).
EVOLUTION OF THE ENDOCRINE SYSTEM
Consideration of the evolution of the endocrine system can help us understand endocrine signaling. Any account of the evolution of the endocrine system must explain the origin of signaling molecules, how the signal came to convey specific information, and how signal transduction proteins arose. At a more complex level,
we must explain diversity of signaling pathways, development of endocrine organs, and complex regulatory networks. Speculations about the evolution of endocrine signals are outlined in the following sections. A schematic representation of events in evolution of the endocrine system is provided in Figure 1-12.
THE ORIGINS OF REGULATORY CHEMICALS & THE METABOLIC CODE
The interaction of hormones with cellular receptors leads to intracellular communication events such as generation of cAMP, phosphorylation, and effects on gene expression or ion transport. In higher organisms, the responses are governed by endocrine, paracrine, juxtacrine, or autocrine signals, whereas similar responses are governed by extracellular signals in bacteria. The common thread is that the cell perceives extracellular signals and utilizes intracellular control networks to respond to the signal. It is likely that the endocrine system had its origins in these types of bacterial signaling systems.
Bacterial regulatory chemicals are typically modified analogs of essential molecules, as noted by Tomkins over 25 years ago in an article entitled “The Metabolic Code.” Tomkins suggested that it would have been impossible for essential molecules to acquire signaling properties in the course of evolution. Thus, ATP functions as an essential mediator of energy balance and would have been unlikely to acquire signaling properties because large variations in ATP concentration would adversely affect cellular function. However, by-products of ATP metabolism could acquire signaling functions. For example, cAMP or related molecules may have been originally generated as by-products of ATP metabolism, such as “idling” of ATP hydrolysis to ADP. Thus, cAMP generation could act as a signal for the idling reaction—and, in turn, for the cellular condition that leads to the idling reaction.
Figure 1-12. Steps in the evolution of the endocrine system, with duplications and mutations of genes that encode essential enzymes and other products that result in new genes that encode products involved in endocrine control.
Proteins transduce regulatory signals and must recognize the signaling molecule and initiate appropriate biologic responses. How could such a multifunctional protein evolve? Proteins consist of a limited number of discrete folded structures or domains, and recombination events can splice diverse domains together and create new functional units. In this regard, the response limb for cAMP in bacteria is a transcription factor whose activity is affected by cAMP binding. This could have arisen, for example, by combining a cAMP-binding protein with a DNA-binding protein. If the new hybrid protein could regulate gene expression—even relatively nonspecifically—then a novel regulatory circuit would have arisen, and if resultant changes in gene expression were favorable to cell survival under the original conditions that led to cAMP generation, then the novel regulatory circuit would become fixed in evolution.
Once molecules like cAMP acquire the capacity to function as a signaling molecule, mechanisms would evolve to generate cAMP more efficiently and ultimately, cAMP could acquire the capacity to become produced and regulated independently of ATP production or hydrolysis, thereby giving the organism flexibility to use the same regulatory system in metabolic control. Indeed, Tomkins referred to cAMP as a “symbol” that is produced in response to a “signal”—glucose deprivation—by bacteria. The symbol cAMP in turn induces enzymes that metabolize alternative substrates such as lactose to overcome the deficiency of glucose.
EVOLUTION OF HORMONES & RECEPTORS
Many hormones are by-products of metabolic reactions that acquired the capacity to symbolize metabolic states of the cell, as described above for cAMP. Thus, peptides are specific breakdown products of larger proteins; steroids are derived from cholesterol and catecholamines; and thyroid hormones are derived from amino acids. Other nuclear receptor ligands are by-products of bile acids and fatty acids. The regulatory roles of these by-products suggest that nuclear receptors
may have first evolved as nutritional sensors and provides support for the generality of the metabolic code.
Primitive endocrine signaling probably arose in early multicellular organisms, where one cell would sense changes in the environment and release signaling molecules, where they would elicit responses in neighboring cells (ie, paracrine signaling). Eventually, this type of signal would permit specialization of cell types that produce the signal or respond to the signal with an appropriate biologic response that would ensure the survival of both cells. Once the primordial hormones—the precursors to today's hormones—were generated, modifications could create additional properties such as specific mechanisms to regulate production, secretion pathways, increased specificity for receptor binding, bioavailability (including binding to transport proteins), and degradation and clearance.
Hormone receptors probably arose by recombining essential functional proteins, as described above for cAMP signaling. In this regard, it is noteworthy that both cell surface and nuclear receptors are composed of discrete functional domains that could have been linked by gene recombination events and acquired novel emergent properties.
EVOLUTION OF MULTIPLE HORMONE RESPONSE SYSTEMS
Once a prototypical hormone response system is in place, it can be expanded to interpret related signals. The genetic events that lead to expansion of signaling capacity would probably resemble those described in the section on genetic disease, ie, gene duplications, rearrangements, recombinations, and specific mutations. The duplications would create related gene families, and subsequent mutational and other events would generate diversity. Novel patterns of gene expression could be created by alteration of the promoter regions of genes, thereby permitting further generation of specialized functions. The peptide hormone genes for GH, PRL, and placental lactogen (chorionic somatomammotropin; CS) genes comprise one family that arose from gene duplication. The glycoprotein hormones share the same α subunit and have different β subunits (albeit with significant homology). An example of a family of genes involved in hormone synthesis are some of those encoding proteins involved in steroid hormone biosynthesis. Examples of receptor families are the seven-transmembrane receptors and nuclear receptors. Indeed, x-ray crystal studies of nuclear receptor family ligand-binding domains reveal that each possesses a similar fold despite wide variations in primary sequence. This suggests that the basic function of the primordial ligand-binding domain has been preserved despite sequence alterations that generate new ligand specificities.
EVOLUTION OF ENDOCRINE GLANDS
Endocrine glands must have evolved from the specialization that resulted in organ systems with a need to carry fluids between them. By default, the primitive cell that released a substance acting in a paracrine way became an endocrine cell. This provided a means for delivery of substances more generally than with the nervous systems, which developed axons to deliver a paracrine signal.
Hormones do not usually regulate a single function and instead elicit coordinated responses in a range of cell types. Furthermore, different hormones play complementary or counterbalancing roles. How might these complex circuits evolve? Once a hormone regulates a given response—eg, glucose metabolism—there would be selective advantages in acquiring the capacity to regulate complementary regulatory processes. Thus, glucocorticoids regulate glucose metabolism and increase glucose production and glycogen storage in response to an “anxiety” stimulus in preparation for starvation, but they have also incorporated complementary actions on lipid and amino acid metabolism. Such networks, once established, would be relatively stable but could become modified and expanded. Hormones with counterbalancing actions could then develop in an analogous fashion.
ENDOCRINE & NERVOUS SYSTEM RELATIONSHIPS: NEUROENDOCRINOLOGY
Neuroendocrinology is the subject area that deals with interactions between the nervous and endocrine systems. The actions of both systems and their interactions underlie practically every regulatory mechanism in the body. There are two major mechanisms of neural regulation of endocrine function. The first, neurosecretion, refers to neurons that secrete hormones into the circulation. The hypothalamus contains neurons that secrete hormones into the general circulation or to blood vessels that communicate with the anterior pituitary. This mechanism is discussed in the sections that follow. The
second is direct autonomic innervation of endocrine tissues, which couples central nervous system signals to hormone release. Examples of this relationship include innervation of the adrenal medulla, kidney, parathyroid gland, and pancreatic islets and are described in chapters on these organs (see Chapter 8, Chapter 10, Chapter 11, and Chapter 17).
Hormones also affect the nervous system, as described in the section on effects of hormones, below.
The primary neuroendocrine interface is at the hypothalamus and pituitary, which form a unit that controls several peripheral endocrine glands and other physiologic activities (Chapter 5). The hypothalamus contains several nuclei of neuronal cells. It communicates with other brain regions and regulates many brain functions, including temperature, appetite, thirst, sexual behavior, defensive reactions such as rage and fear, and body rhythms. However, the hypothalamus is also an endocrine organ that releases hormones. The ventral hypothalamus supplies axons and nerve endings to form the posterior pituitary. Neurohypophysial neurons in the posterior pituitary release vasopressin (ADH) and oxytocin into the general circulation. Hypophysiotropic neurons of the hypothalamus release hormones into the hypothalamic-pituitary blood vessels, which deliver hormones to the anterior pituitary—a major endocrine organ.
The hypophysiotropic neurons of the hypothalamus produce hormones that are secreted into blood vessels which serve the anterior pituitary and regulate hormone release. Stimulating hormones (releasing hormones) include TRH, GnRH, CRH, GHRH, prolactin-releasing factor, and ADH. Inhibitory hormones include somatostatin and dopamine. Some releasing hormones can regulate multiple hormones. For example, TRH stimulates both TSH and prolactin release. In contrast, more than one releasing hormone can affect a single pituitary hormone. ACTH release is stimulated both by CRH and by ADH.
In addition to specialized hormones, the hypothalamus produces neurotransmitters, including bioactive amines, peptides, and amino acids. The bioactive amines include dopamine, norepinephrine, epinephrine, serotonin, acetylcholine, GABA, and histamine. The neuropeptides include VIP, substance P, neurotensin, components of the renin-angiotensin system, cholecystokinin (CCK), opioid peptides, ANP and related peptides, galanin, endothelin, and neuropeptide Y. The amino acids include glutamate and glycine. Some of these neurotransmitters affect the anterior pituitary. As mentioned above, dopamine regulates prolactin release and has complex influences on somatostatin release (seeChapter 5 and below). VIP stimulates the release of several pituitary hormones, including prolactin, growth hormone, and ACTH. Substance P stimulates prolactin and inhibits CRH-stimulated ACTH release. Neurotensin can affect growth hormone and prolactin release. Little is known about the effects of amino acids on endocrine function.
Regulation of Anterior Pituitary Hormone Release
The anterior pituitary produces endocrine hormones (Chapter 5). It has little innervation, and anterior pituitary hormone release is usually regulated by vascular delivery of hypothalamic and peripheral hormones. There are three main patterns of anterior pituitary hormone release (Figure 1-9).
(1) Spontaneous brain rhythms promote pulsatile hypothalamic and pituitary hormone release, as illustrated by the patterns of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release under control of GnRH from the hypothalamus. The amplitude and frequency of the pulses are governed by inputs from the central nervous system and intrinsic properties of the cells. Pulses of LH release can be as frequent as every hour during the follicular phase of the menstrual cycle. Release of other pituitary hormones mostly conforms to circadian rhythms (approximately 24-hour periodicity), which are influenced by the sleep-wake cycle.
(2) Peripheral hormones regulate pituitary hormone release through feedback loops. Thus, cortisol, thyroid hormone, and estrogens inhibit release of their tropic hormones—ACTH, TSH, and LH, respectively. Occasionally, target gland hormones exert positive feedback. Contrary to its usual fast-acting negative effect on LH production, estradiol also initiates a preovulatory surge in LH secretion that requires 48–72 hours of sustained estrogen stimulation. Feedback influences can be directed at the pituitary, the hypothalamus, or—and typically—at both.
(3) Intervening factors such as stress, nutritional influences, illnesses, and other hormones affect hormone release. Thus, stress increases the release of ACTH, growth hormone, and prolactin; and systemic illness can suppress the hypothalamic-pituitary-thyroid axis and the release of gonadotropins. Immunomodulators such as interleukin-1 and interleukin-2 and epinephrine increase CRH and ACTH release; and angiotensin II, interleukin-2, cholecystokinin, and oxytocin can stimulate ACTH release.
The regulation of PRL and GH is different from that of other anterior pituitary hormones. GH and PRL are not subject to the same degree of classic feedback regulatory mechanisms as with some other hormones, although IGF-I, which is produced in response to GH, can feed back to inhibit GH release. Specific releasing hormones (prolactin-releasing factor and GHRH, respectively) and inhibitory hormones (dopamine and somatostatin, respectively) regulate PRL and GH production. The inhibitory hormone is more important for PRL release, whereas the stimulatory hormone is dominant for GH release (Chapter 5).
ENDOCRINE ORGANS & HORMONE PRODUCTION
Individual endocrine organs are described in detail in individual chapters. This section summarizes principles of regulation of these endocrine organs and their signaling responses. The tropic anterior pituitary hormones play an essential role in regulation of hormone production by classic endocrine organs. Thus, hormones like ACTH, FSH, LH, and TSH stimulate hormone release in cognate target glands (adrenals, gonads, and thyroid gland), and their release is regulated by feedback loops as described above. The gonads also produce other hormones—inhibin, follistatin, and activin—that regulate tropic hormone release. These regulatory networks tend to control hormone levels within a narrow range, referred to as the set point. (See the chapters on the various glands or systems.)
There are other modes of regulation of hormone release. Hormone production can be regulated through innervation of the endocrine organ. For example, stress-related catecholamine release by the adrenal medulla is regulated by autonomic innervation. Another specialized case is the pineal gland, which lies at the base of the brain and provides an interface free of the blood-brain barrier between the brain, the cerebral circulation, and the cerebrospinal fluid—and, as such, may be described as part of the neuroendocrine system. The gland receives photosensory information through sympathetic innervation that influences production of melatonin, derived from serotonin, which regulates circadian rhythms and can have antireproductive functions, block GnRH-induced LH release, and have other effects on hormone release. Finally, hormone release can be regulated more directly by environmental signals or nutrient levels. Thus, insulin production is intimately linked to blood glucose levels.
It is also becoming clear that many tissues and organs which are not traditionally considered to be endocrine organs do produce endocrine signals. The kidney is the origin of hormones comprising the renin-angiotensin-aldosterone system. Adipose tissue produces leptin, an important mediator of body weight; and resistin, a peptide that may be involved in the pathology of type 2 diabetes. The heart produces natriuretic peptides. It is likely that many more organs will prove to produce endocrine signals.
ACTIONS OF HORMONES
Hormones have widespread effects that are described in subsequent chapters. Some general patterns are summarized in this section.
Hormones regulate their own production and release and also that of other hormones. These aspects are described in earlier sections onmechanisms of hormone action, synthesis of hormones, and neuroendocrinology.
Hormones exert widespread influences on development. Cretinism resulting from severe hypothyroidism (Chapter 7), dwarfism resulting from growth hormone deficiency (Chapter 6), and inability to develop and survive with a steroid hormone synthesis defect (Chapter 9) illustrate the profound effects of different classes of hormones on development. Hormones influence sexual development, as illustrated by the failure of male sexual development in the androgen-deficient state (Chapter 14).
CELL GROWTH & CANCER
Hormones are important for cell growth. Peptide hormones such as growth hormone, IGF-I, and IGF-II stimulate linear growth and cellular proliferation in other tissues (Chapter 6). Other peptides such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and transforming growth factors α and β (TGFα and TGFβ) are growth factors both in multiple tissues and in endocrine glands. Tropic factors regulate growth of target endocrine glands—eg, ACTH and angiotensin II on the adrenal gland (Chapter 9), TSH on the thyroid gland (Chapter 7), and LH and FSH on
the ovary (Chapter 13) and testis (Chapter 12). Thyroid hormones stimulate growth of several tissues (Chapter 7). Steroid hormones can both inhibit and stimulate cell growth. Glucocorticoids inhibit the growth of several cell types and kill some lymphocyte cell types, whereas estradiol and testosterone and dihydrotestosterone stimulate growth of breast and prostate, respectively.
Hormones also influence cancer. Often the hormone influences the growth of the cancer cell in the same way that it influences the growth of the normal progenitor cell. However, deranged hormone signaling pathways can also cause cancer. Many oncogenes are analogs of growth factors or growth factor receptors, as described above in the section on hormones and oncogenes.
HORMONAL EFFECTS ON INTERMEDIARY METABOLISM
Hormones regulate the metabolism of all major classes of macromolecules. Carbohydrate, fat, protein, amino acid, and nucleic acid metabolism are tightly regulated by insulin, glucagon, somatostatin, growth hormone, catecholamines (epinephrine, norepinephrine), thyroid hormones, glucocorticoids, and other hormones. These interactions are coordinated for finely tuned regulation of intermediary metabolism and numerous conditions such as stress or starvation. Insulin is dominant in lowering blood glucose and in stimulating metabolism of glucose and synthesis of fat, proteins, and nucleic acids. By contrast, cortisol, glucagon, catecholamines, and growth hormone elevate blood glucose by diverse mechanisms. However, these hormones differ in their effects on protein, fat, and nucleic acid metabolism. Hormones affect the uptake of glucose, amino acids, nucleosides, and other small molecules. For example, insulin increases glucose uptake by promoting redistribution of glucose transporters to the plasma membrane. Hormones also affect enzymes involved in metabolism, including, among others, those involved in gluconeogenesis, lipolysis, glycogen synthesis, amino acid metabolism and synthesis, and lipid synthesis.
HORMONAL EFFECTS ON MINERAL & WATER METABOLISM
Hormones affect most aspects of mineral metabolism. Vasopressin regulates serum osmolality and water excretion (Chapter 5) and has numerous effects in the cardiovascular and central nervous systems. The mineralocorticoid aldosterone regulates serum sodium and potassium and to some extent chloride and bicarbonate ion concentrations and balance (Chapter 10). Other hormones, including ANP, insulin, glucagon, catecholamines, angiotensin II, and PTH, also regulate ionic balance.
HORMONAL EFFECTS ON CARDIOVASCULAR & RENAL FUNCTION
The renin-angiotensin system, atrial natriuretic peptide, endothelins, catecholamines, steroid hormones, thyroid hormone, prostaglandins, kinins, vasopressin, cytokines, nitric oxide, substance P, and calcitonin gene-related hormone and urotensin II can profoundly affect these systems. All of these substances can affect heart rate or contractility and constrictor and dilator mechanisms of arteries and veins (seeChapter 10). The growth factor properties of hormones influence cardiovascular development and muscular hyperplasia and hypertrophy and are involved in pathologic processes leading to hypertensive vascular changes, atherosclerosis, heart failure, and cardiac hypertrophy (seeChapter 10). Hormones influence renal function and blood pressure by regulating renal blood flow, glomerular filtration rate, and the transport of ions, water, and other chemicals. Hormones also regulate active and passive transport processes in kidney through activation, redistribution, and stimulation of the synthesis of channels or by generating energy for active transport. Hormones can have effects on lipoprotein metabolism and on cholesterol transport, and this is particularly true for hormone-bound thyroid receptors, peroxisomal proliferator-activated receptors (PPARs), and LXR nuclear receptors. Drugs that block these steps, such as converting enzyme inhibitors, β-adrenergic blockers, and mineralocorticoid hormone antagonists, are used extensively in therapy (Table 1-2).
Table 1-2. Examples of hormone antagonists used in therapy.
HORMONAL EFFECTS ON SKELETAL FUNCTION
Bone is constantly being deposited and resorbed. This process is under complex hormonal control (Chapter 8). Hormones (eg, IGF-I) control growth and mineralization through influences on both the matrix and mineral phase of bone. Cytokines—particularly tumor necrosis factor (TNF)—play a major role in bone remodeling. Osteoprotegerin, a soluble TNF receptor family member, blocks actions of TNF-related proteins in bone cells and thus serves a protective role in preventing the onset of osteoporosis. Examples of other influences on bone remodeling include the effects of PTH and vitamin D on calcium metabolism and of steroids and thyroid hormone on bone matrix. Estrogens promote accrual of bone matrix and prevent the development of osteoporosis while glucocorticoids and thyroid hormone have the opposite effect.
HORMONAL EFFECTS ON REPRODUCTIVE FUNCTION
Gonadotropins regulate ovarian and testicular function and the secretion of hormones from these organs. Testosterone and dihydrotestosterone regulate the development of male sexual characteristics such as penile, muscle, and prostate growth and deepening of the voice and also affect libido and sexual behavior (Chapter 12 and Chapter 14). Female sex steroids regulate functions of female reproductive organs, including the menstrual cycle and ovulation (Chapter 13). Leptin, secreted from adipose tissue, promotes maturation of the reproductive tract and may trigger the onset of puberty.
Pregnancy is regulated, in part, by hormones (Chapter 16). Progesterone is essential for establishing and maintaining pregnancy in humans. It also decreases myometrial sensitivity to oxytocin, leading to suppression of uterine contractile function. Hormones are critical for egg and sperm development, preparation of the uterus for conception and implantation, and development of the fetus. The placenta itself produces a number of hormones, some of which are mostly unique (chorionic somatomammotropin, placental lactogen; CS) and others that are also produced abundantly by other endocrine glands (progesterone and other steroid hormones; Chapter 13 and Chapter 16).
HORMONAL EFFECTS ON THE IMMUNE SYSTEM
Endocrine hormones influence the immune system. Glucocorticoids blunt immunologic and inflammatory responses; these actions form the basis for the use of glucocorticoids in therapy to suppress these responses when they are excessive. Sex steroids usually suppress the immune response. Thus, castration results in enlargement of lymph nodes and spleen, more severe graft-versus-host disease, increased skin graft rejection, and stimulation of T lymphocyte mitogen responsiveness in vitro. These effects target mainly cellular immune responses. However, estrogens may stimulate antibody production, and females tend to have higher levels of the major immunoglobulin classes under both basal and stimulated conditions. Females tend to have a higher incidence of autoimmune diseases than males and more active cellular and humoral immune responses. These differences are not observed before puberty. Thyroid hormone, GH, catecholamines, PRL, and other hormones influence immunologic or inflammatory functions, but their roles are still being defined.
Pregnancy, with its associated hormonal changes, can ameliorate autoimmune diseases through unknown mechanisms. Pregnancy tends to suppress the cellular but not the humoral immune responses. This may be critical to prevent maternal rejection of fetal tissues, though susceptibility to a number of viral and fungal diseases is increased. Immunosuppression is most pronounced in the second and third trimesters and rebounds by about 3–6 months postpartum.
HORMONAL EFFECTS ON THE CENTRAL NERVOUS SYSTEM
The endocrine and nervous systems interact in multiple ways (see Chapter 5). Hormones regulate behavioral and cognitive functions such as mood, appetite, learning, memory, and sexual activity. Hormones such as leptin, which controls satiety, have been identified, and new roles for “old” hormones are continuously being identified. Vasopressin increases affiliative behavior in monogamous voles that express brain V1a receptors, whereas low V1a expression correlates with a lack of response to vasopressin and promiscuous behavior.
Hormones also have secondary influences on the central nervous system through effects on general metabolism. There are a number of examples of mental abnormalities associated with hormone excess or deficiency. These include: depressed mental status that can progress to coma with severe hypothyroidism (Chapter 7); psychosis that can occur with glucocorticoid excess; and coma that can occur with hypoglycemia due to insulin excess (Chapter 18). Thus, hormones, neurotransmitters, and the central nervous system interact extensively, producing results that may shape not only development and physiology but also behavior and cognition.
Beneficial effects of hormones are counterbalanced by deleterious effects. Thus, although glucocorticoids suppress excessive inflammatory and immunologic responses, they also cause osteoporosis, physical disfigurement, and other problems. Estrogens reduce bone loss but can increase the risk for breast and uterine cancer. Thyroid hormones might be used to treat obesity if it were not for the fact that these hormones can have deleterious effects on the heart. These considerations point to a need for improved therapies for these classes of actions. A major goal, therefore, is to achieve selective modulation of receptor action—that is, to simultaneously promote desirable effects and inhibit undesirable ones.
Some of the principles outlined in the previous sections on receptor activity and regulation of hormone levels point to several different ways to obtain selective modulation. As described above, some receptor ligands can behave as partial agonists, mixed agonists-antagonists, or inverse agonists. It may be possible to capitalize on these properties. Tamoxifen and raloxifene show estrogen-like effects on bone but inhibit estrogen action in the breast. Thus, both compounds have advantages over estrogens in hormone replacement therapy; they reverse osteoporosis but should not increase breast cancer risk. However, existing selective modulators do not possess ideal selective properties. Tamoxifen increases uterine cancer risk and raloxifene and tamoxifen exacerbate hot flushes and increase the risk of blood clots and stroke.
Better selective modulation may be achieved in several ways. The selective modulation observed for tamoxifen and raloxifene is due to distortion of the receptor's ligand-binding domain, with consequent differences in the way (relative to estradiol) that it binds various cofactors. Improvements in this profile may be forthcoming. The same hormone can interact with more than one receptor, and isoform-specific ligands could show improvements over nonselective counterparts. Thus, adverse thyroid hormone effects on heart rate are mediated by TRα. TRβ-selective agonists might elicit some of the desirable effects of thyroid hormone without deleterious effects on the heart. Localized delivery systems can achieve selective modulation. Limited examples of this concept are the use of inhalation of glucocorticoid receptor agonists for asthma or application of cortisol creams to the skin. It may be possible to utilize tissue-specific uptake or export systems as a means of targeting drugs to particular tissues. Finally, it may be possible to take advantage of tissue selective metabolism to elicit selective responses. Indeed, idealized selective modulators may in the future make use of combinations of these diverse selective properties.
DISORDERS OF THE ENDOCRINE SYSTEM
The classic disorders of the endocrine system arise from states of excess or deficiency of hormones. However, resistance to hormones also plays a major role in disease. The endocrinologist is also confronted with specific tumors and other problems such as iatrogenic syndromes. The types of abnormalities that in principle can occur are illustrated in Figure 1-13.
Figure 1-13. Causes of hypofunction and hyperfunction of the endocrine system. (Reproduced, with permission, from Baxter JD in:Cecil's Textbook of Medicine. Wyngaarden JB, Smith LH Jr [editors]. Saunders, 1985.)
Destruction of the Gland
A common mechanism for glandular hypofunction is destruction of the gland through autoimmune disease (Chapter 4). This is seen in type 1 diabetes mellitus (Chapter 17), hypothyroidism (Chapter 7), adrenal insufficiency (Chapter 9), and gonadal failure (Chapter 12 and Chapter 13). A polyglandular failure syndrome (Schmidt's syndrome) results from autoimmune destruction of several different endocrine glands in the same patient (Chapter 4). The immunologic damage also results in other abnormalities, including pernicious anemia and vitiligo. Destruction of the pituitary gland is usually due to tumor, ischemia, or autoimmune hypophysitis (Chapter 5). Hypofunction of any of the endocrine glands may result from damage by neoplasms, infection, or hemorrhage.
Endocrine hypofunction can be caused by defects outside traditional endocrine glands. In some cases, these are simply due to damage to tissues that produce hormones or convert hormone precursors to active forms. Thus, renal disease can result in defective conversion of 25(OH)D3 to 1,25(OH)2D3, with consequent abnormalities in calcium and phosphate balance (Chapter 8). Renal disease can also provoke hyporeninemic hypoaldosteronism (Chapter 10) and anemia by damaging the renin-producing juxtaglomerular cells and the erythropoietin-producing cells.
In some cases, factors that influence hormone degradation or sensitivity can precipitate or aggravate hormone deficiency when there is insufficient reserve in the endocrine gland. For example, glucocorticoid therapy, which reduces insulin sensitivity, increases the need for insulin and can precipitate latent diabetes mellitus or aggravate existing diabetes (Chapter 17). Thyroid hormones increase cortisol metabolism, and treatment of hypothyroidism with thyroid hormone can unmask latent adrenal insufficiency (Chapter 9). Treatment with the anticonvulsant phenytoin can accelerate the degradation of glucocorticoids and increase the need for these hormones (Chapter 9).
Defects in Hormone Biosynthesis
Endocrine hypofunction can be due to defects in hormone synthesis. These can be due to defects in genes that encode hormones, regulate hormone production, or encode hormone-producing enzymes or are involved in hormone metabolism. The 21-hydroxylase deficiency syndrome results in defective cortisol production and is one of the most common genetic diseases (Chapter 9, Chapter 14, and Chapter 15). Other adrenal gland defects include 11β-hydroxylase, 17-hydroxylase, and 18-hydroxylase deficiency syndromes (Chapter 9, Chapter 10, and Chapter 14). Dietary iodine deficiency results in deficient thyroid hormone biosynthesis and afflicts millions of people worldwide (Chapter 7). Mutations in genes encoding polypeptide hormones can decrease hormone production or lead to production of defective hormones. Growth deficiency can result from mutations or deletions in the GH gene, defective production of GHRH, or mutations in the gene for the transcription factor Pit-1, which regulates GH synthesis (Chapter 6). A rare form of diabetes mellitus results from a mutation in the insulin gene, with production of abnormal insulin (Chapter 17).
Hyperfunction of endocrine glands results usually from tumors, hyperplasia, or autoimmune stimulation. Endocrine gland tumors can produce excess hormone. Thus, pituitary tumors can overproduce one of the major classes of pituitary hormones (ACTH, GH, PRL, TSH, LH, and FSH;Chapter 5). This leads to stimulation of other glands, as is seen with cortisol excess due to pituitary ACTH-producing tumors or the rare syndrome of hyperthyroidism due to pituitary TSH-producing tumors. Other examples of tumors in endocrine organs resulting in overproduction of hormones are parathyroid glands, PTH (Chapter 8); thyroid parafollicular cells, calcitonin (Chapter 7); thyroid follicular cells, thyroglobulin or thyroid hormone (Chapter 7); pancreatic islets, insulin, or glucagon (Chapter 17); adrenals, cortisol, aldosterone, deoxycorticosterone, androgens, and other steroids (Chapter 9 and Chapter 10); kidney and renin (Chapter 10), or erythropoietin. There are also syndromes of multiple endocrine neoplasia, in which there is a predisposition to develop tumors of several glands (Chapter 22). In contrast, most thyroid gland tumors do not overproduce thyroid hormone (Chapter 7), and it is rare for ovarian or testicular tumors to overproduce steroids or for posterior pituitary tumors to overproduce oxytocin or vasopressin.
There can also be ectopic production of hormones by tumors (Chapter 21). Ectopically produced hormones are usually polypeptide hormones and include ACTH, ADH, and calcitonin. However, other polypeptide hormones such as insulin are rarely if ever expressed ectopically.
Hyperplasia, with increased cellularity and hormone overproduction, can be seen with most endocrine glands. Hyperplasia of the parathyroid glands is seen in renal failure, where depression of serum calcium ion levels stimulates the gland (Chapter 8). Hyperplasia is commonly seen in the adrenal glomerulosa, where it results in aldosterone excess and is a major cause of the
syndrome of primary aldosteronism (Chapter 10). Hyperplasia of the adrenal zonae fasciculata and reticularis results in cortisol excess and Cushing's syndrome (Chapter 9) and is almost always due to a pituitary ACTH-producing tumor. The cause of hyperplasia of the adrenal glomerulosa is not known, and the disorder is therefore referred to as idiopathic hyperplasia or idiopathic aldosteronism. Hyperplasia of the thyroid gland is common and may be due to autoimmune stimulation (see below), iodine deficiency with impaired T4 synthesis and subsequent TSH hypersecretion, or nodular goiter due to genetic biosynthetic abnormalities (Chapter 7). Hyperplasia of the ovaries is very common and results in polycystic ovary syndrome, with abnormalities in ovarian steroid production and insulin resistance; the causes of this syndrome are poorly understood (Chapter 13).
Autoimmune stimulation resulting in hyperfunction is seen most commonly with hyperthyroidism (Chapter 7). In this case, antibodies are produced that bind to and activate the TSH receptor on the gland. Hyperinsulinism due to autoimmune attack on the pancreatic B cells can be seen transiently early in the course of development of type 1 diabetes mellitus (Chapter 17). Otherwise, autoimmune stimulation leading to hyperfunction of endocrine glands is rare.
DEFECTS IN SENSITIVITY TO HORMONES
Genetic and acquired defects in sensitivity to hormones play a crucial role in the pathogenesis of both common and rare disorders. Common disorders include type 2 diabetes mellitus and hypertension. Resistance may be due to a number of different types of defects, eg, in the hormone receptor, in functions distal to the receptor, or in functions extrinsic to the receptor-response pathway.
There are a number of disorders of primary resistance to hormones due to receptor defects. Genetic defects in receptors that cause syndromes of resistance include those for glucocorticoids, thyroid hormones, androgens, vitamin D, leptin, mineralocorticoids, peroxisomal proliferators, PTH, ADH, GH, insulin, and TSH. Defects in hormone response that are due to mutations in postreceptor signaling pathways are less well understood. An exception is the syndrome of pseudohypoparathyroidism, in which mutations occur in the gene encoding the guanylyl nucleotide binding protein that links PTH-receptor binding to activation of adenylyl cyclase (Chapter 3 and Chapter 8). Resistance to thyroid hormone is ordinarily due to mutations in the thyroid hormone receptor (TR); however, in a number of cases, defects in the receptor have not been found, and it is thought that the defect may involve postreceptor loci such as receptor association with coregulatory proteins. Hormone resistance that is due to events distal to the ligand-receptor interaction occurs in type 2 diabetes mellitus, the most common form of that disease (Chapter 17). Weight reduction and diet can normalize these manifestations in some patients, suggesting that the problem is one of impaired adaptation—perhaps due to excessive down-regulation of responsiveness to stimuli. Syndrome X (metabolic syndrome) is characterized by excessive insulin resistance with overlap into type 2 diabetes mellitus, hyperlipidemia with increased triglycerides and cholesterol, and hypertension. This syndrome is commonly observed in obese individuals and accounts for much of the hypertension in Western societies. In hypertension, there are variations in sensitivity to salt, to angiotensin II, to the release of renin in response to various stimuli, and to other effectors. Although mechanisms for the resistance are poorly understood, insights into disorders such as these should come from a better understanding of the physiologic mechanisms that govern sensitivity to hormones. Figure 1-14 is a schematic representation of resistance to hormones at postreceptor loci.
Acquired resistance to hormones can occur when there is frank disease that damages the target tissue and interferes with its ability to respond to the hormone. This can be seen with renal disease and insensitivity to vasopressin and with liver disease and insensitivity to glucagon. It can also occur as a result of excess production of other hormones or substances that promote hormone resistance. Thus, stress responses, hyperglycemia, and states that increase plasma levels of GH, cortisol, or glucagon all lead to insulin resistance that can aggravate or precipitate type 2 diabetes mellitus (Chapter 17). Acquired resistance may also occur in hormone therapy. This is particularly true with GnRH analogs and calcitonin and sometimes occurs with glucocorticoids. In fact, the acquired resistance associated with prolonged exposure to GnRH analogs forms the basis for their use in the treatment of prostate cancer—along with nonsteroidal antiandrogens—to inhibit androgen action. In this clinical context, the GnRH-induced down-regulation of GnRH responsiveness (termed tachyphylaxis) shuts down FSH and LH release with a consequent reduction in testosterone production. Immunologic mechanisms can lead to acquired resistance—as, for example when antibodies are produced to hormones (eg, with insulin or GH therapy) or receptors (eg, insulin receptors; Chapter 17).
The clinical presentations of these syndromes show considerable variations. In classic hormone resistance syndromes, there are elevated or normal hormone levels with clinical manifestations of hormone deficiency and failure of hormonal replacement to correct the disorder. Thus, at worst, the syndromes resemble hormone deficiency states, as is the case with testicular feminization
syndrome (androgen insensitivity; Chapter 14), rickets (vitamin D insensitivity; Chapter 8), and nephrogenic diabetes insipidus (ADH insensitivity; Chapter 5). However, there can be wide variations in the clinical presentations. As an example, manifestations due to mutations of the androgen receptor gene can vary from a phenotypic female with a male genotype to mild androgen deficiency with erectile dysfunction in an otherwise normal-appearing male (Chapter 14).
Figure 1-14. Scheme for generation of hyperresponsiveness or hyporesponsiveness to hormones through excessive or impaired down-regulation. The top row indicates the response network before down-regulation occurs. The second row indicates the magnitude of the response network with normal down-regulation. The third row indicates a situation with excessive down-regulation due to a defect in the effector arm of the response with normal receptor function. The sizes of the circles reflect the influence, and the increased size of the arrow reflects the enhanced down-regulation. The fourth row indicates decreased down-regulation at the effector arm of the response. The dotted arrow reflects the decreased down-regulation and the larger response circles, compared with the second panel, reflect the increased response resulting from the decreased down-regulation.
In many cases, secondary hormone hypersecretion largely compensates for the primary defect, and the clinical presentation is more complex. Elevated hormone levels occur because of absence of the usual feedback inhibition of hormone release resulting from target organ resistance. For example, the clinical presentation of the syndrome of resistance to thyroid hormone (Chapter 7) may have features of euthyroidism, hyperthyroidism (tachycardia, poor attention span), and hypothyroidism (poor growth). In this syndrome, the mutation is in the β form of the TR, but the remaining TRβ encoded by the second allele and the TRα encoded by separate genes are normal. The abnormal β gene causes a decrease in the usual pituitary feedback by thyroid hormone, leading to increases in TSH secretion and hypersecretion of thyroid hormone. This increase in thyroid hormone compensates for the defect by binding the normal β and α receptors and, in some cases, the mutated receptors. Thus, thyroid hormone-regulated functions that are normally mediated through the α receptors are hyperstimulated, and functions mediated through the β receptors can be stimulated normally or can be understimulated in cases where a normal complement of β receptors is required for full activity and the elevated hormone levels cannot overcome the defect in the mutated receptors.
Hyperstimulation of the endocrine glands in resistance syndromes sometimes results in overproduction of other hormones. Thus, mutations that reduce the affinity of the glucocorticoid receptor for cortisol result in compensatory ACTH hypersecretion. Whereas the resulting overproduction of cortisol compensates for the primary defect, parallel increases in other steroids such as deoxycorticosterone and testosterone can promote mineralocorticoid hypertension and hirsutism, respectively.
Primary hormone hyperresponsiveness has rarely been encountered. It may occur in low-renin hypertension in humans and in primary aldosteronism with hyperplasia of the zona glomerulosa, a variant of primary aldosteronism. It has also been encountered in a mineralocorticoid receptor defect where the receptor binds progesterone more tightly than with the wild type receptor. Thus, when progesterone levels are elevated in conditions such as in pregnancy, there is overactivation of the receptor and consequent hypertension. Acquired hypersensitivity can be observed, as with catecholamine hypersensitivity in hyperthyroidism and increased sensitivity to insulin with cortisol deficiency.
SYNDROMES OF HORMONE EXCESS DUE TO ADMINISTRATION OF EXOGENOUS HORMONE
Syndromes of hormone excess can result from deliberate or inadvertent administration of exogenous hormones. Deliberate administration of glucocorticoids in therapy to suppress inflammation may lead to Cushing's syndrome. Use of high doses of thyroid hormone to suppress a malignancy in the gland can lead to hyperthyroidism. Androgen excess with suppression of pituitary
gonadotropins occurs in athletes who take androgens to improve performance. As an example of inadvertent administration, the authors have observed Cushing's syndrome in a patient who was unaware that she had received glucocorticoids. Outbreaks of hyperthyroidism have occurred with consumption of hamburger meat contaminated by thyroid tissue (Chapter 7). Some nasal sprays have contained substances with mineralocorticoid activity that produces a mineralocorticoid excess state (Chapter 10).
NONENDOCRINE PROBLEMS ASSOCIATED WITH ENDOCRINE DISEASE
Endocrine diseases can cause problems unrelated to the endocrine excess or deficiency states. For example, pituitary tumors can cause increased intracranial pressure or neurologic or ocular problems when they extend outside the sella turcica (Chapter 5). Thyroid tumors or large goiters can cause local problems in the neck (Chapter 7).
APPROACH TO THE PATIENT WITH ENDOCRINE DISEASE
Like all other medical specialists, the endocrinologist hopes and tries to prevent diseases and their sequelae or when this is not possible, to detect and treat disease at an early stage. Thus, endocrine diseases are generally most easily recognizable in their more extreme forms, but it is hoped that few patients will progress to that stage. The physician should be aware of subtle early manifestations of endocrine disease and strive for early diagnosis with the aid of laboratory tests. Early diagnosis can also be facilitated by screening for endocrine diseases in certain clinical settings (discussed below).
Several principles should be considered in evaluating endocrine diseases. Symptoms and signs are often vague and attributable to anxiety or depression or to nonendocrine causes. The early presentation of these disorders can be masked even further by compensatory responses. The clinical presentation of a given condition can also differ depending on its chronicity, and a severe deficiency state can present as an acute and severe problem in a patient in whom chronic manifestations of the disorder have not had time to develop. The clinician must decide whether treatment should be instituted immediately, before time-consuming tests leading to definitive diagnosis have been completed. It is sometimes difficult to arrive at a clear diagnosis, and the procedures needed for definitive diagnosis may impose more risk than the disease over a short period of time. In such cases, a decision to follow the patient must be made. For example, this occurs with ACTH-dependent Cushing's syndrome, where the differentiation between an occult carcinoid tumor and a small pituitary adenoma as the source of ACTH hypersecretion may require invasive procedures (Chapter 9).
In today's environment of cost containment, efficiency of diagnosis is a priority. Although modern tests may involve higher cost, they also allow for greater efficiency of diagnosis. Thus, by combining the most efficient use of tests with a careful history and physical examination and sound clinical judgment, diagnosis and management of endocrine disease should be better, quicker, and cheaper than before.
Evidence-based medicine evolved out of concern that physicians were applying clinical impressions to patient care in some cases without sufficient basis in scientific fact. It was observed, for example, that approaches to given diseases differed in various medical centers and regions around the world. It was noted also that in cases where practice activities were not justified, this frequently led to mistakes in therapy based on inferences drawn from subsequently published data. As an example of this, at one time a number of physicians used fluoride to treat osteoporosis based on the observation that fluoride increased bone density. However, subsequent studies found that fluoride did not decrease the rate of bone fractures. In this instance, the use of bone density proved to be an unacceptable marker for fracture risk. A more recent example is the use of estrogen-progestin combinations to prevent cardiovascular disease. This was based on a number of observational studies suggesting that estrogen deficiency in the postmenopausal setting increases cardiovascular risk. However, more rigorous trials showed that estrogen-progestin combinations failed to decrease cardiovascular risk—and actually seemed to increase risk, at least over the first few years of the trial.
Advocates of evidence-based endocrinology acknowledge that physicians are faced with situations where the evidence is not conclusive and yet diagnostic or therapeutic decisions must be made. Guidelines have thus emerged that rank the importance of the evidence. The hierarchy could range downward from well-done randomized trials to meta-analyses, systematic reviews of observational studies, observational studies, physiologic studies, and unsystematic clinical studies. Thus, there is room for clinical judgment. Recommendations based on evidence-based endocrinology can be found in several sources, including UpToDate, the ACP Journal Club, and the Cochrane Database.
How did physicians make decisions prior to “evidence-based endocrinology”? They based them on the evidence that was available, recommendations of opinion
leaders, and their own experience. What is different? First, more recent advances in statistical analyses of data have “raised the bar” concerning the strength of the evidence required to make a recommendation. Second, there has been increased reliance on data related to outcomes. A major result of these changes is that the average physician who reads guidelines from consensus conferences follows treatment plans based on more firmly established evidence.
However, evidence-based endocrinology practiced with higher standards raises additional problems. The rate of scientific progress is accelerating. New methods for diagnosing and treating disease will be developed at a pace much faster than was the rule even a few years ago. It will take many years to prove the efficacy of these developments using today's high standards. For example, recent data from the Pharmaceutical Manufacturers Association states that it takes on average 68 clinical trials, 14 years, and about $500-$800 million to take a drug, once discovered, to approval. These numbers are increasing. This means that it will be many years before many of these developments will be available to most patients, and it may prove impossible to develop many of the newer treatment modalities that will be discovered. How do the opinion leaders deal with this dilemma? They vary in their adherence to established guidelines, often because they are more confident in applying newer modalities. Thus, we can have situations where the leaders in the field are treating patients differently from the generalists who follow the guidelines. Who is right?
Counterbalancing the criticisms outlined earlier justifying the new higher standards are the many examples of treatments that were initiated prior to rigorous proof of efficacy. Examples include the use of histamine H2 receptor blockers for treating peptic ulcer disease, converting enzyme inhibitors for treating heart failure and hypertension, calcium channel blockers for treating hypertension, and β-adrenergic blockers for treating hypertension. A question that emerges is whether opinion leaders are more often right than not. A related question is whether strict adherence to evidence-based endocrinology, with relegation of clinical judgment-based endocrinology to a secondary position in the decision-making process, is likely to provide better or worse care to the endocrine patient. These important questions remain largely unaddressed as this is written.
HISTORY & PHYSICAL EXAMINATION
A carefully performed history and physical examination can provide information that cannot be obtained from laboratory testing. Some diagnoses, such as hypertension, are based on the physical examination alone. Even when the history and physical are unrevealing, they enable the physician to select appropriate laboratory tests and avoid unnecessary testing. Most specialists have a fund of stories about consultations on extensively studied patients where simply elicited symptoms or signs that were overlooked would have led to early diagnosis. Thus, a history and physical examination should address issues that will lead to the diagnosis and to the plan of approach. These activities should also reveal information about how much tissue damage or physical deformity has occurred; how long the disease has been present; the effect of various manifestations on the patient; and relevant data from the social, family, and personal histories that will facilitate evaluation and management.
Manifestations of endocrine disease that are frequently due to nonendocrine or unknown causes (Table 1-3) include fatigue, malaise, weakness, headache, anorexia, depression, weight gain or loss, bruising, and constipation, among others. Thus, weight loss is a common manifestation of hyperthyroidism, though most weight loss is not caused by hyperthyroidism (Chapter 7). Adrenal insufficiency is a rare disease and an even rarer cause of nausea (Chapter 9).
Laboratory evaluations are critical for making and confirming endocrine diagnoses and for ruling out other causes. However, these tests cannot replace good clinical judgment that incorporates all available information in making clinical decisions. Laboratory tests, in general, measure either the level of the hormone in some body fluid, the effects of the hormone, or the sequelae of the process that contributed to the hormonal abnormality. The tests can be performed under random or basal conditions, precisely defined conditions, or in response to some provocative stimulus. In measuring hormone levels, the sensitivity of the assay refers to the lowest concentration of the hormone that can be accurately detected, and the specificity refers to the extent to which cross-reacting species are scored inappropriately in the assay.
Measurements of Hormone Levels: Basal Levels
Immunologic assays are usually utilized for measurements of hormone levels in body fluids. Most measurements use blood or urine samples. The hormone is measured either directly in the samples or following extraction and purification. Most measurements detect active hormone, though measurement of either a metabolite or precursor of the hormone or a concomitantly released substance sometimes provides the best information. Thus, in assessing vitamin D status, it is usually more informative to measure the precursor hormone, 25(OH)D, even though the final active hormone
is 1,25(OH)2D (Chapter 8). In 21-hydroxylase syndrome, the clinical problem is a deficiency of cortisol or aldosterone, but the most sensitive diagnostic measurement is of the plasma 17α-hydroxyprogesterone level, a precursor of cortisol (Chapter 9). In looking for a pheochromocytoma, levels of epinephrine metabolites are sometimes as informative as levels of epinephrine itself (Chapter 11).
Table 1-3. Examples of manifestations of endocrine disease. (The manifestations do not occur in all cases, and the severity can vary markedly.)
Plasma & Urine Assays
Hormone assays only indicate the hormone levels at the time of sampling. For hormones with long half-lives (eg, thyroxine), measurements taken randomly provide an integrated assessment of hormonal status. For hormones with shorter half-lives, such as epinephrine or cortisol, the assay will provide information only for the time of sample collection. Thus, with a pheochromocytoma that episodically releases epinephrine, elevated plasma epinephrine levels would be found only during periods of release and not between them (Chapter 11). Spontaneous Cushing's disease can be associated with an increased number of pulses of cortisol release with normal plasma cortisol levels between pulses (Chapter 9). In early Addison's disease, the number of pulses of cortisol release can be decreased, but occasional releases can result in transient plasma cortisol levels in the normal range (Chapter 9).
Urine assays are generally restricted to measurement of levels of steroid and catecholamine hormones or metabolites and are not useful for polypeptide hormones that are either not cleared or unstable. The collection period can be a random sample or, more often, a 24-hour collection. Interpretations of urinary measurements must account for the fact that urinary levels reflect renal handling of the hormone. Urine measurements were utilized more frequently in the past because larger quantities of the hormone could be obtained. However, with the high sensitivities of today's immunoassays, this advantage is disappearing, and blood measurements are usually preferred. An advantage of urinary assays is that they can provide an integrated assessment of hormonal status. With cortisol, for example, about 1–3% of the hormone released by the adrenal gland appears in the urine, but measurement of the urine cortisol in a 24-hour “urine free cortisol” sample provides an excellent assessment of the integrated cortisol production (Chapter 9). This is important, since cortisol is released episodically, and a random plasma cortisol can be in the normal range in the face of mild to moderate Cushing's disease. Urinary assays are frequently used to document aldosterone excess in primary aldosteronism (Chapter 10) and epinephrine excess in pheochromocytoma (Chapter 11).
Free Hormone Levels
As discussed in an earlier section, many hormones circulate bound to plasma proteins, and the free hormone fraction is generally that which is biologically relevant. Thus, assessment of free hormone levels is more critical than total hormone levels. Tests that measure free hormone levels can utilize equilibrium dialysis, ultrafiltration, competitive binding, and other means. Although such tests are not commonly employed, their use may increase, as evidenced by increasing use of plasma free thyroxine (Chapter 7) and serum ionized Ca2+ measurements (Chapter 8).
Hormone immunoassays utilize animal-derived antibodies with high affinity to the hormone. The antibodies can be polyclonal or monoclonal. In general, a given animal will produce a number of different antibodies to a given antigen, each from a clone of antibody-producing B lymphocytes—thus the term “polyclonal antibodies.” This mixture of antibodies can contain some with extremely high affinities for the hormone, and these will provide a high level of sensitivity in a subsequent radioimmunoassay. Monoclonal antibodies are commonly obtained by injecting the antigen into a mouse or rat or by incubating it with cells in vitro. The animal spleen or incubated cells are then immortalized by fusing them to myeloma cells or transforming them with tumor viruses. This produces a number of clones of antibody-producing cells. The clones are then screened with the antigen until a suitable antibody-producing clone is identified. A major disadvantage of monoclonal antibodies is that many of them have a low affinity for the hormone. In addition, each antibody reacts with only one epitope on the antigen, and these antibodies are not as useful for traditional reagent-limiting assays. However, these antibodies are critical for the “sandwich assays” described below.
In traditional assays, the antigen is labeled in order to detect its binding to the antibody; the label must not block binding of the antigen to the antibody. Early immunoassays traditionally utilized radiolabeled hormones as the antigen. Most commonly the radioisotope was iodine, which can be obtained with a very high specific activity. However, the disadvantages with radioactivity in terms of shelf life and escalating expense for disposal have led to increasing use of nonisotopic means to perform immunoassays. For these, the antigen is linked to an enzyme, fluorescent label, chemiluminescent label, or latex particle that can be agglutinated with the antigen. Enzyme-linked immunosorbent assays (ELISAs) that utilize antibody-coated microtiter plates and an enzyme-labeled reporter antibody can be as sensitive as radioimmunoassays.
In practice, measurement of hormone levels by immunoassay involves incubating the plasma or urine sample or an extract with antisera and then measuring the levels of antigen-antibody complexes by one of several means. The classic immunoassays utilize high-affinity antibodies immobilized (at low concentrations to permit maximal sensitivity) on the surface of a test tube, polystyrene bead, or paramagnetic particle. The unknown sample and the antibody are incubated together, and the labeled antigen is added either at zero time or later. A standard curve is prepared using the antibody and a known concentration of hormone. From this curve, the extent of inhibition of the binding of the labeled hormone by the added hormone is plotted, usually as the ratio of bound to free (B:F) hormone as a function of the log of the total hormone concentration. These plots typically provide a sigmoid curve (Figure 1-15). Alternatively, a log-logit plot can be used to linearize the data (Figure 1-15). The hormone level in the sample is determined by relating the B:F value obtained in the sample to the standard curve.
A modification of immunoassays—termed the sandwich technique—utilizes two different monoclonal antibodies that recognize separate portions of the hormone. This aspect limits the technique, as it is difficult to utilize it for small molecules where separable reactive domains cannot be readily identified. The assay is performed by using the first antibody, attached—preferably
in excess relative to the amount of hormone in the sample—to a solid support matrix to adsorb the hormone to be assayed. After removal of the plasma and washing, the second (labeled) antibody is then incubated with the bound hormone-first antibody complex. The amount of binding of the second antibody is proportionate to the concentration of hormone in the sample. Use of two antibodies results in markedly enhanced specificity with a great reduction in background levels, thus improving both specificity and sensitivity of the assay.
Figure 1-15. Standard curve of hormone radioimmunoassay. (B, counts bound; F, free counts; N, nonspecific count; B0, maximum number of counts bound when only antibody and labeled hormone are incubated.) (Reproduced, with permission, from Vaitukaitis J in:Hormone Assays in Endocrinology and Metabolism, 2nd ed. Felig P et al [editors]. McGraw-Hill, 1987.)
Nonimmunologic assays include chemical assays, which take advantage of chemically reactive groups in the molecule; bioassays, which assess the activity of the hormone incubated with cells or tissues in vitro or following injection into an animal; and receptor-binding and other assays, which exploit the high affinity of the hormone for receptors or other molecules such as plasma-binding proteins. These assays are rarely used because immunoassays are sufficiently robust for most applications. For example, immunoassays are superior to receptor assays because antibodies can be obtained that have much higher affinities for hormones than receptors. One example of a receptor assay uses thyroid tumor cells (FRTL-5 cells) that contain TSH receptors to detect antibodies to these receptors in plasma of patients with Graves' disease.
Diagnosis of Genetic Disease
Methods for diagnosis of genetic diseases using DNA analyses are improving rapidly. Thus, DNA can be obtained from peripheral blood cells; the region of interest can be amplified by PCR; and the gene can be sequenced fairly rapidly. The increased availability of these technologic capacities is rapidly supplanting previous methods such as analysis of restriction fragment length polymorphisms (RFLPs) in diagnosis. In cases where the mutation is known, such a procedure can lead to rapid and accurate diagnosis in the general population. This is the case with sickle cell anemia, in which a single mutation in one codon is present in all individuals who have the disease. It is also the case in kindreds with known mutations, such as those with maturity-onset diabetes of the young (MODY) or glucocorticoid remediable aldosteronism or with mutations in the breast cancer susceptibility genes BRCA1 and BRCA2. It is probable that DNA analysis will be increasingly used for identification of affected family members in kindreds with a shared mutation. Thus, people who have inherited the defect can be advised with regard to appropriate follow-up or preventive surgery, and those without the defect can be spared unnecessary anxiety and testing.
It remains true that in some cases other diagnostic measures are easier to perform in screening of the general population. Multiple mutations can lead to the same disease, and screening for any one mutation within a sequence would potentially miss most affected individuals in the population. For example, 21-hydroxylase deficiency is caused by a number of different mutations, and it has been simpler to measure 17-hydroxyprogesterone levels (Chapter 9) than to examine DNA samples for a large number of mutations. Nevertheless, large scale sequencing of key genes that might contribute to a particular disease phenotype can dramatically improve diagnosis. For example, MODY is caused by monogenic defects in a number of genes (including
glucokinase or a series of pancreatic transcription factors) that lead to glucose intolerance and diabetes-like symptoms. Many of these patients respond well to glucose-lowering drugs without insulin therapy. Large-scale sequencing of possible MODY genes in patients—along with analysis of other family members—can distinguish the subset of patients with MODY from those with type 1 or type 2 diabetes and alter the preferred treatment regimen.
Indirect Measurements of Hormonal Status
Measurement of the effects of hormones can be even more important than measuring the hormone levels and can provide critical complementary information. Even when hormone levels are known, it is common to obtain at least one index of the effects of the hormone in evaluating an endocrine disease. The blood glucose level is generally more useful than the plasma insulin level in diagnosing and treating diabetes mellitus (Chapter 17). Plasma insulin levels can be high in the face of frank hyperglycemia in type 2 diabetes mellitus, and in type 1 diabetes mellitus insulin levels are a much less reliable index of diabetic status than blood glucose (Chapter 17). Measurement of the serum calcium level is critical for evaluating hyperparathyroidism (Chapter 8). Measurement of plasma renin levels in relation to plasma aldosterone levels (aldosterone:renin ratio) is critical for evaluating primary aldosteronism, in which plasma renin levels are relatively suppressed (Chapter 10). The most common causes of elevated aldosterone levels are dehydration, exercise, diuretic therapy, and other conditions that produce secondary aldosteronism; in these settings, the plasma renin levels tend to be high rather than low (Chapter 9).
In many cases, the level of a hormone or parameter affected by a hormone is best interpreted following provocative challenges. For example, with thyroid disease, provocative tests are rarely needed (Chapter 7), whereas with adrenal insufficiency or glucocorticoid excess (Chapter 9), heavy reliance is placed on such tests. With thyroid disease, slow clearance of the hormone results in basal levels of hormone that are highly informative, whereas the pulsatile nature of cortisol release results in fluctuating plasma cortisol levels that need to be measured under more defined conditions. This problem is bypassed in evaluation of adrenal insufficiency by administering an ACTH analog that maximally stimulates the adrenal (Chapter 9). Diagnosis of Cushing's disease reflects a different problem (Chapter 9). Once cortisol hypersecretion has been documented, the cause must be identified. The clinician takes advantage of the fact that ACTH release by pituitary microadenomas—and, consequently, secretion of cortisol by the adrenal glands—are suppressed by the glucocorticoid dexamethasone to a greater extent than is cortisol release by adrenal tumors or ACTH release by ectopic ACTH-producing tumors. Similarly, GnRH analogs (which stimulate FSH and LH release), TRH (which stimulates both prolactin and TSH release), and insulin hypoglycemia (which stimulates the release of ACTH and GH) can be used to evaluate pituitary reserve (Chapter 5). In evaluating primary aldosteronism, provocative stimuli (diuresis, change in posture, inhibition of converting enzyme) are sometimes used to increase renin release (Chapter 10).
Imaging studies are used in diagnosis and follow-up of endocrine diseases. MRI and CT allow visualization of endocrine glands and endocrine tumors at a much greater resolution than in the past. These procedures have been especially useful for evaluation of tumors of the pituitary and adrenals (Chapter 5 and Chapter 9). Scanning of the thyroid gland using radioactive iodine has been useful for evaluation of functioning nodules of this gland (Chapter 7). The endocrinologist can also resort to other sophisticated procedures that involve selective sampling from particular sites. For example, selective venous catheterization of the petrosal sinuses can be particularly useful in detecting ACTH hypersecretion in Cushing's disease (Chapter 9), and selective sampling of the renal veins can be helpful in the diagnosis of renovascular hypertension.
Biopsy procedures are not commonly used for evaluation of endocrine diseases but are occasionally useful to diagnose neoplasia. An exception is the use of fine-needle biopsy of the thyroid gland (Chapter 7), which has had a major impact on evaluation of thyroid nodules.
SCREENING FOR ENDOCRINE DISEASES
Some endocrine diseases are sufficiently common that screening should be part of usual clinical practice. This is true for hypertension and diabetes (Chapter 17). Thus, blood pressure should be measured as part of any physical examination, and when hypertension is present, evaluation for potential causes should be instituted to exclude endocrine disorders (Chapter 10). Thyroid disease has a prevalence of about 3% in women under the age of 60 and an even greater prevalence in both men and women at older ages. The clinical presentations, especially in milder forms, are frequently subtle and missed by the physician. There are no clear recommendations
for screening by measuring serum TSH levels, but such recommendations may ultimately emerge given the increasing awareness of the incidence of thyroid disease and of the detrimental sequelae that can result (Chapter 7). Blood glucose levels should be determined in everyone at some interval. Even though hyperparathyroidism and hypercalcemia of malignancy are of much lower incidence than thyroid disease or diabetes mellitus, determinations of serum calcium ion levels can be easily obtained as part of an automated panel of tests (Chapter 8). Finally, clues to endocrine diseases can be obtained from other abnormalities detected in screening, eg, blood counts and serum electrolyte measurements.
CLINICAL INTERPRETATION OF LABORATORY TESTS
Many salient points in interpreting laboratory tests are mentioned in the preceding sections; these and other points can be summarized as follows:
(1) Any result must be interpreted in light of clinical knowledge about the patient based on the history and physical examination.
(2) Basal levels of hormones or peripheral effects of hormones must be interpreted in light of the way the hormone is released and controlled.
(3) Hormone levels must be interpreted in conjunction with information from other tests that reflect the patient's status: serum PTH levels in conjunction with serum calcium levels (Chapter 8), serum aldosterone levels in conjunction with plasma renin levels (Chapter 9 andChapter 10), serum gonadotropin levels in conjunction with serum estradiol (Chapter 13) or testosterone (Chapter 12) levels, etc.
(4) Occasionally, urinary measurements are superior to plasma tests for assaying the integrated release of hormone. With cortisol, salivary levels are used increasingly.
(5) Ranges of normal values vary from one laboratory to the next. The range for the laboratory utilized should be employed.
(6) Laboratory tests must be interpreted with knowledge of the value of the test, including its sensitivity and specificity (discussed earlier). Reported normal ranges for tests cannot be used as absolute reflections of excess or deficiency states and must be interpreted in light of the clinical situation.
(7) Occasionally, extraneous or contaminating substances interfere with laboratory test results. For example, in illness, plasma lipids sometimes interfere with measurement of thyroid hormone-binding capacity (Chapter 7).
(8) Provocative tests are sometimes necessary.
(9) Imaging studies may help with the diagnosis, especially with respect to the source of hormone hypersecretion.
TREATMENT OF ENDOCRINE DISEASES
Treatment of hormone deficiency states ideally requires replacement with the hormone in a manner that mimics the physiologic setting. In many cases, a reasonable approximation of the physiologic status can be achieved by administering the hormone itself or an analog. Thus, treatment of hypothyroidism with thyroxine, adrenal insufficiency with hydrocortisone, and menopausal symptoms with estrogens have proved effective. In other cases, there are problems with replacement therapy. Whereas recombinant GH is available to treat GH deficiency, it must be injected and is expensive. Whereas PTH has recently been approved by the Food and Drug Administration for treatment of osteoporosis, this peptide hormone is short-acting and may be an inefficient way to treat hypoparathyroidism. Thus, most patients with this disorder will probably continue to be treated with high doses of vitamin D and calcium (Chapter 8). Although insulin therapy controls hyperglycemia and prevents ketoacidosis in most patients with diabetes mellitus, long-term complications still occur with most regimens (Chapter 17). This results at least in part from the fact that we do not replace insulin in an ideal manner. When the hormone is injected subcutaneously, it is not delivered first to the liver; the kinetics of the injected hormone do not mimic accurately enough the physiologic release of insulin; and in many cases the delicate balance between normalization of blood glucose and avoidance of hypoglycemia cannot be achieved. For diseases such as type 1 diabetes mellitus, there is a major need for alternative approaches, as might ultimately be achieved through gene transfer, islet transplantation, mechanical pumps linked to glucose sensors, improved versions of insulin, or other means. Diabetes mellitus also illustrates the fact that the control of conditions such as hyperglycemia or hypertension that usually occur in diabetic patients can have a major effect. Indeed, treating high blood pressure in type 2 diabetes is as important as excellent glycemic control.
Since many cases of type 1 diabetes mellitus, Addison's disease, hypothyroidism, and several other endocrine deficiency states result from autoimmune destruction of the gland, there is a clear need to predict the emergence of the condition and to prevent or limit the damage in the first place. It is already clear that measurement of the levels of certain antibodies with type 1 diabetes mellitus, thyroid disease, and other endocrine deficiency states can predict the development of the disorder before there is major destruction of the
gland. For hormone excess states, treatment is ordinarily directed at the primary cause, usually a tumor, autoimmune condition, or hyperplasia. Tumors are removed when possible. Improvements in surgical techniques have decreased the mortality and morbidity rates associated with surgical removal of tumors of the endocrine system. In addition, alternative and less invasive approaches are supplanting the need for traditional surgery. For example, laparoscopic surgical techniques are being used for removal of tumors inside the abdomen. We cannot yet cure the autoimmune condition that results in hyperthyroidism, so therapy is directed at reducing the secretions of the thyroid gland by pharmacologic blockade, radioiodine therapy, or surgical removal (Chapter 7). Hormone production may also be blocked by pharmacologic means in many other instances. For example, with prolactin hypersecretion, use of the dopamine receptor agonist bromocriptine is preferred to surgical removal of a small prolactinoma (Chapter 5). Octreotide acetate, a somatostatin analog, is sometimes used to block GH hypersecretion (Chapter 6). Inhibitors of steroid production such as ketoconazole are used as an alternative to surgical removal of the steroid-producing tissue (Chapter 9). Mineralocorticoid receptor antagonists (Table 1-2) are used to treat primary aldosteronism, especially when the disorder is due to hyperplasia.
In many cases, it is necessary to control sequelae of hormone excess by alternative means. Thus, β-adrenergic receptor blockers are useful to control sequelae of hyperthyroidism (Chapter 7), α-adrenergic blockers to control sequelae of pheochromocytoma (Chapter 11), mineralocorticoid antagonists to control blood pressure and hypokalemia in primary aldosteronism (Chapter 10), and inhibitors of cholesterol biosynthesis to treat hypercholesterolemia (Chapter 19). With hypertension, a number of modalities are available to block hormone systems. Examples are angiotensin-converting enzyme inhibitors, which block the renin-angiotensin system; calcium channel or β-adrenergic blockers to inhibit second-messenger signaling; or diuretics to lower blood volume.
USES OF HORMONES & SELECTIVE MODULATORS IN THERAPY OF NONENDOCRINE DISEASE
The diverse actions of hormones and hormone antagonists have allowed them to be used extensively in therapy for nonendocrine disease. Hormone action can also be blocked with the use of enzyme inhibitors. Table 1-2 and Table 1-4 list examples of hormone and hormone analog agonists (including eicosanoids) and antagonists. The most extensively used agonists are the glucocorticoids that are given to millions of patients to suppress inflammatory and immunologic responses. That the glucocorticoids would have this application came as a great surprise to the medical world. Hench, Kendall, and Reichstein received the Nobel Prize for this discovery about 1 year after cortisone was first used to treat a patient with rheumatoid arthritis. Other examples include the use of estrogen-progesterone combinations for contraception, GnRH analogs such as leuprolide in combination with nonsteroidal antiandrogens to treat prostate cancer, and antiestrogens to treat breast cancer (Table 1-2). Selective ER modulators such as raloxifene are used for treatment of osteoporosis but may also have benefits in reducing breast cancer incidence. It is likely that the numbers of treatments that are based on modulation or selective modulation of endocrine signaling pathways will increase in the future as new pharmaceuticals become available.
Table 1-4. Hormones used in endocrinologic management for purposes other than replacement.
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