Rhythms govern many endocrine functions. Examples of such rhythmic systems include the insulin-secreting pancreatic beta cell, which regulates blood glucose, and the gonadotropin-releasing hormone (GnRH) neuron, which governs reproductive function. Although serving very different functions within the body, these cell types share many important features. Both GnRH neurons and beta cells, for instance, are hypothesized to generate at least two rhythms endogenously: (1) a burst firing electrical rhythm and (2) a slower rhythm involving intracellular processes. This review details current understanding of these rhythms in each cell type, compares and contrasts the systems, and discusses the relevance of hormone rhythms to both physiology and disease.
Introduction
Rhythmic activity at various frequencies forms the basis of many neural and endocrine systems. The corticothalamic system relating to sleep (1), vasopressin neurons signaling thirst and osmoregulation (2, 3), and PreBoetzinger neurons governing breathing (4), for example, demonstrate rhythmic firing with periods on the order of seconds. Neurons of the suprachiasmatic nucleus, in contrast, generate much slower circadian (~24 hours) rhythms (5, 6), while ultradian rhythms (<24 hours, but typically ~30-120 min) occur in other systems governing, for example, appetite and satiety (7), sleep and dreaming (8-10), cardiac and respiratory function (11), as well as the transcription and translation of several genes (12, 13). Rhythms thus abound in biology as a fundamental means of communication and organization.
This review focuses on two such rhythmic systems: the insulin-secreting pancreatic beta cell, which regulates blood glucose, and the gonadotropin-releasing hormone (GnRH) neuron, which governs reproductive function. Each cell type demonstrates two putatively endogenous rhythms, a rhythm in electrical activity occurring on the order of seconds (‘high frequency’), and a rhythm in secretion on the order of minutes (‘low frequency’). There are also additional rhythmic components to each system (14-19), but as these are not considered to be endogenous to the individual cells, they will not be discussed in this review. The purpose of this introductory review is to describe the intrinsic high and low frequency rhythms of each system, compare their distinct mechanisms and function, and to demonstrate the importance of normal rhythmic function to physiology and dysfunctional rhythmicity to disease.
THE PANCREATIC BETA CELL
Islets of Langerhans are micro-organs that constitute the endocrine portion of the mammalian pancreas and are responsible for regulating blood glucose and body energy metabolism (20). Islets vary in size and shape, and possess their own microvasculature to rapidly carry insulin and other secreted factors out of the islet, as well as to receive nutrients and regulatory factors (21, 22). The islet is composed of several cell types, including glucagon-secreting alpha-cells, insulin-secreting beta cells, somatostatin-secreting delta-cells, and others (23; see Fig.1A). Beta cells compose the vast majority (70-90%) of the total islet mass and synthesize and secrete insulin, a critical regulator of blood glucose (24). In response to a rise in blood glucose concentration following ingestion of a meal, beta cells secrete insulin to stimulate conversion of glucose to glycogen in the liver and the uptake of glucose into insulin target tissues, particularly skeletal muscle and fat (see Fig.1B). When blood glucose levels drop as a result of insulin action, insulin secretion is in turn inhibited, thus completing the regulatory loop. Blood glucose is also regulated by additional factors including glucagon, which counterbalances the effects of insulin by mobilizing glucose from the liver (see Fig.1B).
At the level of the individual beta-cell, the ‘Consensus Model’ provides a detailed description of the cellular response to glucose stimulation (25-27). In this model (see Fig.2), the beta cell is electrically silent at low glucose concentrations (<3 mM, representing fasting conditions), secreting insulin at a low, basal rate. (a) In response to increases in blood glucose (>7 mM, representing conditions following meal ingestion), beta cells take up glucose through the glucose transporter GLUT2 (28, 29). (b) Glucose is then metabolized in the beta cell through a series of biochemical processes, which include increased mitochondrial production of adenosine triphosphate (ATP), resulting in increased cellular energy. For a more detailed description of the complex processes involved in beta cell fuel metabolism, see (30). (c) The resulting increase in the ratio of ATP to ADP (ATP/ADP) closes ATP-sensitive potassium channels (KATP-channels, 31-33). As the dominant resting conductance of the beta cell, KATP-channels normally hyperpolarize the beta-cell membrane under basal glucose conditions to reduce the likelihood of firing action potentials. The closure of KATP-channels following increased glucose metabolism depolarizes the beta cell membrane. (d) This initiates the repetitive firing of calcium-dependent action potentials and the influx of calcium into the beta cell. (e) The resulting increase in intracellular calcium ([Ca2+]i) causes insulin secretion by triggering the exocytosis of insulin containing secretory granules. Once blood glucose is returned to its basal level through insulin action, ATP/ADP levels drop in the beta cell, leading to the reopening of KATP-channels that in turn shut off the glucose-induced electrical activity.
The Consensus Model serves as a good introduction to two important processes in the beta cell: (1) the metabolism of glucose and (2) the regulation of ion channels that generate electrical activity. The inherently rhythmic aspects of both of these processes are discussed below, beginning with beta cell electrical activity. To be complete, we also point out that there are KATP-channel-independent processes that operate in parallel with the Consensus Model to couple glucose uptake and metabolism to granule exocytosis (34-37).
Electrical bursting in beta cells, a high frequency rhythm
Beta cell rhythms were first identified using sharp glass microelectrodes to allow electrophysiological recordings to be made from dissected mouse islets. In 1968, Dean and Matthews showed that mouse islets produce rhythms of repeated action potential firing at a period of ~30 seconds in response to elevated glucose (38). Subsequent studies determined that these rhythms resulted primarily from the coordinated activity of calcium and potassium ion currents (39-43) and that their frequency and duration are glucose-dependent (40, 44, 45). An example of the rhythmic activity of a mouse islet is shown in Fig.3 as measured both by electrical activity (A) and by corresponding changes in [Ca2+]i (B). Bursts of electrical activity have been shown to induce bursts in [Ca2+]i (46, 47), and both electrical activity and [Ca2+]i have been directly correlated with insulin secretion (48-52).
This phenomenon of rhythmic electrical activity is termed ‘bursting’. A burst is described as a train of action potentials that occurs during a ‘plateau’ or ‘active phase’, followed by a period of relative quiescence called the ‘silent phase’ (see Fig.3C). The period of burst firing is measured as the time interval from the start of one burst to the start of the next burst and is also called the ‘burst interval’ or ‘activity cycle’. In beta cells, bursts are also characterized by their ‘plateau fraction’, the relative time spent in the active phase divided by the total burst interval. Plateau fraction is particularly useful for comparing burst firing patterns in beta cells at different glucose concentrations. The duration of time spent in the plateau phase progressively increases with glucose concentration, while the time spent in the silent phase is correspondingly reduced. Because secretion in excitable cells is closely linked to electrical activity and calcium influx, the plateau fraction yields a reasonable estimate of the relative rates of insulin secretion for islets (40, 48-50).
Metabolic oscillations in beta cells, a low frequency rhythm
While the work of Dean and Mathews and others established that mouse pancreatic beta cells are rhythmic, burst firing on the order of seconds does not correspond particularly well with the established period of pulsatile insulin secretion measured in vivo, which in many species ranges from 5-10 min (53-57). A little over a decade after bursting was first reported in islets, two studies reported that islets can also display oscillatory activity with a period about ten times slower than the more classical electrical bursting (58, 59). Subsequent reports using a variety of techniques have shown that islets are in fact capable of a wide variety of oscillatory patterns, which may be slow (Fig.4A), a mixture of fast and slow (B), or fast (C) (47, 60-62). Note that even some fast patterns still can display a small, underlying slow component, as shown in (Fig.4C, lower panel).
As summarized in Table 1, it is now well established that islets generate rhythms in electrical activity, [Ca2+]
i, and a variety of metabolic processes with a period of ~3-5 minutes, in addition to the more rapid bursting activity. Based on these observations from isolated beta cells as well as islets, these low frequency oscillations appear to be endogenous to individual beta cells. Low frequency islet oscillations have also been shown to closely correlate with oscillations in insulin secretion in vitro (50-52) and in vivo (63), suggesting that islet oscillations are indeed important to physiological insulin secretion.
There are two predominant theories about the mechanisms underlying low frequency rhythms in islets. Based on the observation that calcium is required for glucose-stimulated insulin secretion to occur and that each spontaneous oscillation in [Ca2+]i accompanies each oscillation of insulin secretion (60, 79, 80), the ‘calcium hypothesis’ states that oscillations in [Ca2+]i drive insulin secretion. The ‘glycolytic hypothesis’, in contrast, posits that slow oscillations in glycolysis drive oscillations in mitochondrial activity and ATP/ADP, and in turn, KATP-channel conductance, electrical activity, and secretion (81, 82). There is ample evidence for both the glycolytic model (72, 81, 83, 84) and the calcium model (50, 51, 60, 79, 85). Oscillatory processes in glycolysis and [Ca2+]i may even act as independent or inter-related oscillators (82).
THE GNRH NEURON
Unlike the pancreatic beta cell, much less is known about the cellular mechanisms underlying the rhythms of GnRH neurons (also called the luteinizing hormone-releasing hormone or ‘LHRH neurons’). A great deal is known, however, about the function and physiological significance of pulsatile LHRH secretion.
The importance of rhythms to reproductive function
A network of some 800-2000 GnRH neurons is widely distributed throughout the medial basal hypothalamus and preoptic area of the brain (86, 87). Most GnRH neurons target their axons to the median eminence, where GnRH peptide is secreted into the blood steam and carried by the portal vasculature to the anterior pituitary (Fig.5A). Pituitary gonadotropes respond to GnRH signal by secreting both luteinizing hormone (LH) and follicle stimulating hormone (FSH). These pituitary hormones act on the gonads to promote the production of eggs or sperm and the synthesis of steroids (estrogen, testosterone, etc.). The steroids, in turn, provide both positive and negative feedback at the level of the brain and the pituitary to complete a reproductive regulatory loop called the hypothalamic-pituitary-gonadal (HPG) axis (Fig. 5B). The role of GnRH neurons in the HPG-axis is to integrate neural afferent, environmental and steroidal cues related to fertility to form the final common pathway for the central control of reproduction.
The output of this final common pathway is the pulsatile release of GnRH peptide (88-91), a crucial element in maintaining reproductive function. Pulsatile GnRH maintains appropriate levels of LH and FSH, whereas continuous elevation of GnRH suppresses the synthesis and secretion of LH and FSH (92), leading ultimately to infertility (93-95). Modulation of the frequency of GnRH pulses is equally critical (96, 97), as GnRH pulse frequency is a primary determinant of differential pituitary response (98-100). Specifically, higher-frequency pulses favor LH release, whereas lower-frequency pulses favor FSH (Fig.6, 96, 97). To thus ensure the proper development of the ovarian follicle, GnRH neurons must respond to changes in steroid milieu, the stage of the reproductive cycle, and other cues by modulating GnRH pulse intervals from a few minutes to hours.
The GnRH pulse generator, a low frequency rhythm in GnRH neurons
Endogenous secretory rhythms related to reproductive function were first observed in LH at circhoral (~hourly) intervals (101). These hormone pulses were subsequently shown to correlate with episodic volleys of hypothalamic electrical activity (Fig.7, 102, 103). The collection of hypothalamic neurons producing these rhythms has been termed the ‘GnRH pulse generator’.
The molecular basis of the low frequency rhythm comprising the GnRH pulse generator is largely unknown, in part, because GnRH neurons are few in number and widely distributed in a configuration that is neither nuclear nor laminar in organization, making identification of GnRH neurons difficult. Thus, the only electrophysiological report in the first 25 years following the identification of GnRH described results obtained in 4 immunocytochemically identified GnRH neurons out of 102 attempts total using hypothalamic slices (104). Though an impressive effort, this study underscored the need for newer and better methods to identify GnRH neurons. Several strategies have been implemented subsequently, which allow investigators to more efficiently identify and manipulate GnRH neurons experimentally. These include the development of the GT1 and Gn immortalized GnRH cell lines (105, 106), embryonic neuronal cultures (107), and transgenic mice expressing fluorescently tagged GnRH neurons (108-110). Studies utilizing these various techniques have demonstrated pulsatile secretion from cultures of GT1 cells (111-113) (see Figure 8A), embryonic GnRH neurons (114), adult hypothalamic cultures (115, 116), and hypothalamic explants (117). Episodic electrical activity, a correlate to secretion in this system, has also been reported from individual GnRH neurons in brain slices (see Fig.8B) and in dissociated GnRH neurons (91, 118, 119). These findings collectively suggest that the low frequency rhythm observed for GnRH secretion is indeed endogenous to the individual GnRH neuron.
As for the cellular mechanisms of the GnRH pulse generator, there are several possibilities. Autocrine or paracrine feedback of GnRH is one candidate mechanism. GnRH neurons express GnRH-receptors (116, 121, 122), and exogenous GnRH (or agonists) has both positive and/or negative feedback effects on GnRH secretion (116, 123, 124) and electrical activity (122). These findings support a model in which GnRH could act in an autocrine or paracrine feedback loop involving GnRH receptors to modulate secretory pulse frequency through a G-protein-coupled-receptor signaling cascade (122, 125, 126). Another candidate mechanism could involve cycles of gene transcription and translation. As evidence, transcription and translation of GnRH mRNA occurs in a pulsatile fashion in GT1 cells (13, 127), and expression of circadian clock genes appears to be involved in modulating the frequency of GnRH pulses (128). While these studies suggest clear effects on pulsatility, GnRH pulse generation, however, is maintained on the short-term basis of a few hours despite blockade of transcription and translation (113, 127), suggesting that cycles of transcription or translation must not be an integral component of the pulse generator itself. Yet another candidate mechanism, discussed in more detail below, involves cyclic adenosine monophosphate (cAMP) interacting with cyclic-nucleotide-gated-channels (CNG-channels). Though each of these candidate mechanisms shows promise, further evidence is required to determine which mechanism or mechanisms are involved in mediating the underlying oscillatory process responsible for generating GnRH pulses.
Electrical bursting, a high frequency rhythm in GnRH neurons
Although the low frequency rhythm that produces pulsatile GnRH secretion clearly plays an integral role in maintaining reproductive function, much faster rhythms (periods ~5-60 sec) have also been observed. Burst firing from unidentified neurons thought to be GnRH neurons based on their low frequency rhythms was first reported in electrophysiological studies of sheep (102). Spontaneous burst firing of action potentials was later described in detail in GT1 cells (129, Fig.9A) and in GnRH neurons in brain slices (119, 130, 131). Burst firing has also been observed in acutely dissociated GnRH neurons (119, 132, Fig.9B), strongly suggesting that burst firing is an intrinsic property of the GnRH neuron.
By testing the effects of a number of ion channel blockers in cultures of GT1 cells, a critical role for Na+-channels in generating bursts of action potentials and for L-type Ca2+-channels in generating [Ca2+]i oscillations was established (129, 133). Potential modulators of burst firing properties were also identified, including potassium-channels, T-type calcium-channels, and [Ca2+]i stores (129, 134-136). These findings led to a model suggesting that amplitude and/or frequency modulation of burst firing could underlie the secretory patterns in GnRH neurons (129, 136). The presence of many of the same ion channels expressed in GT1 cells has been subsequently confirmed in native GnRH neurons (108, 109, 132, 137, 138).
SIMILARITIES, DIFFERENCES, AND SIGNIFICANCE
Thus far, we have described two rhythmic endocrine systems. In both GnRH neurons and pancreatic beta cells, a low frequency rhythm appears to govern the detectable rate of peptide secretion and circulation of the peptide throughout the body. In both systems, a faster mode of electrical activity also exists. It is interesting that these two different cell types, with very different physiological roles, produce rhythms with remarkably similar qualitative behavior. In this final section, comparisons between these two systems and the relevance of hormone rhythms to both physiology and disease will be the main focus of discussion.
Anatomical organization and communication
One similarity between GnRH neurons and pancreatic islets is their overall anatomical organization. GnRH neurons, as mentioned previously, form a loose network of cells spread throughout the medial basal hypothalamus that act in unison to release GnRH in pulses at the median eminence (Fig.10A). Islets are similarly distributed throughout the pancreas, and although islets are composed of thousands of endocrine cells, each islet acts as a unit to secrete insulin pulses into the vasculature (Fig.10B). It should be noted that while this review has focused on the underlying endogenous rhythms of both beta cells and GnRH neurons, synchronization of many such endogenous oscillators is thought to be requisite for secretory pulses to properly stimulate downstream targets.
The diffuse distribution of many intrinsic oscillators in the body or within specific tissues represents a communications dilemma. How these systems communicate to produce synchronized hormone pulses is not well understood for either GnRH neurons or for islets. It has been proposed that islets can be entrained to a single pancreatic secretory pattern by a number of putative synchronizing mechanisms, such as an intrapancreatic ganglion pacemaker (55, 139, 140), circulating inter-islet factors (141), or feedback interactions with the liver (142, 143), although there is no overwhelming evidence thus far for any of these hypotheses. GnRH neurons, likewise, are separated from one another within the hypothalamus and appear to have some, but not extensive, contact with one another (126, 144, 145). They may not even require physical contact in order to secrete GnRH in a pulsatile fashion, as GnRH pulses can still be observed to occur from acutely dissociated hypothalamic neurons (117). Because most GnRH neurons project their axons the median eminence, this is one potential point of contact or near contact. Of interest in this regard, isolated median eminence tissue containing only GnRH axons and synaptic terminals has been shown to maintain pulsatile GnRH release (146). This suggests that the synchronization of GnRH secretion could occur by any number of methods, including diffusible factors such as nitric oxide (147, 148) and GnRH (122, 125, 126).
The advantage, if any, to having a dispersed distribution of intrinsic oscillators is not known, although one possibility is that this distribution could be a means of gathering different inputs from different brain regions. It is perhaps not unreasonable to suspect that an individual GnRH neuron, for example, might receive certain inputs related to reproduction such as photoperiod (149) or nutrition (150), but not others, based the location of the GnRH neuron in the hypothalamus. Although there is little evidence for this hypothesis at present, GnRH neurons in different hypothalamic regions have been shown to differ in their sensitivity to steroids (118, 151) and in their glutamatergic inputs (152). A network of many widely distributed GnRH neurons might thus collect a wider array of inputs to integrate into the overall reproductive output signal. Islets, likewise, might receive different signals based on anatomical location within the pancreas, although this remains to be established.
Frequency vs. amplitude coding
Although GnRH neurons and pancreatic beta cells may generate rhythms in a seemingly analogous fashion, one interesting difference between the two systems is how their rhythmic hormone release is modulated. GnRH neurons govern reproductive function primarily by altering the frequency of GnRH pulses to code for different responses from the pituitary. This is interesting in that proper reproductive function in females requires numerous processing stages of the follicle during each ovulatory cycle; this requires a precise balance of hormones that are specific to each stage (93, 94). GnRH neurons thus promote continuous changes in the steroidal milieu throughout the ovulatory cycle, which may be most effectively communicated by modulating the frequency of GnRH pulses in response to steroid feedback and cycle stage.
The pancreas, in contrast to GnRH neurons, generates pulses at a fairly steady frequency, and responds to increased glucose loads or to other fuel stimuli by increasing the amplitude of insulin pulses produced, as demonstrated by in vivo studies (80, 153, 154). This use of pulse amplitude modulation instead of frequency modulation could be indicative of a different mechanism of action and purpose (155). Rather than promoting complex shifts in the hormone milieu, the role of islets is to secrete appropriate amounts of insulin on a minute-to-minute basis in order to maintain blood glucose within a tight range. The advantage of amplitude rather than frequency coding in this system is not known at this time, although perhaps the targets of insulin action, liver, muscle and fat tissue, may be better able to respond appropriately to changes in pulse amplitude.
The link between high and low frequency rhythms
For burst patterns resulting from oscillations in plasma membrane potential to interact with slow oscillations in intracellular processes as described above, a mechanism is needed which links plasma membrane ion fluxes to biochemical oscillations in the cytoplasm. As mentioned briefly in the description of the ‘Consensus Model’ of beta-cell function, the KATP-channel can readily provide this link, as first proposed by Cook and Hales (156). KATP-channels were first shown to be metabolically sensitive in beta cells by demonstrating the direct effects of exogenous ATP on single channel activity in inside-out patches (25, 156), or by exposing beta cells to bath glucose while recording KATP-channel activity in cell-attached patches (157). Spontaneous changes in single-channel KATP conductance in situ were later observed to occur at similar intervals as low frequency metabolic oscillations (77, 78), implying that the activity of KATP-channels may be responsive to endogenous changes in beta-cell fuel metabolism.
Although the KATP-channel appears to be the dominant link between metabolic processes and the plasma membrane of the beta cell, low frequency islet rhythms in electrical activity persist in islets from SUR1 -/- knockout mice, which lack functional KATP channels (158). This suggests metabolic oscillations must also influence other electrical mechanisms. A number of other ion channels are thought to be metabolically sensitive in beta cells, such as chloride-channels (159), L-type Ca2+-channels (160), and Kslow channels (161, 162), any of which might in theory be able to function as redundant or secondary mechanisms in the event of the loss of the KATP-channel. The majority of evidence, nevertheless, points to the KATP-channel as the dominant player in coupling rhythmic processes in the pancreatic beta cell under physiological conditions.
For GnRH neurons, an analogous mechanism for linking intracellular processes to changes in the membrane potential has been proposed involving cAMP and cyclic nucleotide gated nonselective channels or ‘CNG-channels’. In the cAMP model, an increase in neuronal cAMP opens the CNG-channels, resulting in cell depolarization, increased electrical activity, and GnRH secretion. Rising cAMP levels also subsequently activate a protein kinase A pathway that in turn reduces cAMP, thus closing the CNG-channels and reducing secretion (163, 164). As evidence for this mechanism, CNG-channel subunits have been identified in GT1 cells (164) and in rat GnRH neurons (165). Treatment with cAMP or analogs activate CNG-channels in GT1 cells (166), increases spontaneous [Ca2+]i oscillations and burst firing (166), and increases GnRH secretion (167). Further, blocking CNG-channels eliminates the stimulatory effects of cAMP (166), demonstrating that CNG-channels play a key role in carrying out the effects of cAMP. More recently, a study of GT1 cells overexpressing phosphodiesterase to lower cAMP levels reduced GnRH pulse frequency (168), suggesting that cAMP may be capable of generating, or at least modulating, low frequency rhythms in GnRH neurons.
The relevance of rhythms to physiology and disease
Pulsatility in the GnRH system, as stated earlier, is crucial to proper reproductive function. An imbalance in steroids, stress, or a significant change in energy balance towards obesity or anorexia can result in disruptions or arrest of the pulsatile GnRH signal. The inability to modulate GnRH pulse frequency can also result in the loss of reproductive function. Hyperprolacteinemia and hypothalamic amenorrhea, for example, are associated with chronically low frequency GnRH pulses (169, 170), and polycystic ovarian syndrome (PCOS) is associated with persistent high frequency GnRH pulses (171). These reproductive disorders thus demonstrate pulsatility and pulse frequency coding in the GnRH system are crucial to fertility. A summary of reproductive disorders related to dysfunctional GnRH pulsatility can be found in (94).
Pulsatility also appears to be important to the regulation of blood glucose by insulin. Delivering intermittent insulin to healthy individuals results in a more effective hypoglycemic response than continuous insulin in most (172-175), but not all, studies (176, 177). In addition, patients with diabetes were found to respond more effectively to pulsatile insulin delivery than to continuous delivery (175, 178-180). Loss of pulsatile secretion also seems to be an early stage of Type 2 diabetes, with reduction in the amplitude and possibly frequency of the insulin pulses being linked to diabetic patients (181-185). The close relatives of diabetic patients also demonstrate considerable degradation of pulsatile insulin secretion despite reporting clinically normal responses to glucose challenges and normal levels of insulin resistance (186, 187). Aberrant insulin pulsatility has also been reported in specific forms of diabetes and in other metabolic disorders, including mature onset diabetes of the young Type 2 (MODY2) (188), Tauri’s disease or glucogenosis type-VII (183), and maternally inherited diabetes and deafness (MIDD) (189), as well as other conditions such as hypertension (190) and obesity (191, 192). Thus, considerable evidence suggests pulsatile insulin secretion is important to the proper regulation of blood glucose and a potential early warning sign of an emerging disease state. A review of metabolic disorders related to dysfunction in pulsatile insulin secretion can be found in (80).
Conclusions
Several common themes have emerged from this comparison of the GnRH and insulin endocrine systems. First, rhythmic activity takes on several forms, including a low frequency rhythm synonymous with secretory pulses and also a higher frequency burst pattern, which may facilitate secretory efficiency and/or communication with downstream targets. Second, these rhythms appear to be functionally connected. Third, secretory rhythms from GnRH neurons and pancreatic beta cells are functionally important to their respective systems, as evidenced by the disorders and disease states that can result from the loss of pulsatile activity. The GnRH neuron and the pancreatic beta cell may thus serve as models for study of other endocrine systems.
Acknowledgments.
Supported by NIH grants RO1 DK46409 to L.S.S. and F32 DK065462 to C.S.N. Thanks to Drs. Suzanne Moenter and my family for valuable editorial comments and Min Zhang for providing unpublished data.
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and Leslie S. Satin Islets of Langerhans are micro-organs that constitute the endocrine portion of the mammalian pancreas and are responsible for regulating blood glucose and body energy metabolism (20). Islets vary in size and shape, and possess their own microvasculature to rapidly carry insulin and other secreted factors out of the islet, as well as to receive nutrients and regulatory factors (21, 22). The islet is composed of several cell types, including glucagon-secreting alpha-cells, insulin-secreting beta cells, somatostatin-secreting delta-cells, and others (23; see Fig.1A). Beta cells compose the vast majority (70-90%) of the total islet mass and synthesize and secrete insulin, a critical regulator of blood glucose (24). In response to a rise in blood glucose concentration following ingestion of a meal, beta cells secrete insulin to stimulate conversion of glucose to glycogen in the liver and the uptake of glucose into insulin target tissues, particularly skeletal muscle and fat (see Fig.1B). When blood glucose levels drop as a result of insulin action, insulin secretion is in turn inhibited, thus completing the regulatory loop. Blood glucose is also regulated by additional factors including glucagon, which counterbalances the effects of insulin by mobilizing glucose from the liver (see Fig.1B).
At the level of the individual beta-cell, the ‘Consensus Model’ provides a detailed description of the cellular response to glucose stimulation (25-27). In this model (see Fig.2), the beta cell is electrically silent at low glucose concentrations (<3 mM, representing fasting conditions), secreting insulin at a low, basal rate. (a) In response to increases in blood glucose (>7 mM, representing conditions following meal ingestion), beta cells take up glucose through the glucose transporter GLUT2 (28, 29). (b) Glucose is then metabolized in the beta cell through a series of biochemical processes, which include increased mitochondrial production of adenosine triphosphate (ATP), resulting in increased cellular energy. For a more detailed description of the complex processes involved in beta cell fuel metabolism, see (30). (c) The resulting increase in the ratio of ATP to ADP (ATP/ADP) closes ATP-sensitive potassium channels (KATP-channels, 31-33). As the dominant resting conductance of the beta cell, KATP-channels normally hyperpolarize the beta-cell membrane under basal glucose conditions to reduce the likelihood of firing action potentials. The closure of KATP-channels following increased glucose metabolism depolarizes the beta cell membrane. (d) This initiates the repetitive firing of calcium-dependent action potentials and the influx of calcium into the beta cell. (e) The resulting increase in intracellular calcium ([Ca2+]i) causes insulin secretion by triggering the exocytosis of insulin containing secretory granules. Once blood glucose is returned to its basal level through insulin action, ATP/ADP levels drop in the beta cell, leading to the reopening of KATP-channels that in turn shut off the glucose-induced electrical activity.
As summarized in Table 1, it is now well established that islets generate rhythms in electrical activity, [Ca2+] i, and a variety of metabolic processes with a period of ~3-5 minutes, in addition to the more rapid bursting activity. Based on these observations from isolated beta cells as well as islets, these low frequency oscillations appear to be endogenous to individual beta cells. Low frequency islet oscillations have also been shown to closely correlate with oscillations in insulin secretion in vitro (50-52) and in vivo (63), suggesting that islet oscillations are indeed important to physiological insulin secretion.
The output of this final common pathway is the pulsatile release of GnRH peptide (88-91), a crucial element in maintaining reproductive function. Pulsatile GnRH maintains appropriate levels of LH and FSH, whereas continuous elevation of GnRH suppresses the synthesis and secretion of LH and FSH (92), leading ultimately to infertility (93-95). Modulation of the frequency of GnRH pulses is equally critical (96, 97), as GnRH pulse frequency is a primary determinant of differential pituitary response (98-100). Specifically, higher-frequency pulses favor LH release, whereas lower-frequency pulses favor FSH (Fig.6, 96, 97). To thus ensure the proper development of the ovarian follicle, GnRH neurons must respond to changes in steroid milieu, the stage of the reproductive cycle, and other cues by modulating GnRH pulse intervals from a few minutes to hours.
The molecular basis of the low frequency rhythm comprising the GnRH pulse generator is largely unknown, in part, because GnRH neurons are few in number and widely distributed in a configuration that is neither nuclear nor laminar in organization, making identification of GnRH neurons difficult. Thus, the only electrophysiological report in the first 25 years following the identification of GnRH described results obtained in 4 immunocytochemically identified GnRH neurons out of 102 attempts total using hypothalamic slices (104). Though an impressive effort, this study underscored the need for newer and better methods to identify GnRH neurons. Several strategies have been implemented subsequently, which allow investigators to more efficiently identify and manipulate GnRH neurons experimentally. These include the development of the GT1 and Gn immortalized GnRH cell lines (105, 106), embryonic neuronal cultures (107), and transgenic mice expressing fluorescently tagged GnRH neurons (108-110). Studies utilizing these various techniques have demonstrated pulsatile secretion from cultures of GT1 cells (111-113) (see Figure 8A), embryonic GnRH neurons (114), adult hypothalamic cultures (115, 116), and hypothalamic explants (117). Episodic electrical activity, a correlate to secretion in this system, has also been reported from individual GnRH neurons in brain slices (see Fig.8B) and in dissociated GnRH neurons (91, 118, 119). These findings collectively suggest that the low frequency rhythm observed for GnRH secretion is indeed endogenous to the individual GnRH neuron.
Although the low frequency rhythm that produces pulsatile GnRH secretion clearly plays an integral role in maintaining reproductive function, much faster rhythms (periods ~5-60 sec) have also been observed. Burst firing from unidentified neurons thought to be GnRH neurons based on their low frequency rhythms was first reported in electrophysiological studies of sheep (102). Spontaneous burst firing of action potentials was later described in detail in GT1 cells (129, Fig.9A) and in GnRH neurons in brain slices (119, 130, 131). Burst firing has also been observed in acutely dissociated GnRH neurons (119, 132, Fig.9B), strongly suggesting that burst firing is an intrinsic property of the GnRH neuron.