Signal transduction in the hypothalamic corticotropin-releasing factor system and its clinical implications.

Corticotropin-releasing factor (CRF) is a major regulatory peptide in the hypothalamic-pituitary-adrenal (HPA) axis under stress conditions. In response to stress, CRF is produced in the hypothalamic paraventricular nucleus. Forskolin- or pituitary adenylate cyclase-activating polypeptide-stimulated CRF gene transcription is mediated by the cyclic AMP (cAMP) response element on the CRF 5'-promoter region. Estrogens enhance activation of the CRF gene in stress, while inducible cAMP-early repressor suppresses the stress response via inhibition of the cAMP-dependent CRF gene. Glucocorticoid-dependent repression of cAMP-stimulated CRF promoter activity is mediated by both the negative glucocorticoid-response element and the serum-response element, while interleukin-6 (IL-6) stimulates the CRF gene. Suppressor of cytokine signaling-3, stimulated by IL-6 and cAMP, is involved in the negative regulation of CRF gene expression. Such complex mechanisms contribute to stress responses and homeostasis in the hypothalamus. Moreover, disruption of the HPA axis may cause a number of diseases related to stress. For example, CRF-induced p21-activated kinase 3 mRNA expression may be related to the proliferation of corticotrophs in Nelson's syndrome. A higher molecular weight form of immunoreactive ß-endorphin, putative proopiomelanocortin (POMC), is increased in CRF-knockout mice, suggesting the important role of CRF in the processing of POMC through changes in prohormone convertase type-1 expression levels.

Ghrelin stimulates corticotropin-releasing factor and vasopressin gene expression in rat hypothalamic 4B cells.

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) play a central role in regulating the stress response. In response to stress, CRF and AVP neurons in the hypothalamic paraventricular nucleus secrete the peptides to stimulate the release of adrenocorticotropic hormone from the anterior pituitary. Ghrelin, an endogenous ligand of the growth hormone-releasing peptide receptors (GHSR), has been shown to stimulate the release of CRF and AVP by rat hypothalamic explants. However, little is known about the ability of the ghrelin signaling pathways to activate the CRF and AVP genes in the hypothalamus. In the present study, we examined the direct effect of ghrelin on CRF and AVP gene expression in hypothalamic 4B cells, which show the characteristics of the hypothalamic parvocellular paraventricular nucleus neurons. Cells were transfected with CRF or AVP promoter to examine the activity of each promoter. Ghrelin stimulated the promoter activities and mRNA levels for both CRF and AVP. The involvement of a protein kinase pathway was examined using inhibitors. Protein kinase A and phospholipase C pathways were shown to be involved in ghrelin-induced increases in both CRF and AVP promoter activities. GHSR type 1a (GHSR1a) mRNA levels were also increased by ghrelin, and these ghrelin-induced levels were suppressed by a GHSR1a antagonist. Thus, ghrelin-dependent pathways are involved in the regulation of CRF and AVP gene expression in the hypothalamus: ghrelin, an orexigenic hormone, stimulates CRF, an anorexigenic/anxiogenic factor in the hypothalamus, resulting in hypothalamic-pituitary-adrenal axis activation to stimulate the release of glucocorticoids.

The frequency of type 2 diabetic patients who meet the endocrinological screening criteria of subclinical Cushing's disease.

Cushing's syndrome, including its mild form/state of adrenal-dependent subset (subclinical Cushing's syndrome; subCS), is known to enhance glucose intolerance, hypertension and obesity. Recently, subclinical Cushing's disease (subCD) has been identified, but its prevalence and the extent of consequent metabolic derangement are unclear. We screened 90 type 2 diabetic patients hospitalized in our department for subCD, according to the diagnostic guideline proposed by the working group of Japanese Ministry of Health, Welfare and Labor in 2006. Plasma ACTH and cortisol levels in the morning and at midnight were determined, and overnight 0.5 mg dexamethasone suppression test (DST) was performed. Those who showed poor cortisol suppression in DST underwent the desmopressin (DDAVP) test. Fifty-seven patients (63.3%) demonstrated abnormally high midnight cortisol levels (>or=2.5 microg/dL), while only nine of them failed to suppress plasma cortisol levels to <3 microg/dL after DST. Although none of the eight patients who underwent the DDAVP test demonstrated the anticipated paradoxical rise in plasma ACTH, these eight patients (8.9%) endocrinologically met the screening criteria of subCD. Since a considerable percentage of pituitary adenomas causing overt Cushing's disease are not identifiable in magnetic resonance imaging, many of those causing subCD may also be unidentifiable. Further follow-up studies including confirmatory testing and pituitary imaging are necessary.

[Disorder of cholesterol metabolism: regulation of intracellular cholesterol and membrane trafficking].

It has been clarified that several transcription factors and functioning proteins play important roles regulating intracellular cholesterol levels. They bind to the ER membrane and sense changes in cholesterol levels in the membrane through SSD. An important membrane-binding transcription factor, SREBP, is retained in the ER membrane, forming an SREBP/SCAP/INSIG trimer when cellular cholesterol levels are abundant. This complex blocks the transport of SREBPs to the Golgi apparatus, thus preventing subsequent transcriptional activation. When cellular cholesterol levels are low, the ER cholesterol concentration is below a threshold value ( <5 mol %). Under these conditions, SCAP escorts SREBPs from the ER to Golgi apparatus by binding to a component of the CopII protein coat. Once in the Golgi apparatus, the SREBPs are proteolytically processed to generate their nuclear form, the bHLH leucine zipper, that activates genes for cholesterol synthesis and uptake. HMG-CoA reductase is also post-transcriptionally regulated by sterol, with INSIG binding of the protein leading to its proteosomal degradation. We demonstrated that Tangier disease and Niemann-Pick disease type B and type C are metabolic disorders of membrane cholesterol. These diseases are not so common in clinical medicine; however, it is very important to understand membrane lipid metabolism, especially in the ER. It will be clarified in the near future disorders of membrane cholesterol trafficking contribute to the pathogeneses of many kinds of disease affecting through ER functioning.

HMG-CoA reductase inhibitor, simvastatin improves reverse cholesterol transport in type 2 diabetic patients with hyperlipidemia.

AIM: ApoA-I and HDL promote cellular cholesterol efflux in the early stages of the reverse cholesterol transport (RCT) pathway. A low plasma HDL-C level is characteristic of atherogenic dyslipidemia in patients with type 2 diabetes. We evaluated plasma lipid levels and the expression of factors related to RCT in type 2 diabetic patients, and the effects of an HMG-CoA reductase inhibitor, simvastatin, were studied. METHODS: Messenger RNA (mRNA) expression in circulating mononuclear cells was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), focusing on the following factors: liver X receptor alpha (LXR alpha), ATP-binding cassette A1 (ABCA1), scavenger receptor class B type 1 (SR-B1), apolipoprotein E (ApoE), apolipoprotein A-1 (ApoA-1), caveolin, and cholesterol ester transfer protein (CETP). Type 2 diabetic subjects (n=29) were divided into three subgroups: patients with normolipidemia (DM group, n=11), patients with untreated hyperlipidemia (DMHL group, n=10), and those with hyperlipidemia treated with simvastatin 5-10mg/day (DMST group, n=8). The control group (CNT group) included seven healthy volunteers. RESULTS: Simvastatin treatment significantly increased plasma levels of ApoA-I compared to the other three groups. Simvastatin treatment improved the expression of mRNA for LXRalpha, ABCA1, and ApoA-I compared with DMHL or control groups. CONCLUSION: Our data suggest that RCT may be reduced in type 2 diabetic patients with hyperlipidemia, and simvastatin may be able to improve reverse cholesterol transport for this population of diabetic patients.

Possible Insulinotropic Action of Apolipoprotein A-I Through the ABCA1/Cdc42/cAMP/PKA Pathway in MIN6 Cells.

Aims/Introduction: We studied the mechanisms for the possible insulinotropic action of apolipoprotein (Apo) A-I in mouse insulinoma (MIN6) cells. Materials and Methods: The effects of ApoA-I on cAMP production and glucose-stimulated insulin secretion (GSIS), and the dose dependency (ApoA-I at 5, 10, 25, and 50 µg/ml) were determined using MIN6 cells. The effects of the small-interference ribonucleic acid (siRNA) of ATP-binding cassette transporter A1(ABCA1) and Cell division control protein 42 homolog (Cdc42) on the insulinotropic action of ApoA-I was studied, as well as mRNA and protein levels of ABCA1 and Cdc42. Then, the influence of cAMP inhibitor SQ22536, and the cAMP-dependent protein kinase inhibitor Rp-cAMPS on ApoA-I action were studied. Results: Addition of ApoA-I produced cAMP and increased insulin secretion, dose-dependently in high glucose concentration (25 mmmol/l). and ABCA1 protein and Cdc42 mRNA and protein were also enhanced. Specific ABCA1 and Cdc42 siRNA significantly decreased the effects of ApoA-I on insulin secretion compared with negative controls. Manifestations of ABCA1 and Cdc42 mRNA and protein were less than that of the negative control group. Both cAMP inhibiror (SQ22536) and protein kinases inhibitor (Rp-cAMPS) strongly inhibited the effects of ApoA-I on insulin secretion. Conclusions: We demonstrated that ApoA-I enhances glucose-stimulated insulin release in high glucose at least partially through the ABCA1/Cdc42/cAMP/ Protein kinase A (PKA) pathway.

[Clinical analysis of diabetes mellitus with infectious diseases].

The frequency of diabetes mellitus has risen in Japan as the traditional diet has become increasing Americanized and society has aged. With this has come a rise in infectious diseases and complications elderly diabetic patients and a growing need for appropriate management to maintain their quality of life (QOL) and minimize medical measures. Subjects were 98 diabetic patients-60 men and 38 women hospitalized for intravenous antibiotic treatment of infectious disease from 2002 to 2005. We studied plasma glucose control, plasma and urinary protein levels related to nephropathy, and inflammatory responses to treatment. Subjects were divided into good (under 6.5%), fair (from 6.5% to 8.0%), poor (over 8.0%) and severe (over 10%) groups by HbA1c level on admission. We then compared white blood cell counts, CRP levels and the antibacterial medication periods. Those with poorly controlled plasma glucose control, hypoalbuminuria and interrupted or untreated diabetes required significantly longer antibacterial administration. Insulin was increased by the complications of infection, and decreased as infection ameliorated. Appropriate antibiotic administration is essential for diabetic patients with infectious disease, in addition to early intervention, strict plasma glucose control, continuous treatment, and maintenance of good nutrition. Such treatment improves QOL, shortens antibiotic administration, staves off antibiotic-resistant bacteria, and cuts medical costs.

Glucose-stimulated insulin response in non-diabetic patients with lipoprotein lipase deficiency and hypertriglyceridemia.

Elevations in plasma triglyceride (TG) and free fatty acid (FFA) concentrations are generally thought to play a role in the pathogenesis of insulin-resistant diabetes. The objective of this study was to investigate the relationship between hypertriglyceridemia and glucose-stimulated insulin responsiveness in non-diabetic patients. Forty subjects were divided into three BMI-matched groups as follows: one group consisted of 8 patients with a lipoprotein lipase (LPL) deficiency, another consisted of 12 patients with hypertriglyceridemia and a third consisted of 20 subjects with normal TG levels. In response to a 75 g oral glucose tolerance test, plasma insulin levels in the LPL-deficient subjects were higher (106+/-11 microU/ml) than those in the hypertriglyceridemic (69+/-16 microU/ml) and normolipidemic (29+/-3 microU/ml) subjects, at 30 min. On the other hand, their plasma glucose levels (127+/-6 mg/dl) were less than those seen in the normolipidemic group (165+/-9 mg/dl) after 90 min. Thus, LPL-deficient subjects with hypertriglyceridemia displayed an enhanced glucose-stimulated insulin response as well as lower blood glucose levels, the latter of which is not generally seen in those with hypertriglyceridemia and normolipidemia.

Clinical Significance of Determining Plasma MicroRNA33b in Type 2 Diabetic Patients with Dyslipidemia.

AIM: Sterol regulatory element-binding protein (SREBP)-1c is the dominant liver insulin-stimulated isoform and strongly correlates with diabetic dyslipidemia characterized by hyperinsulinemia [i.e., high-density lipoprotein cholesterol (HDL-C) levels and hypertriglyceridemia]. MicroRNA (miRNA) 33b is harbored in the intron of SREBP-1c and represses ATP-binding cassette, sub-family A, and member 1 (ABCA1) expression, essential for HDL formation. We measured plasma miRNA33b levels as possible biomarkers for diabetic dyslipidemia in patients with type 2 diabetes mellitus (T2DM) showing insulin resistance. METHODS: The participants included 50 patients with T2DM (M/F 31/19) enrolled in an educational program for controlling blood glucose levels at Hirosaki University Hospital. HbA1c, fasting plasma glucose, insulin, and lipid levels were determined. Plasma miRNA33b, miRNA33a and miRNA148a were quantified using a TaqMan® MicroRNA Assay, and values were corrected with reference to miRNA16. RESULTS: Mean BMI of participants were 28.2±6.6 (kg/m2) and the Homeostasis Model Assessment of Insulin Resistance was 4.3±2.7. Patients' laboratory findings indicated diabetic dyslipidemia with insulin resistance. Plasma miRNA33b/16 levels revealed a positive correlation with plasma insulin level (r=0.326, P=0.021), serum C-peptide (r=0.280, P=0.049), and triglyceride (r=0.351, P=0.012), but no association with HDL-C (r=-0.210, P=0.143). The blood level of miRNA33a was approximately 1/150th of that of miRNA33b and was not correlated with the above parameters. CONCLUSION: We postulated that plasma miRNA33b may be useful as a new metabolic biomarker of dyslipidemia in patients with T2DM as well as metabolic syndrome via an insulin/SREBP-1c/miRNA33b/ABCA1 pathway.

The Evaluation of Adrenal Function in Two Cases of Hypocortisolism Accompanied by Liver Cirrhosis.

Adrenal insufficiency may occur in patients with liver cirrhosis. The assessment of hypothalamus-pituitary-adrenal function is important in such patients, but there is no consensus as to how it should be performed. We herein report the results of our evaluation of the adrenal function in two patients with hypocortisolism accompanied by liver cirrhosis. The patients lacked the typical features of hypocortisolism. One was diagnosed with hypocortisolism accompanied by liver cirrhosis while the other had secondary adrenal insufficiency caused by a hypothalamic disorder. Hypocortisolism accompanied by liver cirrhosis should be evaluated by endocrine tests to determine its pathogenesis. A low-dose adrenocorticotropic hormone test may be appropriate for non-critically ill cirrhotic patients.

Evaluation of apolipoprotein E secretion by macrophages in type 2 diabetic patients: role of HDL and apolipoprotein A-I.

It has been shown that apolipoprotein A-I (ApoA-I) stimulates the secretion of apolipoprotein E (ApoE) from human macrophages. ApoA-I is a major protein constituent of HDL which because of its role in reverse cholesterol transport, has been implicated in the prevention of atherosclerosis. We herein investigated the ability of monocyte-derived macrophages (MDMs) in 42 patients with type 2 diabetes to secrete ApoE; these patients commonly have low plasma HDL and ApoA-I levels. Our data showed that ApoE secretion from these cells was reduced in patients with low plasma HDL and ApoA-I levels; there were positive correlation between ApoE secretion from MDMs and plasma HDL (r2=0.33, p=0.03) and ApoA-I (r2=0.31, p=0.03). Furthermore, we found that ApoE secretion increased concomitantly with an increase in HDL or ApoA-I in treated diabetics (n=24) from 1.99+/-1.86 to 3.40+/-1.77 ng/mg cell protein. These findings suggest another possible link between HDL and ApoA-I metabolism and atherosclerosis in patients with type 2 diabetes.

Clinical characteristics of Japanese youth-onset type 2 diabetes with ketonuria.

The study analyzed the clinical background and eating habits of Japanese youth-onset type 2 diabetes. Thirty-six patients with type 2 diabetes (22 males, 14 females) with onset in less than 20-year-old were studied. All patients were negative for anti-glutamic acid decarboxylase (GAD) antibody and islet cell antibody. Cases diagnosed as having abnormalities in the mitochondrial gene, maturity onset diabetes of the young (MODY), and apparent type 1 diabetes were excluded from the study. Urinary ketone was detected positive in 11 cases among 36 patients at the onset of diabetes. We compared the clinical characteristics and food compositions between the patients with ketonuria and those without ketonuria. Age and urinary C-peptide secretion did not show any significant difference between both groups. In the patients with ketonuria, male to female ratio was remarkably high (10:1) compared with the group without ketonuria (12:13). Positive diabetic family history was predominantly higher in the group with ketonuria (11/11) than that in the group without ketonuria (17/25). All these were identical to previously reported characteristics of soft-drink ketosis. However, we in this study, revealed the difference of total calorie intake and dietary composition between youth-onset type 2 diabetes with and without ketonuria. As a result dietary contents such as carbohydrate, fat and confectionery in the former group were also 1.5, 1.4-2.4 times higher, respectively, than those in the latter group.

Effects of fluvastatin in type 2 diabetic patients with hyperlipidemia: reduction in cholesterol oxidation products and VCAM-1.

The purpose of this study was to investigate the lipid-lowering and anti-oxidative effects of fluvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, in type 2 diabetic patients. Six patients (3 men and 3 women, mean age = 56.2) took 20 mg of fluvastatin once daily (at night) for 12 weeks. Several markers of oxidative stress were then measured in these patients including plasma cholesterol oxidation products, i.e. oxysterols, and the levels of circulating adhesion molecules. Plasma total cholesterol levels were reduced by 12.3% in these individuals after 4 weeks of treatment, with levels remaining below 220 mg/dl for the entire treatment period. LDL levels were significantly reduced at 4 (18.1%) and 12 weeks (16.1%), and triglyceride levels were significantly reduced after 8 (22.5%) and 12 (37.7%) weeks of treatment. HDL-C levels increased from 50.7 +/- 15.4 prior to treatment to 63.8 +/- 24.3 mg/dl after 12 weeks of treatment, though this increase was not statistically significant. Lipid hydroperoxide, thiobarbituric acid-reactive substance (TBARS), and oxysterol levels were also reduced, suggesting that fluvastatin also had anti-oxidative effects. Finally, VCAM-1 levels were similarly reduced by fluvastatin treatment. We conclude that fluvastatin safely improves the plasma lipid profile in type 2 diabetic patients with hyperlipidemia. We speculate that this drug might be doubly effective in reducing atherosclerosis and cardiac events in these patients as a result of its demonstrated anti-oxidative effects and its ability to reduce VCAM-1 levels.

LDL particle size and lipid composition are risk factors for microalbuminuria in normotensive and normocholesterolemic patients with type 2 diabetes.

The study was to evaluate the influence of particle size and lipid composition of low-density lipoprotein (LDL) on urinary albumin excretion and oxidative susceptibility of LDL, and to define association between LDL particle size and alpha-tocopherol content in LDL from normotensive and normocholesterolemic patients with type 2 diabetes. Twenty-three patients with type 2 diabetes (13 males, 10 females) were studied, and none of these patients had hypertension, hypercholesterolemia and overt proteinuria. The baseline body mass index of all patients was less than 28 kg/m2. All patients were hospitalized in Hirosaki University Hospital and took dietary therapy whose total intake was restricted to less than 30 kcal/kg of ideal body weight for 3 weeks. Their plasma glucose levels were controlled within fasting plasma glucose <140 mg/dl and 2-h postprandial plasma glucose <200 mg/dl. LDL particle size was evaluated by using high-resolution polyacrylamide gel electrophoresis (Lipoprint LDL System) and expressed by Rf value. LDL was incubated with 0.25 microM CuSO4 for 20 h, and the degree of LDL oxidation was determined by malondialdehide analysis. Twenty-four-hour urinary C-peptide excretion and plasma triglyceride concentration in patients with microalbuminuria were significantly higher than those in normoalbuminuric patients. Rf values in microalbuminuric patients were significantly greater than those in normoalbuminuric patients. There were significantly inverse correlations between Rf value and alpha-tocopherol content in LDL, and between Rf value and LDL-free cholesterol/LDL-total cholesterol. Thiobarbituarte-reactive substance level in LDL had a tendency to correlate with Rf value and significantly inverse correlation to alpha-tocopherol content in LDL. In type 2 diabetics without hypertension, hypercholesterolemia and obvious obesity, smaller LDL particle size, accompanied by mild hyperinsulinemia and mild hypertriglyceridemia seems to be one of the important factors responsible for microalbuminuria. In addition, the present study suggests that the decrease of alpha-tocopherol content in small LDL particle is associated with oxidative susceptibility to Cu2+-induced oxidation.

Reduction of paraoxonase-1 activity may contribute the qualitative impairment of HDL particles in patients with type 2 diabetes.

AIMS: Cholesterol efflux with high-density lipoprotein (HDL) particles has an important role in the first step of reverse cholesterol transport (RCT). However, HDL function in type 2 diabetes has not been well investigated thoroughly. We measured cholesterol efflux in 36 patients with type 2 diabetes compared with 9 controls without diabetes. METHODS: The HDL fraction was separated with polyacrylamide gel and recovered using the protein recovery system. Concentration adjusted HDL fraction was used to determine HDL-mediated cholesterol efflux (Efflux-hdl) from THP-1 derived macrophages. We measured paraoxonase-1 (PON 1) activity to determine antioxidation capacity, serum amyloid A protein (SAA) to determine inflammatory response, and carboxymethyl-lysin (CML) to determine antiglucoxidative capacity. RESULTS: Efflux-hdl demonstrated no correlation with plasma apoprotein A-1 (ApoA-I) or HDL-cholesterol in patients with diabetes. PON1 activity in the patients' HDL fraction was positively correlated with Efflux-hdl (r=0.39, p=0.02), and showed a negative tendency with HbA1c levels (r=-0.28, p=0.10). SAA and CML levels did not demonstrate correlation with Efflux-hdl in patients with diabetes. CONCLUSION: We confirmed the functional changes in HDL particles in the patients. Efflux-hdl from macrophages was reduced depending upon the decrease in PON1 activity, which was inversely related to HbA1c levels.

Regulation and roles of urocortins in the vascular system.

Urocortins (Ucns) are members of the corticotropin-releasing factor (CRF) family of peptides. Ucns would have potent effects on the cardiovascular system via the CRF receptor type 2 (CRF(2) receptor). Regulation and roles of each Ucn have been determined in the vascular system. Ucns have more potent vasodilatory effects than CRF. Human umbilical vein endothelial cells (HUVECs) express Ucns1-3 mRNAs, and the receptor, CRF(2a) receptor mRNA. Ucns1-3 mRNA levels are differentially regulated in HUVECs. Differential regulation of Ucns may suggest differential roles of those in HUVECs. Ucn1 and Ucn2 have strong effects on interleukin (IL)-6 gene expression and secretion in rat aortic smooth muscle A7r5 cells. The increase that we observed in IL-6 levels following Ucn treatment of A7r5 cells suggests that smooth muscle cells may be a source of IL-6 secretion under physiological stress conditions. Ucns are important and unique modulators of vascular smooth muscle cells and act directly or indirectly as autocrine and paracrine factors in the vascular system.

Differential regulation of gonadotropin-releasing hormone by corticotropin-releasing factor family peptides in hypothalamic N39 cells.

Corticotropin-releasing factor (CRF) is involved in a variety of physiological functions including regulation of hypothalamo-pituitary-adrenal axis activity during stressful periods. Urocortins (Ucns) are known to be members of the CRF family peptides. CRF has a high affinity for CRF receptor type 1 (CRF(1) receptor). Both Ucn2 and Ucn3 have very high affinity for CRF receptor type 2 (CRF(2) receptor) with little or no binding affinity for the CRF(1) receptor. Gonadotropin-releasing hormone (GnRH) is known to be involved in the regulation of the stress response. Gonadotropin-inhibitory hormone (GnIH) neurons interact directly with GnRH neurons, and the action of GnIH is mediated by a novel G-protein coupled receptor, Gpr147. This study aimed to explore the possible function of CRF family peptides and the regulation of GnRH mRNA in hypothalamic GnRH cells. Both mRNA and protein expression of the CRF(1) receptor and CRF(2) receptor were found in hypothalamic GnRH N39 cells. CRF suppressed GnRH mRNA levels via the CRF(1) receptor, while Ucn2 increased the levels via the CRF(2) receptor. Both CRF and Ucn2 increased Gpr147 mRNA levels. The results indicate that CRF and Ucn2 can modulate GnRH mRNA levels via each specific CRF receptor subtype. Finally, CRF suppressed GnRH protein levels, while Ucn2 increased the levels. Differential regulation of GnRH by CRF family peptides may contribute to the stress response and homeostasis in GnRH cells.