Chronic reduction of insulin receptors in the ventromedial hypothalamus produces glucose intolerance and islet dysfunction in the absence of weight gain.

Insulin is believed to regulate glucose homeostasis mainly via direct effects on the liver, muscle, and adipose tissues. The contribution of insulin's central nervous system effects to disorders of glucose metabolism has received less attention. To evaluate whether postnatal reduction of insulin receptors (IRs) within the ventromedial hypothalamus (VMH), a brain region critical for glucose sensing, contributes to disorders of peripheral glucose metabolism, we microinjected a lentiviral vector expressing an antisense sequence to knockdown IRs or a control lentiviral vector into the VMH of nonobese nondiabetic rats. After 3-4 mo, we assessed 1) glucose tolerance, 2) hepatic insulin sensitivity, and 3) insulin and glucagon secretion, using the glucose clamp technique. Knockdown of IRs locally in the VMH caused glucose intolerance without altering body weight. Increments of plasma insulin during a euglycemic clamp study failed to suppress endogenous glucose production and produced a paradoxical rise in plasma glucagon in the VMH-IR knockdown rats. Unexpectedly, these animals also displayed a 40% reduction (P < 0.05) in insulin secretion in response to an identical hyperglycemic stimulus (∼220 mg/dl). Our data demonstrate that chronic suppression of VMH-IR gene expression is sufficient to impair glucose metabolism as well as α-cell and ß-cell function in nondiabetic, nonobese rats. These data suggest that insulin resistance within the VMH may be a significant contributor to the development of type 2 diabetes.

Glucose prevents the fall in ventromedial hypothalamic GABA that is required for full activation of glucose counterregulatory responses during hypoglycemia.

Local delivery of glucose into a critical glucose-sensing region within the brain, the ventromedial hypothalamus (VMH), can suppress glucose counterregulatory responses to systemic hypoglycemia. Here, we investigated whether this suppression was accomplished through changes in GABA output in the VMH. Sprague-Dawley rats had catheters and guide cannulas implanted. Eight to ten days later, microdialysis-microinjection probes were inserted into the VMH, and they were dialyzed with varying concentrations of glucose from 0 to 100 mM. Two groups of rats were microdialyzed with 100 mM glucose and microinjected with either the K(ATP) channel opener diazoxide or a GABA(A) receptor antagonist. These animals were then subjected to a hyperinsulinemic-hypoglycemic glucose clamp. As expected, perfusion of glucose into the VMH suppressed the counterregulatory responses. Extracellular VMH GABA levels positively correlated with the concentration of glucose in the perfusate. In turn, extracellular GABA concentrations in the VMH were inversely related to the degree of counterregulatory hormone release. Of note, microinjection of either diazoxide or the GABA(A) receptor antagonist reversed the suppressive effects of VMH glucose delivery on counterregulatory responses. Some GABAergic neurons in the VMH respond to changes in local glucose concentration. Glucose in the VMH dose-dependently stimulates GABA release, and this in turn dose-dependently suppresses the glucagon and epinephrine responses to hypoglycemia. These data suggest that during hypoglycemia a decrease in glucose concentration within the VMH may provide an important signal that rapidly inactivates VMH GABAergic neurons, reducing inhibitory GABAergic tone, which in turn enhances the counterregulatory responses to hypoglycemia.

Testicular regulation of neuronal glucose and monocarboxylate transporter gene expression profiles in CNS metabolic sensing sites during acute and recurrent insulin-induced hypoglycemia.

Recurrent insulin-induced hypoglycemia (RIIH) impairs glucose counter-regulatory function in male humans and rodents and, in the latter, diminishes neuronal activation in CNS structures that monitor metabolic homeostasis, including the lateral hypothalamic area (LHA) and dorsal vagal complex (DVC). We investigated whether habituated neuronal reactivity in CNS sensing sites to hypoglycemia is correlated with modified monocarboxylate and/or glucose uptake by using quantitative real-time RT-PCR to analyze neuronal monocarboxylate transporter (MCT2) and glucose transporter variant (GLUT and GLUT4) gene expression profiles in the microdissected LHA, ventromedial nucleus hypothalamus (VMH), and DVC after one or multiple insulin injections. Because orchidectomy (ORDX) maintains uniform glycemic responses to RIIH in male rats, we also examined whether regional gene response patterns are testes dependent. In the intact male rat DVC, MCT2, GLUT3, and GLUT4 gene expression was not altered by acute hypoglycemia but was enhanced by RIIH. MCT2 and GLUT3 mRNA levels in the ORDX rat DVC did not differ among groups, but GLUT4 transcripts were progressively increased by acute and recurrent hypoglycemia. Precedent hypoglycemia decreased or increased basal MCT2 and GLUT4 gene expression, respectively, in the intact rat LHA; LHA GLUT3 transcription was augmented by RIIH in intact rats only. Acute hypoglycemia suppressed MCT2, GLUT3, and GLUT4 gene expression in the intact rat VMH, a response that was abolished by RIIH. In ORDX rats, VMH gene transcript levels were unchanged in response to one dose of insulin but were selectively diminished during RIIH. These data demonstrate site-specific, testes-dependent effects of acute and recurrent hypoglycemia on neuronal metabolic substrate transporter gene expression in characterized rat brain metabolic sensing loci and emphasize the need to assess the impact of potential alterations in glucose and lactate uptake during RIIH on general and specialized, e.g., metabolic monitoring, functions of neurons in those sites.

Vagal complex monocarboxylate transporter-2 expression during hypoglycemia.

Astrocytic provision of lactate provision to neurons may be a critical indicator of substrate fuel availability in metabolic sensing sites in the brain, including the hindbrain dorsal vagal complex. We examined the hypothesis that vagal complex monocarboxylate transporter protein levels are gender dependent and estrogen dependent, and that estrogen influences adaptation of these protein responses during repeated insulin-induced hypoglycemia. Western blot analyses showed that male and estrogen-treated ovariectomized female rats exhibit opposite changes in monocarboxylate transporter-2 levels after one insulin injection, as well as divergent patterns of adaptation to this metabolic challenge. The data suggest that sex differences in hypoglycemic patterns in vagal complex lactate transport may underlie disparate signaling of cellular energy imbalance.

Type II glucocorticoid receptor involvement in habituated activation of lateral hypothalamic area orexin-A-immunopositive neurons during recurring insulin-induced hypoglycemia.

Neurons that synthesize the potent orexigenic neuropeptide, orexin-A (ORX-A) are confined to the lateral hypothalamic area (LHA) and adjacent structures, and project throughout the central neuroaxis to structures that govern central nervous system responses to energy imbalance. Insulin-induced hypoglycemia (IIH) upregulates prepro-orexin mRNA and Fos immunostaining of LHA ORX-A neurons. These neurons apparently become desensitized to this metabolic challenge, since both responses are diminished by recurrent insulin-induced hypoglycemia (RIIH). Recent studies implicate central type II glucocorticoid receptors (GR) in RIIH-associated glucose counterregulatory collapse and decline in Fos labeling of central metabolic loci, including the LHA. The present studies evaluated the role of GR in patterns of LHA ORX-A neuronal transcriptional activation during RIIH. Groups of adult male rats were injected subcutaneously with one or four doses of the intermediate-acting insulin, Humulin NPH, on as many days, or with diluent alone. Rats injected with four doses of insulin were pretreated by intracerebroventricular (icv) administration of the selective GR antagonist, CP-472555, or the vehicle, propylene glycol, prior to insulin administration on days 1-3. All animals were sacrificed by transcardial perfusion 2h after injections on day 4. Processing of LHA tissue sections for dual-immunoperoxidase staining of ORX-A- and Fos-immunoreactivity (-ir) showed that colabeling of ORX-A neurons for Fos was increased by a single injection of NPH, whereas this genomic response was diminished by RIIH. Icv administration of CP-472555 during antecedent hypoglycemia prevented RIIH-associated reductions in Fos expression by these neurons. Antagonist treatment of diluent-injected controls did not alter mean numbers of ORX-A- plus Fos-ir neurons. Total numbers of ORX-A-immunopositive neurons were not different among treatment groups. These data demonstrate that precedent central GR blockade prevents adaptation of LHA ORX-A neuronal reactivity to RIIH. These results provide unique pharmacological evidence that hypoglycemic hypercorticosteronemia diminishes activation of this neurotransmitter phenotype in this critical metabolic structure to subsequent hypoglycemia via central GR-dependent mechanisms.

Habituation of insulin-induced hypoglycemic transcription activation of lateral hypothalamic orexin-A-containing neurons to recurring exposure.

A CNS component of glucose counterregulatory collapse is supported by evidence for nonuniform genomic responsiveness of neurons in characterized central autonomic loci during recurring insulin-induced hypoglycemia (IIH). We have reported that exacerbated hypoglycemia and attenuated patterns of glucagon and epinephrine secretion in rats treated by daily sc injection of the intermediate-acting insulin formulation, Humulin NPH (NPH), are correlated with diminished immunodemonstrability of the AP-1 transcription factor, Fos, in several components of the central metabolic regulatory circuitry, including the lateral hypothalamic area (LHA). Neurons that synthesize the potent orexigenic peptide neurotransmitter, orexin-A, are restricted to the LHA and adjacent hypothalamic loci, and project throughout the central neuroaxis to structures that govern autonomic and behavioral motor output. Dual-label immunocytochemical and real-time RT-PCR techniques were utilized here to evaluate the functional status of this LHA phenotype during a single versus repetitive exposure to prolonged IIH. Tissue sections were collected at predetermined rostrocaudal levels of the LHA after acute or repeated NPH administration, and processed for nuclear Fos- and cytoplasmic orexin-A-immunoreactivity (-ir). Mean numbers of orexin-A-ir neurons were not different between treatment groups. Colabeling of these cells for Fos was increased relative to controls following a single injection of insulin, but numbers of Fos-ir-positive orexin-A neurons were significantly reduced after treatment with four versus one dose of insulin. Prepro-orexin mRNA levels in microdissected LHA tissue were upregulated during acute hypoglycemia, but were returned to control levels by repeated IIH. These data corroborate previous evidence that IIH is an activational stimulus for orexin-A-synthesizing neurons in the LHA, and further demonstrate that induction of cfos and prepro-orexin gene expression by acute hypoglycemia is attenuated by precedent exposure to hypoglycemia. The current results thus provide unique evidence for neurotransmitter-specific habituation of LHA neuronal sensitivity to IIH.

Lactate-induced release of GABA in the ventromedial hypothalamus contributes to counterregulatory failure in recurrent hypoglycemia and diabetes.

Suppression of GABAergic neurotransmission in the ventromedial hypothalamus (VMH) is crucial for full activation of counterregulatory responses to hypoglycemia, and increased γ-aminobutyric acid (GABA) output contributes to counterregulatory failure in recurrently hypoglycemic (RH) and diabetic rats. The goal of this study was to establish whether lactate contributes to raising VMH GABA levels in these two conditions. We used microdialysis to deliver artificial extracellular fluid or L-lactate into the VMH and sample for GABA. We then microinjected a GABAA receptor antagonist, an inhibitor of lactate transport (4CIN), or an inhibitor of lactate dehydrogenase, oxamate (OX), into the VMH prior to inducing hypoglycemia. To assess whether lactate contributes to raising GABA in RH and diabetes, we injected 4CIN or OX into the VMH of RH and diabetic rats before inducing hypoglycemia. L-lactate raised VMH GABA levels and suppressed counterregulatory responses to hypoglycemia. While blocking GABAA receptors did not prevent the lactate-induced rise in GABA, inhibition of lactate transport or utilization did, despite the presence of lactate. All three treatments restored the counterregulatory responses, suggesting that lactate suppresses these responses by enhancing GABA release. Both RH and diabetic rats had higher baseline GABA levels and were unable to reduce GABA levels sufficiently to fully activate counterregulatory responses during hypoglycemia. 4CIN or OX lowered VMH GABA levels in both RH and diabetic rats and restored the counterregulatory responses. Lactate likely contributes to counterregulatory failure in RH and diabetes by increasing VMH GABA levels.

Influence of insulin in the ventromedial hypothalamus on pancreatic glucagon secretion in vivo.

OBJECTIVE: Insulin released by the beta-cell is thought to act locally to regulate glucagon secretion. The possibility that insulin might also act centrally to modulate islet glucagon secretion has received little attention. RESEARCH DESIGN AND METHODS: Initially the counterregulatory response to identical hypoglycemia was compared during intravenous insulin and phloridzin infusion in awake chronically catheterized nondiabetic rats. To explore whether the disparate glucagon responses seen were in part due to changes in ventromedial hypothalamus (VMH) exposure to insulin, bilateral guide cannulas were inserted to the level of the VMH and 8 days later rats received a VMH microinjection of either 1) anti-insulin affibody, 2) control affibody, 3) artificial extracellular fluid, 4) insulin (50 microU), 5) insulin receptor antagonist (S961), or 6) anti-insulin affibody plus a gamma-aminobutyric acid A (GABA(A)) receptor agonist muscimol, prior to a hypoglycemic clamp or under baseline conditions. RESULTS: As expected, insulin-induced hypoglycemia produced a threefold increase in plasma glucagon. However, the glucagon response was fourfold to fivefold greater when circulating insulin did not increase, despite equivalent hypoglycemia and C-peptide suppression. In contrast, epinephrine responses were not altered. The phloridzin-hypoglycemia induced glucagon increase was attenuated (40%) by VMH insulin microinjection. Conversely, local VMH blockade of insulin amplified glucagon twofold to threefold during insulin-induced hypoglycemia. Furthermore, local blockade of basal insulin levels or insulin receptors within the VMH caused an immediate twofold increase in fasting glucagon levels that was prevented by coinjection to the VMH of a GABA(A) receptor agonist. CONCLUSIONS: Together, these data suggest that insulin's inhibitory effect on alpha-cell glucagon release is in part mediated at the level of the VMH under both normoglycemic and hypoglycemic conditions.

Site-specific habituation of insulin-induced hypoglycemic induction of fos immunoreactivity in glucocorticoid receptor: immunopositive neurons in the male rat brain.

Current studies show that type II glucocorticoid receptor (GR) stimulation during recurring insulin-induced hypoglycemia (RIIH) results in diminished hypoglycemic activation of neurons in discrete CNS metabolic structures, namely the lateral hypothalamic area (LHA), hypothalamic paraventricular (PVH) and dorsomedial (DMH) nuclei, and nucleus of the solitary tract (NTS). The present work utilized immunofluorescence histochemistry to evaluate the reactivity of GR-expressing neurons in characterized hypothalamic, thalamic, and hindbrain metabolic structures to glucoprivation, and to determine if antecedent hypoglycemic stimulation of central GR decreases Fos protein expression by these neurons. Groups of adult male rats were injected subcutaneously with one or four doses of the intermediate-acting insulin, Humulin NPH, on as many days, while controls received diluent only. Rats injected with four doses of insulin were pretreated by intracerebroventricular administration of the selective GR antagonist, CP-475222, or vehicle alone prior to insulin doses 1-3. All animals were sacrificed by trancardial perfusion 2 h after injections on day four of the study. Mean numbers of GR-immunoreactive (-ir) neurons did not differ between groups injected with diluent versus one dose of insulin in each structure evaluated, but were significantly elevated above baseline on the fourth day of RIIH in the LHA and DMH, but not the PVH, VMH, ARC, thalamic paraventricular (PVT), or NTS. Counts of GR-ir-positive neurons in each site were similar between groups treated with CP-475222 or vehicle icv during RIIH. While mean numbers of GR-plus Fos-ir neurons in the PVH, DMH, LHA, and NTS, but not the PVT were significantly elevated after one dose of NPH, this increase was abolished in each site by RIIH. Pharmacological antagonism of central GR during antecedent hypoglycemia prevented RIIH-associated habituation of Fos colabeling of GR-expressing neurons in the PVH, DMH, and LHA. These data show that RIIH increases nuclear immunolabeling for GR in discrete CNS metabolic structures, evidence that recurring metabolic stress may amplify receptor-mediated genomic regulatory function in local neurons. The results also demonstrate that GR-containing neurons in the LHA, DMH, PVH, and NTS react to hypoglycemia by induction of the Fos stimulus-transcription cascade, and that precedent stimulation of central GR is critical for RIIH-associated habituation of this functional response in the former three sites.