Wanling Zhu

Activation of AMP-Activated Protein Kinase Within the Ventromedial Hypothalamus Amplifies Counterregulatory Hormone Responses in Rats With Defective Counterregulation

Defective counterregulatory responses (CRRs) to hypoglycemia are associated with a marked increase in the risk of severe hypoglycemia. The mechanisms leading to the development of defective CRRs remain largely unknown, although they are associated with antecedent hypoglycemia. Activation of AMP-activated protein kinase (AMPK) in the ventromedial hypothalamus (VMH) amplifies the counterregulatory increase in glucose production during acute hypoglycemia. To examine whether activation of AMPK in the VMH restores defective CRR, controlled hypoglycemia (∼2.8 mmol/l) was induced in a group of 24 Sprague-Dawley rats, all of which had undergone a 3-day model of recurrent hypoglycemia before the clamp study. Before the acute study, rats were microinjected to the VMH with either 5-aminoimidazole-4-carboxamide (AICAR; n = 12), to activate AMPK, or saline (n = 12). In a subset of rats, an infusion of H3-glucose was additionally started to calculate glucose turnover. Stimulation of AMPK within the VMH was found to amplify hormonal CRR and increase endogenous glucose production. In addition, analysis of tissue from both whole hypothalamus and VMH showed that recurrent hypoglycemia induces an increase in the gene expression of AMPK α1 and α2. These findings suggest that the development of novel drugs designed to selectively activate AMPK in the VMH offer a future therapeutic potential for individuals with type 1 diabetes who have defective CRRs to hypoglycemia.

Influence of Insulin in the Ventromedial Hypothalamus on Pancreatic Glucagon Secretion In Vivo

OBJECTIVE Insulin released by the β-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 μU), 5) insulin receptor antagonist (S961), or 6) anti-insulin affibody plus a γ-aminobutyric acid A (GABAA) 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 GABAA receptor agonist. CONCLUSIONS Together, these data suggest that insulins inhibitory effect on α-cell glucagon release is in part mediated at the level of the VMH under both normoglycemic and hypoglycemic conditions.

ATP-Sensitive K+ Channels Regulate the Release of GABA in the Ventromedial Hypothalamus During Hypoglycemia

OBJECTIVE—To determine whether alterations in counterregulatory responses to hypoglycemia through the modulation of ATP-sensitive K+ channels (KATP channels) in the ventromedial hypothalamus (VMH) are mediated by changes in GABAergic inhibitory tone in the VMH, we examined whether opening and closing KATP channels in the VMH alter local GABA levels and whether the effects of modulating KATP channel activity within the VMH can be reversed by local modulation of GABA receptors. RESEARCH DESIGN AND METHODS—Rats were cannulated and bilateral guide cannulas inserted to the level of the VMH. Eight days later, the rats received a VMH microinjection of either 1) vehicle, 2) the KATP channel opener diazoxide, 3) the KATP channel closer glybenclamide, 4) diazoxide plus the GABAA receptor agonist muscimol, or 5) glybenclamide plus the GABAA receptor antagonist bicuculline methiodide (BIC) before performance of a hypoglycemic clamp. Throughout, VMH GABA levels were measured using microdialysis. RESULTS—As expected, diazoxide suppressed glucose infusion rates and increased glucagon and epinephrine responses, whereas glybenclamide raised glucose infusion rates in conjunction with reduced glucagon and epinephrine responses. These effects of KATP modulators were reversed by GABAA receptor agonism and antagonism, respectively. Microdialysis revealed that VMH GABA levels decreased 22% with the onset of hypoglycemia in controls. Diazoxide caused a twofold greater decrease in GABA levels, and glybenclamide increased VMH GABA levels by 57%. CONCLUSIONS—Our data suggests that KATP channels within the VMH may modulate the magnitude of counterregulatory responses by altering release of GABA within that region.

Increased GABAergic Tone in the Ventromedial Hypothalamus Contributes to Suppression of Counterregulatory Reponses After Antecedent Hypoglycemia

OBJECTIVE—We have previously demonstrated that modulation of γ-aminobutyric acid (GABA) inhibitory tone in the ventromedial hypothalamus (VMH), an important glucose-sensing region in the brain, modulates the magnitude of glucagon and sympathoadrenal responses to hypoglycemia. In the current study, we examined whether increased VMH GABAergic tone may contribute to suppression of counterregulatory responses after recurrent hypoglycemia. RESEARCH DESIGN AND METHODS—To test this hypothesis, we quantified expression of the GABA synthetic enzyme, glutamic acid decarboxylase (GAD), in the VMH of control and recurrently hypoglycemic rats. Subsequently, we used microdialysis and microinjection techniques to assess changes in VMH GABA levels and the effects of GABAA receptor blockade on counterregulatory responses to a standardized hypoglycemic stimulus. RESULTS—Quantitative RT-PCR and immunoblots in recurrently hypoglycemic animals revealed that GAD65 mRNA and protein were increased 33 and 580%, respectively. Basal VMH GABA concentrations were more than threefold higher in recurrently hypoglycemic animals. Furthermore, whereas VMH GABA levels decreased in both control and recurrently hypoglycemic animals with the onset of hypoglycemia, the fall was not significant in recurrently hypoglycemic rats. During hypoglycemia, recurrently hypoglycemic rats exhibited a 49–63% reduction in glucagon and epinephrine release. These changes were reversed by delivery of a GABAA receptor antagonist to the VMH. CONCLUSIONS—Our data suggest that recurrent hypoglycemia increases GABAergic inhibitory tone in the VMH and that this, in turn, suppresses glucagon and sympathoadrenal responses to subsequent bouts of acute hypoglycemia. Thus, hypoglycemia-associated autonomic failure may be due in part to a relative excess of the inhibitory neurotransmitter, GABA, within the VMH.

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.

Increased GABAergic Output in the Ventromedial Hypothalamus Contributes to Impaired Hypoglycemic Counterregulation in Diabetic Rats

OBJECTIVE Impaired glucose counterregulation during hypoglycemia is well documented in patients with type 1 diabetes; however, the molecular mechanisms underlying this defect remain uncertain. We reported that the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in a crucial glucose-sensing region within the brain, the ventromedial hypothalamus (VMH), plays an important role in modulating the magnitude of the glucagon and epinephrine responses to hypoglycemia and investigated whether VMH GABAergic tone is altered in diabetes and therefore might contribute to defective counterregulatory responses. RESEARCH DESIGN AND METHODS We used immunoblots to measure GAD65 protein (a rate-limiting enzyme in GABA synthesis) and microdialysis to measure extracellular GABA levels in the VMH of two diabetic rat models, the diabetic BB rat and the streptozotocin (STZ)-induced diabetic rat, and compared them with nondiabetic controls. RESULTS Both diabetic rat models exhibited an ~50% increase in GAD65 protein as well as a twofold increase in VMH GABA levels compared with controls under baseline conditions. Moreover, during hypoglycemia, VMH GABA levels did not change in the diabetic animals, whereas they significantly declined in nondiabetic animals. As expected, glucagon responses were absent and epinephrine responses were attenuated in diabetic rats compared with their nondiabetic control counterparts. The defective counterregulatory response in STZ-diabetic animals was restored to normal with either local blockade of GABAA receptors or knockdown of GAD65 in the VMH. CONCLUSIONS These data suggest that increased VMH GABAergic inhibition is an important contributor to the absent glucagon response to hypoglycemia and the development of counterregulatory failure in type 1 diabetes.

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 insulins 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 β-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.

Modulation of β-Adrenergic Receptors in the Ventromedial Hypothalamus Influences Counterregulatory Responses to Hypoglycemia

OBJECTIVE Norepinephrine is locally released into the ventromedial hypothalamus (VMH), a key brain glucose-sensing region in the response to hypoglycemia. As a result, this neurotransmitter may play a role in modulating counterregulatory responses. This study examines whether norepinephrine acts to promote glucose counterregulation via specific VMH β-adrenergic receptors (BAR). RESEARCH DESIGN AND METHODS Awake male Sprague-Dawley rats received, via implanted guide cannulae, bilateral VMH microinjections of 1) artificial extracellular fluid, 2) B2AR agonist, or 3) B2AR antagonist. Subsequently, a hyperinsulinemic-hypoglycemic clamp study was performed. The same protocol was also used to assess the effect of VMH delivery of a selective B1AR or B3AR antagonist. RESULTS Despite similar insulin and glucose concentrations during the clamp, activation of B2AR in the VMH significantly lowered by 32% (P < 0.01), whereas VMH B2AR blockade raised by 27% exogenous glucose requirements during hypoglycemia (P < 0.05) compared with the control study. These changes were associated with alternations in counterregulatory hormone release. Epinephrine responses throughout hypoglycemia were significantly increased by 50% when the B2AR agonist was delivered to the VMH (P < 0.01) and suppressed by 32% with the B2AR antagonist (P < 0.05). The glucagon response was also increased by B2AR activation by 63% (P < 0.01). Neither blockade of VMH B1AR nor B3AR suppressed counterregulatory responses to hypoglycemia. Indeed, the B1AR antagonist increased rather than decreased epinephrine release (P < 0.05). CONCLUSIONS Local catecholamine release into the VMH enhances counterregulatory responses to hypoglycemia via stimulation of B2AR. These observations suggest that B2AR agonists might have therapeutic benefit in diabetic patients with defective glucose counterregulation.

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.

Improvement in hepatic insulin sensitivity after Roux-en-Y gastric bypass in a rat model of obesity is partially mediated via hypothalamic insulin action

Aims/hypothesisRoux-en-Y gastric bypass (RYGB) surgery, an effective treatment for morbid obesity, commonly leads to near complete resolution of type 2 diabetes. The underlying mechanisms, however, remain unclear and factors other than weight loss alone may be involved.MethodsTo determine whether increased hypothalamic insulin sensitivity after RYGB drives the rapid improvement in glucose metabolism, high-fat-fed rats received either an insulin receptor (IR) antisense vector or a control lentiviral vector that was microinjected into the ventromedial hypothalamus (VMH). Six weeks later, rats underwent RYGB or control gastrointestinal surgery.ResultsFour weeks after surgery, weight loss was comparable in RYGB and surgical controls. Nevertheless, only RYGB rats that received the control vector demonstrated both improved hepatic and peripheral insulin sensitivity. Insulin suppressed hepatic glucose production (HGP) by 50% (p < 0.05) with RYGB, whereas the effect of insulin on HGP was completely absent in VMH IR knockdown (IRkd) rats. By contrast, both RYGB groups displayed an identical twofold increase in insulin-stimulated peripheral glucose uptake. The animals that underwent control gastrointestinal surgery failed to show any improvement in either hepatic or peripheral insulin sensitivity; VMH IRkd did not influence the magnitude of insulin resistance.Conclusions/interpretationOur findings demonstrate that RYGB surgery in high-fat-fed obese rats enhances hepatic and peripheral insulin sensitivity independently of weight loss. The improved hepatic, but not the peripheral, response to insulin is mediated centrally at the level of the VMH. These data provide direct evidence that the metabolic benefits of RYGB surgery are not simply a consequence of weight loss but likely in part involve the central nervous system.