Yuyan Ding

Key role for AMP-activated Protein Kinase in the Ventromedial Hypothalamus in Regulating Counterregulatory Hormone Responses to Acute Hypoglycemia

OBJECTIVE—To examine in vivo in a rodent model the potential role of AMP-activated protein kinase (AMPK) within the ventromedial hypothalamus (VMH) in glucose sensing during hypoglycemia. RESEARCH DESIGN AND METHODS—Using gene silencing technology to selectively downregulate AMPK in the VMH, a key hypothalamic glucose-sensing region, we demonstrate a key role for AMPK in the detection of hypoglycemia. In vivo hyperinsulinemic-hypoglycemic (50 mg dl−1) clamp studies were performed in awake, chronically catheterized Sprague-Dawley rats that had been microinjected bilaterally to the VMH with an adeno-associated viral (AAV) vector expressing a short hairpin RNA for AMPKα. RESULTS—In comparison with control studies, VMH AMPK downregulation resulted in suppressed glucagon (∼60%) and epinephrine (∼40%) responses to acute hypoglycemia. Rats with VMH AMPK downregulation also required more exogenous glucose to maintain the hypoglycemia plateau and showed significant reductions in endogenous glucose production and whole-body glucose uptake. CONCLUSIONS—We conclude that AMPK in the VMH plays a key role in the detection of acute hypoglycemia and initiation of the glucose counterregulatory response.

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.

The Medial Amygdalar Nucleus: A Novel Glucose-Sensing Region That Modulates the Counterregulatory Response to Hypoglycemia

OBJECTIVE To determine whether the medial amygdalar nucleus (MAN) represents a novel brain glucose-sensing region involved in the detection of hypoglycemia and generation of a counterregulatory hormone response. RESEARCH DESIGN AND METHODS Fura-2 calcium imaging was used to assess glucose responsivity in neurons isolated from the MAN and single-cell real-time reverse transcription PCR used to examine gene expression within glucose-responsive neurons. In vivo studies with local MAN perfusion of the glucoprivic agent, 2-deoxyglucose (2-DG), under normal and hypoglycemic conditions and also after MAN lesioning with ibotenic acid, were used to examine the functional role of MAN glucose sensors. In addition, retrograde neuronal tracer studies were used to examine reciprocal pathways between the MAN and the ventromedial hypothalamus (VMH). RESULTS The MAN contains a population of glucose-sensing neurons (13.5%), which express glucokinase, and the selective urocortin 3 (UCN3) receptor CRH-R2, but not UCN3 itself. Lesioning the MAN suppressed, whereas 2-DG infusion amplified, the counterregulatory response to hyperinsulinemic hypoglycemia in vivo. However, 2-DG infusion to the MAN or VMH under normoglycemic conditions had no systemic effect. The VMH is innervated by UCN3 neurons that arise mainly from the MAN, and ∼1/3 of MAN UCN3 neurons are active during mild hypoglycemia. CONCLUSIONS The MAN represents a novel limbic glucose-sensing region that contains characteristic glucokinase-expressing glucose-sensing neurons that respond directly to manipulations of glucose availability both in vitro and in vivo. Moreover, UCN3 neurons may provide feedback inhibitory regulation of the counterregulatory response through actions within the VMH and the MAN.

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.

Hypothalamic AMP-activated protein kinase activation with AICAR amplifies counterregulatory responses to hypoglycemia in a rodent model of type 1 diabetes

In nondiabetic rodents, AMP-activated protein kinase (AMPK) plays a role in the glucose-sensing mechanism used by the ventromedial hypothalamus (VMH), a key brain region involved in the detection of hypoglycemia. However, AMPK is regulated by both hyper- and hypoglycemia, so whether AMPK plays a similar role in type 1 diabetes (T1DM) is unknown. To address this issue, we used four groups of chronically catheterized male diabetic BB rats, a rodent model of autoimmune T1DM with established insulin-requiring diabetes (40 +/- 4 pmol/l basal c-peptide). Two groups were subjected to 3 days of recurrent hypoglycemia (RH), while the other two groups were kept hyperglycemic [chronic hyperglycemia (CH)]. All groups subsequently underwent hyperinsulinemic hypoglycemic clamp studies on day 4 in conjunction with VMH microinjection with either saline (control) or AICAR (5-aminoimidazole-4-carboxamide) to activate AMPK. Compared with controls, local VMH application of AICAR during hypoglycemia amplified both glucagon [means +/- SE, area under the curve over time (AUC/t) 144 +/- 43 vs. 50 +/- 11 ng.l(-1).min(-1); P < 0.05] and epinephrine [4.27 +/- 0.96 vs. 1.06 +/- 0.26 nmol.l(-1).min(-1); P < 0.05] responses in RH-BB rats, and amplified the glucagon [151 +/- 22 vs. 85 +/- 22 ng.l(-1).min(-1); P < 0.05] response in CH-BB rats. We conclude that VMH AMPK also plays a role in glucose-sensing during hypoglycemia in a rodent model of T1DM. Moreover, our data suggest that it may be possible to partially restore the hypoglycemia-specific glucagon secretory defect characteristic of T1DM through manipulation of VMH AMPK.

Amplified Hormonal Counterregulatory Responses to Hypoglycemia in Rats After Systemic Delivery of a SUR-1–Selective K+ Channel Opener?

OBJECTIVE— In glucose-sensing neurons, ATP-sensitive K+ channels (KATP channels) are thought to translate metabolic signals into an alteration in neuronal firing rates. Because these neurons express the Kir6.2/SUR-1 isoform of the KATP channel, we sought to examine the therapeutic potential of the SUR-1–selective potassium channel opener (KCO), NN414, to amplify counterregulatory response to hypoglycemia. RESEARCH DESIGN AND METHODS— In vivo dose-response studies with NN414 delivered intravenously to normal Sprague-Dawley rats before the induction of controlled hypoglycemia were performed. Based on these studies, the potential for NN414 to restore counterregulatory responses in chronically cannulated nondiabetic and diabetic BB rats was explored using the in vivo hyperinsulinemic-hypoglycemic clamp technique. RESULTS— NN414 delivered systemically amplified epinephrine responses during acute hypoglycemia and showed a persisting effect to amplify the epinephrine response when given 24 h before the hypoglycemic study. Local delivery of a potassium-channel blocker to the ventromedial hypothalamus reversed the effects of systemic NN414. In addition, NN414 amplified the epinephrine response to hypoglycemia in both nondiabetic and diabetic BB rats with defective hormonal counterregulation. CONCLUSIONS— These studies demonstrate in a variety of rodent models that systemic delivery of Kir6.2/SUR-1–selective KCOs enhance the glucose counterregulatory response to insulin-induced hypoglycemia. Future studies in human subjects are now required to determine their potential as a therapy for hypoglycemia-associated autonomic failure in type 1 diabetes.

Reduction in SGLT1 mRNA Expression in the Ventromedial Hypothalamus Improves the Counterregulatory Responses to Hypoglycemia in Recurrently Hypoglycemic and Diabetic Rats

The objective of this study was to determine whether the sodium-glucose transporter SGLT1 in the ventromedial hypothalamus (VMH) plays a role in glucose sensing and in regulating the counterregulatory response to hypoglycemia, and if so, whether knockdown of in the VMH can improve counterregulatory responses to hypoglycemia in diabetic rats or rats exposed to recurrent bouts of hypoglycemia (RH). Normal Sprague-Dawley rats as well as RH or streptozotocin (STZ)-diabetic rats received bilateral VMH microinjections of an adenoassociated viral vector containing either the SGLT1 short hairpin RNA (shRNA) or a scrambled RNA sequence. Subsequently, these rats underwent a hypoglycemic clamp to assess hormone responses. In a subgroup of rats, glucose kinetics was determined using tritiated glucose. The shRNA reduced VMH SGLT1 expression by 53% in nondiabetic rats, and this augmented glucagon and epinephrine responses and hepatic glucose production during hypoglycemia. Similarly, SGLT1 knockdown improved the glucagon and epinephrine responses in RH rats and restored the impaired epinephrine response to hypoglycemia in STZ-diabetic animals. These findings suggest that SGLT1 in the VMH plays a significant role in the detection and activation of counterregulatory responses to hypoglycemia. Inhibition of SGLT1 may offer a potential therapeutic target to diminish the risk of hypoglycemia in diabetes.

GPR119 Agonism Increases Glucagon Secretion During Insulin-Induced Hypoglycemia

Insulin-induced hypoglycemia in diabetes is associated with impaired glucagon secretion. In this study, we tested whether stimulation of GPR119, a G-protein–coupled receptor expressed in pancreatic islet as well as enteroendocrine cells and previously shown to stimulate insulin and incretin secretion, might enhance glucagon secretion during hypoglycemia. In the study, GPR119 agonists were applied to isolated islets or perfused pancreata to assess insulin and glucagon secretion during hypoglycemic or hyperglycemic conditions. Insulin infusion hypoglycemic clamps were performed with or without GPR119 agonist pretreatment to assess glucagon counterregulation in healthy and streptozotocin (STZ)-induced diabetic rats, including those exposed to recurrent bouts of insulin-induced hypoglycemia that leads to suppression of hypoglycemia-induced glucagon release. Hypoglycemic clamp studies were also conducted in GPR119 knockout (KO) mice to evaluate whether the pharmacological stimulatory actions of GPR119 agonists on glucagon secretion during hypoglycemia were an on-target effect. The results revealed that GPR119 agonist-treated pancreata or cultured islets had increased glucagon secretion during low glucose perfusion. In vivo, GPR119 agonists also significantly increased glucagon secretion during hypoglycemia in healthy and STZ-diabetic rats, a response that was absent in GPR119 KO mice. In addition, impaired glucagon counterregulatory responses were restored by a GPR119 agonist in STZ-diabetic rats that were exposed to antecedent bouts of hypoglycemia. Thus, GPR119 agonists have the ability to pharmacologically augment glucagon secretion, specifically in response to hypoglycemia in diabetic rodents. Whether this effect might serve to diminish the occurrence and severity of iatrogenic hypoglycemia during intensive insulin therapy in patients with diabetes remains to be established.

Leptin acts in the brain to influence hypoglycemic counterregulation: disparate effects of acute and recurrent hypoglycemia on glucagon release.

Leptin has been shown to diminish hyperglycemia via reduced glucagon secretion, although it can also enhance sympathoadrenal responses. However, whether leptin can also inhibit glucagon secretion during insulin-induced hypoglycemia or increase epinephrine during acute or recurrent hypoglycemia has not been examined. To test whether leptin acts in the brain to influence counterregulation, hyperinsulinemic hypoglycemic (∼45 mg/dl) clamps were performed on rats exposed to or not exposed to recurrent hypoglycemia (3 days, ∼40 mg/dl). Intracerebroventricular artificial cerebral spinal fluid or leptin was infused during the clamp. During acute hypoglycemia, leptin decreased glucagon responses by 51% but increased epinephrine and norepinephrine by 24 and 48%, respectively. After recurrent hypoglycemia, basal plasma leptin levels were undetectable. Subsequent brain leptin infusion during hypoglycemia paradoxically increased glucagon by 45% as well as epinephrine by 19%. In conclusion, leptin acts within the brain to diminish glucagon secretion during acute hypoglycemia but increases epinephrine, potentially limiting its detrimental effects during hypoglycemia. Exposure to recurrent hypoglycemia markedly suppresses plasma leptin, whereas exogenous brain leptin delivery enhances both glucagon and epinephrine release to subsequent hypoglycemia. These data suggest that recurrent hypoglycemia may diminish counterregulatory responses in part by reducing brain leptin action.

Glycemic Variability and Brain Glucose Levels in T1DM

The impact of glycemic variability on brain glucose transport kinetics among individuals with type 1 diabetes mellitus (T1DM) remains unclear. Fourteen individuals with T1DM (age 35 ± 4 years; BMI 26.0 ± 1.4 kg/m2; HbA1c 7.6 ± 0.3) and nine healthy control participants (age 32 ± 4; BMI 23.1 ± 0.8; HbA1c 5.0 ± 0.1) wore a continuous glucose monitor (Dexcom) to measure hypoglycemia, hyperglycemia, and glycemic variability for 5 days followed by 1H MRS scanning in the occipital lobe to measure the change in intracerebral glucose levels during a 2-h glucose clamp (target glucose concentration 220 mg/dL). Hyperglycemic clamps were also performed in a rat model of T1DM to assess regional differences in brain glucose transport and metabolism. Despite a similar change in plasma glucose levels during the hyperglycemic clamp, individuals with T1DM had significantly smaller increments in intracerebral glucose levels (P = 0.0002). Moreover, among individuals with T1DM, the change in brain glucose correlated positively with the lability index (r = 0.67, P = 0.006). Consistent with findings in humans, streptozotocin-treated rats had lower brain glucose levels in the cortex, hippocampus, and striatum compared with control rats. These findings that glycemic variability is associated with brain glucose levels highlight the need for future studies to investigate the impact of glycemic variability on brain glucose kinetics.