EphA5-EphrinA5 Interactions Within the Ventromedial Hypothalamus Influence Counterregulatory Hormone Release and Local Glutamine/Glutamate Balance During Hypoglycemia

Activation of β-cell EphA5 receptors by its ligand ephrinA5 from adjacent β-cells has been reported to decrease insulin secretion during hypoglycemia. Given the similarities between islet and ventromedial hypothalamus (VMH) glucose sensing, we tested the hypothesis that the EphA5/ephrinA5 system might function within the VMH during hypoglycemia to stimulate counterregulatory hormone release as well. Counterregulatory responses and glutamine/glutamate concentrations in the VMH were assessed during a hyperinsulinemic-hypoglycemic glucose clamp study in chronically catheterized awake male Sprague-Dawley rats that received an acute VMH microinjection of ephrinA5-Fc, chronic VMH knockdown, or overexpression of ephrinA5 using an adenoassociated viral construct. Local stimulation of VMH EphA5 receptors by ephrinA5-Fc or ephrinA5 overexpression increased, whereas knockdown of VMH ephrinA5 reduced counterregulatory responses during hypoglycemia. Overexpression of VMH ephrinA5 transiently increased local glutamate concentrations, whereas ephrinA5 knockdown produced profound suppression of VMH interstitial fluid glutamine concentrations in the basal state and during hypoglycemia. Changes in ephrinA5/EphA5 interactions within the VMH, a key brain glucose-sensing region, act in concert with islets to restore glucose homeostasis during acute hypoglycemia, and its effect on counterregulation may be mediated by changes in glutamate/glutamine cycling.Lowering glucose levels toward normal in insulin-treated patients with type 1 and type 2 diabetes diminishes the risk of long-term complications (1,2). The degree to which this can be achieved in clinical practice is often limited by the increased risk of hypoglycemia (3). In nondiabetic individuals, a fall in blood glucose is rapidly detected, and a series of compensatory responses occur to prevent or limit hypoglycemia and to restore euglycemia (4–6). These responses include the secretion of glucagon, epinephrine, and norepinephrine along with the suppression of endogenous insulin secretion, which together promote endogenous glucose production, reduce glucose utilization, and generate typical warning symptoms. These protective responses are often disrupted in type 1 diabetic patients receiving intensive insulin therapy who have a history of hypoglycemia (7). As a result, the fear of hypoglycemia is the major factor limiting the benefits of intensive insulin treatment (8).Activation of counterregulation requires effective detection of falling glucose levels. Although a complex network of glucose sensors has been described in the central nervous system (9–11) and peripherally (12), the brain appears to have the dominant role during hypoglycemia and, specifically, the ventromedial region of the hypothalamus or VMH (13,14). Interestingly, VMH neurons contain much of the same glucose-sensing machinery (e.g., glucokinase [15], ATP-sensitive K⁺ channels [16–18]) as pancreatic β-cells, suggesting that parallels exist between the molecular mechanisms used by them and those used by β-cells. In keeping with this idea, EphA5/ephrinA5, members of synaptically localized cell adhesion molecules (19), are also specifically expressed in β-cells and have been shown to regulate insulin secretion (20).The Eph receptor tyrosine kinases and their membrane-anchored ephrin ligands play a critical role in modulating neuronal synaptic structure and its physiological properties (21). Eph receptors and their ligands, the ephrins, are membrane-bound proteins that have been divided into A and B subclasses that preferentially bind to their corresponding subclass. In the brain they play an important role in cell–cell interactions (22). Eph/ephrin interactions are bidirectional (23). Ligand binding to the Eph receptor induces “forward signaling,” mostly through phosphotyrosine-mediated pathways; however, ephrins can also signal into their host cell via receptor binding, which is referred to as “reverse signaling” (19,24).Historically, these proteins were thought to mainly function as regulators of nervous system development (25). Specifically, they were thought to primarily play a role in axon guidance during the assembly of the neural circuitry (26,27). However, many Eph receptors and their ephrin ligands are present in the adult brain and are enriched in glutamate excitatory synapses (28). Moreover, a growing body of evidence now indicates that Eph receptors are expressed in synaptic terminals where they influence synaptic plasticity via binding to glial-derived ephrins (21). These interactions between neurons and glia at the level of the synapse may serve to modulate the transmission of neurochemical signals at the synapse (29). Whether hypoglycemia per se induces local changes in the VMH affecting both neuronal synapses and surrounding glia cells is unknown, but alterations in neuron–glia interactions could potentially modulate neurotransmission within brain glucose-sensing regions. Expression of ephrinA5 has been shown to be present in the VMH (30) as well as in a variety of other brain regions (31,32).This study tests the hypothesis that stimulation of crosstalk between EphA5 receptors and ephrinA5 within the VMH might regulate the magnitude of counterregulatory responses to hypoglycemia and that reductions in the capacity of ephrinA5 to activate EphA5 receptors in the VMH might impair glucose counterregulation. It is noteworthy in this regard that in β-cells, EphA/ephrinA is also bidirectional; EphA5 forward signaling inhibits insulin secretion, whereas ephrinA5 reverse signaling stimulates insulin secretion after glucose stimulation (20).     

Ventromedial Hypothalamic Nitric Oxide Production Is Necessary for Hypoglycemia Detection and Counterregulation

OBJECTIVE

The response of ventromedial hypothalamic (VMH) glucose-inhibited neurons to decreased glucose is impaired under conditions where the counterregulatory response (CRR) to hypoglycemia is impaired (e.g., recurrent hypoglycemia). This suggests a role for glucose-inhibited neurons in the CRR. We recently showed that decreased glucose increases nitric oxide (NO) production in cultured VMH glucose-inhibited neurons. These in vitro data led us to hypothesize that NO release from VMH glucose-inhibited neurons is critical for the CRR.

RESEARCH DESIGN AND METHODS

The CRR was evaluated in rats and mice in response to acute insulin-induced hypoglycemia and hypoglycemic clamps after modulation of brain NO signaling. The glucose sensitivity of ventromedial nucleus glucose-inhibited neurons was also assessed.

RESULTS

Hypoglycemia increased hypothalamic constitutive NO synthase (NOS) activity and neuronal NOS (nNOS) but not endothelial NOS (eNOS) phosphorylation in rats. Intracerebroventricular and VMH injection of the nonselective NOS inhibitor N^G-monomethyl-l-arginine (l-NMMA) slowed the recovery to euglycemia after hypoglycemia. VMH l-NMMA injection also increased the glucose infusion rate (GIR) and decreased epinephrine secretion during hyperinsulinemic/hypoglycemic clamp in rats. The GIR required to maintain the hypoglycemic plateau was higher in nNOS knockout than wild-type or eNOS knockout mice. Finally, VMH glucose-inhibited neurons were virtually absent in nNOS knockout mice.

CONCLUSIONS

We conclude that VMH NO production is necessary for glucose sensing in glucose-inhibited neurons and full generation of the CRR to hypoglycemia. These data suggest that potentiating NO signaling may improve the defective CRR resulting from recurrent hypoglycemia in patients using intensive insulin therapy.Intensive insulin therapy significantly reduces the onset and progression of hyperglycemia-related complications in patients with type 1 and advanced type 2 diabetes. However, intensive insulin therapy also causes a clinically adverse effect: hypoglycemia (1). Powerful neuroendocrine and autonomic counterregulatory mechanisms protect the brain from hypoglycemia (2,3). These protective mechanisms, known as the counterregulatory response (CRR) to hypoglycemia, involve the release of hormones (e.g., glucagon, epinephrine) that restore euglycemia by stimulating hepatic glucose production and inhibiting peripheral glucose uptake (3). Although the physiology of the CRR is well understood, the underlying cellular mechanisms by which the brain senses hypoglycemia and initiates the CRR remain elusive.During hypoglycemia, central and peripheral glucose sensors detect declining glucose levels (4). In the brain, the ventromedial hypothalamus, which includes the arcuate nucleus and the ventromedial nucleus (VMN), is important in the initiation of the CRR (5–7). This region contains specialized glucose-sensing neurons (GSNs). Ventromedial hypothalamic (VMH) GSN electrical activity is regulated by physiologically relevant changes in extracellular glucose levels (8–11). Glucose-excited neurons decrease, whereas glucose-inhibited neurons increase, their input resistance, membrane potential, and action potential frequency when extracellular glucose is reduced (10). Many studies suggest that VMH glucose-inhibited neurons play a critical role in the control of the CRR (4). For example, the response of VMH glucose-inhibited neurons to decreased glucose is impaired under conditions where the CRR is impaired (e.g., recurrent hypoglycemia) (12,13).Nitric oxide (NO) is a gaseous messenger produced by NO synthase (NOS). Two classes of NOS have been identified in the brain: the inducible NOS (iNOS) and the constitutive NOS, which includes the neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms (14). Hypothalamic NO is involved in the regulation of food intake and glucose homeostasis (15–18). In support of this, we have recently shown that VMH glucose-inhibited neurons produce NO via nNOS in response to decreased extracellular glucose levels (19,20). Therefore, in this study, we test the hypothesis that NO production by VMH glucose-inhibited neurons is necessary for the CRR to hypoglycemia. We tested this hypothesis using a combination of in vivo and in vitro techniques in wild-type rats and mice as well as in transgenic nNOS and eNOS knockout mice.     

Human Brain Glycogen Metabolism During and After Hypoglycemia

OBJECTIVE

We tested the hypotheses that human brain glycogen is mobilized during hypoglycemia and its content increases above normal levels (“supercompensates”) after hypoglycemia.

RESEARCH DESIGN AND METHODS

We utilized in vivo ¹³C nuclear magnetic resonance spectroscopy in conjunction with intravenous infusions of [¹³C]glucose in healthy volunteers to measure brain glycogen metabolism during and after euglycemic and hypoglycemic clamps.

RESULTS

After an overnight intravenous infusion of 99% enriched [1-¹³C]glucose to prelabel glycogen, the rate of label wash-out from [1-¹³C]glycogen was higher (0.12 ± 0.05 vs. 0.03 ± 0.06 μmol · g⁻¹ · h⁻¹, means ± SD, P < 0.02, n = 5) during a 2-h hyperinsulinemic-hypoglycemic clamp (glucose concentration 57.2 ± 9.7 mg/dl) than during a hyperinsulinemic-euglycemic clamp (95.3 ± 3.3 mg/dl), indicating mobilization of glucose units from glycogen during moderate hypoglycemia. Five additional healthy volunteers received intravenous 25–50% enriched [1-¹³C]glucose over 22–54 h after undergoing hyperinsulinemic-euglycemic (glucose concentration 92.4 ± 2.3 mg/dl) and hyperinsulinemic-hypoglycemic (52.9 ± 4.8 mg/dl) clamps separated by at least 1 month. Levels of newly synthesized glycogen measured from 4 to 80 h were higher after hypoglycemia than after euglycemia (P ≤ 0.01 for each subject), indicating increased brain glycogen synthesis after moderate hypoglycemia.

CONCLUSIONS

These data indicate that brain glycogen supports energy metabolism when glucose supply from the blood is inadequate and that its levels rebound to levels higher than normal after a single episode of moderate hypoglycemia in humans.Glucose is the primary fuel for the adult brain. During euglycemia and hyperglycemia, the brain receives more glucose from the blood than it utilizes and normal metabolism can be maintained. However, how the energy needs of the brain are met during hypoglycemia has been a matter of debate. Mobilization of glucose stored in the form of glycogen is one potential mechanism that could support brain metabolism when blood glucose is low. Glycogen content of the brain has been measured at 3–10 μmol/g (1–4), an amount much higher than brain glucose at euglycemia (1–1.5 μmol/g) (5). Although brain glycogen content is much lower than liver (200–400 μmol/g) (6) and muscle (80 μmol/g) (7), we have previously estimated that it can augment cerebral energy needs during short periods of glucose deficit in humans (4). In the current study, we addressed this question in normal human volunteers using nuclear magnetic resonance (NMR) methodology first developed in rats (8) and then translated to humans (9,10). With this technique, [¹³C]glucose is administered intravenously and its incorporation into and wash-out from brain glycogen is tracked (9,10). [1-¹³C]glucose has been the substrate of choice since the NMR signal of [1-¹³C]glucose in glycogen is well resolved from those of free [1-¹³C]glucose and other glucosyl positions. The ¹³C NMR measurement of brain glycogen was recently validated by comparing glycogen concentrations obtained in vivo in rats to those measured in extracted tissue by a standard biochemical assay (11).Using ¹³C NMR, we recently estimated that 3–4 μmol/g glucose is stored in the form of glycogen in the awake human brain (4). This is in agreement with a measurement of 5–6 μmol/g in normal gray and white matter obtained by biopsies during surgery of patients with epilepsy (12) because anesthesia is known to trigger glycogen accumulation (13). Based on these studies, the glycogen content of the brain represents a significant glucose reservoir relative to free glucose. We found that human brain glycogen turns over very slowly relative to the cerebral rate of glucose utilization (CMR_glc) under normal physiology (4), similar to what has been observed in the rodent brain (1,8,14). Namely, at euglycemia and hyperglycemia, bulk brain glycogen turns over at a rate that is ∼1–2% of CMR_glc (15–18) in both humans and rodents. Importantly, glycogen synthesis and breakdown rates can be altered by many factors, such as nutrients, neurotransmitters, and hormones, including glucose and insulin (19–22). The low metabolic rate of glycogen under normal physiology, together with the capacity to acutely regulate glycogen synthase and phosphorylase in response to nutritional and hormonal state, indicate that glycogen may serve as an emergency reservoir when glucose supply from the blood is inadequate. Indeed, brain glycogen is mobilized during hypoglycemia in the rodent brain (23–26), but whether a similar event occurs in humans during hypoglycemia is unknown.In rodents, brain glycogen was observed to rebound to levels higher than normal, a phenomenon termed “supercompensation,” after a single hypoglycemic episode (23). This led to the hypothesis that glycogen may be involved in the pathogenesis of hypoglycemia unawareness by supplying extra fuel to the brain during episodes experienced soon after the initial hypoglycemia (23,27). Glycogen supercompensation has not yet been studied in the human brain.The aims of the current study were 1) to assess glycogen mobilization in the human brain during moderate hypoglycemia and 2) to determine if the glycogen synthesis rate is increased after a hypoglycemic episode indicating supercompensation in the human brain.     

A Mouse Model of Human Hyperinsulinism Produced by the E1506K Mutation in the Sulphonylurea Receptor SUR1

Loss-of-function mutations in the K_ATP channel genes KCNJ11 and ABCC8 cause neonatal hyperinsulinism in humans. Dominantly inherited mutations cause less severe disease, which may progress to glucose intolerance and diabetes in later life (e.g., SUR1-E1506K). We generated a mouse expressing SUR1-E1506K in place of SUR1. K_ATP channel inhibition by MgATP was enhanced in both homozygous (homE1506K) and heterozygous (hetE1506K) mutant mice, due to impaired channel activation by MgADP. As a consequence, mutant β-cells showed less on-cell K_ATP channel activity and fired action potentials in glucose-free solution. HomE1506K mice exhibited enhanced insulin secretion and lower fasting blood glucose within 8 weeks of birth, but reduced insulin secretion and impaired glucose tolerance at 6 months of age. These changes correlated with a lower insulin content; unlike wild-type or hetE1506K mice, insulin content did not increase with age in homE1506K mice. There was no difference in the number and size of islets or β-cells in the three types of mice, or evidence of β-cell proliferation. We conclude that the gradual development of glucose intolerance in patients with the SUR1-E1506K mutation might, as in the mouse model, result from impaired insulin secretion due a failure of insulin content to increase with age.Congenital hyperinsulinism of infancy (HI) is a rare genetic disorder characterized by enhanced insulin secretion that leads to persistent hypoglycemia soon after birth (1,2). It occurs in ∼1 in 50,000 live births within the general population and at higher levels in communities that practice consanguineous marriage. The severity of the disease varies from a mild form, which responds to treatment with drugs (such as diazoxide or octreotide), to a severe drug-resistant form, which may require removal of most of the pancreas. Early diagnosis is important to avoid irreversible brain damage due to the hypoglycemia. Loss-of-function mutations in either the pore-forming (Kir6.2, encoded by KCNJ11) or regulatory (SUR1, ABCC8) subunit of the β-cell plasma membrane ATP-sensitive potassium (K_ATP) channel are the most common causes of HI (3–6).The K_ATP channel plays a crucial role in insulin secretion by coupling the energy state of the β-cell to the plasma membrane potential (5). It achieves this by sensing changes in intracellular adenine nucleotides, being inhibited by ATP binding to Kir6.2 and activated by MgATP (and MgADP) binding and hydrolysis at the nucleotide-binding domains of SUR1 (7,8). As a consequence, the K_ATP channel is open when metabolism is low, keeping the β-cell membrane hyperpolarized and preventing insulin secretion. An increase in β-cell metabolism, consequent on elevation of the plasma glucose concentration, leads to an increase in intracellular ATP and K_ATP channel closure. This produces a membrane depolarization that opens voltage-gated Ca²⁺ channels, stimulating Ca²⁺ influx and exocytosis of insulin granules. Loss-of-function mutations in either Kir6.2 or SUR1 result in permanent membrane depolarization, persistent Ca²⁺ influx, and thus continuous, unregulated insulin release (2–6).Most reported mutations in KCNJ11 and ABCC8 that cause HI are inherited recessively; these mutations cause the most severe form of the disease. However, a few dominantly expressed mutations have also been described (9–15). Children with these mutations generally have a milder phenotype than those with recessive mutations, and their hypoglycemia is well controlled by the K_ATP channel opener diazoxide. Members of one family, who are heterozygous carriers of the SUR1-E1506K mutation, have mild neonatal HI but are at increased risk of diabetes in middle age (9,14); 4 out of 11 had overt diabetes, and 5 of those without diabetes showed impaired glucose tolerance. Similarly, a child with a heterozygous SUR1-R370S mutation causing neonatal hyperinsulinism developed diabetes at 10 years of age (15). Studies of other dominantly inherited mutations have not shown a link between HI mutations and late-onset diabetes, although the disease severity may diminish later in life as many patients no longer require diazoxide therapy and become normoglycemic (12).Despite their impaired glucose tolerance, blood glucose levels were normal in heterozygous carriers of the SUR1-E1506K mutation without diabetes, and only slightly increased in those with diabetes (14). Electrophysiological studies indicate that the E1506K mutation does not impair membrane trafficking but results in channels that are no longer activated by MgATP (9,16). As a consequence, homozygous whole-cell K_ATP currents are absent, and heterozygous K_ATP currents are 30–50% smaller than wild type (WT). The smaller K_ATP currents would be expected to result in membrane depolarization, thus accounting for the increased insulin secretion in human neonates. Why this translates into reduced insulin secretion later in life is unclear, as is why impaired insulin secretion was observed in all carriers of the E1506K mutation but diabetes in only some of them.Unexpectedly, genetic deletion of SUR1 in mice did not mimic human hyperinsulinism (6,17–20). SUR1^−/− mice exhibited hypoglycemia on the first day of life but normal blood glucose levels subsequently (17), and by 12 weeks of age, they showed impaired glucose tolerance (17,18). The β-cell resting membrane potential was depolarized (17), and the basal intracellular calcium concentration was elevated (18), as expected if K_ATP channels were blocked. Reported data on insulin secretion from SUR1^−/− islets are controversial. Early studies suggested that basal insulin secretion was not elevated (17) and that glucose-induced insulin secretion from isolated islets (17) and perfused pancreas (18) was severely impaired. However, subsequent studies showed that basal insulin secretion is elevated (as expected from the raised [Ca²⁺]_i), that after overnight culture in 10 mmol/L glucose, glucose-stimulated insulin release is greater than from WT islets (19,20), and that the amplifying effect of glucose on insulin secretion is intact. What remains unclear, however, is why SUR1^−/− mice have a different phenotype from the human disease, and why some human mutations cause diabetes later in life.To address these questions, we generated a mouse carrying a human HI mutation, SUR1-E1506K, which causes neonatal hypoglycemia and predisposes to diabetes late in life (9,14). We show here that both homozygous and heterozygous mice secrete more insulin than their WT littermates early in life, but with age, homozygous mice secrete less insulin and become increasingly glucose intolerant.     

Evidence That Hyperglycemia After Recovery From Hypoglycemia Worsens Endothelial Function and Increases Oxidative Stress and Inflammation in Healthy Control Subjects and Subjects With Type 1 Diabetes

Currently there is debate on whether hypoglycemia is an independent risk factor for atherosclerosis, but little attention has been paid to the effects of recovery from hypoglycemia. In normal control individuals and in people with type 1 diabetes, recovery from a 2-h induced hypoglycemia was obtained by reaching normoglycemia or hyperglycemia for another 2 h and then maintaining normal glycemia for the following 6 h. Hyperglycemia after hypoglycemia was also repeated with the concomitant infusion of vitamin C. Recovery with normoglycemia is accompanied by a significant improvement in endothelial dysfunction, oxidative stress, and inflammation, which are affected by hypoglycemia; however, a period of hyperglycemia after hypoglycemia worsens all of these parameters, an effect that persists even after the additional 6 h of normoglycemia. This effect is partially counterbalanced when hyperglycemia after hypoglycemia is accompanied by the simultaneous infusion of vitamin C, suggesting that when hyperglycemia follows hypoglycemia, an ischemia–reperfusion-like effect is produced. This study shows that the way in which recovery from hypoglycemia takes place in people with type 1 diabetes could play an important role in favoring the appearance of endothelial dysfunction, oxidative stress, and inflammation, widely recognized cardiovascular risk factors.Recent evidence suggests that hypoglycemia may play an important role in the vascular complications of diabetes (1). Hypoglycemia causes oxidative stress (2), inflammation (3), and endothelial dysfunction (4). Oxidative stress is considered the key player in the pathogenesis of diabetes complications (5). During hyperglycemia, oxidative stress is produced at the mitochondrial level (5), similarly as in hypoglycemia (2). Therefore, oxidative stress might be considered the common factor linking hyperglycemia, hypoglycemia, and the vascular complications of diabetes. Consistent with this hypothesis is the evidence that hyperglycemia (6) and hypoglycemia both produce endothelial dysfunction and inflammation through the generation of oxidative stress (4,7). Endothelial dysfunction and inflammation are well-recognized pathogenic factors for vascular disease, particularly in diabetes (8).There is, however, evidence that free radical production rises, not only during hypoglycemia but particularly during glucose reperfusion after hypoglycemia (9). In both mice and cultured neurons, hypoglycemia, followed by different concentrations of glucose reperfusion, has been linked to a degree of superoxide production and neuronal death that increased proportionally with glucose concentrations during the reperfusion period (9).Until now, little attention has been given to studying the effects of recovery from hypoglycemia. The aim of this study was to evaluate these effects and, in particular, to determine if the level of glycemia reached during recovery could have a different impact, in vivo, on oxidative stress generation, inflammation, and endothelial function.     

Effects of Differing Antecedent Increases of Plasma Cortisol on Counterregulatory Responses During Subsequent Exercise in Type 1 Diabetes

OBJECTIVE

Antecedent hypoglycemia can blunt neuroendocrine and autonomic nervous system responses to next-day exercise in type 1 diabetes. The aim of this study was to determine whether antecedent increase of plasma cortisol is a mechanism responsible for this finding.

RESEARCH DESIGN AND METHODS

For this study, 22 type 1 diabetic subjects (11 men and 11 women, age 27 ± 2 years, BMI 24 ± 1 kg/m², A1C 7.9 ± 0.2%) underwent four separate randomized 2-day protocols, with overnight normalization of blood glucose. Day 1 consisted of morning and afternoon 2-h hyperinsulinemic- (9 pmol · kg⁻¹ · min⁻¹) euglycemic clamps (5.1 mmol/l), hypoglycemic clamps (2.9 mmol/l), or euglycemic clamps with a physiologic low-dose intravenous infusion of cortisol to reproduce levels found during hypoglycemia or a high-dose infusion, which resulted in further twofold greater elevations of plasma cortisol. Day 2 consisted of 90-min euglycemic cycling exercise at 50% Vo_2max.

RESULTS

During exercise, glucose levels were equivalently clamped at 5.1 ± 0.1 mmol/l and insulin was allowed to fall to similar levels. Glucagon, growth hormone, epinephrine, norepinephrine, and pancreatic polypeptide responses during day 2 exercise were significantly blunted following antecedent hypoglycemia, low- and high-dose cortisol, compared with antecedent euglycemia. Endogenous glucose production and lipolysis were also significantly reduced following day 1 low- and high-dose cortisol.

CONCLUSIONS

Antecedent physiologic increases in cortisol (equivalent to levels occurring during hypoglycemia) resulted in blunted neuroendocrine, autonomic nervous system, and metabolic counterregulatory responses during subsequent exercise in subjects with type 1 diabetes. These data suggest that prior elevations of cortisol may play a role in the development of exercise-related counterregulatory failure in those with type 1 diabetes.The Diabetes Control and Complications Trial has definitively demonstrated that intensive glycemic control can reduce microvascular complications in those with type 1 diabetes (1). However, intensive therapy is associated with an increased incidence of hypoglycemia (2). Exercise is a cornerstone of diabetes management. It improves insulin sensitivity, helps in body weight maintenance, and lowers the risk of cardiovascular disease. Unfortunately, exercise is associated with an increased prevalence of hypoglycemia in patients with diabetes. Furthermore, fear of hypoglycemia results in a serious limitation to the widespread implementation of intensive glycemic control and exercise.Previous studies have shown that antecedent hypoglycemia can blunt neuroendocrine, autonomic nervous system (ANS), and metabolic counterregulatory responses to subsequent exercise (3,4). Reciprocally, antecedent exercise can also blunt homeostatic counterregulatory responses to subsequent hypoglycemia (5,6). Therefore, vicious cycles can be created in type 1 diabetes where an episode of hypoglycemia or exercise can downregulate counterregulatory responses to a subsequent episode of either stress, thereby increasing the risk for further hypoglycemia. However, the mechanisms responsible for exercise-associated counterregulatory failure are not known. Multiple studies have demonstrated that antecedent increases of corticosteroids can blunt subsequent ANS and neuroendocrine responses to a wide spectrum of differing antecedent stress (7–21). Previous studies have also demonstrated that pharmacologic antecedent increases of cortisol can blunt counterregulatory responses to subsequent hypoglycemia in normal subjects (a stress that defends against a falling plasma glucose with similar counterregulatory responses compared with exercise) (7–8). Although, the question of whether physiologic levels of cortisol can blunt counterregulatory responses to subsequent hypoglycemia is still undecided (15,22–24). However, studies specifically investigating the effects of prior elevations of corticosteroids on counterregulatory mechanisms during exercise are lacking. Furthermore, no study has investigated the mechanisms responsible for exercise-related counterregulatory failure in the clinically relevant group of those with type 1 diabetes.Therefore, the specific aim of this present study was to test the hypothesis that antecedent physiologic or pharmacologic elevations of cortisol could blunt counterregulatory responses during subsequent submaximal exercise in type 1 diabetic individuals. To test this hypothesis, hydrocortisone was administered intravenously on day 1 during hyperinsulinemic-euglycemic clamps and responses to subsequent euglycemic exercise were studied during the following day.     

Insulin Reciprocally Regulates Glucagon Secretion in Humans

OBJECTIVE

We tested the hypothesis that an increase in insulin per se, i.e., in the absence of zinc, suppresses glucagon secretion during euglycemia and that a decrease in insulin per se stimulates glucagon secretion during hypoglycemia in humans.

RESEARCH DESIGN AND METHODS

We measured plasma glucagon concentrations in patients with type 1 diabetes infused with the zinc-free insulin glulisine on three occasions. Glulisine was infused with clamped euglycemia (∼95 mg/dl [5.3 mmol/l]) from 0 to 60 min on all three occasions. Then, glulisine was discontinued with clamped euglycemia or with clamped hypoglycemia (∼55 mg/dl [3.0 mmol/l]) or continued with clamped hypoglycemia from 60 to 180 min.

RESULTS

Plasma glucagon concentrations were suppressed by −13 ± 3, −9 ± 3, and −12 ± 2 pg/ml (−3.7 ± 0.9, −2.6 ± 0.9, and −3.4 ± 0.6 pmol/l), respectively, (all P < 0.01) during zinc-free hyperinsulinemic euglycemia over the first 60 min. Glucagon levels remained suppressed following a decrease in zinc-free insulin with euglycemia (−14 ± 3 pg/ml [−4.0 ± 0.9 pmol/l]) and during sustained hyperinsulinemia with hypoglycemia (−14 ± 2 pg/ml [−4.0 ± 0.6 pmol/l]) but increased to −3 ± 3 pg/ml (−0.9 ± 0.9 pmol/l) (P < 0.01) following a decrease in zinc-free insulin with hypoglycemia over the next 120 min.

CONCLUSIONS

These data indicate that an increase in insulin per se suppresses glucagon secretion and a decrease in insulin per se, in concert with a low glucose concentration, stimulates glucagon secretion. Thus, they document that insulin is a β-cell secretory product that, in concert with glucose and among other signals, reciprocally regulates α-cell glucagon secretion in humans.The regulation of pancreatic islet α-cell glucagon secretion by nutrients, hormones, neurotransmitters, and drugs is complex and incompletely understood (1–8). It involves direct signaling of α-cells (1) and indirect signaling of α-cells by β-cell (2–4) and δ-cell (5) secretory products, the autonomic nervous system (6,7), and gut incretins (8). Among the intraislet mechanisms, there is evidence that indirect reciprocal β-cell–mediated signaling of α-cells normally predominates over direct α-cell signaling in the regulation of glucagon secretion in humans (9–13). The physiological concept is as follows: 1) A β-cell secretory product, or products, tonically restrains α-cell glucagon secretion during postabsorptive euglycemia. 2) A decrease in β-cell secretion, in concert with a low α-cell glucose concentration, signals an increase in α-cell glucagon secretion during hypoglycemia (9–12). 3) An increase in β-cell secretion negates direct α-cell stimulation and thus results in no change or even suppression of α-cell glucagon secretion following a mixed meal (13).Among the various candidate signaling molecules—insulin, zinc, γ-aminobutyric acid, and amylin among others (4)—there is evidence that insulin is a β-cell secretory product that normally restrains basal α-cell glucagon secretion (14). First, administered insulin suppresses glucagon secretion in several species (4,14,15) including humans (16). However, most available insulin preparations contain zinc and zinc is cosecreted with insulin from β-cells and it has been reported that a decrease in pancreatic arterial zinc, but not in insulin depleted of zinc, increases glucagon secretion during hypoglycemia in streptozotocin diabetic rats, leading the authors to conclude that the inhibitory β-cell secretory product is zinc—not insulin (17). Second, perfusion of the rat (3) and the human (18) pancreas (and incubation of rat islets [15]) with an antibody to insulin increases glucagon release. Because insulin circulates as a zinc-free monomer (19), that finding seemingly implicates insulin directly as an α-cell inhibitory factor and suggests that zinc is not the only β-cell product that reciprocally regulates α-cell glucagon secretion. However, it could be reasoned that the antibody binds insulin-zinc complexes before they dissociate at physiological pH and with dilution. Third, findings—that siRNA-mediated knockdown of insulin receptors prevented the effect of low glucose concentrations to increase glucagon release from isolated mouse islets (20) and that blockade of insulin signaling with the phosphatidylinositol 3-kinase inhibitor wortmannin prevented the effect of high glucose concentrations to decrease glucagon release from isolated rat and human islets (21)—also appear to implicate insulin as the relevant β-cell secretory product.We tested the hypothesis that an increase in insulin per se, i.e., in the absence of an increase in zinc, suppresses glucagon secretion during euglycemia and that a decrease in insulin per se, in concert with low glucose concentrations, stimulates glucagon secretion in humans. To do so, we studied patients with type 1 diabetes, individuals with essentially no endogenous insulin secretion and no α-cell glucagon secretory response to hyperinsulinemic hypoglycemia (22–29) but viable α-cells as evidenced by a glucagon secretory response to administered amino acids (22,30–32), on three separate occasions. Plasma glucagon concentrations were measured during infusion of the zinc-free insulin glulisine with clamped euglycemia over 60 min on all three occasions. Then, glucagon concentrations were measured following discontinuation of glulisine, to produce a sharp decrease in systemic and therefore α-cell zinc-free insulin levels, over 120 min with clamped euglycemia on one occasion or clamped hypoglycemia on another occasion. On a third occasion, glucagon levels were measured during sustained glulisine infusion with clamped hypoglycemia. The data document 1) a decrease in plasma glucagon during zinc-free hyperinsulinemic euglycemia and 2) an increase in plasma glucagon following a decrease in zinc-free insulin during hypoglycemia but not following a decrease in insulin without hypoglycemia or during hypoglycemia without a decrease in insulin.     

Additive Effects of Genetic Variation in GCK and G6PC2 on Insulin Secretion and Fasting Glucose

OBJECTIVE

Glucokinase (GCK) and glucose-6-phosphatase catalytic subunit 2 (G6PC2) regulate the glucose-cycling step in pancreatic β-cells and may regulate insulin secretion. GCK rs1799884 and G6PC2 rs560887 have been independently associated with fasting glucose, but their interaction on glucose-insulin relationships is not well characterized.

RESEARCH DESIGN AND METHODS

We tested whether these variants are associated with diabetes-related quantitative traits in Mexican Americans from the BetaGene Study and attempted to replicate our findings in Finnish men from the METabolic Syndrome in Men (METSIM) Study.

RESULTS

rs1799884 was not associated with any quantitative trait (corrected P > 0.1), whereas rs560887 was significantly associated with the oral glucose tolerance test 30-min incremental insulin response (30′ Δinsulin, corrected P = 0.021). We found no association between quantitative traits and the multiplicative interaction between rs1799884 and rs560887 (P > 0.26). However, the additive effect of these single nucleotide polymorphisms was associated with fasting glucose (corrected P = 0.03) and 30′ Δinsulin (corrected P = 0.027). This additive association was replicated in METSIM (fasting glucose, P = 3.5 × 10⁻¹⁰ 30′ Δinsulin, P = 0.028). When we examined the relationship between fasting glucose and 30′ Δinsulin stratified by GCK and G6PC2, we noted divergent changes in these quantitative traits for GCK but parallel changes for G6PC2. We observed a similar pattern in METSIM.

CONCLUSIONS

Our data suggest that variation in GCK and G6PC2 have additive effects on both fasting glucose and insulin secretion.Genome-wide association (GWA) studies have identified several loci for type 2 diabetes (1–5) and type 2 diabetes–related quantitative traits (6–20). Two of these loci, glucokinase (GCK) (21–23) and glucose-6-phosphatase catalytic subunit 2 (G6PC2) (9,10), regulate the critical glucose-sensing mechanism within pancreatic β-cells. Mutations in GCK confer susceptibility to maturity-onset diabetes of the young (MODY)-2 (24–26), and a −30 GCK promoter variant (rs1799884) has been shown to be associated with β-cell function (21), fasting glucose, and birth weight (23). Chen et al. (9) demonstrated an association between the G6PC2 region and fasting glucose, an observation replicated by Bouatia-Naji et al. (10). Although fasting glucose levels are associated with both GCK and G6PC2, there has been no evidence that genetic variation at these two loci contribute a risk for type 2 diabetes, suggesting contribution to mild elevations in glycemia.GCK phosphorylates glucose to glucose-6-phosphate, whereas G6PC2 dephosphorylates glucose-6-phosphate back to glucose, forming a glucose cycle previously demonstrated to exist within pancreatic β-cells (27,28). The important role of GCK in glucose sensing by pancreatic islets has been demonstrated by numerous studies, and other studies suggest a role for glucose cycling in insulin secretion and diabetes (28–30), implying that the balance between GCK and G6PC2 activity is important for determining glycolytic flux, ATP production, and subsequent insulin secretion. This was validated by a demonstration that direct manipulation of glucose cycling alters insulin secretion (31,32).BetaGene is a study in which we are performing detailed phenotyping of Mexican American probands with recent gestational diabetes mellitus (GDM) and their family members to obtain quantitative estimates of body composition, insulin sensitivity (S_I), acute insulin response (AIR), and β-cell compensation (disposition index) with the goal of identifying genes influencing variations in type 2 diabetes–related quantitative traits (33–35). Based on the evidence that variation in GCK (rs1799884) and G6PC2 (rs560887) are independently associated with fasting glucose concentrations and both are crucial to glucose cycling in β-cells, we hypothesized that interaction between these loci may be associated not just with fasting glucose but also with measures of insulin secretion or β-cell function. We tested this hypothesis in the BetaGene Study and, for replication, in a separate sample of Finnish men participating in the METabolic Syndrome in Men (METSIM) Study (36).     

Severe Hypoglycemia–Induced Lethal Cardiac Arrhythmias Are Mediated by Sympathoadrenal Activation

For people with insulin-treated diabetes, severe hypoglycemia can be lethal, though potential mechanisms involved are poorly understood. To investigate how severe hypoglycemia can be fatal, hyperinsulinemic, severe hypoglycemic (10–15 mg/dL) clamps were performed in Sprague-Dawley rats with simultaneous electrocardiogram monitoring. With goals of reducing hypoglycemia-induced mortality, the hypotheses tested were that: 1) antecedent glycemic control impacts mortality associated with severe hypoglycemia; 2) with limitation of hypokalemia, potassium supplementation could limit hypoglycemia-associated deaths; 3) with prevention of central neuroglycopenia, brain glucose infusion could prevent hypoglycemia-associated arrhythmias and deaths; and 4) with limitation of sympathoadrenal activation, adrenergic blockers could prevent hypoglycemia-induced arrhythmic deaths. Severe hypoglycemia–induced mortality was noted to be worsened by diabetes, but recurrent antecedent hypoglycemia markedly improved the ability to survive an episode of severe hypoglycemia. Potassium supplementation tended to reduce mortality. Severe hypoglycemia caused numerous cardiac arrhythmias including premature ventricular contractions, tachycardia, and high-degree heart block. Intracerebroventricular glucose infusion reduced severe hypoglycemia–induced arrhythmias and overall mortality. β-Adrenergic blockade markedly reduced cardiac arrhythmias and completely abrogated deaths due to severe hypoglycemia. Under conditions studied, sudden deaths caused by insulin-induced severe hypoglycemia were mediated by lethal cardiac arrhythmias triggered by brain neuroglycopenia and the marked sympathoadrenal response.The rate of sudden death in young people with type 1 diabetes is 10-fold higher than in age-matched control subjects (1). Sudden nocturnal deaths account for up to 27% of all unexplained deaths in people with type 1 diabetes (2,3). The aptly named “dead in bed syndrome” identifies otherwise healthy young individuals with type 1 diabetes who were found dead in bed with no clear cause of death (1–9). It has been speculated that their deaths were due to excess insulin administration that resulted in severe hypoglycemia (3,5–11). However, the mechanisms by which severe hypoglycemia can be lethal is unclear.During hypoglycemia, lack of glucose supply to neurons can lead to confusion, brain damage (12), seizures (13,14), and even death. Indeed, 6–10% of deaths in young people with type 1 diabetes are directly attributable to hypoglycemia (15–17). It has been hypothesized that abrupt cardiac arrhythmias contribute to severe hypoglycemia–induced sudden death. Clinical studies using electrocardiograms (ECGs) have found that cardiac arrhythmias associated with corrected QT (QTc) prolongation occur during moderate hypoglycemia (18–21). QTc prolongation represents dispersion of ventricle depolarization and can lead to increased risk of fatal cardiac arrhythmias (22–24). In the setting of insulin-induced severe hypoglycemia, it remains unknown whether fatal cardiac arrhythmias are mediated by brain neuroglycopenia per se or systemic factors acting directly on the heart. Systemically, both the insulin-induced decrement in potassium levels and the counterregulatory-induced increase in catecholamine levels have been speculated to contribute to arrhythmias (25–27). It has been shown previously that the administration of potassium or β-blockers can reduce mild cardiac arrhythmias during moderate hypoglycemia (∼45 mg/dL) (26). However, it remains to be determined whether the prevention of hypokalemia or blocking the actions of the catecholamines protects against lethal arrhythmias and cardiorespiratory arrest during severe hypoglycemia.In order to determine preventable causes of death during hypoglycemia, severe hypoglycemic clamps were performed in Sprague-Dawley rats with simultaneous arterial blood sampling and ECG monitoring. It was hypothesized that brain neuroglycopenia and the marked sympathoadrenal response during severe hypoglycemia trigger fatal cardiac arrhythmias.     

Recurrent Moderate Hypoglycemia Ameliorates Brain Damage and Cognitive Dysfunction Induced by Severe Hypoglycemia

OBJECTIVE

Although intensive glycemic control achieved with insulin therapy increases the incidence of both moderate and severe hypoglycemia, clinical reports of cognitive impairment due to severe hypoglycemia have been highly variable. It was hypothesized that recurrent moderate hypoglycemia preconditions the brain and protects against damage caused by severe hypoglycemia.

RESEARCH DESIGN AND METHODS

Nine-week-old male Sprague-Dawley rats were subjected to either 3 consecutive days of recurrent moderate (25–40 mg/dl) hypoglycemia (RH) or saline injections. On the fourth day, rats were subjected to a hyperinsulinemic (0.2 units · kg⁻¹ · min⁻¹) severe hypoglycemic (∼11 mg/dl) clamp for 60 or 90 min. Neuronal damage was subsequently assessed by hematoxylin-eosin and Fluoro-Jade B staining. The functional significance of severe hypoglycemia–induced brain damage was evaluated by motor and cognitive testing.

RESULTS

Severe hypoglycemia induced brain damage and striking deficits in spatial learning and memory. Rats subjected to recurrent moderate hypoglycemia had 62–74% less brain cell death and were protected from most of these cognitive disturbances.

CONCLUSIONS

Antecedent recurrent moderate hypoglycemia preconditioned the brain and markedly limited both the extent of severe hypoglycemia–induced neuronal damage and associated cognitive impairment. In conclusion, changes brought about by recurrent moderate hypoglycemia can be viewed, paradoxically, as providing a beneficial adaptive response in that there is mitigation against severe hypoglycemia–induced brain damage and cognitive dysfunction.Hypoglycemia is the major obstacle in achieving tight glycemic control in people with diabetes (1). Intensive insulin therapy increases the risk of iatrogenic hypoglycemia (2). Episodes of both moderate and severe hypoglycemia have long-term clinical consequences. Recurrent moderate hypoglycemia induces a maladaptive response that limits symptoms of hypoglycemia (hypoglycemia unawareness), limits the counterregulatory response to subsequent hypoglycemia (hypoglycemia-associated autonomic failure), and thus jeopardizes patient safety (1). By depriving the brain of glucose, more severe hypoglycemia causes brain damage in animal studies and leads to long-term impairments in learning and memory (3,4). However, studies examining the effect of severe hypoglycemia in humans are conflicting. Severe hypoglycemia has been shown to alter brain structure (5–7) and cause significant cognitive damage in many (5,7–12) but not all (13–16) studies. Reasons for the discrepancy between human and animal studies are unknown, but a major contributing factor may be the extent of glycemia control (including recurrent hypoglycemia) prior to the episode of severe hypoglycemia.In other models of brain damage, such as ischemic stroke, brief, mild episodes of antecedent brain ischemia has been shown to cause a beneficial adaptation that protects the brain against a subsequent episode of more severe ischemia (a phenomena known as ischemic preconditioning) (17). In a similar fashion, antecedent, recurrent episodes of moderate hypoglycemia were hypothesized to protect the brain against damage caused by a subsequent episode of more severe hypoglycemia.To investigate this hypothesis, recurrent moderately hypoglycemic (25–40 mg/dl) rats (RH rats) and control saline-injected rats (CON rats) were subjected to hyperinsulinemic, severe hypoglycemic clamps (10–15 mg/dl). One group of rats was killed 1 week after severe hypoglycemia to quantify brain damage, while a second group of rats was evaluated by behavioral and cognitive tests 6–8 weeks after the severe hypoglycemia. The results demonstrated that recurrent antecedent moderate hypoglycemia preconditioned the brain and protected it against neurological damage and cognitive defects induced by an episode of severe hypoglycemia.     

Amelioration of Hypoglycemia Via Somatostatin Receptor Type 2 Antagonism in Recurrently Hypoglycemic Diabetic Rats

Selective antagonism of somatostatin receptor type 2 (SSTR2) normalizes glucagon and corticosterone responses to hypoglycemic clamp in diabetic rats. The purpose of this study was to determine whether SSTR2 antagonism (SSTR2a) ameliorates hypoglycemia in response to overinsulinization in diabetic rats previously exposed to recurrent hypoglycemia. Streptozotocin diabetic rats (n = 19), previously subjected to five hypoglycemia events over 3 days, received an insulin bolus (10 units/kg i.v.) plus insulin infusion (50 mU/kg/min i.v.) until hypoglycemia ensued (≤3.9 mmol/L) (experimental day 1 [Expt-D1]). The next day (Expt-D2), rats were allocated to receive either placebo treatment (n = 7) or SSTR2a infusion (3,000 nmol/kg/min i.v., n = 12) 60 min prior to the same insulin regimen. On Expt-D1, all rats developed hypoglycemia by ∼90 min, while on Expt-D2, hypoglycemia was attenuated with SSTR2a treatment (nadir = 3.7 ± 0.3 vs. 2.7 ± 0.3 mmol/L in SSTR2a and controls, P < 0.01). Glucagon response to hypoglycemia on Expt-D2 deteriorated by 20-fold in the placebo group (P < 0.001) but improved in the SSTR2a group (threefold increase in area under the curve [AUC], P < 0.001). Corticosterone response deteriorated in the placebo-treated rats on Expt-D2 but increased twofold in the SSTR2a group. Catecholamine responses were not affected by SSTR2a. Thus, SSTR2 antagonism after recurrent hypoglycemia improves the glucagon and corticosterone responses and largely ameliorates insulin-induced hypoglycemia in diabetic rats.The management of type 1 diabetes mellitus is impeded by the constant threat of hypoglycemia, caused by the inability to achieve physiological insulin replacement and because of a failure in the hormone counterregulation of hypoglycemia (1). Recurrent hypoglycemia increases the susceptibility to subsequent hypoglycemia, since it contributes to both defective hormone counterregulation and reduced symptom recognition (2). The reduction in symptom recognition for hypoglycemia has a profound impact on patient quality of life, and this population fears hypoglycemia more than long-term complications (3,4). The elevated risk of recurrent hypoglycemia, often precipitated by intensive insulin therapy, frequently necessitates a relaxation in management, which ultimately places the individual at risk for earlier complications (3). Currently, there are few prophylactic strategies that limit the risk of developing insulin-induced hypoglycemia (5), perhaps because the neuroendocrine mechanism(s) of impairment has yet to be fully elucidated. None of these treatments would be considered a preventative pharmacological approach.With repeated exposure to hypoglycemia, there are impairments in the neuroendocrine and autonomic responses to subsequent hypoglycemia (6–9), perhaps because of defects in the regions of the central nervous system that detect and respond to hypoglycemia (1). In addition to numerous neuroendocrine deficiencies related to glucose sensing and blunted counterregulatory responses because of central deficiencies (7,10–14), elevation in circulating somatostatin levels in type 1 diabetes mellitus has long been thought to impair the counterregulatory response to insulin-induced hypoglycemia (15–20).Somatostatin acts on various receptor subtypes (somatostatin receptor type [SSTR]1–5), being both a regulator of hormone secretion (typically inhibitory) and a neurotransmitter (21). With respect to glucose counterregulatory hormones, somatostatin release in the brain lowers pituitary growth hormone secretion indirectly via hypothalamic suppression of growth hormone–releasing hormone release and directly by acting on somatotrophs via SSTR2 and -5 (22). In the adrenal gland, somatostatin inhibits acetylcholine stimulated medullary catecholamine secretion and inhibits corticosteroid secretion predominantly via SSTR2 (23). In humans, somatostatin lowers pancreatic glucagon and insulin release through SSTR2 (24). In rats, somatostatin inhibits insulin secretion predominantly through SSTR5 (25) and glucagon secretion exclusively through SSTR2 (21).Paradoxically, somatostatin concentrations are elevated at baseline and rise further during hypoglycemia in patients with type 1 diabetes mellitus who are on exogenous insulin (19). Various animal models of type 1 diabetes mellitus (7,17,18,26) and isolated islet studies in healthy rats (27) have demonstrated that elevations in somatostatin limit the glucagon response to hypoglycemia or arginine stimulation via SSTR2 activation. Since somatostatin also inhibits the release of all of the key hormones involved in glucose counterregulation (i.e., cortisol, growth hormone, catecholamines) (21,28), an elevation in somatostatin levels in type 1 diabetes mellitus may be one of the reasons why glucose counterregulation fails. Accordingly, the systemic administration of a somatostatin receptor agonist exacerbates severe hypoglycemia in patients with type 1 diabetes mellitus (29), likely because of reductions in glucose counterregulatory hormone levels to ensuing insulin-induced hypoglycemia. Thus, the use of a SSTR2 antagonist (SSTR2a) may be helpful in improving glucose counterregulation in this patient population. In support of this, we recently demonstrated that SSTR2a (PRL-2903) normalizes the glucagon and corticosterone responses to hypoglycemic clamp in diabetic rats (26). Since these were glucose clamp experiments, it was not possible to determine whether hypoglycemia could be prevented with SSTR2 antagonism. It is also unclear whether the improvement in the counterregulatory hormone response caused by SSTR2a would have favorable effects on glucoregulation in diabetes. In this present work, we tested the hypothesis that hypoglycemia can be prevented/attenuated with SSTR2 antagonism treatment in animals previously exposed to repeated hypoglycemic challenge by enhancing counterregulatory responses. We demonstrate here that the glucagon and corticosterone responses improve by SSTR2 antagonism and that the depth and duration of hypoglycemia are ameliorated in diabetic rats previously exposed to recurrent hypoglycemia.     

Why Do SGLT2 Inhibitors Inhibit Only 30–50% of Renal Glucose Reabsorption in Humans?

Sodium glucose cotransporter 2 (SGLT2) inhibition is a novel and promising treatment for diabetes under late-stage clinical development. It generally is accepted that SGLT2 mediates 90% of renal glucose reabsorption. However, SGLT2 inhibitors in clinical development inhibit only 30–50% of the filtered glucose load. Why are they unable to inhibit 90% of glucose reabsorption in humans? We will try to provide an explanation to this puzzle in this perspective analysis of the unique pharmacokinetic and pharmacodynamic profiles of SGLT2 inhibitors in clinical trials and examine possible mechanisms and molecular properties that may be responsible.Type 2 diabetes is a serious global health issue that has reached epidemic proportions in both developed and developing countries over the last two decades (1). With currently available medicines, many diabetic patients fail to achieve optimal glycemic control (HbA_1c <6.5–7.0%). With the exception of the glucagon-like peptide 1 analogs and the thiazolidinediones (2), other antidiabetic medications lose their effectiveness to control hyperglycemia over time, partially due to the progressive decline of β-cell function (2–4). As a consequence, many patients receive multiple antidiabetic medicines and eventually require insulin therapy, which often fails to achieve the desired glycemic goal and is associated with weight gain and hypoglycemia (5,6). Failure to achieve glycemic targets is the primary factor responsible for the microvascular complications (retinopathy, neuropathy, nephropathy) and, to a lesser extent, macrovascular complications (2,7). In addition, the majority of diabetic patients are overweight or obese, and many of the current therapies are associated with weight gain, which causes insulin resistance and deterioration in glycemic control (2).Given the difficulty in achieving optimal glycemic control (8,9) for many diabetic patients using current therapies, there is an unmet medical need for new antidiabetic agents. Although it has been known for 50 years (10,11) that renal glucose reabsorption is increased in type 2 diabetic patients, only recently have the clinical therapeutic implications of this observation been recognized (2,12). Inhibition of renal tubular glucose reabsorption, leading to a reduction in blood glucose concentration through enhanced urinary glucose excretion, provides a novel insulin-independent therapy (2,12) that in animal models of diabetes has been shown to reverse glucotoxicity and improve insulin sensitivity and β-cell function (13,14). The majority (∼80–90%) of filtered plasma glucose is reabsorbed in the early proximal tubule by the high-capacity, low-affinity sodium glucose cotransporter (SGLT) 2 (15,16). The remaining 10–20% of filtered glucose is reabsorbed by the high-affinity, low-capacity SGLT1 transporter in the more distal portion of the proximal tubule. After glucose is actively reabsorbed by SGLT2 and SGLT1 into the proximal tubular cells, it is diffused out of the cells from the basolateral side into blood through facilitative GLUT 2 and 1 (15). Because the majority of glucose reabsorption occurs via the SGLT2 transporter, pharmaceutical companies have focused on the development of SGLT2 inhibitors, and multiple SGLT2 inhibitors currently are in human phase II and III clinical trials (17). This class of antidiabetic medication effectively lowers blood glucose levels and offers additional benefits, including weight loss, low propensity for causing hypoglycemia, and reduction in blood pressure. The SGLT2 inhibitors are effective as monotherapy and in combination with existing therapies (2,12,14,15,17), including insulin (18). Because of their unique mechanism of action (12,15), which is independent of the severity of insulin resistance and β-cell failure, type 2 diabetic individuals with recent-onset diabetes (<1 year) respond equally well as type 2 diabetic patients with long-standing diabetes (>10 years) (19).Dapagliflozin is the most advanced SGLT2 inhibitor in clinical trials (12,17,20). In addition, multiple other SGLT2 inhibitors are in phase II to III trials (Fig. 1) (17,21). However, none of these SGLT2 inhibitors are able to inhibit >30–50% of the filtered glucose load, despite in vitro studies indicate that 100% inhibition of the SGLT2 transporter should be achieved at the drug concentrations in humans (22,23). In this perspective, we shall examine potential explanations for this apparent paradox. Resolution of the paradox has important clinical implications with regard to the efficacy of this class of drugs and the development of more efficacious SGLT2 inhibitors.Open in a separate windowFIG. 1.SGLT2 inhibitors in late-stage clinical trials.     

Distinct Effects of Leptin and a Melanocortin Receptor Agonist Injected Into Medial Hypothalamic Nuclei on Glucose Uptake in Peripheral Tissues

OBJECTIVE

The medial hypothalamus mediates leptin-induced glucose uptake in peripheral tissues, and brain melanocortin receptors (MCRs) mediate certain central effects of leptin. However, the contributions of the leptin receptor and MCRs in individual medial hypothalamic nuclei to regulation of peripheral glucose uptake have remained unclear. We examined the effects of an injection of leptin and the MCR agonist MT-II into medial hypothalamic nuclei on glucose uptake in peripheral tissues.

RESEARCH DESIGN AND METHODS

Leptin or MT-II was injected into the ventromedial (VMH), dorsomedial (DMH), arcuate nucleus (ARC), or paraventricular (PVH) hypothalamus or the lateral ventricle (intracerebroventricularly) in freely moving mice. The MCR antagonist SHU9119 was injected intracerebroventricularly. Glucose uptake was measured by the 2-[³H]deoxy-d-glucose method.

RESULTS

Leptin injection into the VMH increased glucose uptake in skeletal muscle, brown adipose tissue (BAT), and heart, whereas that into the ARC increased glucose uptake in BAT, and that into the DMH or PVH had no effect. SHU9119 abolished these effects of leptin injected into the VMH. Injection of MT-II either into the VMH or intracerebroventricularly increased glucose uptake in skeletal muscle, BAT, and heart, whereas that into the PVH increased glucose uptake in BAT, and that into the DMH or ARC had no effect.

CONCLUSIONS

The VMH mediates leptin- and MT-II–induced glucose uptake in skeletal muscle, BAT, and heart. These effects of leptin are dependent on MCR activation. The leptin receptor in the ARC and MCR in the PVH regulate glucose uptake in BAT. Medial hypothalamic nuclei thus play distinct roles in leptin- and MT-II–induced glucose uptake in peripheral tissues.Leptin is an adipocyte hormone that inhibits food intake and increases energy expenditure (1). The hypothalamus is a principal target of leptin in its regulation of energy metabolism (2–5). The arcuate nucleus (ARC) is the most well characterized of hypothalamic nuclei in terms of its role in the central effects of leptin (2–5). The ARC contains two populations of leptin-responsive neurons: pro-opiomelanocortin (POMC)-expressing neurons, which release the potent anorexic peptide α-melanocyte–stimulating hormone, and neurons that release two potent orexigenic peptides, agouti-related peptide (AgRP) and neuropeptide Y (NPY) (2–5). α-Melanocyte–stimulating hormone activates the melanocortin receptor (MCR), whereas AgRP competitively inhibits this receptor and NPY functionally antagonizes MCR signaling (6). Both sets of neurons project to second-order MCR-expressing neurons within the hypothalamus, including the paraventricular (PVH), ventromedial (VMH), dorsomedial (DMH), and lateral hypothalamus, as well as to other brain regions such as the brain stem (2,4,7,8). Leptin inhibits food intake through reciprocal regulation of POMC and AgRP/NPY neurons in the ARC and consequent activation of MCR in hypothalamic nuclei, including the PVH (5,6,7,9). Mice lacking the melanocortin 3 (MC3R) or 4 (MC4R) receptor show increased adiposity and feeding efficiency (4). Restoration of MC4R expression in certain sets of PVH neurons prevented hyperphagia and reduced body weight in MC4R-null mice (9). In addition to that in the ARC, the leptin receptor Ob-Rb in other hypothalamic nuclei has also been shown to regulate energy intake and adiposity. Neurons positive for steroidogenic factor 1 (SF1; also known as Ad4BP) (10,11) are largely restricted to the VMH in the adult brain. Leptin depolarizes these neurons, and specific ablation of the leptin receptor in SF1-positive cells induced obesity and increased susceptibility to a high-fat diet in mice (12).The leptin receptor in the brain also regulates glucose metabolism in certain peripheral tissues (13–17). Treatment with leptin ameliorates diabetes in lipodystrophic mice and humans (18,19). Intravenous or intracerebroventricular administration of leptin markedly increased whole-body glucose turnover and glucose uptake by certain tissues in mice without any substantial change in plasma insulin or glucose levels (13). We have also previously shown that microinjection of leptin into the medial hypothalamus, such as into the VMH, but not into the lateral hypothalamus, preferentially increased glucose uptake in skeletal muscle, heart, and brown adipose tissue (BAT) (14–16). Restoration of Ob-Rb expression in the ARC and the VMH of the Ob-Rb–mutated Koletsky rat by adenovirus- or adeno-associated virus–mediated gene transfer improved peripheral insulin sensitivity and reduced plasma glucose concentration (17,20). Ablation of suppressor of cytokine signaling 3 (SOCS3) in SF1-positive cells (10,11) improved glucose homeostasis in mice fed a high-fat diet (21). Furthermore, intracerebroventricular injection of the MCR agonist (MT-II) increased whole-body glucose turnover and expression of GLUT4 in skeletal muscle (22). Ob-Rb in the ARC and the VMH as well as the brain melanocortin pathway are thus implicated in the regulation of glucose uptake in peripheral tissues as well as in energy metabolism. However, little is known about the contributions of the leptin receptor and MCR in individual medial hypothalamic nuclei to regulation of glucose uptake in peripheral tissues, as opposed to their roles in the regulation of food intake and leanness.We have now examined the acute effects of microinjection of leptin and MT-II into the VMH, ARC, DMH, and PVH, all of which express Ob-Rb, MC3R, and MC4R at a high level (3–7,23–25), on glucose uptake in peripheral tissues of mice in vivo. Our results suggest that the VMH mediates stimulatory actions of leptin and MT-II on glucose uptake in skeletal muscle, heart, and BAT, whereas the leptin receptor in the ARC as well as MCRs in PVH regulate glucose uptake in BAT. The medial hypothalamic nuclei thus appear to play distinct roles in the regulation of glucose uptake in peripheral tissues by leptin and MT-II.     

Medium-Chain Fatty Acids Improve Cognitive Function in Intensively Treated Type 1 Diabetic Patients and Support In Vitro Synaptic Transmission During Acute Hypoglycemia

OBJECTIVE

We examined whether ingestion of medium-chain triglycerides could improve cognition during hypoglycemia in subjects with intensively treated type 1 diabetes and assessed potential underlying mechanisms by testing the effect of β-hydroxybutyrate and octanoate on rat hippocampal synaptic transmission during exposure to low glucose.

RESEARCH DESIGN AND METHODS

A total of 11 intensively treated type 1 diabetic subjects participated in stepped hyperinsulinemic- (2 mU · kg⁻¹ · min⁻¹) euglycemic- (glucose ∼5.5 mmol/l) hypoglycemic (glucose ∼2.8 mmol/l) clamp studies. During two separate sessions, they randomly received either medium-chain triglycerides or placebo drinks and performed a battery of cognitive tests. In vitro rat hippocampal slice preparations were used to assess the ability of β-hydroxybutyrate and octanoate to support neuronal activity when glucose levels are reduced.

RESULTS

Hypoglycemia impaired cognitive performance in tests of verbal memory, digit symbol coding, digit span backwards, and map searching. Ingestion of medium-chain triglycerides reversed these effects. Medium-chain triglycerides also produced higher free fatty acids and β-hydroxybutyrate levels compared with placebo. However, the increase in catecholamines and symptoms during hypoglycemia was not altered. In hippocampal slices β-hydroxybutyrate supported synaptic transmission under low-glucose conditions, whereas octanoate could not. Nevertheless, octanoate improved the rate of recovery of synaptic function upon restoration of control glucose concentrations.

CONCLUSIONS

Medium-chain triglyceride ingestion improves cognition without adversely affecting adrenergic or symptomatic responses to hypoglycemia in intensively treated type 1 diabetic subjects. Medium-chain triglycerides offer the therapeutic advantage of preserving brain function under hypoglycemic conditions without causing deleterious hyperglycemia.Maintaining plasma glucose (PG) at near-normal levels in individuals with type 1 diabetes reduces the risk for developing long-term microvascular complications (1). However, intensive insulin therapy increases the risk of severe hypoglycemia, which can cause rapid deterioration of cognitive function and often occurs without warning symptoms (1,2). As a result, hypoglycemia limits the ability of patients to achieve target glycemic goals because the immediate fear of hypoglycemia exceeds the fear of long-term complications. Therefore, new strategies to protect the brain from hypoglycemia-induced injury are essential for optimizing the benefits of insulin therapy.Although the brain relies primarily on glucose, it can use alternative fuels such as monocarboxylic acids, lactate, and ketones to maintain energy homeostasis (3–7). Exposure to prolonged fasting or hypoglycemia causes adaptive changes in the brain, including an enhanced ability to utilize alternative fuels (3,8,9). Thus, patients with intensively managed type 1 diabetes, by virtue of their increased exposure to hypoglycemia, may develop an enhanced ability to use alternate fuels, which, in turn, might provide neuroprotection during hypoglycemia.Medium-chain triglycerides, constituents of coconut and palm kernel oils, are medium-chain fatty acid esters of glycerol. Medium-chain triglycerides have a favorable safety profile and are used to treat a variety of disorders (10–12). They offer a readily available noncarbohydrate fuel source because they are rapidly absorbed and quickly metabolized into medium-chain fatty acids (10). Medium-chain fatty acids do not require chylomicrons for transport or carnitine for entry into mitochondria (10). As a result, metabolism of medium-chain fatty acids promotes the generation of ketones (10). Furthermore, animal data suggest that medium-chain fatty acids can readily cross the blood-brain barrier (BBB) and be oxidized by the brain (13). Thus, medium-chain fatty acids may provide both a direct and an indirect brain fuel source via the generation of ketones, offering type 1 diabetic patients a prophylactic treatment strategy to preserve brain function during hypoglycemic episodes without raising blood glucose levels.To explore this possibility, we evaluated whether oral medium-chain triglycerides could improve cognitive performance during acute insulin-induced hypoglycemia in intensively treated type 1 diabetic subjects. In addition, an in vitro hippocampal slice preparation from nondiabetic rats was used to assess the ability of β-hydroxybutyrate and octanoate to support neuronal activity when the glucose supply is deficient.     

Increased Brain Lactate Concentrations Without Increased Lactate Oxidation During Hypoglycemia in Type 1 Diabetic Individuals

Previous studies have reported that brain metabolism of acetate is increased more than twofold during hypoglycemia in type 1 diabetic (T1D) subjects with hypoglycemia unawareness. These data support the hypothesis that upregulation of blood-brain barrier monocarboxylic acid (MCA) transport may contribute to the maintenance of brain energetics during hypoglycemia in subjects with hypoglycemia unawareness. Plasma lactate concentrations are ∼10-fold higher than acetate concentrations, making lactate the most likely alternative MCA as brain fuel. We therefore examined transport of [3-¹³C]lactate across the blood-brain barrier and its metabolism in the brains of T1D patients and nondiabetic control subjects during a hypoglycemic clamp using ¹³C magnetic resonance spectroscopy. Brain lactate concentrations were more than fivefold higher (P < 0.05) during hypoglycemia in the T1D subjects compared with the control subjects. Surprisingly, we observed no increase in the oxidation of blood-borne lactate in the T1D subjects, as reflected by similar ¹³C fractional enrichments in brain glutamate and glutamine. Taken together, these data suggest that in addition to increased MCA transport at the blood-brain barrier, there may be additional metabolic adaptations that contribute to hypoglycemia unawareness in patients with T1D.Despite the increased availability of improved methods for managing glycemic control (i.e., continuous glucose monitoring), failing counterregulation and hypoglycemia unawareness still present a real burden in the daily life of type 1 diabetic (T1D) and advanced (insulin-deficient) type 2 diabetic patients (1,2). Recurrent episodes of hypoglycemia are considered to induce both the failure in counterregulatory hormone release and hypoglycemia unawareness, a concept known as hypoglycemia-associated autonomic failure (3,4).Although the exact mechanisms of hypoglycemia unawareness are still unknown, studies have predominantly focused on adaptations related to nutrient transport into the brain and changes in brain energy metabolism. For example, changes in the transport of plasma glucose across the blood-brain barrier and consequently the brain glucose levels have been the topic of various studies (5–10). Other studies have focused on glycogen supercompensation, a hypothesis suggesting increased storage of glucose in astroglial glycogen after recurrent hypoglycemic events (11–13). The increased astroglial glycogen would function as a glucose reserve during hypoglycemia. However, during a 50-h wash-in and wash-out study of [1-¹³C]glucose, control subjects showed higher levels of newly synthesized brain glycogen than hypoglycemia-unaware T1D subjects (11). Öz et al. (11) consequently concluded that glycogen supercompensation did not contribute to hypoglycemia unawareness in T1D patients.Previously we have reported that brain transport and metabolism of acetate is increased more than twofold in intensively treated T1D subjects with hypoglycemia unawareness (14). These data support the hypothesis that upregulation of blood-brain barrier monocarboxylic acid (MCA) transport via MCA transporter 1 (15,16) may be a hallmark of hypoglycemia unawareness in T1D patients. In contrast to acetate, which circulates in plasma at relatively low concentrations (∼0.1 mmol/L), plasma lactate concentrations are ∼10-fold higher during hypoglycemia (17), making it a primary candidate for an alternative brain fuel (18–21).Lactate metabolism can play a central role in neuroenergetics, as suggested by the astrocyte-neuron lactate shuttle (22). The astrocyte-neuron lactate shuttle models the compartmentalized metabolism of glucose in astrocytes and neurons. It describes how glucose is metabolized through glycolysis in astrocytes, producing lactate. Lactate is then shuttled to neighboring neurons where it is oxidized. The astrocyte-neuron lactate shuttle is analogous to the intercellular lactate shuttle that was proposed earlier and describes skeletal muscle lactate metabolism (23).We have shown in healthy subjects that there is sufficient lactate transport activity to supply ∼10% of the brain’s energy needs at physiological lactate concentrations (24). Increased blood-brain barrier transport capacity of MCAs, and thus lactate, could contribute to the maintenance of brain energetics during hypoglycemia, providing the brain with an increased influx of alternative substrates (14). However, to our knowledge, there is no direct evidence of increased brain transport and oxidation of plasma lactate in T1D patients. We therefore examined transport of lactate over the blood-brain barrier and its metabolic fate in healthy T1D patients and nondiabetic control subjects during a hypoglycemic clamp by measuring ¹³C label incorporation from intravenously administered [3-¹³C]lactate into brain lactate, glutamate (Glu), and glutamine (Gln) by ¹³C magnetic resonance spectroscopy (MRS).     

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 (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 α-cell glucagon release is in part mediated at the level of the VMH under both normoglycemic and hypoglycemic conditions.The pancreatic β-cell is the primary regulator of glucose homeostasis and glucagon secretion (1,2). In the presence of changes in blood glucose concentration, local changes in insulin together with zinc and/or γ-aminobutyric acid (GABA) release from the β-cell are thought to act in concert to regulate α-cell glucagon secretion (3,4). However, during hypoglycemia the central nervous system (5,6), and the VMH in particular, also plays a critical role in stimulating the release of glucagon and other counterregulatory hormones, via changes in the activity of specialized glucose-sensing neurons (7–10). Moreover, mice with selective loss of glutamate release in steroidogenic factor 1 neurons, which are expressed exclusively in the VMH, show a markedly impaired glucagon secretory response to acute hypoglycemia (11). These and other studies suggest that central regulation of pancreatic α-cell glucagon secretion may play a greater role than previously anticipated, at least during insulin-induced hypoglycemia (2,6–8).The fact that the VMH may directly influence α-cell glucagon release, and that glucose-sensing neurons in the VMH appear to use similar mechanisms for detecting fluctuations in glucose to the pancreatic α- and β-cells (7,8), suggested that insulin might play a similar regulatory role through a central mechanism. The possibility that insulin might exert a central effect is consistent with evidence that brain insulin levels mirror those in the circulation (12), that glucose-sensing neurons express insulin receptors, and that insulin alters the firing rate of these neurons (8,13–15). Moreover, insulin has been reported to act on the hypothalamus to regulate hepatic glucose production (16). However, studies in humans evaluating whether the level of circulating insulin during exogenous insulin administration influences counterregulatory hormone responses have yielded conflicting results. It has been reported that higher levels of plasma insulin may amplify (17,18) or inhibit (19–21) epinephrine responses during hypoglycemia. One study noted a significant effect of circulating insulin on accompanying glucagon responses, specifically an inhibitory action (20). Whether insulin acts centrally to influence glucose counterregulation is also uncertain. In animal studies, insulin delivered directly into the brain in very large doses has been reported to amplify counterregulatory hormone secretion (22–24) or to have no direct effect (25).This study was undertaken to determine whether insulin acts within the ventromedial hypothalamus (VMH) to modulate the secretion of glucoregulatory hormones. For this purpose, we either blocked insulin action or administered insulin to the VMH, during hypoglycemia produced in the presence or absence of elevated circulating insulin levels, respectively. In addition, we examined whether insulin acts locally in the VMH under basal conditions to regulate glucoregulatory hormone secretion in the absence of elevated levels of insulin. Our findings provide in vivo evidence for the first time that insulin acts within the VMH to influence the release of glucagon from α-cells both during hypoglycemia and under basal conditions.     

SH2B1 Enhances Insulin Sensitivity by Both Stimulating the Insulin Receptor and Inhibiting Tyrosine Dephosphorylation of Insulin Receptor Substrate Proteins

OBJECTIVE

SH2B1 is a SH2 domain-containing adaptor protein expressed in both the central nervous system and peripheral tissues. Neuronal SH2B1 controls body weight; however, the functions of peripheral SH2B1 remain unknown. Here, we studied peripheral SH2B1 regulation of insulin sensitivity and glucose metabolism.

RESEARCH DESIGN AND METHODS

We generated TgKO mice expressing SH2B1 in the brain but not peripheral tissues. Various metabolic parameters and insulin signaling were examined in TgKO mice fed a high-fat diet (HFD). The effect of SH2B1 on the insulin receptor catalytic activity and insulin receptor substrate (IRS)-1/IRS-2 dephosphorylation was examined using in vitro kinase assays and in vitro dephosphorylation assays, respectively. SH2B1 was coexpressed with PTP1B, and insulin receptor–mediated phosphorylation of IRS-1 was examined.

RESULTS

Deletion of peripheral SH2B1 markedly exacerbated HFD-induced hyperglycemia, hyperinsulinemia, and glucose intolerance in TgKO mice. Insulin signaling was dramatically impaired in muscle, liver, and adipose tissue in TgKO mice. Deletion of SH2B1 impaired insulin signaling in primary hepatocytes, whereas SH2B1 overexpression stimulated insulin receptor autophosphorylation and tyrosine phosphorylation of IRSs. Purified SH2B1 stimulated insulin receptor catalytic activity in vitro. The SH2 domain of SH2B1 was both required and sufficient to promote insulin receptor activation. Insulin stimulated the binding of SH2B1 to IRS-1 or IRS-2. This physical interaction inhibited tyrosine dephosphorylation of IRS-1 or IRS-2 and increased the ability of IRS proteins to activate the phosphatidylinositol 3-kinase pathway.

CONCLUSIONS

SH2B1 is an endogenous insulin sensitizer. It directly binds to insulin receptors, IRS-1 and IRS-2, and enhances insulin sensitivity by promoting insulin receptor catalytic activity and by inhibiting tyrosine dephosphorylation of IRS proteins.Insulin decreases blood glucose both by promoting glucose uptake into skeletal muscle and adipose tissue and by suppressing hepatic glucose production. In type 2 diabetes, the ability of insulin to reduce blood glucose is impaired (insulin resistance) because of a combination of genetic and environmental factors, resulting in hyperglycemia. Insulin resistance is not only the hallmark but also a determinant of type 2 diabetes.Insulin binds to and activates the insulin receptor. Insulin receptor tyrosyl phosphorylates insulin receptor substrates (IRS-1, -2, -3, and -4). IRS proteins, particularly IRS-1 and IRS-2, initiate and coordinate multiple downstream pathways, including the phosphatidylinositol 3-kinase/Akt pathway (1). Genetic deletion of IRS-1, IRS-2, or Akt2 causes insulin resistance in mice, indicating that the IRS protein/phosphatidylinositol 3-kinase/Akt2 pathway is required for regulation of glucose homeostasis by insulin (2–5). Insulin receptor and IRS proteins are negatively regulated by various intracellular molecules, including PTP1B, Grb10, Grb14, SOCS1, SOCS3, JNK, PKCθ, S6K, and IKKβ (6–23). The relative contribution of these negative regulators to the progression of insulin resistance has been extensively studied (6–24). However, insulin signaling is likely to also be modulated by positive regulators. In this study, we demonstrate that SH2B1 is a novel endogenous insulin sensitizer.SH2B1 is a member of the SH2B family of adapter proteins that also includes SH2B2 (APS) and SH2B3 (Lnk). SH2B1 and SH2B2 are expressed in multiple tissues, including insulin target tissues (e.g., skeletal muscle, adipose tissue, liver, and the brain); by contrast, SH2B3 expression is restricted to hematopoietic tissue (25,26). Structurally, SH2B family members have an NH₂-terminal dimerization domain, a central pleckstrin homology domain, and a COOH-terminal Src homology 2 (SH2) domain. The dimerization domain mediates homodimerization or heterodimerization between different SH2B proteins (27). SH2B1 and SH2B2 bind via their SH2 domains to a variety of tyrosine phosphorylated proteins, including JAK2 and insulin receptor, in cultured cells (28). Genetic deletion of SH2B1 results in marked leptin resistance, obesity, insulin resistance, and type 2 diabetes in mice, demonstrating that SH2B1 is required for the maintenance of normal body weight, insulin sensitivity, and glucose metabolism (29–32). Surprisingly, SH2B2-null mice have normal body weight and slightly improved insulin sensitivity (32,33), suggesting that SH2B1 and SH2B2 have distinct functions in vivo. However, it remains unclear whether SH2B1 cell autonomously regulates insulin sensitivity in peripheral insulin target tissues because systemic deletion of SH2B1 causes obesity, which may cause insulin resistance in SH2B1-null mice.We generated a mouse model in which recombinant SH2B1 is specifically expressed in the brain of SH2B1-null mice (TgKO) using transgenic approaches (31). Neuron-specific restoration of SH2B1 corrects both leptin resistance and obesity, suggesting that neuronal SH2B1 regulates energy balance and body weight by enhancing leptin sensitivity (31). Consistent with these conclusions, polymorphisms in the SH2B1 loci are linked to leptin resistance and obesity in humans (34–36). In this work, we demonstrate that deletion of SH2B1 in peripheral tissues impairs insulin sensitivity independent of obesity in TgKO mice. Moreover, we demonstrate that SH2B1 directly promotes insulin responses by stimulating insulin receptor catalytic activity and by protecting IRS proteins from tyrosine dephosphorylation.     

Gliotransmission and Brain Glucose Sensing: Critical Role of Endozepines

Hypothalamic glucose sensing is involved in the control of feeding behavior and peripheral glucose homeostasis, and glial cells are suggested to play an important role in this process. Diazepam-binding inhibitor (DBI) and its processing product the octadecaneuropeptide (ODN), collectively named endozepines, are secreted by astroglia, and ODN is a potent anorexigenic factor. Therefore, we investigated the involvement of endozepines in brain glucose sensing. First, we showed that intracerebroventricular administration of glucose in rats increases DBI expression in hypothalamic glial-like tanycytes. We then demonstrated that glucose stimulates endozepine secretion from hypothalamic explants. Feeding experiments indicate that the anorexigenic effect of central administration of glucose was blunted by coinjection of an ODN antagonist. Conversely, the hyperphagic response elicited by central glucoprivation was suppressed by an ODN agonist. The anorexigenic effects of centrally injected glucose or ODN agonist were suppressed by blockade of the melanocortin-3/4 receptors, suggesting that glucose sensing involves endozepinergic control of the melanocortin pathway. Finally, we found that brain endozepines modulate blood glucose levels, suggesting their involvement in a feedback loop controlling whole-body glucose homeostasis. Collectively, these data indicate that endozepines are a critical relay in brain glucose sensing and potentially new targets in treatment of metabolic disorders.To regulate energy homeostasis, the brain integrates peripheral signals delivered by the blood, including metabolites and hormones, and generates appropriate responses by modulating food intake and peripheral organ activity (1). The arcuate nucleus of the hypothalamus is a major site for integration of energy status. It possesses two interconnected populations of neurons, one producing the orexigenic neuropeptide Y (NPY), and the other one producing the anorexigenic peptide α-melanocyte–stimulating hormone (α-MSH), a processing product of proopiomelanocortin (POMC) (2,3). Moreover, agouti-related protein (AgRP), a potent orexigenic peptide, is coexpressed with NPY in most NPYergic arcuate neurons and acts as an endogenous antagonist of the melanocortin (MC) receptors (2).Direct glucose sensing by the central nervous system has been extensively demonstrated. It is noteworthy that central administration of glucose reduces NPY expression, increases POMC expression, and markedly reduces feeding (4). Conversely, central glucoprivation using 2-deoxyglucose (2-DG) elicits food intake and activates neurohumoral counterregulatory responses similar to those observed during systemic hypoglycemia (4–7). Importantly, alteration in brain glucose sensing is associated with obesity and diabetes (8,9). The cellular mechanisms underlying central glucose sensing are far from being understood. By analogy to pancreatic β-cells, it may involve GLUT2 and glucokinase (10–12).Several studies suggest that astroglial cells play an important role in glucose sensing. First, tanycytes, specialized ependymal cells located in the floor of the third ventricle, were found to be glucose-sensitive elements, and stimulation of their cell bodies by glucose evokes calcium waves (13,14). Second, selective destruction of tanycytes impairs feeding and hyperglycemia responses induced by 2-DG (15). Third, genetic inactivation of GLUT2 impairs glucagon secretion induced by hypoglycemia, and re-expression of GLUT2 in glia restores this response (5). Fourth, selective stimulation of glucose metabolism in hypothalamic glia by overexpression of GLUT1 normalizes plasma glucose levels in diabetic rats (16). Together, these studies strengthen the emerging concept that glial cells can detect changes in nutrient availability and interact with neurons to regulate energy homeostasis.Diazepam-binding inhibitor (DBI) and its peptide fragments, including the octadecaneuropeptide (ODN), which are known to bind benzodiazepine receptors, are collectively termed endozepines (17). These peptides are specifically produced by glial cells in the central nervous system, and hypothalamic astrocytes and tanycytes express high levels of endozepines (18,19). Numerous data indicate that endozepines are secreted from astroglial cells and, in line with well-characterized gliotransmitters, this process is regulated by physiological stimuli (20–22). A role for endozepines in the control of energy homeostasis has been demonstrated by central administration of ODN, or its COOH-terminal octapeptide (OP) fragment, which markedly inhibits food intake and reduces body weight in rodents (23,24). Pharmacological experiments revealed that the anorexigenic effects of ODN and OP are mediated through activation of a metabotropic receptor distinct from benzodiazepine receptors (24). Moreover, the intracerebroventricular injection of ODN increases POMC mRNA levels and decreases NPY mRNA levels in the arcuate nucleus (25). Finally, acute food deprivation markedly reduces hypothalamic DBI mRNA levels in mice, indicating that endozepine expression correlates with energy status (18). Altogether, these data led us to hypothesize that endozepines may be released as a function of glucose status and act as a relay in brain glucose sensing.