Objective

A number of recent studies suggest that leptin has effects on glucose metabolism and pancreatic hormone secretion. Therefore, the effect of leptin administration on circulating glucose, insulin and glucagon in fed and fasted mice was investigated. The potential contribution of the sympathetic nervous system to the effects of leptin was also examined.

Design

Recombinant human or murine leptin was administered intraperitoneally (300 microg/mouse per 12 h over 24 h) to fed or fasted, normal or chemically sympathectomized NMRI mice. Blood samples were collected at baseline and after 24 h.

Measurements

Plasma concentrations of glucose, insulin and glucagon.

Results

In the fed state (n = 24), leptin administration did not affect glucose, insulin or glucagon concentrations after 24 h. Fasting (n = 24) reduced body weight by 2.2+/-0.4 g, plasma glucose by 3.7+/-0.4 mmol/l, plasma insulin by 138+/-35 pmol/l, and plasma glucagon by 32+/-7 pg/ml. In fasted mice, human leptin (n = 24) increased plasma glucose by 1.5+/-0.2 mmol/l (P = 0.041), plasma insulin by 95+/-22 pmol/l (P = 0.018), and plasma glucagon by 16+/-3 pg/ml (P = 0.025), relative to saline-injected control animals. Murine leptin exerted similar stimulating effects on circulating glucose (+1.0+/-0.2 mmol/l, P = 0.046), insulin (+58+/-17 pmol/l, P = 0.038) and glucagon (+24+/-9 pg/ml, P = 0.018) as human leptin in fasted mice (n = 12) with no significant effect in fed mice (n = 12). Human leptin did not affect circulating glucose, insulin or glucagon in fasted mice after chemical sympathectomy with 6-hydroxydopamine (40 mg/kg iv 48 h prior to fasting; n = 12).

Conclusion

Leptin increases circulating glucose, insulin and glucagon in 24 h fasted mice by a mechanism requiring intact sympathetic nerves.

Objective

The pharmacokinetic characteristics of human leptin were examined in rhesus monkeys and in C57BL/6J mice fed a normal chow or a high-fat diet.

Design

For the monkey study, in nine rhesus monkeys (body weight 12.4 +/- 2.4 kg; mean +/- s.d.), recombinant met-human leptin was injected intravenously or subcutaneously (1 mg/kg). For the mouse study, after 6 months of feeding C57BL/6J mice a high-fat diet (body weight 32.9 +/- 3.6 g; n = 8) or a control diet (24.5 +/- 1.2 g; n = 6), recombinant met-human leptin was administered intraperitoneally (10 microg/g). Blood samples were collected for leptin measurement at specific time points after leptin administration.

Measurements

Plasma leptin concentrations were determined by radioimmunoassay and pharmacokinetic analysis was performed.

Results

Disposition of human leptin in rhesus monkeys was biphasic following intravenous administration, with a terminal phase half-life of 96.4 +/- 16.5 min and clearance of 1.8 +/- 0.2 ml/min/kg. Subcutaneously administered leptin was absorbed slowly, perhaps by a zero-order process as leptin levels appeared to plateau and remained elevated throughout the 8 h sampling period. In C57BL/6J mice, the absorption and elimination of human leptin were both first-order following intraperitoneal administration. Pharmacokinetic parameters did not differ between normal-weight mice fed a chow diet and obese mice fed a high-fat diet. The elimination half-life was 47.0 +/- 26.4 min in mice fed a high-fat diet and 49.5 +/- 12.0 min in mice fed a control diet.

Conclusion

The kinetics of leptin in rhesus monkeys were biphasic and clearance was similar to values previously reported in humans. The estimated half-life was 96.4 min in rhesus monkeys and 49.5 min in normal weight mice. The was no difference in leptin kinetics between high-fat fed and control mice, suggesting that the increased baseline leptin levels in the obese mice are due to increased leptin production and secretion.

This article examines the role of the autonomic nervous system in mediating the increase of glucagon secretion observed during insulin-induced hypoglycemia (IIH). In the first section, we briefly review the importance of the alpha-cell response in recovery from hypoglycemia under both physiologic conditions and pathophysiologic conditions, such as type 1 diabetes. We outline three possible mechanisms that may contribute to increased glucagon secretion during hypoglycemia but emphasize autonomic mediation. In the second section, we review the critical experimental data in animals, nonhuman primates, and humans suggesting that, in the absence of diabetes, the majority of the glucagon response to IIH is mediated by redundant autonomic stimulation of the islet alpha-cell. Because the glucagon response to hypoglycemia is often impaired in patients with type 1 diabetes, in the third section, we examine the possibility that autonomic impairment contributes to the impairment of the glucagon response in these patients. We review two different types of autonomic impairment. The first is a slow-onset and progressive neuropathy that worsens with duration of diabetes, and the second is a rapid-onset, but reversible, autonomic dysfunction that is acutely induced by antecedent hypoglycemia. We propose that both types of autonomic dysfunction can contribute to the impaired glucagon responses in patients with type 1 diabetes. In the fourth section, we relate restoration of these glucagon responses to restoration of the autonomic responses to hypoglycemia. Finally, in the fifth section, we summarize the concepts underlying the autonomic hypothesis, the evidence for it, and the implications of the autonomic hypothesis for the treatment of type 1 diabetes.

Acylation stimulating protein (ASP) is a hormone produced by adipocytes and is of importance for the storage of energy as fat. We examined whether ASP might also have effects on islet function. In clonal INS-1 cells, ASP dose-dependently augmented glucose-stimulated insulin secretion. The lowest effective dose of ASP at 10 mmol/l glucose was 5 micro mol/l. The effect was glucose-dependent because ASP did not increase insulin secretion at 1 mmol/l glucose but had clear effect at 10 and 20 mmol/l glucose. Similarly, ASP augmented glyceraldehyde-induced insulin secretion but the hormone did not enhance insulin secretion in response to depolarization by 20 mmol/l of KCl. ASP-induced insulin secretion was completely abolished by competitive inhibition of glucose phosphorylation by glucokinase with 5-thio-glucose and was partially inhibited by the calcium channel blocker, nifedipine, and by the protein kinase C inhibitor, GF109203. Furthermore, thapsigargin, an inhibitor of Ca(2+)-ATPase in the endoplasmic reticulum, did not affect ASP-induced insulin secretion. ASP (>5 micro mol/l) also augmented glucose-stimulated insulin secretion from islets isolated from C57BL/6J mice, and intravenous administration of ASP (50 nmol/kg) augmented the acute (1 and 5 min) insulin response to intravenous glucose (1 g/kg) in C57BL/6J mice. This was accompanied by an increased rate of glucose disposal. Minimal model analyses of data derived from the intravenous glucose tolerance test revealed that whereas ASP augmented insulin secretion, the hormone did not affect insulin sensitivity (S(I)) or glucose effectiveness (S(G)). We conclude that ASP augments glucose-stimulated insulin secretion through a direct action on the islet beta cells. The effect is dependent on glucose phosphorylation, calcium uptake and protein kinase C. Stimulation of insulin secretion by ASP in vivo results in augmented glucose disposal.

Glucagon response to insulin-induced hypoglycaemia is impared in diabetes, but the mechanism is not established. Pancreatic A cell hyporesponsiveness to adrenergic or cholinergic stimulation could contribute to the impairment. We therefore compared the plasma glucagon responses to intravenous infusion of adrenaline (1200 ng kg(-1) min(-1) for 20 min) or to intravenous injection of the cholinergic agonist carbachol (50 micrograms kg(-1)) in chloral hydrate-anaesthetized rats made diabetic with the use of streptozotocin (80 mg kg(-1) subcutaneously) 6 weeks before and in anaesthetized control rats. Insulin was infused intravenously to reduce plasma glucose levels to below 1.8 mmol L(-1). As expected, the plasma glucagon response was reduced by approximately 45% in streptozotocin-diabetic rats compared with controls (P = 0.045). During adrenaline infusion, plasma glucagon levels increased by 277 +/- 92 pg mL(-1) in controls (P = 0.009) and by 570 +/- 137 pg mL(-1) in the diabetic rats (P = 0.002). Thus, the plasma glucagon response to adrenaline was approximately doubled in the diabetic rats (P = 0.045). Following carbachol injection, plasma glucagon levels were raised by 1211 +/- 208 pg mL(-1) (P < 0.001) in controls but only by 555 +/- 242 pg mL(-1) in the diabetic rats (P = 0.049). Thus, the plasma glucagon response to carbachol was impared by approximately 58% in the diabetic rats (P = 0.028). We conclude that carbachol-stimulated glucagon secretion is impared concomitantly with the impared glucagon response to hypoglycaemia in streptozotocin-diabetic rats, whereas adrenaline-induced glucagon secretion is exaggerated. We suggest that a reduced pancreatic A cell responsiveness to cholinergic stimulation could contribute to the impairment of the glucagon response to insulin-induced hypoglycaemia in diabetes.

In summary, this article briefly reviews the evidence that three separate autonomic inputs to the islet are capable of stimulating glucagon secretion and that each is activated during IIH. We have reviewed our evidence that these autonomic inputs mediate the glucagon response to IIH, both in non-diabetic animals and humans. Finally, we outline our new preliminary data suggesting an eSIN in an autoimmune animal model of T1DM. We conclude that the glucagon response to IIH is autonomically mediated in non-diabetic animals and humans. We further suggest that at least one of these autonomic inputs, the sympathetic innervation of the islet, is diminished in autoimmune T1DM. These data raise the novel possibility that an autonomic defect contributes to the loss of the glucagon response to IIH in T1DM.

Mechanisms regulating circulating leptin are incompletely understood. We developed a radioimmunoassay for mouse leptin to examine the influence of age, dietary fat content, and fasting on plasma concentrations of leptin in the background strain for the ob/ob mouse, the C57BL/6J mouse. Plasma leptin increased with age [5.3 +/- 0.6 ng/ml at 2 mo (n = 23) vs. 14.2 +/- 1.6 ng/ml at 11 mo (n = 15), P < 0.001]. Across all age groups (2-11 mo, n = 160), log plasma leptin correlated with body weight (r = 0.68, P < 0.0001), plasma insulin (r = 0.38, P < 0.001), and amount of intra-abdominal fat (r = 0.90, P < 0.001), as revealed by magnetic resonance imaging. Plasma leptin was increased by a high-fat diet (58% fat for 10 mo) and reduced by fasting for 48 h. The reduction of plasma leptin was correlated with the reduction of plasma insulin (r = 0.43, P = 0.012) but not with the initial body weight or the change in body weight. Moreover, the reduction in plasma leptin by fasting was impaired by high-fat diet. Thus plasma leptin in C57BL/6J mice 1) increases with age or a high-fat diet; 2) correlates with body weight, fat content, and plasma insulin; and 3) is reduced during fasting by an action inhibited by high-fat diet and related to changes of plasma insulin.

Neural control of pancreatic polypeptide (PP) release has not been previously investigated in the mouse. In addition, it is not known to what extent increased glucagon secretion during hypoglycemia in mice is neurally mediated vs. an effect of hypoglycemia to directly stimulate glucagon secretion at the level of the islet. Feeding or the cholinergic agonist carbachol increased plasma PP levels in conscious mice (+74 +/- 18 pg/ml vs. fasted mice and +141 +/- 17 pg/ml vs. control, respectively). Neuroglucopenia induced by 2-deoxy-D-glucose or insulin-induced hypoglycemia also increased plasma PP (+79 +/- 18 and +89 +/- 11 pg/ml vs. control, respectively). These increases were abolished by hexamethonium and reduced by atropine methylnitrate (atropine). Hypoglycemia-induced hyperglucagonemia (+1,243 +/- 275 pg/ml) was reduced to 31 +/- 7% of control by atropine (+382 +/- 85 pg/ml), to 48 +/- 9% of control by combined adrenergic blockade (+601 +/- 112 pg/ml), and nearly abolished by atropine plus combined blockade (+143 +/- 41 pg/ml; 11 +/- 3% of control) or hexamethonium (+151 +/- 38 pg/ml; 12 +/- 3% of control). We conclude the following in the mouse. 1) Feeding or cholinergic agonists increase plasma PP. 2) During neuroglucopenia or hypoglycemia, plasma PP is increased via nicotinic and muscarinic mechanisms. 3) The glucagon response to hypoglycemia is predominantly the result of autonomic activation and is mediated by both muscarinic and adrenergic mechanisms.

Our laboratory has investigated the role of the neuropeptide galanin in the sympathetic neural control of both the canine endocrine pancreas and liver. Galanin mRNA and peptide were found in the neuronal cell bodies of the celiac ganglion, which projects fibers to both organs. Galanin fibers formed dense networks around the islets. Galanin was released from these nerves and the amount released appeared sufficient to markedly inhibit basal insulin secretion. We therefore propose that galanin is a sympathetic neurotransmitter in canine endocrine pancreas. Galanin was also found in hepatic nerves usually co-localized with tyrosine hydroxylase, a sympathetic marker. Further, intraportal administration of the sympathetic neurotoxin, 6-hydroxydopamine, abolished galanin staining in the hepatic parenchyma. We evaluated the role of galanin in mediating the actions of sympathetic nerves to increase hepatic glucose production and decrease hepatic arterial conductance. Local infusion of synthetic galanin had little effect by itself, but it did potentiate the action of norepinephrine to stimulate hepatic glucose production, demonstrating a neuromodulatory action. In contrast, galanin had no effect on hepatic arterial blood flow. We therefore propose that in the liver galanin functions as a neuromodulator of norepinephrine's metabolic action.