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

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

Peroral gene therapy of lactose intolerance using an adeno-associated virus vector.

Gene therapy is usually reserved for severe and medically refractory disorders because of the toxicity, potential long-term risks and invasiveness of most gene transfer protocols. Here we show that an orally administered adeno-associated viral vector leads to persistent expression of a beta-galactosidase transgene in both gut epithelial and lamina propria cells, and that this approach results in long-term phenotypic recovery in an animal model of lactose intolerance. A gene 'pill' associated with highly efficient and stable gene expression might be a practical and cost-effective strategy for even relatively mild disorders, such as lactase deficiency.

Control of autoimmune diabetes in NOD mice by GAD expression or suppression in beta cells.

Glutamic acid decarboxylase (GAD) is a pancreatic beta cell autoantigen in humans and nonobese diabetic (NOD) mice. beta Cell-specific suppression of GAD expression in two lines of antisense GAD transgenic NOD mice prevented autoimmune diabetes, whereas persistent GAD expression in the beta cells in the other four lines of antisense GAD transgenic NOD mice resulted in diabetes, similar to that seen in transgene-negative NOD mice. Complete suppression of beta cell GAD expression blocked the generation of diabetogenic T cells and protected islet grafts from autoimmune injury. Thus, beta cell-specific GAD expression is required for the development of autoimmune diabetes in NOD mice, and modulation of GAD might, therefore, have therapeutic value in type 1 diabetes.

Effect of starvation on the turnover and metabolic response to leucine.

UNLABELLED: l-Leucine was administered as a primed continuous 3-4-h infusion in nonobese and obese subjects in the postabsorptive state and for 12 h in obese subjects after a 3-day and 4-wk fast. In nonobese and obese subjects studied in the post-absorptive state, the leucine infusion resulted in a 150-200% rise in plasma leucine above preinfusion levels, a small decrease in plasma glucose, and unchanged levels of plasma insulin and glucagon and blood ketones. Plasma isoleucine (60-70%) and valine (35-40%) declined to a greater extent than other amino acids (P < 0.001). After 3 days and 4 wk of fasting, equimolar infusions of leucine resulted in two- to threefold greater increments in plasma leucine as compared to post-absorptive subjects, a 30-40% decline in other plasma amino acids, and a 25-30% decrease in negative nitrogen balance. Urinary excretion of 3-methylhistidine was however, unchanged. Plasma glucose which declined in 3-day fasted subjects after leucine administration, surprisingly rose by 20 mg/100 ml after 4 wk of fasting. The rise in blood glucose occurred in the absence of changes in plasma glucagon and insulin and in the face of a 15% decline in endogenous glucose production (as measured by infusion of [3-(3)H]glucose). On the other hand, fractional glucose utilization fell by 30% (P < 0.001), thereby accounting for hyperglycemia. The estimated metabolic clearance rate of leucine fell by 48% after 3 days of fasting whereas the plasma delivery rate of leucine was unchanged, thereby accounting for a 40% rise in plasma leucine during early starvation. After a 4-wk fast, the estimated metabolic clearance rate of leucine declined further to 59% below base line. Plasma leucine nevertheless fell to postabsorptive levels as the plasma delivery rate of leucine decreased 65% below postabsorptive values. CONCLUSIONS: (a) Infusion of exogenous leucine in prolonged fasting results in a decline in plasma levels of other amino acids, improvement in nitrogen balance and unchanged excretion of 3-methylhistidine, thus suggesting stimulation of muscle protein synthesis, (b) leucine infusion also reduces glucose production and to an even greater extent, glucose consumption, thereby raising blood glucose concentration; and (c) the rise in plasma leucine in early starvation results primarily from a decrease in leucine clearance which drops progressively during starvation.

Synergistic interactions of physiologic increments of glucagon, epinephrine, and cortisol in the dog: a model for stress-induced hyperglycemia.

To evaluate the role of anti-insulin hormone actions and interactions in the pathogenesis of stress-induced hyperglycemia, the counterregulatory hormones, glucagon, epinephrine, and cortisol were infused alone as well as in double and triple combinations into normal conscious dogs in doses that were designed to simulate changes observed in severe stress. Infusion of glucagon, epinephrine, or cortisol alone produced only mild or insignificant elevations in plasma glucose concentration. In contrast, the rise in plasma glucose produced by combined infusion of any two counterregulatory hormones was 50-215% greater (P < 0.005-0.001) than the sum of the respective individual infusions. Furthermore, when all three hormones were infused simultaneously, the increment in plasma glucose concentration (144+/-2 mg/dl) was two- to fourfold greater than the sum of the responses to the individual hormone infusions or the sum of any combination of double plus single hormone infusion (P < 0.001). Infusion of glucagon or epinephrine alone resulted in a transient rise in glucose production (as measured by [3-(3)H]glucose). While glucagon infusion was accompanied by a rise in glucose clearance, with epinephrine there was a sustained, 20% fall in glucose clearance. When epinephrine was infused together with glucagon, the rise in glucose production was additive, albeit transient. However, the inhibitory effect of epinephrine on glucose clearance predominated, thereby accounting for the exaggerated glycemic response to combined infusion of glucagon and epinephrine. Although infusion of cortisol alone had no effect on glucose production, the addition of cortisol markedly accentuated hyperglycemia produced by glucagon and(or) epinephrine primarily by sustaining the increases in glucose production produced by these hormones. The combined hormonal infusions had no effect on beta-hydroxybutyrate concentration. It is concluded that (a) physiologic increments in glucagon, epinephrine, and cortisol interact synergistically in the normal dog so as to rapidly produce marked fasting hyperglycemia; (b) in this interaction, epinephrine enhances glucagon-stimulated glucose output and interferes with glucose uptake while cortisol sustains elevations in glucose production produced by epinephrine and glucagon; and (c) these data indicate that changes in glucose metabolism in circumstances in which several counterregulatory hormones are elevated (e.g., "stress hyperglycemia") are a consequence of synergistic interactions among these hormones.

Impaired hormonal responses to hypoglycemia in spontaneously diabetic and recurrently hypoglycemic rats. Reversibility and stimulus specificity of the deficits.

To evaluate the roles of iatrogenic hypoglycemia and diabetes per se in the pathogenesis of defective hormonal counterregulation against hypoglycemia in insulin-dependent diabetes mellitus (IDDM), nondiabetic, and spontaneously diabetic BB/Wor rats were studied using a euglycemic/hypoglycemic clamp. In nondiabetic rats, recurrent (4 wk) insulin-induced hypoglycemia (mean daily glucose, MDG, 59 mg/dl) dramatically reduced glucagon and epinephrine responses by 84 and 94%, respectively, to a standardized glucose fall from 110 to 50 mg/dl. These deficits persisted for > 4 d after restoring normoglycemia, and were specific for hypoglycemia, with normal glucagon and epinephrine responses to arginine and hypovolemia, respectively. After 4 wk of normoglycemia, hormonal counterregulation increased, with the epinephrine, but not the glucagon response reaching control values. In diabetic BB rats (MDG 245 mg/dl with intermittent hypoglycemia), glucagon and epinephrine counterregulation were reduced by 86 and 90%, respectively. Chronic iatrogenic hypoglycemia (MDG 52 mg/dl) further suppressed counterregulation. Prospective elimination of hypoglycemia (MDG 432 mg/dl) improved, but did not normalize hormonal counterregulation. In diabetic rats, the glucagon defect appeared to be specific for hypoglycemia, whereas deficient epinephrine secretion also occurred during hypovolemia. We concluded that both recurrent hypoglycemia and the diabetic state independently lead to defective hormonal counterregulation. These data suggest that in IDDM iatrogenic hypoglycemia magnifies preexisting counterregulatory defects, thereby increasing the risk of severe hypoglycemia.

Effect of ketone infusions on amino acid and nitrogen metabolism in man.

To evaluate the role of hyperketonemia in the hypoalaninemia and decreased protein catabolism of prolonged starvation, Na dl-beta-hydroxybutyrate was administered as a primed continuous 3-6-h infusion in nonobese subjects and in obese subjects in the postabsorptive state and after 3 days and 3-5 1/2 wk of starvation. An additional obese group received 12-h ketone infusions on 2 consecutive days after 5-10 wk of fasting. The ketone infusion in nonobese and obese subjects studied in the postabsorptive state resulted in total blood ketone acid levels of 1.1-1.2 mM, a 5-15 mg/100 ml decrease in plasma glucose, and unchanged levels of insulin, glucagon, lactate, and pyruvate. Plasma alanine fell by 21% (P smaller than 0.001) in 3 h. In contrast, other amino acids were stable or varied by less than 10%. Infusions lasting 6 h reduced plasma alanine by 37%, reaching levels comparable to those observed in prolonged starvation. Equimolar infusions of NaC1 and/or administration of NaHCO3 failed to alter plasma alanine levels. During prolonged fasting, plasma alanine, which had fallen by 40% below prefast levels, fell an additional 30% in response to the ketone infusion. In association with repeated prolonged (12 h) infusions in subjects fasted 5-10 wk, urinary nitrogen excretion fell by 30%, returning to base line after cessation of theinfusions and paralleling the changes in plasma alanine. Ketone infusins resulted in two- to fourfold greater increments in blood ketone acids in fasted as compared to postabsorptive subjects. It is concluded that increased blood ketone acid levels induced by infusions of Na DL-beta-hydroxybutyrate result in hypoalaninemia and in nitrogen conservation in starvation. These data suggest that hyperketonemia may be a contributory factor in the decreased availability or circulating alanine and reduction in protein catabolism characteristic of prolonged fastings9

Role of counterregulatory hormones in the catabolic response to stress.

Patients with major injury or illness develop protein wasting, hypermetabolism, and hyperglycemia with increased glucose flux. To assess the role of elevated counterregulatory hormones in this response, we simultaneously infused cortisol (6 mg/m2 per h), glucagon (4 ng/kg per min), epinephrine (0.6 microgram/m2 per min), and norepinephrine (0.8 micrograms/m2 per min) for 72 h into five obese subjects receiving only intravenous glucose (150 g/d). Four obese subjects received cortisol alone under identical conditions. Combined infusion maintained plasma hormone elevations typical of severe stress for 3 d. This caused a sustained increase in plasma glucose (60-80%), glucose production (100%), and total glucose flux (40%), despite persistent hyperinsulinemia. In contrast, resting metabolic rate changed little (9% rise, P = NS). Urinary nitrogen excretion promptly doubled and remained increased by approximately 4 g/d, reflecting increased excretion of urea and ammonia. Virtually all plasma amino acids declined. The increment in nitrogen excretion was similar in three additional combined infusion studies performed in 3-d fasted subjects not receiving glucose. Cortisol alone produced a smaller glycemic response (20-25%), an initially smaller insulin response, and a delayed rise in nitrogen excretion. By day 3, however, daily nitrogen excretion was equal to the combined group as was the elevation in plasma insulin. Most plasma amino acids rose rather than fell. In both infusion protocols nitrogen wasting was accompanied by only modest increments in 3-methylhistidine excretion (approximately 20-30%) and no significant change in leucine flux. We conclude: (a) Prolonged elevations of multiple stress hormones cause persistent hyperglycemia, increased glucose turnover, and increased nitrogen loss; (b) The sustained nitrogen loss is no greater than that produced by cortisol alone; (c) Glucagon, epinephrine, and norepinephrine transiently augment cortisol-induced nitrogen loss and persistently accentuate hyperglycemia; (d) Counterregulatory hormones contribute to, but are probably not the sole mediators of the massive nitrogen loss, muscle proteolysis, and hypermetabolism seen in some clinical settings of severe stress.

Glucose modulates rat substantia nigra GABA release in vivo via ATP-sensitive potassium channels.

Glucose modulates beta cell insulin secretion via effects on ATP-sensitive potassium (KATP) channels. To test the hypothesis that glucose exerts a similar effect on neuronal function, local glucose availability was varied in awake rats using microdialysis in the substantia nigra, the brain region with the highest density of KATP channels. 10 mM glucose perfusion increased GABA release by 111 +/- 42%, whereas the sulfonylurea, glipizide, increased GABA release by 84 +/- 20%. In contrast, perfusion of the KATP channel activator, lemakalim, or depletion of ATP by perfusion of 2-deoxyglucose with oligomycin inhibited GABA release by 44 +/- 8 and 45 +/- 11%, respectively. Moreover, the inhibition of GABA release by 2-deoxyglucose and oligomycin was blocked by glipizide. During systemic insulin-induced hypoglycemia (1.8 +/- 0.3 mM), nigral dialysate GABA concentrations decreased by 49 +/- 4% whereas levels of dopamine in striatal dialysates increased by 119 +/- 18%. We conclude that both local and systemic glucose availability influences nigral GABA release via an effect on KATP channels and that inhibition of GABA release may in part mediate the hyperexcitability associated with hypoglycemia. These data support the hypothesis that glucose acts as a signaling molecule, and not simply as an energy-yielding fuel, for neurons.

Insulin binding to monocytes and insulin action in human obesity, starvation, and refeeding.

Insulin binding to monocytes and insulin action in vivo was examined in 14 obese subjects during the postabsorptive state and after starvation and refeeding. Tissue sensitivity to insulin was evaluated with the euglycemic insulin clamp technique. The plasma insulin concentration is acutely raised and maintained 100 muU/ml above the fasting level, and plasma glucose is held constant by a variable glucose infusion. The amount of glucose infused is a measure of tissue sensitivity to insulin and averaged 285+/-15 mg/m(2) per min in controls compared to 136+/-13 mg/m(2) per min in obese subjects (P <0.001). (125)I-Insulin binding to monocytes averaged 8.3+/-0.4% in controls vs. 4.6+/-0.5% in obese subjects (P < 0.001). Insulin binding and insulin action were highly correlated in both control (r = 0.86, P < 0.001) and obese (r = 0.94, P < 0.001) groups. Studies employing tritiated glucose to measure glucose production indicated hepatic as well as extrahepatic resistance to insulin in obesity. After 3 and 14 days of starvation, insulin sensitivity in obese subjects decreased to 69+/-4 and 71+/-7 mg/m(2) per min, respectively, whereas (125)I-insulin binding increased to 8.8+/-0.7 and 9.0+/-0.4%. In contrast to the basal state, there was no correlation between insulin binding and insulin action. After refeeding, tissue sensitivity increased to 168+/-14 mg/m(2) per min (P < 0.001) whereas insulin binding fell to 5.0+/-0.3%. We conclude that (a) in the postabsorptive state insulin binding to monocytes provides an index of in vivo insulin action in nonobese and obese subjects and, (b) during starvation and refeeding, insulin binding and insulin action changes in opposite directions suggesting that postreceptor events determine in vivo insulin sensitivity.

Acute effects of insulin-like growth factor I on glucose and amino acid metabolism in the awake fasted rat. Comparison with insulin.

To elucidate the acute metabolic actions of insulin-like growth factor I (IGF-I), we administered a primed (250 micrograms/kg), continuous (5 micrograms/kg.min) infusion of human recombinant (Thr 59) IGF-I or saline to awake, chronically catheterized 24-h fasted rats for 90 min. IGF-I was also infused while maintaining euglycemia (glucose clamp technique) and its effects were compared to those of insulin. IGF-I infusion caused a twofold rise in IGF-I levels and a 75-85% decrease in plasma insulin. When IGF-I alone was given, plasma glucose fell by 30-40 mg/dl (P less than 0.005) due to a transient twofold increase (P less than 0.05) in glucose uptake; hepatic glucose production and plasma FFA levels remained unchanged. IGF-I infusion with maintenance of euglycemia produced a sustained rise in glucose uptake and a marked stimulation of [3-3H]glucose incorporation into tissue glycogen, but still failed to suppress glucose production and FFA levels. IGF-I also produced a generalized 30-40% reduction in plasma amino acids, regardless of whether or not hypoglycemia was prevented. This was associated with a decrease in leucine flux and a decline in the incorporation of [1-14C]leucine into muscle and liver protein (P less than 0.05). When insulin was infused in a dosage that mimicked the rise in glucose uptake seen with IGF-I, nearly identical changes in amino acid metabolism occurred. However, insulin suppressed glucose production by 65% and FFA levels by 40% (P less than 0.001). Furthermore, insulin was less effective than IGF-I in promoting glycogen synthesis. We conclude that (a) IGF-I produces hypoglycemia by selectively enhancing glucose uptake; (b) IGF-I is relatively ineffective in suppressing hepatic glucose production or FFA levels; and (c) IGF-I, like insulin, lowers circulating amino acids by reducing protein breakdown rather than by stimulating protein synthesis. Thus, IGF-I's metabolic actions in fasted rats are readily distinguished from insulin.

Effect of insulin-like growth factor-1 on the responses to and recognition of hypoglycemia in humans. A comparison with insulin.

Recombinant human insulin-like growth factor-1 (rhIGF-1) lowers blood glucose in humans but its effect on counterregulatory responses has not been established. We therefore compared infusions of rhIGF-1 (0.7 micrograms/kg per min) and insulin (0.8 mU/kg.min) for 120 min in 10 healthy volunteers (glucose allowed to fall freely). With both, glucose fell rapidly because of stimulation of glucose uptake and suppression of hepatic glucose production. Despite similar plasma glucose nadirs (2.6 +/- 0.1 vs. 2.7 +/- 0.1 mM), the glucagon response was absent (P < 0.005), growth hormone release was attenuated (P < 0.03), and norepinephrine levels were increased (P < 0.05) by rhIGF-1 compared with insulin. Absent glucagon responses were associated with a blunting of the rebound increase in glucose production (P < 0.05 vs. insulin). After stopping the infusions, glucose recovery was delayed with rhIGF-1 (P < 0.001 vs. insulin). To further evaluate the effects of rhIGF-1 during a standard hypoglycemic stimulus, eight additional healthy subjects received rhIGF-1 or insulin while glucose was clamped at 2.8 mM. Again the rise in glucagon during insulin-induced hypoglycemia was totally abolished by rhIGF-1. Growth hormone responses were delayed, whereas increases in norepinephrine, heart rate, and symptomatic awareness of hypoglycemia were greater with rhIGF-1 compared with insulin (P < 0.05). It was concluded that rhIGF-1 suppression of glucagon release during hypoglycemia impairs glucose recovery. Paradoxically, awareness of hypoglycemia is enhanced with rhIGF-1 in part due to stimulation of the sympathetic activity.

Mechanisms of epinephrine-induced glucose intolerance in normal humans.

To evaluate the role of the splanchnic bed in epinephrine-induced glucose intolerance, we selectively assessed the components of net splanchnic glucose balance, i.e., splanchnic glucose uptake and hepatic glucose production, and peripheral glucose uptake by combining infusion of [3-(3)H]glucose with hepatic vein catheterization. Normal humans received a 90-min infusion of either glucose alone (6.5 mg/kg(-1) per min(-1)) or epinephrine plus glucose at two dose levels: (a) in amounts that simulated the hyperglycemia seen with glucose alone (3.0 mg/kg(-1) per min(-1)); and (b) in amounts identical to the control study. During infusion of glucose alone, blood glucose rose twofold, insulin levels and net posthepatic insulin release increased three- to fourfold, and net splanchnic glucose output switched from a net output (1.65+/-0.12 mg/kg(-1) per min(-1)) to a net uptake (1.56+/-0.18). This was due to a 90-95% fall (P < 0.001) in hepatic glucose production and a 100% rise (P < 0.001) in splanchnic glucose uptake (from 0.86+/-0.14 to 1.71+/-0.12 mg/kg(-1) per min(-1)), which in the basal state amounted to 30-35% of total glucose uptake. Peripheral glucose uptake rose by 170-185% (P < 0.001). When epinephrine was combined with the lower glucose dose, blood glucose, insulin release, and hepatic blood flow were no different from values observed with glucose alone. However, hepatic glucose production fell only 40-45% (P < 0.05 vs. glucose alone) and, most importantly, the rise in splanchnic glucose uptake was totally blocked. As a result, splanchnic glucose clearance fell by 50% (P < 0.05), and net splanchnic glucose uptake did not occur. The rise in peripheral glucose uptake was also reduced by 50-60% (P < 0.001). When epinephrine was added to the same dose of glucose used in the control study, blood glucose rose twofold higher (P < 0.001). The initial rise in splanchnic glucose uptake was totally prevented; however, beyond 30 min, splanchnic glucose uptake increased, reaching levels seen in the control study when severe hyperglycemia occurred. Splanchnic glucose clearance, nevertheless, remained suppressed throughout the entire study (40%-50%, P < 0.01). It is concluded that (a) the splanchnic bed accounts for one-third of total body glucose uptake in the basal state in normal humans; (b) epinephrine markedly inhibits the rise in splanchnic glucose uptake induced by infusion of glucose; and (c) this effect does not require a fall in insulin and is modulated by the level of hyperglycemia. Our data indicate that the splanchnic bed is an important site of glucose uptake in post-absorptive humans and that epinephrine impairs glucose tolerance by suppressing glucose uptake by both splanchnic and peripheral tissues, as well as by its well known stimulatory effect on endogenous glucose production.

Local ventromedial hypothalamus glucose perfusion blocks counterregulation during systemic hypoglycemia in awake rats.

The ventromedial hypothalamic nucleus (VMH) is necessary for the integrated hormonal response to hypoglycemia. To determine the role of the VMH as a glucose sensor, we performed experiments designed to specifically prevent glucopenia in the VMH, while producing hypoglycemia elsewhere. We used awake chronically catheterized rats, in which local VMH glucose perfusion (100 mM or 15 mM of D-glucose) was combined with a sequential euglycemic-hypoglycemic clamp. In two control groups the VMH was perfused either with (a) an iso-osmotic solution lacking glucose, or with (b) nonmetabolizable L-glucose (100 mM). During systemic hypoglycemia glucagon and catecholamine concentrations promptly increased in the control animals perfused with either 100 mM L-glucose or the iso-osmotic solution lacking glucose. In contrast, glucagon, epinephrine and norepinephrine release was inhibited in the animals in which the VMH was perfused with D-glucose; hormonal secretion was partially suppressed by the VMH perfusion with 15 mM D-glucose and suppressed by approximately 85% when the VMH was perfused with 100 mM D-glucose, as compared with the control groups. We conclude that the VMH must sense hypoglycemia for full activation of catecholamine and glucagon secretion and that it is a key glucose sensor for hypoglycemic counterregulation.

Influence of basal insulin and glucagon secretion on potassium and sodium metabolism. Studies with somatostatin in normal dogs and in normal and diabetic human beings.

To examine the role of basal insulin and glucagon secretion in potassium and sodium homeostasis, somatostatin, a potent inhibitor of insulin and glucagon secretion, was infused for 5 h into healthy human subjects, maturity-onset diabetes, juvenile-onset diabetics, and normal dogs. Infusion of somatostatin resulted in an increase in serum potassium (0.5-0.6 meq/liter) in normal subjects and maturity-onset diabetics, but not in juvenile-onset diabetics despite equivalent reductions in plasma glucagon in all three groups. A similar rise in serum potassium was observed in normal conscious dogs given somatostatin and was reversed by insulin replacement. Urinary excretion of potassium was unaffected by somatostatin. In dogs given intravenous potassium chloride in doses (0.375 meq/kg per h) which do not alter basal insulin levels, the rise in serum potassium (0.6 meq/liter in controls) increased 100% when somatostatin was administered together with the KCl infusion. Addition of replacement doses of insulin to the somatostatin infusion resulted in increments in serum potassium which were comparable to infusion of KCl alone. Urinary potassium excretion rose after KCl administration and was unchanged by the addition of somatostatin. Serum sodium concentration was unaffected by somatostatin administration in both the human and dog studies. However, urinary sodium excretion displayed a biphasic response falling by 20-60% within the first 2 h of somatostatin administration and then rising to values 50-80% above basal levels at 3-4 h. Inulin and p-aminohippurate clearances were unaffected by somatostatin. It is concluded that (a) potassium homeostasis is influenced by basal insulin levels in the absence of which serum potassium concentration rises and potassium tolerance declines; (b) this effect of insulin is mediated via extrarenal mechanisms of potassium disposal; (c) somatostatin has a biphasic effect on urinary sodium secretion, the mechanism of which remains to be established.

Insulin control of glucose metabolism in man: a new kinetic analysis.

Analyses of the control of glucose metabolism by insulin have been hampered by changes in bloog glucose concentration induced by insulin administration with resultant activation of hypoglycemic counterregulatory mechanisms. To eliminate such mechanisms, we have employed the glucose clamp technique which allows maintenance of fasting blood glucose concentration during and after the administration of insulin. Analyses of six studies performed in young healthy men in the postabsorptive state utilizing the concurrent administration of [14C]glucose and 1 mU/kg per min (40 mU/m2 per min) porcine insulin led to the development of kinetic models for insulin and for glucose. These models account quantitatively for the control of insulin on glucose utilization and on endogenous glucose production during nonsteady states. The glucose model, a parallel three-compartment model, has a central compartment (mass = 68 +/- 7 mg/kg; space of distribution = blood water volume) in rapid equilibrium with a smaller compartment (50 +/- 17 mg/kg) and in slow equilibrium with a larger compartment (96 +/-21 mg/kg). The total plasma equivalent space for the glucose system averaged 15.8 liters or 20.3% body weight. Two modes of glucose loss are introduced in the model. One is a zero-order loss (insulin and glucose independent) from blood to the central nervous system; its magnitude was estimated from published data. The other is an insulin-dependent loss, occurring from the rapidly equilibrating compartment and, in the basal period, is smaller than the insulin-independent loss. Endogenous glucose production averaged 1.74 mg/kg per min in the basal state and enters the central compartment directly. During the glucose clamp experiments plasma insulin levels reached a plateau of 95 +/-8 microU/ml. Over the entire range of insulin levels studied, glucose losses were best correlated with levels of insulin in a slowly equilibrating insulin compartment of a three-compartment insulin model. A proportional control by this compartment on glucose utilization was adequate to satisfy the observed data. Insulin also rapidly decreased the endogenous glucose production to 33% of its basal level (0.58 mg/kg per min), this suppression being maintained for at least 40 min after exogenous insulin infusion was terminated and after plasma insulin concentrations had returned to basal levels. The change in glucose utilization per unit change in insulin in the slowly equilibrating insulin compartment is proposed as a new measure for insulin sensitivity. This defines insulin effects more precisely than previously used measures, such as plasma glucose/plasma insulin concentration ratios. Glucose clamp studies and the modeling of the coupled kinetics of glucose and insulin offers a new and potentially valuable tool to the study of altered states of carbohydrate metabolism.

Comparison of the metabolic effects of recombinant human insulin-like growth factor-I and insulin. Dose-response relationships in healthy young and middle-aged adults.

The actions of recombinant human insulin-like growth factor-I (rhIGF-I) and insulin were compared in 21 healthy young (24 +/- 1 yr) and 14 healthy middle-aged (48 +/- 2 yr) subjects during 3-h paired euglycemic clamp studies using one of three doses (rhIGF-I 0.2, 0.4, and 0.8 micrograms/kg.min and insulin 0.2, 0.4, and 0.8 mU/kg.min, doses chosen to produce equivalent increases in glucose uptake). In younger subjects, rhIGF-I infusions suppressed insulin by 19-33%, C-peptide by 47-59% and glucagon by 33-47% (all, P < 0.02). The suppression of C-peptide was less pronounced with insulin than with rhIGF-I (P < 0.007). The metabolic responses to rhIGF-I and insulin were remarkably similar: not only did both hormones increase glucose uptake and oxidation in a nearly identical fashion, but they also produced similar suppression of glucose production, free fatty acid levels, and fat oxidation rates. In contrast, rhIGF-I had a more pronounced amino acid-lowering effect than did insulin (P < 0.004). In middle-aged subjects, basal IGF-I levels were 44% lower (P < 0.0001) whereas basal insulin and C-peptide were 20-25% higher than in younger subjects. Age did not alter the response to rhIGF-I. However, insulin-induced stimulation of glucose uptake was blunted in older subjects (P = 0.05). Our data suggest that absolute IGF-I and relative insulin deficiency contribute to adverse metabolic changes seen in middle age.

Adrenergic blockade alters glucose kinetics during exercise in insulin-dependent diabetics.

We investigated the effects of alpha and/or beta adrenergic blockade (with phentolamine and/or propranolol) on glucose homeostasis during exercise in six normal subjects and in seven Type I diabetic subjects. The diabetics received a low dose insulin infusion (0.07 mU/kg X min) designed to maintain plasma glucose at approximately 150 mg/dl. In normals, neither alpha, beta, nor combined alpha and beta adrenergic blockade altered glucose production, glucose uptake, or plasma glucose concentration during exercise. In diabetics, exercise alone produced a decline in glucose concentration from 144 to 116 mg/dl. This was due to a slightly diminished rise in hepatic glucose production in association with a normal increase in glucose uptake. When exercise was performed during beta adrenergic blockade, the decline in plasma glucose was accentuated. An exogenous glucose infusion (2.58 mg/kg X min) was required to prevent glucose levels from falling below 90 mg/dl. The effect of beta blockade was accounted for by a blunted rise in hepatic glucose production and an augmented rise in glucose utilization. These alterations were unrelated to changes in plasma insulin and glucagon levels, which were similar in the presence and absence of propranolol. In contrast, when the diabetics exercised during alpha adrenergic blockade, plasma glucose concentration rose from 150 to 164 mg/dl. This was due to a significant increase in hepatic glucose production and a small decline in exercise-induced glucose utilization. These alterations also could not be explained by differences in insulin and glucagon levels. We conclude that the glucose homeostatic response to exercise in insulin-dependent diabetics, in contrast to healthy controls, is critically dependent on the adrenergic nervous system.

Ventromedial hypothalamic lesions in rats suppress counterregulatory responses to hypoglycemia.

The central nervous system has been implicated in the activation of counterregulatory hormone release during hypoglycemia. However, the precise loci involved are not established. To determine the role of the ventromedial hypoglycemia, we performed hypoglycemic clamp studies in conscious Sprague-Dawley rats with bilateral VMH lesions produced by local ibotenic acid injection 2 wk earlier. Rats with lesions in the lateral hypothalamic area, frontal lobe, sham operated (stereotaxic needle placement into hypothalamus without injection), and naive animals served as control groups. The clamp study had two phases. For the first hour plasma glucose was fixed by a variable glucose infusion at euglycemia (approximately 5.9 mM). Thereafter, for an additional 90 min, glucose was either allowed to fall to (a) mild hypoglycemia (approximately 3.0 mM) or (b) more severe hypoglycemia (approximately 2.5 mM). Glucagon and catecholamine responses of lateral hypothalamic area-, frontal lobe-lesioned, sham operated, and naive animals were virtually identical at each hypoglycemic plateau. In contrast, glucagon, epinephrine, and norepinephrine responses in the VMH-lesioned rats were markedly inhibited; hormones were diminished by 50-60% during mild and by 75-80% during severe hypoglycemia as compared with the other groups. We conclude that the VMH plays a crucial role in triggering the release of glucagon and catecholamines during hypoglycemia.

Induction of insulitis by glutamic acid decarboxylase peptide-specific and HLA-DQ8-restricted CD4(+) T cells from human DQ transgenic mice.

Insulin-dependent diabetes mellitus in humans is linked with specific HLA class II genes, e.g., HLA-DQA1*0301/ DQB1*0302 (DQ8). To investigate the roles of HLA-DQ8 molecules and glutamic acid decarboxylase (GAD) in disease development, we generated DQ8(+)/I-Abo transgenic mice expressing functional HLA-DQ8 molecules and devoid of endogenous mouse class II. DQ8(+)/I-Abo mice produced antigen-specific antibodies and formed germinal centers after immunization with GAD65 peptides. Two GAD peptide-specific (247-266 and 509-528), DQ8 restricted Th1 CD4(+) T cell lines, were generated from immunized DQ8(+)/I-Abo mice. They induced severe insulitis after adoptive transfer into transgene positive (but not negative) mice who were treated with a very low dose of streptozotocin that alone caused no apparent islet pathology. In addition to CD4, islet mRNA from these mice also showed expression of CD8, IFNgamma, TNFalpha, Fas, and Fas ligand. Our data suggest that a mild islet insult in the presence of HLA-DQ8 bearing antigen-presenting cells promotes infiltration of GAD peptide reactive T cells into the islet.