U Keller

The role of insulin and glucagon in the regulation of basal glucose production in the postabsorptive dog.

The aim of the present experiments was to determine the role of insulin and glucagon in the regulation of basal glucose production in dogs fasted overnight. A deficiency of either or both pancreatic hormones was achieved by infusin somatostatin (1 mug/kg per min), a potent inhibitor of both insulin and glucagon secretion, alone or in combination with intraportal replacement infusions of either pancreatic hormone. Infusion of somatostatin alone caused the arterial levels of insulin and glucagon to drop rapidly by 72+/-6 and 81+/-8%, respectively. Intraportal infusion of insulin and glucagon at rates of 400 muU/kg per min and 1 ng/kg per min, respectively, resulted in the maintenance of the basal levels of each hormone. Glucose production was measured using tracer (primed constant infusion of [3-3H]glucose) and arteriovenous difference techniques. Isolated glucagon deficiency resulted in a 35+/-5% (P less than 0.05) rapid and sustained decrease in glucose production which was abolished upon restoration of the plasma glucagon level. Isolated insulin deficiency resulted in a 52+/-16% (P less than 0.01) increase in the rate of glucose production which was abolished when the insulin level was restored. Somatostatin had no effect on glucose production when the changes in the pancreatic hormone levels which it normally induces were prevented by simultaneous intraportal infusion of both insulin and glucagon. In conclusion, in the anesthetized dog fasted overnight; (a) basal glucagon is responsible for at least one-third of basal glucose production, (b) basal insulin prevents the increased glucose production which would result from the unrestrained action of glucagon, and (c) somatostatin has no acute effects on glucose turnover other than those it induces through perturbation of pancreatic hormone secretion. This study indicates that the opposing actions of the two pancreatic hormones are important in the regulation of basal glucose production in the postabsorptive state.

Evidence for an important role of glucagon in the regulation of hepatic glucose production in normal man.

To investigate the role of glucagon in regulating hepatic glucose production in man, selective glucagon deficiency was produced in four normal men by infusing somatostatin (0.9 mg/h) and regular pork insulin (150-muU/kg per min) for 2 h. Exogenous glucose was infused to maintain euglycemia. Arterial plasma glucagon levels fell by greater than 50% whereas plasma insulin levels were maintained in the range of 10-14 muU/ml. In response to these hormonal changes, net splanchnic glucose production (NSGP) fell by 75% and remained suppressed for the duration of the study. In contrast, when somatostatin alone was administered to normal men, resulting in combined insulin and glucagon deficiency (euglycemia again maintained), NSGP fell markedly but only transiently, reaching its nadir at 15 min. Thereafter, NSGP rose progressively, reaching the basal rate at 105 min. These data indicate that the induction of selective glucagon deficiency in man (with basal insulin levels maintained) is associated with a marked and sustained fall in hepatic glucose production. We conclude, therefore, that basal glucagon plays an important role in the maintenance of basal hepatic glucose production in normal man.

Effect of glucose, independent of changes in insulin and glucagon secretion, on alanine metabolism in the conscious dog.

To study the effects of hyperglycemia on the metabolism of alanine and lactate independent of changes in plasma insulin and glucagon, glucose was infused into five 36-h-fasted dogs along with somatostatin and constant replacement amounts of both insulin and glucagon. Hepatic uptakes of alanine and lactate were calculated using the arteriovenous difference technique. [14C]Alanine was infused to measure the conversion of alanine and lactate into glucose. Hyperglycemia (delta 115 mg/dl) of 2 h duration caused the plasma alanine level to increase by over 50%. This change was caused by an increase in the inflow of alanine into plasma since the net hepatic uptake of the amino acid did not change. Taken together, the above findings indicate that glucose per se can significantly impair the fractional extraction of alanine by the liver. Hepatic extraction of lactate was also affected by hyperglycemia and had fallen to zero within 90 min of starting the glucose infusion. This fall was associated with a doubling of arterial lactate level. Conversion of [14C]-alanine and [14C]lactate into [14C]glucose was suppressed by 60 +/- 11% after 2 h of hyperglycemia, and because this fall could not be entirely accounted for by decreased lactate extraction an inhibitory effect of glucose on gluconeogenesis within the liver is suggested. These studies indicate that the plasma glucose level per se can be an important determinant of the level of alanine and lactate in plasma as well as the rate at which they are converted to glucose.

Prevention of Hypophosphatemia by Phosphate Infusion During Treatment of Diabetic Ketoacidosis and Hyperosmolar Coma

To assess the effect of phosphate replacement therapy on the course of diabetic coma, 24 patients with severe diabetic ketoacidosis and 16 patients with non-ketotic hyperosmolar coma were randomly assigned either to standardized conventional treatment alone or combined with phosphate infusions. Insulin and fluid therapy produced a rapid fall of plasma phosphorus; almost all patients not receiving phosphate infusions developed marked hypophosphatemia within 12 h. Hypophosphatemia was prevented by administration of 62 ± 5 mmol (range 35–140) sodium phosphate. Initial red blood cell 2,3-diphosphoglycerate (2,3-DPG) concentrations were markedly decreased in ketoacidotic patients. The recovery of 2,3-DPG upon institution of therapy was accelerated when phosphate replacement infusions were given. The increase in 2,3-DPG during the first 48 h was 56% greater (P < 0.02) when phosphate was administered, but later the difference between the two treatment groups disappeared. Non-ketotic hyperosmolar coma patients revealed normal 2,3-DPG concentrations on admission, and a similar decline of plasma phosphorus occurred, as in ketoacidosis, during treatment. 2,3-DPG levels remained unaffected by phosphate therapy. While plasma calcium levels declined during the initial 48 h in both groups, transient postinfusion hyperphosphatemia was noted in 7 of 17 patients. A favorable effect of phosphate therapy on the clinical course of diabetic ketoacidosis or hyperosmolar coma could not be demonstrated.

The Roles of Insulin, Glucagon, and Free Fatty Acids in the Regulation of Ketogenesis in Dogs

The roles of basal insulin, glucagon, and free fatty acids in the regulation of ketogenesis were studied in three-day-fasted anesthetized dogs. Four protocols were employed: (1) infusion of somatostatin, resulting in combined insulin and glucagon deficiency, (2) somatostatin and intraportal glucagon replacement (1 ng./kg./min.) to produce insulin deficiency, (3) somatostatin combined with intraportal insulin replacement (350 μU./kg./min.) to produce glucagon deficiency, and (4) saline controls. Simultaneous blood sampling from the femoral artery and portal and hepatic veins allowed determination of hepatic uptake or production of free fatty acids, glycerol, ketone bodies, and glucose. When combined deficiency of insulin and glucagon was produced, no significant effect on hepatic ketone production was noted (108 ± 6 per cent of mean basal), whereas the induction of selective Insulin deficiency (basal glucagon level maintained) resulted in a rise of net hepatic ketone production (185 ± 24 per cent of mean basal, P < 0.01). Isolated glucagon lack (basal insulin maintained) did not alter net hepatic ketone production. To assess the effect of increased substrate supply on ketogenesis, all experiments included a period of acute elevation of free fatty adds (FFA), produced by infusion of Intralipld and heparin. FFA elevation in saline controls caused only a moderate stimulation of ketone production (149 ± 16 per cent of mean basal, P < 0.01) despite a threefold increase in FFA uptake. However, the combination of elevation of FFA and selective insulin deficiency (glucagon maintained) resulted in a greatly increased hepatic ketone production (357 ± 58 per cent of mean basal, P < 0.01 vs. controls), which was also significantly higher than during combined insulin and glucagon deficiency (P < 0.05). Lipolysis, as indicated by arterial glycerol levels, as well as hepatic FFA uptake was not affected by acute suppression of pancreatic hormone levels. The study demonstrated that basal insulin plays an important role in repressing ketogenesis and that basal amounts of glucagon, while ineffective in the presence of insulin, exert a stimulatory effect on ketogenesis when insulin is deficient. Glucagons stimulatory effect on ketogenesis was due to an effect in the liver rather than to an increase in lipolysis or in hepatic FFA uptake. Increasing FFA supply per se was associated with only limited stimulation of ketogenesis, whereas the combination of insulin deficiency, basal concentrations of glucagon, and increased FFA load produced a synergistic augmentation of hepatic ketone production.

Effects of insulin at two dose levels on gluconeogenesis from alanine in fasting man

We have determined the effect of insulin infused at 1 and 5 mU/kg/min on gluconeogenesis from alanine in 48-hr fasted men. The conversion of alanine to glucose was measured by the arterial-hepatic venous catheterization technique combined with the infusion of 14C-alanine. During insulin infusion, euglycemia was maintained by variable glucose infusion. When insulin was infused at 1 mU/kg/min the net splanchnic production of 14C-glucose was suppressed by 80% but glucagon infused at the end of the study resulted in substantial release of 14C-glucose from the liver suggesting marked accumulation of labeled glucose in glycogen. When insulin was infused at 5 mU/kg/min the splanchnic release of 14C-glucose was also markedly suppressed but in contrast to the lower insulin dose very little labeled glucose accumulated in glycogen. Neither the high nor the low dose insulin infusion had any effect on net splanchnic alanine uptake and plasma glucagon levels fell by 35% in both protocols. These data demonstrate that in 48-hr fasted man, (1) a small increment in insulin concentration will suppress glucose production but mostly by diverting the newly formed glucose into glycogen; (2) at higher concentrations, insulin will inhibit glucose production mainly by suppressing glucoeogenesis; and (3) this insulin-induced suppression of gluconeogenesis is due to an intrahepatic effect rather than an effect on the splanchnic extraction of alanine.

Effect of hyperglycemia independent of changes in insulin or glucagon on lipolysis in the conscious dog

In the face of fixed basal levels of insulin (9 microunits/ml) and glucagon (63 pg/ml) maintained by the infusion of somatostatin and replacement amounts of the two pancreatic hormones, the mean arterial plasma glucose concentration was elevated from 102 to 217 mg/dl by continuous glucose infusion. Hyperglycemia resulted in a significant decrease in the arterial blood glycerol (35%) and plasma free fatty acid concentrations (46%). The drop in the blood glycerol level was paralleled by a decline in hepatic glycerol uptake indicating that hyperglycemia did not alter the fractional extraction of glycerol by the liver. These results support the view that glucose has a direct antilipolytic effect in vivo.

The Roles of Insulin and Glucagon in the Regulation of Gluconeogenesis in the Postabsorptive Dog

The aim of the present study was to determine the role of insulin and glucagon in the regulation of basal gluconeogenesis in overnight-fasted anesthetized dogs. A deficiency of either or both pancreatic hormones was achieved by infusing somatostatin (1 μg./kg./min.), a potent inhibitor of both insulin and glucagon secretion, alone or in combination with intraportal replacement infusions of either pancreatic hormone. Infusion of somatostatin alone caused the arterial levels of insulin and glucagon to drop rapidly by 72±6 per cent and 81±8 per cent, respectively. Intraportal infusions of insulin and glucagon at rates of 400 μU./kg./min. and 1 ng./kg./min., respectively, resulted in the maintenance of the basal levels of each hormone. Gluconeogenesis was assessed by measuring both the splanchnic extraction of alanine and the conversion of 141C-alanine into l4C-glucose. Isolated glucagon deficiency (basal insulin level maintained) resulted in a 44±9 per cent (P <0.05) decrease in the conversion rate of alanine into glucose. Isolated insulin deficiency (basal glucagon level maintained) resulted in a 74±7 per cent (P <0.01) increase in the conversion rate of alanine into glucose. Somatostatin had no effect on alanine conversion into glucose when the changes in the pancreatic hormone levels that it normally induces were prevented by simultaneous intraportal infusion of both insulin and glucagon. A deficiency of either hormone for up to 60 minutes failed to alter the splanchnic extraction rate of alanine. In conclusion, in the overnight-fasted anesthetized dog, (1) basal glucagon plays a significant role in the stimulation of gluconeogenesis from alanine, (2) basal insulin exerts a potent inhibitory effect on the rate of conversion of alanine into glucose, with the result that glucagons gluconeogenic potential is markedly enhanced during insulin deficiency, and (3) somatostatin has no acute effects on gluconeogenesis from alanine other than those it induces through perturbation of pancreatic hormone secretion. This study indicates that the opposing actions of the two pancreatic hormones are important for the fine regulation of gluconeogenesis.

Effects of Fasting on Gluconeogenesis from Alanine in Nondiabetic Man

The present study was designed to determine the effect of fasting on gluconeogenesis from alanine. This process was measured by combining the infusion of l4C-alanine with the A-V difference technique. Normal subjects were studied after 12 and 48 h of fasting and obese subjects were studied after 18 days of therapeutic starvation. After a 12-h fast the hepatic venous 14C-alanine specific activity was 34% lower than the arterial 14C-alanine specific activity, which was consistent with a dynamic exchange of alanine between the intestine and the plasma and with a net release of unlabeled alanine by the intestine. Under these conditions the net splanchnic alanine uptake (106 μmol/min) would underestimate the actual hepatic extraction of alanine, which could be estimated as at least 147 μmol/min. After 48 h and 18 days of fasting, the 14C-alanine specific activity equaled that in the arterial plasma, indicating cessation of intestinal release of alanine into the portal circulation. Under these circumstances, net splanchnic alanine uptake would equal net hepatic alanine extraction. Thus, actual hepatic alanine ëxtraction rates at 48 h and 18 days of fasting were 134 and 94 μmol/min, respectively. In the face of a decreasing hepatic alanine extraction with fasting, gluconeogenesis from alanine increased by 100% (from 41 to 82 μmol/min) after 48 h of fasting. After 18 days of starvation 51 /μmol/min of alanine was converted to glucose, a rate still 25% higher than after a 12-h fast. We concluded: (a) fasting is associated with a gradual decrease in hepatic alanine extraction; (b) however, gluconeogenesis from alanine is increased after 48 h and 18 days of fasting due to a more efficient intrahepatic conversion of alanine to glucose.

Effect of Norepinephrine on Ketogenesis, Fatty Acid Oxidation, and Esterification in Isolated Rat Hepatocytes

Recent studies in man demonstrated a marked ketogenic effect of increased plasma norepinephrine concentrations as observed in diabetic ketoacidosis. Since this effect may have been due either to increased substrate supply for ketogenesis (lipolysis) or to direct hepatic activation of ketogenesis, the latter mechanism was examined in isolated rat hepatocytes. Incubation of hepatocytes with norepinephrine (10−7 to 10−4 M) resulted in a dose-dependent increase in conversion of the long-chain fatty acid [1−14C]palmitate into ketone bodies and CO2. Norepinephrine decreased [1-14C]palmitate conversion into triglycerides without affecting fatty acid uptake. Norepinephrine enhanced ketogenesis from [1-14C]palmitate in a physiologic range of fatty acid concentrations (0.5–2.5 mM), but failed to affect fatty acid esterification to phospholipids or mono- and diglycerides. In contrast to long-chain fatty acids, oxidation of the medium-chain fatty acid [1-14C]octanoate to ketone bodies was not enhanced by norepinephrine, whereas CO2 production increased. The effect of norepinephrine on [1-14C]fatty acid oxidation was blocked by the a, receptor blocker prazosine. The results demonstrate that norepinephrine diverts long-chain fatty acids into the pathways of oxidation and ketogenesis away from esterification, suggesting enhanced carnitine-dependent mitochondrial fatty acid uptake. The studies using octanoate indicated that norepinephrine also enhanced fatty acid oxidation by increasing the flux of acetyl-CoA through the Krebs cycle. The data suggest that stress-associated sympathetic activation and norepinephrine discharge, as observed in diabetic ketoacidosis, result in direct activation of ketogenesis in the liver.