J E Liljenquist

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.

Glucose disposal during insulinopenia in somatostatin-treated dogs. The roles of glucose and glucagon.

The first aim of this study was to determine whether the plasma glucose level can regulate hepatic glucose balance in vivo independent of its effects on insulin and glucagon secretion. To accomplish this, glucose was infused into conscious dogs whose basal insulin and glucagon secretion had been replaced by exogenous intraportal insulin and glucagon infusion after somatostatin inhibition of endogenous pancreatic hormone release. The acute induction of hyperglycemia (mean increment of 121 mg/dl) in the presence of basal levels of insulin (7+/-1 muU/ml) and glucagon (76+/-3 pg/ml) resulted in a 56% decrease in net hepatic glucose production but did not cause net hepatic glucose uptake. The second aim of the study was to determine whether a decrease in the plasma glucagon level would modify the effect of glucose on the liver. The above protocol was repeated with the exception that glucagon was withdrawn (83% decrease in plasma glucagon) coincident with the induction of hyperglycemia. Under this circumstance, with the insulin level basal (7+/-1 muU/ml) and the glucagon levels reduced (16+/-2 pg/ml), hyperglycemia (mean increment of 130 mg/dl) promoted marked net hepatic glucose uptake (1.5+/-0.2 mg/kg per min) and glycogen deposition. In conclusion, (a) physiological increments in the plasma glucose concentration, independent of their effects on insulin and glucagon secretion, can significantly reduce net hepatic glucose production in vivo but at levels as high as 230 mg/dl cannot induce net hepatic glucose storage and (b) in the presence of basal insulin the ability of hyperglycemia to stimulate net hepatic glucose storage is influenced by the plasma glucagon concentration.

Differential Sensitivity of Glycogenolysis and Gluconeogenesis to Insulin Infusions in Dogs

The suppressive effect of insulin on hepatic glucose production is generally recognized. Though it is well established that this effect is at least partially due to inhibition of glycogenolysis, controversy still exists about insulins effect on gluconeogenesis. The present study was undertaken to determine whether insulin could affect gluconeogenesis from alanine in the intact dog and to compare the effect of insulin on glycogenolysis and gluconeogenesis. In anesthetized dogs fasted overnight, blood samples were drawn simultaneously from a femoral artery and hepatic vein. Alanine-U-14C, 10 μCi./kg., was infused over 110 minutes. A constant insulin infusion at either 1 or 5 mU./kg./min. was begun at 50 minutes, and blood glucose concentration was maintained by a variable glucose infusion. When insulin was infused at 1 mU./kg./min., resulting in plasma immunoreactive insulin (IRI) levels of 73 ± 10 μU./ml., the net splanchnic glucose production (NSGP) was suppressed from 2.7 ± 2 mg./kg./min. to virtually zero. In constrast, this small increment in insulin concentration had no demonstrable effect on the net splanchnic uptake of alanine or on the conversion of plasma alanine to glucose (7.9 ± 0.3 μmol/min.). Insulin infused at 5 mU./kg./min. resulted in IRI levels of 240 ± 25 muU./ml. This higher insulin concentration was associated with a marked suppression of both the NSGP (100 per cent) and the conversion of plasma alanine to glucose (90 per cent) but did not affect the extraction of alanine by the splanchnic bed. Doses of both 1 and 5 mU./kg./min. were associated with a 35 per cent fall in immunoreactive glucagon levels. These data demonstrate that (1) glycogenolysis is more sensitive than gluconeogenesis to the inhibitory effect of small increments in insulin concentrations, (2) gluconeogenesis could be suppressed by insulin but only at higher insulin concentrations, (3) this suppression of gluconeogenesis from alanine by insulin was due to an intrahepatic effect rather than an effect on the splanchnic extraction of alanine, and finally, (4) that insulin can suppress glucagon in the absence of hyperglycemia.

Effects of Glucagon on Lipolysis and Ketogenesis in Normal and Diabetic Men

The effect of glucagon (50 ng/kg/min) on arterial glycerol concentration and net splanchnic production of total ketones and glucose was studied after an overnight fast in four normal and five insulin-dependent diabetic men. Brachial artery and hepatic vein catheters were inserted and splanchnic blood flow determined using indocyanine green. The glucagon infusion resulted in a mean circulating plasma level of 4,420 pg/ml. In the normal subjects, the glucagon infusion resulted in stimulation of insulin secretion indicated by rising levels of immunoreactive insulin and C-peptide immunoreactivity. Arterial glycerol concentration (an index of lipolysis) declined markedly and net splanchnic total ketone production was virtually abolished. In contrast, the diabetic subjects secreted no insulin (no rise in C-peptide immunoreactivity) in response to glucagon. Arterial glycerol and net splanchnic total ketone production in these subjects rose significantly (P=<0.05) when compared with the results in four diabetics who received a saline infusion after undergoing the same catheterization procedure.Net splanchnic glucose production rose markedly during glucagon stimulation in the normals and diabetics despite the marked rise in insulin in the normals. Thus, the same level of circulating insulin which markedly suppressed lipolysis and ketogenesis in the normals failed to inhibit the glucagon-mediated increase in net splanchnic glucose production. It is concluded (a) that glucagon at high concentration is capable of stimulating lipolysis and ketogenesis in insulin-deficient diabetic man; (b) that insulin, mole for mole, has more antilipolytic activity in man than glucagon has lipolytic activity; and (c) that glucagon, on a molar basis, has greater stimulatory activity than insulin has inhibitory activity on hepatic glucose release.

Gluconeogenesis from Alanine in Normal Postabsorptive Man: Intrahepatic Stimulatory Effect of Glucagon

Although the stimulatory effect of glucagon on gluconeogenesis has been well demonstrated in certain systems in vitro, this effect has never been established in man. The present study was undertaken, therefore, to determine whether glucagon could stimulate gluconeogenesis from alanine in normal fasting man. Glucagon might stimulate this process by increasing the hepatic alanine uptake and/or by shunting the extracted alanine within the liver into the gluconeogenic pathway. In order to be able to examine these two aspects of gluconeogenesis, we combined the hepatic veinbrachial artery catheterization technic with an isotopic infusion of alanine-14C. Alanine-14C specific activity was measured in whole blood and plasma by use of a rapid chromatographic technic. Since plasma contributed 93 per cent of the alanine extracted by the splanchnic bed with a specific activity three times that of the red blood cells, plasma alanine specific activity was used to study the conversion of alanine to glucose. A constant infusion of alanine-14C achieved a relatively stable arterial specific activity by forty minutes. The administration of glucagon by constant infusion (15–50 ng./kg./min.) had no effect on the splanchnic extraction of alanine. Net splanchnic glucose-14C production, however, doubled during the glucagon infusion, and the conversion of alanine to glucose increased from 30 ± 2 to 58 ± 9 μmol/min. These data (1) demonstrate that in normal man fasted twelve to fourteen hours, glucagon at supraphysiblogic levels can double the rate of gluconeogenesis from alanine and (2) indicate that this stimulatory effect of glucagon is exerted within the liver by shunting the extracted alanine toward new glucose formation rather than by increasing the hepatic extraction of alanine.

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.

Transient Stimulatory Effect of Sustained Hyperglucagonemia on Splanchnic Glucose Production in Normal and Diabetic Man

Insulin can modulate glucagon-stimulated hepatic glucose production and is considered to be the major factor acting in vivo to exert a counterregulatory action to glucagon. The insulindependent diabetic, therefore, might be especially vulnerable to enhanced hepatic glucose production promoted by glucagon. To investigate this hypothesis, low-dose glucagon infusions were administered to normal and diabetic men to compare the effects of glucagon on net splanchnic glucose production (NSGP). Four normal and three insulin-dependent, ketosis-prone, hyperglycemic diabetic men (insulin withheld for 24 hours) underwent brachialartery-hepatic-vein catheterization. Each received a 90-minute glucagon infusion at 5 ng./kg./min. Glucagon levels rose four-tofivefold in both groups, plateauing at 300–600 pg./ml. In the normals, NSGP rose from 92 ± 12 to 211 ± 31 mg./min. at 15 minutes and returned to basal levels by 45 minutes. Insulin measured in the hepatic vein rose from 19 ± 6 to 33 ± 11 /μU./ml., while plasma glucose rose 17 mg./dl. In the insulin-dependent diabetics, NSGP rose from 78 ± 24 to a peak of 221 ± 33 mg./min. at 30 minutes and then fell sharply to 113 ± 15 mg./min. at 60 minutes despite continuing hyperglucagonemia. Plasma glucose in the diabetics rose 21 mg./dl. These data suggest a mechanism that acts to rapidly diminish glucagon-induced hepatic glucose production in diabetic man but does not appear to be mediated by increased insulin secretion.

Effect of Glucagon on Net Splanchnic Cyclic AMP Production in Normal and Diabetic Men

Glucagon activates hepatic adenylate cyclase, thereby increasing acutely the liver content of cyclic AMP (cAMP) as well as the release of cAMP into the hepatic vein. Insulin, on the other hand, antagonizes this glucagon-mediated cAMP production, thus providing a hypothetical mechanism through which insulin might correct some of the metabolic abnormalities of diabetes. To study this hormonal interaction in man, net splanchnic cAMP production (NScAMPP) was investigated in normal and insulin-dependent diabetic men under basal conditions and in response to intravenous glucagon, 50 ng/kg/min for 2 h. In normals (n=19), basal hepatic vein cAMP concentration was 23.6+/-1.1 nM and NScAMPP was 1.7+/-0.6 nmol/min. Glucagon stimulated NScAMPP in four normal subjects to a peak of 99.6+/-43 nmol/min at 25 min with a subsequent fall to 12.4+/-5.1 nmol/min by 90 min despite continuing glucagon infusion. Endogenous insulin secretion was stimulated as indicated by rising levels of immunoreactive insulin and C-peptide (connecting peptide) immunoreactivity, raising the possibility that endogenous insulin might be responsible for the fall in NScAMPP that followed the initial spike. In the diabetics (n=8), basal hepatic vein cAMP concentration was 24.7+/-1.2 nM and NScAMPP was undetectable. Glucagon stimulated NScAMPP in five diabetics to a peak of 169.9+/-42.6 with a subsequent fall to 17.4+/-3.9 nmol/min by 90 min even though endogenous insulin secretion was not stimulated (no rise in C-peptide immunoreactivity). Although the mean increase in NScAMPP was greater in the diabetics, the two groups did not differ significantly.Conclusions. In normal resting man the liver is a significant source of circulating cAMP. Diabetics do not release abnormally large amounts of hepatic cAMP under basal conditions. Glucagon markedly enhances hepatic cAMP release with a spike-decline pattern in both normal and diabetic men. The decline in hepatic cAMP release despite continuing glucagon stimulation is due to factors other than a stimulation of insulin secretion.

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.