Effects of glucagon and pentagastrin on glibenclamide-induced insulin release

It was recently reported that administration of sulphonylureas may lead to a stimulation of the release of glucagon and a gastrin-like peptide. These peptides may then eventually influence the insulin releasing action of the sulphonylureas. Therefore, the effects of glucagon and pentagastrin on basal and glibenclamide-induced insulin secretion were studied. It was found that at a dose of 4.25 nmol/kg the two peptides induced a significant stimulation of basal insulin secretion. However, pentagastrin at the dose of 4.25 nmol/kg did not influence the insulin release induced by glibenclamide, whereas an equimolar dose of glucagon potentiated the insulin secretory response to this sulphonylurea drug by about 40%. Glucagon in contrast to pentagastrin thus positively modulates the insulin secretory pathway stimulated by the sulphonylurea drug glibenclamide.     

Different effects of glibenclamide and the structural analogue HB 699 on the 45Ca2+ uptake by ob/ob-mouse islets

The effects on 45Ca2+ uptake of HB 699, an acyl-amino-alkyl benzoic acid derivative, was compared to those of glibenclamide in incubations using the La3+ wash technique. HB 699 enhanced the 45Ca2+ net uptake in a concentration range (10-200 microM) where insulin release was also stimulated. Glibenclamide showed maximum stimulation of 45Ca2+ net uptake already at 1 microM. HB 699 did not clearly stimulate the short-term 45Ca2+ uptake whether or not the islets were preincubated with the drug. It is suggested that HB 699-induced insulin release is mediated, at least partly, by increased mobility of beta-cell Ca2+.     

Application of (13)C-filtered (1)H NMR to evaluate drug action on gluconeogenesis and glycogenolysis simultaneously in isolated rat hepatocytes

The effects of two inhibitors of hepatic glucose production, AICAR (5-aminoimidazole-4-carboxamide riboside) and metformin, whose precise mechanisms of action are a matter of some controversy, have been investigated in isolated rat hepatocytes by application of a novel NMR-based method whereby effects on metabolic flow from the two glucose-producing pathways, glycogenolysis and gluconeogenesis, and also lactate production, can be studied simultaneously. Hepatocytes were pre-incubated for 24 h with 15 mM 1-(13)C-glucose to load the cells with labeled glycogen, which under subsequent glycogenolytic conditions would yield predominantly 1-(13)C glucose and 3-(13)C-lactate, followed (after washing) by incubation in media with 2-(13)C-glycerol, which under subsequent gluconeogenic conditions would yield 2,5-(13)C-glucose, or if metabolized to lactate, 2-(13)C-lactate. Glucose production was then stimulated by glucagon for 3 h in the absence or presence of the inhibitors and then incubation media were analyzed by (13)C-HSQC (heteronuclear single quantum coherence)-filtered (1)H NMR spectra. The results show that metformin only inhibits glucose production by inhibition of gluconeogenesis, but also that it increases lactate production from both glycogenolysis and from glycerol, whereas, and contrary to expectations, AICAR inhibits glucose production by inhibiting both gluconeogenesis and glycogenolysis, and also increases lactate production from glycerol. The data show that application of this methodology can be used to answer important questions about drug action on hepatic metabolism that are not readily accessible by alternative means.     

Effects of PYY and PP on endocrine pancreas

Novel intestinal polypeptide YY (PYY) and pancreatic polypeptide (PP) were infused in fed anaesthetized rats. The peptides (10 and 100 pmol/kg · min) were administered during 30 min, alone, together with glucose or together with arginine. Plasma concentrations of glucose, insulin and glucagon were measured. At the dose of 10 pmol/kg · min the peptides had no effect. PP at the dose of 100 pmol/kg · min slightly augmented basal, but had no effect on stimulated insulin and glucagon release. PYY at the dose of 100 pmol/kg · min was without effect on basal insulin and glucagon levels and on glucose-induced insulin release, but exerted an inhibitory effect on arginine-induced secretion of both insulin and glucagon. It is unlikely that PYY and PP can affect secretion of insulin and glucagon via blood circulation. The potential capability of high doses of PP to affect insulin and glucagon secretion suggests that this peptide may exert direct (paracrine) effects on the pancreatic A-and B-cells     

Effects of glucagon and insulin on net hepatic metabolism of glucose precursors in sheep.

The net hepatic metabolism of amino glycerol, lactate, and pyruvate was determined in conscious fed sheep by multiplying the venoarterial concentration differences by the hepatic blood or plasma flow. In each experiment several sets of control blood samples were taken; glucagon or insulin then was infused intraportally for 2 h during which additional samples were taken. Four types of experiments were performed: 1) glucagon infusion (150 mug/h) into normal sheep, 2) glucagon infusion (100 mug/h) into insulin-treated alloxanized sheep, 3) insulin infusion (1.17 U/h) into normal sheep, and 4) insulin plus glucose infusion (12.3 mmol/h) into normal sheep. The second group of experiments was performed to prevent reflex hyperinsulinemia, and the fourth was performed to prevent reflex hyperglucagonemia. Glucagon directly stimulated the net hepatic uptake of alanine, glycine, glutamine, arginine, asparagine, threonine, serine, and lactate. Glucagon also stimulated lipolysis in adipose tissue. Insulin, on the other hand, appeared to have a lipogenic effect on adipose tissue and to stimulate directly the uptake of valine, isoleucine, leucine, tyrosine, lysine, and alanine only at extrahepatic sites. The study showed that, in sheep, the effects of glucagon primarily are on liver, and insulin's effects primarily are on skeletal muscle and adipose tissue where it promotes protein and lipid synthesis.     

Role of gluconeogenesis in epinephrine-stimulated hepatic glucose production in humans

To evaluate the contribution of gluconeogenesis to epinephrine-stimulated glucose production, we infused epinephrine (0.06 micrograms X kg-1 X min-1) for 90 min into normal humans during combined hepatic vein catheterization and [U-14C]alanine infusion. Epinephrine infusion produced a rise in blood glucose (50-60%) and plasma insulin (30-40%), whereas glucagon levels increased only at 30 min (19%, P less than 0.05). Net splanchnic glucose output transiently increased by 150% and then returned to base line by 60 min. In contrast, the conversion of labeled alanine and lactate into glucose increased fourfold and remained elevated throughout the epinephrine infusion. Similarly, epinephrine produced a sustained increase in the net splanchnic uptake of cold lactate (four- to fivefold) and alanine (50-80%) although the fractional extraction of both substrates by splanchnic tissues was unchanged. We conclude that a) epinephrine is a potent stimulator of gluconeogenesis in humans, and b) this effect is primarily mediated by mobilization of lactate and alanine from extrasplanchnic tissues. Our data suggest that the initial epinephrine-induced rise in glucose production is largely due to activation of glycogenolysis. Thereafter, the effect of epinephrine on glycogenolysis (but not gluconeogenesis) wanes, and epinephrine-stimulated gluconeogenesis becomes the major factor maintaining hepatic glucose production.     

Effects of short-term and prolonged infusions of somatostatin on endocrine pancreas, body weight and food intake in rats

Infusion of cyclic somatostatin (700 mg/kg/min) for 4 h in rat fed and libitum suppressed basal insulin but not glucagon release. It was accompanied by hypoglycemia during the first hour whereas, at the end of the infusion, hyperglycemia was present. The same dose of somatostatin applied 60 min prior to and during a 30 min load of glucose or arginine significantly inhibited their effects on insulin and glucagon release. In contrast, when this dose of somatostatin was given during a 24 h period by the i.v. route it did not inhibit glucose induced insulin release though circulating somatostatin levels were constantly and markedly elevated. Furthermore, in rats continuously infused with somatostatin for 4 days, no effect was found either on plasma concentrations of glucose, insulin, glucagon, growth hormone and cyclic AMP, or on body weight gain, food consumption or water intake. The pancreases of these animals showed normal concentrations of insulin and glucagon and a normal nuclear area of D-cells. Our experiments demonstrate that, in short-term experiments in rats, somatostatin influences insulin and glucagon release as well as glucose homeostasis. Furthermore, they suggest that during prolonged i.v. administration of somatostatin, rats develop mechanisms counteracting the effect of the peptide, e.g., peripheral tachyphylaxis.     

Increased glucagon secretion in protein-fed rats: lack of relationship to plasma amino acids.

This investigation was designed to test the hypothesis that protein feeding stimulated glucagon secretion because amino acids liberated during protein digestion function as glucagon secretagogues. Rats were fed high-protein (HP) or control diets for 9--10 days and blood taken from the aorta or portal vein (PV) at 0800, 1300, 1700, 1900, 2100, and 2300 for determination of amino acids, glucose, insulin, and glucagon. Glucose, insulin, and glucagon of control rats showed little change. In HP rats, PV glucose rose during fasting (0800-1700) and declined during feeding (1700-0800), changes that reflected alterations of glucagon and insulin secretion. PV glucagon in HP rats that was elevated 2--4 times rose during fasting, whereas PV and arterial amino acids declined. HP feeding caused enhanced glucagon release that was associated with increased amino acids in PV and arterial plasma, especially the branched-chain group. Although these findings suggest that protein feeding promotes glucagon release because branched-chain amino acids are elevated, these amino acids are known to have little effect on alpha cell function. Thus, we conclude that protein feeding influences glucagon secretion through some mechanism other than increased blood amino acid levels.     

Effect of leukocytic endogenous mediators on endocrine pancreas secretory responses.

Crude mediators from stimulated rabbit peritoneal leukocytes (LEM) engender numerous physiologic alterations in rats, which are similar to those observed during infection. One hour after the intraperitoneal injection of crude LEM, plasma insulin and glucagon concentrations are elevated; at 2 h the hormonal alterations are manifested by a 30% increase in hepatic cyclic adenosine 3',5'-monophosphate (cAMP), glycogen depression, and uptake of 14C-labeled nonmetabolizable amino acid analogues (AA). Plasma hormone concentrations reach maximum levels by 5 h and decline by 24 h. The hepatic concentrations of AA parallel the insulin and glucagon responses and correlate with the inverse of insulin/glucagon molar ratio. In spite of mobilization of hepatic glycogen evident at 5 h, plasma glucose concentrations were transiently depressed. Plasma insulin, glucagon, and hepatic AA concentrations were dose dependent. Plasma insulin and glucagon responses to crude LEM may explain increases in hepatic cAMP, uptake of AA, and glycogenolysis as well as hypoglycemia. These data partially characterize the role of crude LEM, provide an explanation for the stimuli-inducing hyperglucagonemia and hyperinsulinemia during infection. They implicate the endocrine pancreas as a factor regulating the host's metabolic response to infection.     

Sulphonylurea (glubenclamide) enhances somatostatin and inhibits glucagon release induced by arginine

Arginine significantly stimulated the release of insulin, glucagon and somatostatin from the isolated perfused rat pancreas. A sulphonylurea, glibenclamide, markedly enhanced the effect of arginine on somatostatin release and inhibited its effect on glucagon release. Insulin release was not modulated by addition of glibenclamide. These findings support the idea of a paracrine interaction of islets hormones.     

Effect of vasoactive intestinal polypeptide on glycogen metabolism in rat hepatocytes

The effects of vasoactive intestinal polypeptide (VIP) on several enzymes of glycogen metabolism in rat hepatocytes were compared with those of glucagon and of vasopressin (ADH). VIP caused phosphorylase activation and glycogenolysis in hepatocytes from fed rats. In hepatocytes from fasted rats incubated with glucose, lactate, and pyruvate, VIP inhibited net glycogen deposition, inactivated glycogen synthase, and activated phosphorylase. VIP was about 100-fold less potent than glucagon and 1,000-fold less potent than ADH in causing activation of phosphorylase. The ability of VIP to activate phosphorylase was not altered by chelation of the calcium in the medium. The half maximal effective doses of VIP for both phosphorylase activation and stimulation of glycogenolysis were 10-30 nM. Treatment with VIP, ADH, or glucagon did not decrease phosphorylase phosphatase activity. Each of these hormones, however, lengthened the lag time before synthase phosphatase activity was expressed in vitro. Other gut hormones tested did not affect hepatocyte glycogen metabolism. These results do not support the concept of physiologic control of hepatic glycogen metabolism by VIP or by other gut hormones.     

Compartmentation of glycolysis and glycogenolysis in the perfused rat heart

Developing methods that can detect compartmentation of metabolic pathways in intact tissues may be important for understanding energy demand and supply. In this study, we investigated compartmentation of glycolysis and glycogenolysis in the isolated perfused rat heart using (13)C NMR isotopomer analysis. Rat hearts previously depleted of myocardial glycogen were perfused with 5.5 mm [U-(13)C]glucose plus 50 mU/mL insulin until newly synthesized glycogen recovered to new steady-state levels ( approximately 60% of pre-depleted values). After a short wash-out period, the perfusate glucose was then switched to [1-(13)C]glucose, and glycolysis and glycogenolysis were stimulated by addition of glucagon (1 microg/ml). A (13)C NMR multiplet analysis of the methyl resonance of lactate provided an estimate of pyruvate derived from glucose vs glycogen while a multiplet analysis of the C4 resonance of glutamate provided an estimate of acetyl-CoA derived from glycolytic pyruvate vs glycogenolytic pyruvate. These two indices were not equivalent and their difference was further magnified in the presence of insulin during the stimulation phase. These combined observations are consistent with functional compartmentation of glycolytic and glycogenolytic enzymes that allows pyruvate generated by these two processes to be distinguished at the level of lactate and acetyl-CoA.     

The effect of different diets and of insulin on the hormonal response to prolonged exercise

The importance of carbohydrate availability during exercise for metabolism and plasma hormone levels was studied. Seven healthy men ran on a treadmill at 70% of individual maximal oxygen uptake having eaten a diet low (F) or high (CH) in carbohydrate through 4 days. At exhaustion the subjects were encouraged to continue to run while glucose infusion increased plasma glucose to preexercise levels. Forearm venous blood, biopsies from vastus muscle and expiratory gas were analyzed. Time to exhaustion was longer in CH- (106 +/- 5 min (S.E.)) than in F-expts. (64 +/- 6). During exercise, overall carbohydrate combustion rate, muscular glycogen depletion and glucose and lactate concentrations, carbohydrate metabolites in plasma, and estimated rate of hepatic glucose production were higher, fat metabolites lower, and the decrease in plasma glucose slower in CH- than in F-expts. Plasma norepinephrine increased and insulin decreased similarly in CH- and F-expts., whereas the increase in glucagon, epinephrine, growth hormone and cortisol was enhanced in F-expts. Glucose infusion eliminated hypoglycemic symptoms but did not substantially increase performance time. During the infusion epinephrine decreased markedly and glucagon even to preexercise levels. Infusion of insulin (to 436% of preexercise concentration) in addition to glucose in F-expts. did not change the plasma levels of the other hormones more than infusion of glucose only but reduced fat metabolites in plasma. At exhaustion muscular glycogen depletion was slow, and the glucose gradient between plasma and sarcoplasma as well as the muscular glucose 6-phosphate concentration had decreased. Conclusions: The preceding diet modifies the energy depots, the state of which (as regards size, receptors and enzymes) is of prime importance for metabolism during prolonged exercise. Plentiful carbohydrate stores favor both glucose oxidation and lactate production. During exercise norepinephrine increases and insulin decreases independent of plasma glucose changes whereas receptors sensitive to glucose privation but not to acute changes in insulin levels enhance the exercise-induced secretion of glucagon, epinephrine, growth hormone and cortisol. Abolition of cerebral hypoglycemia does not inevitably increase performance time, because elimination of the hypoglycemia may not abolish muscular energy lack.     

Effects of somatostatin on glucagon-stimulated glycogenolysis and gluconeogenesis in hepatocytes cultured in vitro

The effects of somatostatin (SS-14) on glycogenolysis and gluconeogenesis in rat hepatocytes cultured in vitro in a serum-free medium were investigated. Somatostatin (122 nmol 1^-1) did not significantly change the basal glucose production with or without pyruvate (10 mmol 1^-1). Glucagon strongly (over 100%) increased the glucose production in hepatocytes incubated in a medium supplemented with 10 mmol 1^-1 pyruvate. This increase in glucose production is the result of increased rates of gluconeogenesis and glycogenolysis. Somatostatin partially inhibited the glucagon stimulated increase in glucose production. Glucagon also significantly increased the glucose production in a glucose-free medium without pyruvate, which resulted from an increase of glycogenolysis. Somatostatin did not inhibit the increase in glucose production in these conditions. After a 4 h ‘fast’, glycogen in hepatocytes fell to a very low level. Glucose production was minimal. After the addition of pyruvate, there was a increase in gluconeogenesis and glucose production. Glucagon stimulated the rate of gluconeogenesis. Somatostatin completely inhibited this glucagon-stimulated increase in gluconeogenesis.     

Effects of somatostatin on glucagon-stimulated glycogenolysis and gluconeogenesis in hepatocytes cultured in vitro.

The effects of somatostatin (SS-14) on glycogenolysis and gluconeogenesis in rat hepatocytes cultured in vitro in a serum-free medium were investigated. Somatostatin (122 nmol l-1) did not significantly change the basal glucose production with or without pyruvate (10 mmol l-1). Glucagon strongly (over 100%) increased the glucose production in hepatocytes incubated in a medium supplemented with 10 mmol l-1 pyruvate. This increase in glucose production is the result of increased rates of gluconeogenesis and glycogenolysis. Somatostatin partially inhibited the glucagon stimulated increase in glucose production. Glucagon also significantly increased the glucose production in a glucose-free medium without pyruvate, which resulted from an increase of glycogenolysis. Somatostatin did not inhibit the increase in glucose production in these conditions. After a 4 h 'fast', glycogen in hepatocytes fell to a very low level. Glucose production was minimal. After the addition of pyruvate, there was a increase in gluconeogenesis and glucose production. Glucagon stimulated the rate of gluconeogenesis. Somatostatin completely inhibited this glucagon-stimulated increase in gluconeogenesis.     

Vitamin D3 stimulates calcium-45 uptake by isolated mouse islets in vitro

Islets isolated from ob/ob mice which had been fed a vitamin D-deficient diet released significantly less insulin in response to glucose than did vitamin D-replete islets but showed normal net 45Ca2+ uptake. To determine whether vitamin D3 has a direct effect on the pancreatic B cell, islets from ob/ob mice on a normal diet were exposed to vitamin D3 in vitro for 1 week or only 3 h, and then glucose-stimulated 45Ca2+ uptake and insulin release were measured. Exposure to 1 nM or 1 microM vitamin D3 for 1 week stimulated 45Ca2+ uptake in the presence of 3 mM, but not 20 mM glucose, and did not affect insulin release. Exposure to vitamin D3 for 3 h did not significantly increase net 45Ca2+ uptake although there was a tendency to such an effect (P = 0.10). In conclusion, vitamin D-deficiency in vivo suppressed subsequent glucose-stimulated insulin release in vitro and this effect may be due to a direct effect of the sterol (or one of its metabolites) on calcium handling by the B cell.     

Impaired insulin-dependent glucose metabolism in granulosa-lutein cells from anovulatory women with polycystic ovaries

BACKGROUND: Insulin resistance and hyperinsulinaemia are well-recognized characteristics of anovulatory women with polycystic ovary syndrome (PCOS) but, paradoxically, steroidogenesis by PCOS granulosa cells remains responsive to insulin. The hypothesis to be tested in this study is that insulin resistance in the ovary is confined to the metabolic effects of insulin (i.e. glucose uptake and metabolism), whereas the steroidogenic action of insulin remains intact. METHODS: Granulosa-lutein cells were obtained during IVF cycles from seven women with normal ovaries, six ovulatory women with PCO (ovPCO) and seven anovulatory women with PCO (anovPCO). Mean body mass index was in the normal range in all three groups. Granulosa-lutein cells were cultured with insulin (1, 10, 100 and 1000 ng/ml) and LH (1, 2.5 and 5 ng/ml). Media were sampled at 24 and 48 h and analysed for glucose uptake, lactate production and (48 h only) progesterone production. RESULTS: Insulin-stimulated glucose uptake by cells from anovPCO was attenuated at higher doses of insulin (100 and 1000 ng/ml) compared with that by cells from either ovPCO (P=0.02) or controls (P=0.02). Insulin and LH stimulated lactate production in a dose-dependent manner, but insulin-dependent lactate production was markedly impaired in granulosa-lutein cells from anovPCO compared with either normal (P=0.002) or ovPCO (P<0.0001). By contrast, there was no difference in insulin-stimulated progesterone production between granulosa-lutein cells from the three ovarian types. CONCLUSIONS: Granulosa-lutein cells from women with anovPCOS are relatively resistant to the effects of insulin-stimulated glucose uptake and utilization compared with those from normal and ovPCO, whilst maintaining normal steroidogenic output in response to physiological doses of insulin. These studies support the probability of a post-receptor, signalling pathway-specific impairment of insulin action in PCOS.     

Effects of glucagon on hepatic glycogen and smooth endoplasmic reticulum

The action of glucagon on hepatic glycogen and smooth endoplasmic reticulum (SER) was studied in samples of liver taken sequentially from anesthetized rats. The physiological state of each animal was assessed by continuously monitoring aortic blood pressure and blood lactate/pyruvate ratios. High hepatic glycogen levels were established by using 10–12 hr fasted control-fed rats infused continuously with glucose. In rats receiving glucose only, hepatic glycogen levels remained above 5.0% during the 4-hr period of glucose administration. Centrilobular hepatocytes displayed an abundance of glycogen which often appeared dispersed with elements of SER between the glycogen particles. Periportal cells had dense clumps of glycogen with few vesicles of SER restricted to the periphery of the glycogen masses. The addition of glucagon to the glucose infusate caused a marked stimulation of glycogenolysis. In these rats, the hepatic glycogen level (x?±SE) was 6.71±.15% 1 hr after glucose and declined after initiation of glucagon infusion as follows: 5.86±.29% (15 min), 4.89±.26% (1 hr), 2.16±.40% (2 hr), and 1.66±.29% (3 hr). The fine structure of hepatocytes showed a dramatic response to the administration of glucagon. The glycogen regions of the cells were noticeably decreased in size and number of glycogen granules 3 hr after initiation of glucagon infusion, and SER was abundant in both periportal and centrilobular hepatocytes. The interpretation offered is that glucagon induces the formation of new SER membranes which participate in glycogen breakdown and/or glucose release from hepatocytes.     

Regulation of glucose production from lactate in experimental sepsis

Gluconeogenic and oxidative capabilities with lactate as a substrate were studied in perfused livers isolated from rats in late sepsis. Glucose release in the presence of 5 mM lactate was significantly depressed in livers from septic rats. When gluconeogenesis was stimulated by phenylephrine, livers from septic rats exhibited both a decreased sensitivity and lower maximal rate of glucose release when compared with livers from sham-operated rats. Oxygen consumption (VO2) by perfused livers from septic rats was also depressed under the above conditions. The addition of lysine in concentrations greater than 0.5 mM restored glucose production in livers from septic rats to a rate not different from sham-operated controls but did not restore VO2. However, inclusion of lysine (5 mM) in the perfusate was not able to restore sensitivity to stimulation by phenylephrine in livers from septic rats. Although hepatic ATP levels were depressed in sepsis, the decrease was not sufficient to explain the decreased rates of glucose production. We conclude from these results that primary cellular defects in gluconeogenic and oxidative capabilities occur during sepsis that are independent of inadequate perfusion.     

Hormonal and metabolic response to physical exercise,fasting and cold exposure in the rat

Summary Four groups of rats were subjected to the following conditions: (1) 48 h fasting, (2) 48 h of 4°C cold exposure, (3) 5 h treadmill running, (4) 48 h fasting with 4°C cold exposure. The groups were compared to fed control rats in order to study hormonal and metabolic responses in blood and tissue samples. Isolated hepatocytes were used to evaluate the rate of ketogenesis. Decreases in liver glycogen and increases in blood free fatty acids (FFA) confirmed that glycogenolysis and lipolysis occur in these situations of metabolic stress. Increases in the glucagon/insulin plasma ratio were also noted. Plasma catecholamine levels were only enhanced after running and after cold exposure. Production of blood ketone bodies was stimulated more by running and by fasting than by cold exposure. The low ketone body production observed after cold exposure seems to be linked to increases liver glycogen levels and decreased FFA availability. Liver cells isolated after cold exposure exhibited higher ketogenesis than these isolated after running. This difference in ketogenic capacity could result both from the longer hormonal stimulation by high glucagon/insulin plasma ratios and from the metabolic state of the liver.