S.R. Wagle

Studies on the inhibition of insulin release, glycogenolysis and gluconeogenesis by somatostatin in the rat islets of Langerhans and isolated hepatocytes

Abstract The effect of somatostatin on insulin release, glycogenolysis and gluconeogenesis was studied in isolated islets of Langerhans and hepatocytes. Addition of somatostatin (0.2 μg – 100 μg) to isolated islets of Langerhans inhibited insulin release from 30 to 90 percent. Studies with isolated hepatocytes showed that somatostatin inhibited both glucagon-stimulated glycogenolysis and gluconeogenesis by 40–50 percent, whereas it had no effect on epinephrine-stimulated glycogenolysis.

Studies on the effects of insulin and acetylcholine on activation of glycogen synthase and on glycogenesis in hepatocytes isolated from normal fed rats

Abstract The addition of insulin (4.0 × 10 −11 M) or acetylcholine (10 −6 M) to isolated hepatocytes stimulated glycogen accumulation and this stimulation was more pronounced when the medium glucose was raised from 50 to 300 mg percent. Studies with [ 14 C]-glucose showed a two-fold stimulation in glycogen synthesis by the addition of insulin (4.0 × 10 −11 M) or acetylcholine (10 −6 M). A sixteen percent increase in the activity of glycogen synthase was observed in cells incubated for 10 minutes with insulin (4.0 × 10 −11 M) or acetylcholine (10 −6 M), whereas at one hour incubation a 40 percent increase in activity was observed with the same concentration of insulin or acetylcholine. The effects of insulin and acetylcholine were not additive.

Studies on the in vitro effects of insulin on glycogen synthesis and ultra-structure in isolated rat liver hepatocytes☆

Abstract Rat liver hepatocytes were isolated by collagenase in vitro perfusion technique and effect of insulin on glycogen synthesis and ultra-structure was studied. Addition of insulin stimulated glycogen synthesis and maintained better cellular structure. Synthesis of glycogen was linear in isolated hepatocytes when incubated with various concentrations of glucose (0–800 mg%) reaching initial levels. Concanavaline A inhibited epinephrine stimulated glycogenolysis but had no effect on glucagon stimulated glycogenolysis. These studies indicate that insulin is required for glycogen synthesis and for maintaining hepatocytes ultrastructure. Furthermore, isolated hepatocytes retain various receptors and that different hormones utilize different receptor sites.

Stimulation of glycogenolysis by epinephrine and glucagon and its inhibition by insulin in isolated rat liver hepatocytes

Abstract Rat liver hepatocytes were isolated by collagenase in vitro perfusion technique and the effect of epinephrine, glucagon and insulin on glycogenolysis was studied. Both glucagon and epinephrine at the concentration of 10−6M, stimulated gluconeogenesis by 80–100%. Addition of insulin (33 μUnits/ml) completely abolished the epinephrine-stimulated glycogenolysis whereas only 50% inhibition was observed with insulin in glucagon stimulated glycogenolysis. This stimulation was observed within 2–5 min after the addition of the hormones. These results suggest that hepatocytes isolated with low concentrations of collagenase retain glucagon, epinephrine and insulin receptor sites.

Somatostatin: a metabolic regulator

Somatostatin, the hypothalamic release-inhibiting factor, has been found to stimulate gluconeogenesis in rat kidney cortical slices. Stimulation by somatostatin was linear and dose-dependent. Other bioactive peptides such as cholecystokinin, gastrointestinal peptide, secretin, neurotensin, vasoactive intestinal peptide, pancreatic polypeptide, beta endorphin and substance P did not affect the renal gluconeogenic activity. Somatostatin-induced gluconeogenesis was blocked by phentolamine (alpha adrenergic antagonist) and prazosin (alpha1 adrenergic antagonist) but not by propranolol (beta adrenergic antagonist) and yohimbine (alpha2 adrenergic antagonist) suggesting that the effect is via alpha1 adrenergic stimuli. Studies on the involvement of Ca2+ revealed that tissue depletion and omission of Ca2+ from the reaction mixture would abolish the stimulatory effect of somatostatin. Furthermore, somatostatin enhanced the uptake of 45calcium in renal cortical slices which could be blocked by lanthanum, an inhibitor of Ca2+ influx. It is proposed that the stimulatory effect of somatostatin on renal gluconeogenesis is mediated by alpha1 adrenergic receptors, or those which functionally resemble alpha1 receptors and that the increased influx of Ca2+ may be the causative factor for carrying out the stimulus.

Somatostatin inhibition of glucagon-stimulated adenosine 3'-5'-monophosphate accumulation in isolated hepatocytes.

Summary The cyclic form of somatotropin release inhibitor factor over a concentration range from 0.50 to 8.0 μg/ml was found to inhibit glucagon-stimulated cyclic AMP accumulation in isolated rat hepato-cytes. The authors are grateful to Nancy Bridgforth, Ann Miller, Sue Wills, and Lucinda Carr for skilled technical assistance and to Deborah Barr and Carol Grimme for typing of the manuscript. The authors are also very grateful to Dr. R. Deghenghi of Ayerst Laboratories, Montreal, Quebec, Canada, for the supply of cyclic form somatostatin, Lot No. Ay-24,910.

Interrelationship of insulin and glucagon ratios on carbohydrate metabolism in isolated hepatocytes containing high glycogen

Abstract The effect of physiological concentrations of glucagon and insulin on glycogenolysis was studied in the presence and absence of substrates in isolated hepatocytes containing high glycogen. In the absence of substrates glucagon stimulated glycogenolysis at 10−14M concentration, and addition of 100 μunits of insulin partially inhibited glucagon stimulated glycogenolysis (10−14M to 10−11M). However, in the presence of substrates, insulin completely inhibited glucagon stimulated glycogenolysis (10−14M to 10−11M), indicating that molar glucagon and insulin ratios control carbohydrate metabolism in liver. Additional studies showed incorporation of amino acid into protein was linear for only 3 to 4 hr in cells containing low glycogen, whereas in cells containing high glycogen, incorporation was linear for 8 to 10 hr.

Studies on Gluconeogenesis in Galactosamine Induced Hepatitis

Summary Hepatitis was induced in animals treated with D-GalN-HCl (1.5 g/kg) in vitro. Incorporation of alanine-UL-14C, pyruvate-2-14C, and glutamic-3,4-14C acid into glucose by liver slices from GalN-treated animals was found decreased to 20-25% of the control values. The CO2 production from these substrates was impaired by 50%. Activity of gluconeogenic enzymes was measured, PEP carboxykinase and pyruvate carboxylase exhibited the most important changes and FDPase and G-6-Pase were also significantly decreased but failed to respond to fasting. It is suggested that this GalN-induced hepatitis might be a good model to use to reproduce a study of hypoglycemia found sometimes in human viral hepatitis and other liver damage.

Interrelationship of somatostatin, insulin, and calcium in the control of gluconeogenesis in kidney cortex slices

Summary The effects of somatostatin and insulin on renal gluconeogenesis and the involvement of Ca ++ in somatostatin action were investigated. Addition of somatostatin stimulated gluconeogenesis in the renal cortex and this stimulation was inhibited by insulin. Maximum stimulatory effect by somatostatin was observed at 30 minutes. No stimulatory effect of somatostatin was observed in calcium depleted kidney cortex slices. However, addition of Ca ++ completely restored the stimulatory effect of somatostatin. These results suggest that insulin and somatostatin may be playing important physiological roles in renal gluconeogenesis and that Ca ++ is required for the somatostatin-induced enhancement in gluconeogenic activity.

Studies on the differential response to insulin on the stimulation of amino acid incorporation into protein in isolated hepatocytes containing different levels of glycogen

Abstract Effect of insulin on amino acid incorporation into protein by isolated rat liver hepatocytes was studied. A two to three-fold increase in the incorporation of U- 14 C-Leucine and U- 14 C-Phenylalanine into protein by insulin (100 μUnits) was observed in isolated hepatocytes containing high glycogen. This effect was abolished by the addition of glucagon (3 × 10 −6 M). No stimulation in amino acid incorporation by insulin was observed when isolated hepatocytes contained low or no glycogen. Electron micrographs of incubated cells show that in the presence of insulin more normal parallel strands of polyribosomes are maintained as compared to control cell preparation.