Chandra Mohan

The intracellular site of action of insulin: the mitochondrial Krebs cycle.

Publisher Summary This chapter discusses the intracellular site of action of insulin involving the mitochondrial Krebs cycle. To begin with, there is at present no coherent model that accounts, through the process of partial phosphorylation or dephosphorylation of enzymes or other proteins, for all of the manifold actions of insulin on cells. It is known that insulin stimulates the uptake of glucose most effectively in fat cells in which mitochondria are close to the plasma membrane. Also, much evidence has accumulated to show that insulin reverses the net catabolism of protein, glycogen, and fat and promotes their build up and storage. Furthermore, insulin stimulates the incorporation into protein of primarily those pyruvate carbons that are converted to glutamic acid through the Krebs cycle. Insulin has a far greater stimulatory effect on the incorporation into protein of the methylene carbons of succinate than the carboxyl carbons. This can be attributed to the fact that carbons 2 and 3 of succinate either reenter the cycle as acetyl-CoA or remain in the mitochondrial Krebs cycle for a second or third opportunity to be converted to glutamate by the transamination of α-KG.

Inhibition of protein and lipid synthesis in muscle by 2,4-dinitrofluorobenzene, an inhibitor of creatine phosphokinase.

The incorporation of [3H]-valine into protein and [14C]-acetate into lipid was measured in rat diaphragm and hepatocytes after pretreatment of the tissue with 2,4-dinitrofluorobenzene (FDNB), an inhibitor of creatine phosphokinase (CPK) (EC2.7.3.2). The activity of CPK was also measured. Increasing concentrations of FDNB inhibited protein and lipid synthesis in muscle in parallel with the inhibition of CPK activity. In hepatocytes, which have little CPK activity, similar concentrations of FDNB had no effect on protein synthesis and little effect on lipid synthesis. The possible role of CPK and the creatine phosphate shuttle in muscle metabolism is discussed.

Insulin as a probe of mitochondrial metabolism in situ

Our previous studies of insulin action have led us to the finding that insulin acts specifically on the mitochondrial Krebs cycle to stimulate, by 30%, the oxidation of carbons 2 and 3 of pyruvate to CO2. Insulin also stimulates the oxidation of both carbons of acetate. These carbons can be converted to CO2 only after passing through all of the reactions of the Krebs cycle more than once. Carboxyl groups, such as number 1 of pyruvate, are oxidized to CO2 without any effect of insulin, and can be converted to CO2 by extramitochondrial enzyme. We conclude that insulin must act on the complete intramitochondrial cycle and not on the four enzymes of the Krebs cycle which are present in the cytoplasm. The path taken by those carbons affected by insulin is traced through the complete Krebs cycle, and the necessity for this effect to be mitochondrial has been verified by demonstration of the same specific effect of insulin on the oxidation of the 2 and 3 carbons of succinate. The use of this phenomenon is proposed for the study not only of human diabetes, but of all mitochondrial disorders, by using 14C specifically labeled tracers in culture or biopsy material, or 13C labeled tracer material in vivo. (Mol Cell Biochem 174: 91–96, 1997)

Insulin “inhibition” of gluconeogenesis by stimulation of protein synthesis

Abstract The effect of insulin on gluconeogenesis in diabetic rabbits has been studied by measurement of both pool size and specific activity of alanine, lactate, glucose, and protein during the infusion of labeled alanine, acetate, glycerol, and lactate. Insulin administration causes an immediate net drop in the pool sizes of alanine, lactate, glucose, pyruvate, and glycerol. The radioactivity of tracer alanine is found to be shunted from glucose to protein on insulin administration concurrent with the fall in the plasma levels of three-carbon compounds measured. This is consistent with the net pool size experiments and suggests that the major carbon flow is directed away from glucose toward protein synthesis by insulin. The recovery of most of the counts from alanine which did not go into glucose in protein also supports the proposition that the major effect of insulin on gluconeogenesis is to stimulate protein synthesis. Further experiments with [14C]acetate which can be only incorporated into glucose through the activity of PC and PEPCK show that although its incorporation into protein is increased significantly by insulin its incorporation into glucose is unaffected. All these results are consistent with the conclusion that insulin “inhibits” gluconeogenesis by stimulating resynthesis of amino acids into protein before they are converted to glucose. In this way insulin modulates hyperglycemia induced by gluconeogenetic hormones.

Circadian Rhythms in the Central Cholinergic System in Aging Animals

A number of changes in cyclically varying parameters during aging have been reported. These have included phase shifts in the diurnal distribution of spontaneous locomotor activity of rats (Mohan, 1975), mitotic index in the rat ear epithelium (Bullough, 1949), body temperature (Yunnis et al., 1974), urinary excretion of catecholamines (Descovich et al., 1974), eosinophil count (Halberg et al., 1955) and several other physiological parameters (Scheving et al., 1974).

Effect of insulin on the metabolic distribution of carbons 1, 2, and 3 of pyruvate

It has long been known that the carbons of pyruvate are converted to CO2 at different points in the metabolic process. This report deals with the observation that insulin affects the oxidation of carbons 2 and 3 primarily and has little effect on the oxidation of the carboxyl carbon. Oxidation of different carbons of pyruvate and their incorporation into various metabolic components was studied in isolated rat hepatocytes. Insulin stimulated the 14CO2 production from [2-14C]- and [3-14C]pyruvate and from [U-14C]alanine. However, it had little or no effect on the activity of the pyruvate dehydrogenase complex as measured by the evolution of 14CO2 from [1-14C]pyruvate or [1-14C] alanine. Insulin also stimulated the incorporation of carbons 2 and 3 of pyruvate into protein but had no effect on the incorporation of carbon 1. Incorporation of [1-14C]- and [U-14C]alanine into protein was differentially enhanced by insulin in a manner similar to that of the pyruvate carbons. The fact that insulin stimulates the incorporation of [1-14C]alanine into protein but not [1-14C]pyruvate suggests the possibility of a compartmentation of pyruvate metabolism in the isolated hepatocytes. These studies show that the stimulation of [2-14C]- and [3-14C]pyruvate incorporation into protein involves the stimulatory effect of insulin on the activity of the Krebs cycle which is evident from the fact that insulin did not stimulate the pyruvate carbons to enter protein via alanine but the incorporation via glutamate was increased by about 40%.

Differential effects of insulin, epinephrine, and glucagon on rat hepatocyte mitochondrial activity.

Oxidation of [2,3-14C]succinate carbons in the mitochondrial Krebs cycle was used as a probe to investigate the effects of insulin, epinephrine, glucagon, and 2,4-dinitrophenol (2,4-DNP) on isolated rat hepatocytes. Epinephrine, glucagon, and 2,4-DNP had a far greater stimulatory effect on 14CO2 formation from [2,3-14C]succinate than insulin. Unlike insulin, epinephrine and glucagon had no significant effect on the anabolic utilization of succinate carbons for protein synthesis. Our results suggest that although epinephrine, glucagon, and 2,4-DNP enhance the movement of tracer carbons through the Krebs cycle, only insulin is capable of enhancing amphibolite utilization for protein synthesis.

In vitro protein degradation measured by differential loss of labeled methionine and 3-methylhistidine: The effect of insulin

A new technique for studying the effect of insulin on protein degradation is reported. The method is based on measuring the parallel release of a reutilizable and a nonreutilizable amino acid from muscle protein. Animals are prelabeled in vivo with [Me-3H]methionine which labels both the nonreutilizable 3-methylhistidine and the reutilizable methionine of tissue protein. The data presented show that insulin has only a trivial effect on the loss of 3-methylhistidine from muscle protein, while it substantially diminishes the efflux of methionine. The analysis of muscle protein confirms the observation that insulin causes the reincorporation of methionine and has a minimum effect on the loss of 3-methylhistidine. This supports the view that the major inhibitory effect of insulin on gluconeogenesis is the diversion of the flow of amino acids away from the gluconeogenesis pathway back toward protein synthesis.

Lack of insulin stimulation on Percoll-prepared or high-speed-centrifuged rat liver hepatocytes

Rat liver hepatocytes isolated from a 30-31% percoll density gradient at 10,000g are refractory toward insulin stimulation of 14CO2 formation and 14C-incorporation into protein from [2,3-14C]succinate. Basal hepatocyte oxidation of succinate was not impaired by the presence of 5% percoll in the incubation medium nor was it impaired when percoll-free hepatocytes were used that had been isolated after centrifugation at 9000g; however, in both instances the stimulatory effect of insulin was lost. Hepatocyte damage may have occurred in these processes. This is in contrast to previous work which shows that insulin (10 mU/ml) will stimulate [2,3-14C]succinate oxidation and [2,3-14C]succinate carbon incorporation into protein in non-percoll-treated hepatocytes (isolated by centrifugation at 10g) by about 29%. We conclude that the latter procedure although more time consuming is the more gentle method of choice and leaves the hepatocyte in a form more closely related to an in vivo state than does treatment with a percoll density gradient at 10,000g.

Ammonia Inhibits Insulin Stimulation of the Krebs Cycle: Further Insight Into Mechanism of Hepatic Coma*

Oxidation of [2,3-14C]succinate in the intramitochondrial Krebs cycle was used as a probe to investigate the effect of ammonia on protein incorporation and Krebs cycle oxidation of succinate carbons in isolated rat hepatocytes. At low concentrations of ammonium chloride (0.1 to 0.5 mM) a slight increase in14CO2 formation from [2,3-14C]succinate was observed, however, the stimulatory effect of insulin was significantly reduced. Insulin failed to cause any stimulation of succinate carbons incorporation into hepatocyte protein in the presence of ammonium chloride. Addition of ammonium chloride also depressed the movement of tracer carbons into the gluconeogenesis pathway. The activity of the amphibolic amino acid pool was significantly enhanced by ammonia. The data presented in this paper lend strong support to the Krebs-cycle depletion theory of hepatic coma. They also suggest that reduced mitochondrial Krebs cycle activity caused by increased amphibolic depletion of substrates results in loss of insulin sensitivity in ammonia toxicity.