Leonard A. Fahien

Feasibility of Pathways for Transfer of Acyl Groups from Mitochondria to the Cytosol to Form Short Chain Acyl-CoAs in the Pancreatic Beta Cell

The mitochondria of pancreatic beta cells are believed to convert insulin secretagogues into products that are translocated to the cytosol where they participate in insulin secretion. We studied the hypothesis that short chain acyl-CoA (SC-CoAs) might be some of these products by discerning the pathways of SC-CoA formation in beta cells. Insulin secretagogues acutely stimulated 1.5–5-fold increases in acetoacetyl-CoA, succinyl-CoA, malonyl-CoA, hydroxymethylglutaryl-CoA (HMG-CoA), and acetyl-CoA in INS-1 832/13 cells as judged from liquid chromatography-tandem mass spectrometry measurements. Studies of 12 relevant enzymes in rat and human pancreatic islets and INS-1 832/13 cells showed the feasibility of at least two redundant pathways, one involving acetoacetate and the other citrate, for the synthesis SC-CoAs from secretagogue carbon in mitochondria and the transfer of their acyl groups to the cytosol where the acyl groups are converted to SC-CoAs. Knockdown of two key cytosolic enzymes in INS-1 832/13 cells with short hairpin RNA supported the proposed scheme. Lowering ATP citrate lyase 88% did not inhibit glucose-induced insulin release indicating citrate is not the only carrier of acyl groups to the cytosol. However, lowering acetoacetyl-CoA synthetase 80% partially inhibited glucose-induced insulin release indicating formation of SC-CoAs from acetoacetate in the cytosol is important for insulin secretion. The results indicate beta cells possess enzyme pathways that can incorporate carbon from glucose into acetyl-CoA, acetoacetyl-CoA, and succinyl-CoA and carbon from leucine into these three SC-CoAs plus HMG-CoA in their mitochondria and enzymes that can form acetyl-CoA, acetoacetyl-CoA, malonyl-CoA, and HMG-CoA in their cytosol.

Normal Thyroid Thermogenesis but Reduced Viability and Adiposity in Mice Lacking the Mitochondrial Glycerol Phosphate Dehydrogenase

The mitochondrial glycerol phosphate dehydrogenase (mGPD) is important for metabolism of glycerol phosphate for gluconeogenesis or energy production and has been implicated in thermogenesis induced by cold and thyroid hormone treatment. mGPD in combination with the cytosolic glycerol phosphate dehydrogenase (cGPD) is proposed to form the glycerol phosphate shuttle, catalyzing the interconversion of dihydroxyacetone phosphate and glycerol phosphate with net oxidation of cytosolic NADH. We made a targeted deletion inGdm1 and produced mice lacking mGPD. On a C57BL/6J background these mice showed a 50% reduction in viability compared with wild-type littermates. Uncoupling protein-1 mRNA levels in brown adipose tissue did not differ between mGPD knockout and control pups, suggesting normal thermogenesis. Pups lacking mGPD had decreased liver ATP and slightly increased liver glycerol phosphate. In contrast, liver and muscle metabolites were normal in adult animals. Adult mGPD knockout animals had a normal cold tolerance, normal circadian rhythm in body temperature, and demonstrated a normal temperature increase in response to thyroid hormone. However, they were found to have a lower body mass index, a 40% reduction in the weight of white adipose tissue, and a slightly lower fasting blood glucose than controls. The phenotype may be secondary to consequences of the obligatory production of cytosolic NADH from glycerol metabolism in the mGPD knockout animal. We conclude that, although mGPD is not essential for thyroid thermogenesis, variations in its function affect viability and adiposity in mice.

Glyceraldehyde Phosphate and Methyl Esters of Succinic Acid: Two “New” Potent Insulin Secretagogues

We discovered that two physiologically occurring metabolic intermediates, glyceraldehyde phosphate and succinate, are potent insulin secretagogues. No other glycolytic intermediate besides glyceraldehyde phosphate was insulinotropic. Succinate, when added to islets as either its monomethyl or dimethyl ester to increase its cellular permeability, was also insulinotropic. In islets, as in other cell types, these esters are apparently hydrolyzed intracellulary to succinate. Unesterified succinate and other unesterified citric acid–cycle intermediates did not stimulate insulin release. Initiation of insulin release by esters of succinate suggests that mitochondrial metabolism alone is sufficient to initiate and support insulin release. However, this is specific for succinate in that esters of fumarate, pyruvate, and citrate were not insulinotropic.

[117] l-glutamate dehydrogenase (frog and tadpole liver)

Publisher Summary This chapter describes the assay, purification, and properties of L-glutamate dehydrogenase. Frog-liver glutamate dehydrogenase can be conveniently prepared in crystalline form. The frog-liver enzyme is prepared from a mitochondrial fraction by solubilizing with detergent and fractionating and crystallizing with ammonium sulfate. After three recrystallizations, the enzyme is pure on the basis of polyacrylamide-gel electrophoresis and sedimentation experiments. The tadpole-liver enzyme has been purified several fold (specific activity equal to that of pure frog-liver enzyme) when a similar procedure is combined with chromatography on diethylaminoethyl (DEAE)-Sephadex. The rate of oxidation of diphosphopyridine nucleotide (DPNH) is measured spectrophotometrically at 340 m μ . The frog-liver enzyme is stable for several months when stored at 4° as a crystalline suspension. Many mono- and dicarboxylic L-amino acids are oxidized at significant rates by frog-liver glutamate dehydrogenase when dinitrophenyl (DPN) is the coenzyme. Adenosine diphosphate (ADP) and adenosine triphosphate (ATP) increase the rate of DPN reduction. ADP also has a similar effect on the rate of DPNH oxidation.