High level of ATP citrate lyase expression in human and rat pancreatic islets.

A comparison of an enzyme's level in pancreatic islets with its level in other body tissues can give clues about the importance of a metabolic pathway in the islets. ATP citrate lyase plays a key role in the pyruvate citrate shuttle, as well as for the synthesis of short chain acyl-CoAs and lipid, and its level in human and rat pancreatic islets relative to other tissues has not been previously reported. We compared the level of ATP citrate lyase mRNA and enzyme activity in pancreatic islets of humans and rats and the INS-1 832/13 cell line to levels in liver, a lipid synthesizing organ, and also kidney. The mRNA level was much higher in human islets and rat islets than in liver and kidney of the same genus and the enzyme activity was 8-fold and 12-fold higher in islets of humans and rats, respectively, compared to liver of the same genus. These data support other evidence that indicates ATP citrate lyase is important for the pyruvate citrate shuttle and lipid synthesis in insulin secretion.

Sphingosine kinase 1 knockdown reduces insulin synthesis and secretion in a rat insulinoma cell line.

To evaluate the role of sphingosine kinase 1 (SphK1) in insulin secretion, we used stable transfection to knock down the expression of the Sphk1 gene in the rat insulinoma INS-1 832/13 cell line. Cell lines with lowered Sphk1 mRNA expression and SphK1 enzyme activity (SK11 and SK14) exhibited lowered glucose- and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine-stimulated insulin release and low insulin content associated with decreases in the mRNA of the insulin 1 gene. Overexpression of the rat or human Sphk1 cDNA restored insulin secretion and total insulin content in the SK11 cell line, but not in the SK14 cell line. The Sphk1 cDNA-transfected SK14 cell line expressed significantly less SphK1 activity than the Sphk1 cDNA-transfected SK11 cells suggesting that the shRNA targeting SK14 was more effective in silencing the exogenous rat Sphk1 mRNA. The results indicate that SphK1 activity is important for insulin synthesis and secretion.

Decreased levels of metabolic enzymes in pancreatic islets of patients with type 2 diabetes.

AIMS/HYPOTHESIS: Glucose-stimulated insulin secretion is defective in patients with type 2 diabetes. We sought to acquire new information about enzymes of glucose metabolism, with an emphasis on mitochondrial enzymes, by comparing pancreatic islets of type 2 diabetes patients with those of non-diabetic controls. METHODS: Expression of genes encoding 13 metabolic enzymes was estimated with microarrays and activities of up to nine metabolic enzymes were measured. RESULTS: The activities of the mitochondrial enzymes, glycerol phosphate dehydrogenase, pyruvate carboxylase (PC) and succinyl-CoA:3-ketoacid-CoA transferase (SCOT) were decreased by 73%, 65% and 92%, respectively, in the diabetic compared with the non-diabetic islets. ATP citrate lyase, a cytosolic enzyme of the mitochondrial citrate pyruvate shuttle, was decreased 57%. Activities of propionyl-CoA carboxylase, NADP-isocitrate dehydrogenase, cytosolic malic enzyme, aspartate aminotransferase and malate dehydrogenase were not significantly different from those of the control. The low activities of PC and SCOT were confirmed with western blots, which showed that their protein levels were low. The correlation of relative mRNA signals with enzyme activities was good in four instances, moderate in four instances and poor in one instance. In diabetic islets, the mRNA signal of the islet cell-enriched transcription factor musculoaponeurotic fibrosarcoma oncogene homologue A, which regulates expression of islet genes, including the PC gene, was decreased to 54% of the control level. PC activity and protein levels in the non-diabetic islets were significantly lower than in islets from non-diabetic rodents. CONCLUSIONS/INTERPRETATION: Low levels of certain islet metabolic enzymes, especially mitochondrial enzymes, are associated with human type 2 diabetes.

Recovery of soluble dietary fiber is dependent on the method of analysis.

The effects of different methods on the distribution of total neutral sugars (TNS), uronic acids (UA), and beta-glucans (beta G) between the soluble (S) and insoluble (I) fractions of dietary fiber (DF) were determined for peas, kidney beans, oat bran, rice, and macaroni. Incorporation of a protease step into the Theander method "A" modestly increased, and addition of a pepsin digestion further increased the proportion of total fiber recovered in the S fraction. The effect of extraction method on the distribution of TNS, UA, and beta G between the S and I fractions varied with the food. The three methods measured the same total DF in a food and 1-3% starch in the I fraction of peas and kidney beans. Use of dimethyl sulfoxide to solubilize starch, or elevated temperature to extract S components, had no effect on the distribution of DF between S and I fractions of peas and macaroni. Incomplete protein hydrolysis did not always lower Klason lignin and excluding lignin from the fiber complex did not always substantially increase the S fraction.

Oat fiber: composition versus physiological function in rats.

The effect of processing on the ability of oat fiber to lower plasma and liver cholesterol concentrations in rats was studied. Male Sprague-Dawley rats were fed diets containing 6% dietary fiber as cellulose, oat bran, high fiber oat flour or one of four processed high fiber oat flours for 3 wk. All diets also contained 1.0% cholesterol and 0.2% cholic acid. At the conclusion plasma and liver concentrations of cholesterol and triglycerides were measured. All of the oat products significantly lowered plasma and liver cholesterol without depressing food intake or weight gain. As little as 4% dietary fiber in a processed oat flour significantly lowered cholesterol concentrations. Detailed fiber analysis of all of the oat fiber products revealed that processing increased the proportion of the total fiber that was soluble. The proportions recovered as total beta-glucans and total neutral sugars also increased, in part because the proportion recovered as Klason lignin decreased in all of the processed oat flours except the one prepared by a high pressure extrusion process.