Impaired Anaplerosis and Insulin Secretion in Insulinoma Cells Caused by Small Interfering RNA-mediated Suppression of Pyruvate Carboxylase

Abstract

Anaplerosis, the synthesis of citric acid cycle intermediates, by pancreatic beta cell mitochondria has been proposed to be as important for insulin secretion as mitochondrial energy production. However, studies designed to lower the rate of anaplerosis in the beta cell have been inconclusive. To test the hypothesis that anaplerosis is important for insulin secretion, we lowered the activity of pyruvate carboxylase (PC), the major enzyme of anaplerosis in the beta cell. Stable transfection of short hairpin RNA was used to generate a number of INS-1 832/13-derived cell lines with various levels of PC enzyme activity that retained normal levels of control enzymes, insulin content, and glucose oxidation. Glucose-induced insulin release was decreased in proportion to the decrease in PC activity. Insulin release in response to pyruvate alone, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine, or methyl succinate plus ␤-hydroxybutyrate was also decreased in the PC knockdown cells. Consistent with a block at PC, the most PC-deficient cells showed a metabolic crossover point at PC with increased basal and/or glucose-stimulated pyruvate plus lactate and decreased malate and citrate. In addition, in BCH plus glutamine-stimulated PC knockdown cells, pyruvate plus lactate was increased, whereas citrate was severely decreased, and malate and aspartate were slightly decreased. The incorporation of 14 C into lipid from [U-14 C]glucose was decreased in the PC knockdown cells. The results confirm the central importance of PC and anaplerosis to generate metabolites from glucose that support insulin secretion and even suggest PC is important for insulin secretion stimulated by noncarbohydrate insulin secretagogues.Glucose is the most potent physiological insulin secretagogue in the pancreatic beta cell, and it stimulates insulin secretion via its metabolism by aerobic glycolysis. Pyruvate, the terminal product of glycolysis, is metabolized in mitochondria to make ATP to power intracellular processes. However, somewhat surprisingly, in the beta cell, a large amount of glucosederived pyruvate, equal to one-half the total pyruvate entering mitochondrial metabolism, is carboxylated in the pyruvate carboxylase reaction to form oxaloacetate (1-5). The oxaloacetate can combine with pyruvate-derived acetyl-CoA to form citrate enabling the beta cell mitochondrion to increase the rate of synthesis of any citric acid cycle intermediate (anaplerosis) (6 -9). The rate of pyruvate carboxylation correlates with the glucose concentration applied to islets and is thus correlated with the rate of insulin secretion (2). The level of pyruvate carboxylase in the pancreatic islet beta cell is higher than in most body tissues (4, 10 -13) and is equal to the levels in gluconeogenic tissues, such as liver and kidney (4), which possess very high levels of the enzyme. However, the beta cell does not possess the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (11, 14, 15) or fructose-1,6-bisphosphatase (8), and this explains why it ...

Adaptation and failure of pancreatic β cells in murine models with different degrees of metabolic syndrome

“…Expression of the gene pyruvate carboxylase (PCX), which is another target gene of PPAR (Jitrapakdee et al, 2005), is decreased in PPAR2 KO islets. As expected, the level of mRNA encoding PCX is also significantly decreased in islets from POKO mice compared with ob/ob mice (Hasan et al, 2008).…”

Supplement of TCA cycle intermediates protects against high glucose/palmitate-induced INS-1 beta cell death

“…In particular, our experiments showing that enhanced anaplerosis by BCH and reduced cataplerosis by BTA restored HG/PA-induced reduction of oxidation rate and ATP level suggest that the impaired energy metabolism in HG/PA-treated cells is partly due to insufficient supply of TCA cycle intermediates. Interestingly, imbalanced mitochondrial energy production in HG/ PA-treated cells may explain beta cell dysfunctions observed in type 2 diabetes, such as impaired glucose-stimulated insulin secretion and impaired expression of insulin gene [49][50][51].…”

Liver X Receptor Agonists Augment Human Islet Function through Activation of Anaplerotic Pathways and Glycerolipid/Free Fatty Acid Cycling

“…In further support of LXRmediated activation of anaplerotic pathways, we observed an increase in pyruvate carboxylase enzyme activity in human islets treated with 1 M TO-901317. Previous studies have shown that siRNA-mediated knockdown of IDH1 or pyruvate carboxylase leads to a significant decrease in glucose-stimulated insulin secretion in INS-1 cells and rat islets, with decreased flux through these alternative NADPH producing pathways (36,49).…”