Discovery of a Genetic Metabolic Cause for Mauriac Syndrome in Type 1 Diabetes.

A mechanistic cause for Mauriac syndrome, a syndrome of growth failure and delayed puberty associated with massive liver enlargement from glycogen deposition in children with poorly controlled type 1 diabetes, is unknown. We discovered a mutation in the catalytic subunit of liver glycogen phosphorylase kinase in a patient with Mauriac syndrome whose liver extended into his pelvis. Glycogen phosphorylase kinase activates glycogen phosphorylase, the enzyme that catalyzes the first step in glycogen breakdown.

Mitochondrial malic enzyme 3 is important for insulin secretion in pancreatic β-cells.

Pancreatic β-cells with severely knocked down cytosolic malic enzyme (ME1) and mitochondrial NAD(P) malic enzyme (ME2) show normal insulin secretion. The mitochondrial NADP malic enzyme (ME3) is very low in pancreatic β-cells, and ME3 was previously thought unimportant for insulin secretion. Using short hairpin RNAs that targeted one or more malic enzyme mRNAs in the same cell, we generated more than 25 stable INS-1 832/13-derived insulin cell lines expressing extremely low levels of ME1, ME2, and ME3 alone or low levels of two of these enzymes in the same cell line.

Knockdown of both mitochondrial isocitrate dehydrogenase enzymes in pancreatic beta cells inhibits insulin secretion.

Background: There are three isocitrate dehydrogenases (IDHs) in the pancreatic insulin cell; IDH1 (cytosolic) and IDH2 (mitochondrial) use NADP(H). IDH3 is mitochondrial, uses NAD(H) and was believed to be the IDH that supports the citric acid cycle.

Methods: With shRNAs targeting mRNAs for these enzymes we generated cell lines from INS-1 832/13 cells with severe (80%-90%) knockdown of the mitochondrial IDHs separately and together in the same cell line.

Knockdown of pyruvate carboxylase or fatty acid synthase lowers numerous lipids and glucose-stimulated insulin release in insulinoma cells.

We previously showed that knockdown of the anaplerotic enzyme pyruvate carboxylase in the INS-1 832/13 insulinoma cell line inhibited glucose-stimulated insulin release and glucose carbon incorporation into lipids. We now show that knockdown of fatty acid synthase (FAS) mRNA and protein also inhibits glucose-stimulated insulin release in this cell line. Levels of numerous phospholipids, cholesterol esters, diacylglycerol, triglycerides and individual fatty acids with C14-C24 side chains were acutely lowered about 20% in glucose-stimulated pyruvate carboxylase knockdown cells over a time course that coincides with insulin secretion.

Differences between human and rodent pancreatic islets: low pyruvate carboxylase, atp citrate lyase, and pyruvate carboxylation and high glucose-stimulated acetoacetate in human pancreatic islets.

Anaplerosis, the net synthesis in mitochondria of citric acid cycle intermediates, and cataplerosis, their export to the cytosol, have been shown to be important for insulin secretion in rodent beta cells. However, human islets may be different. We observed that the enzyme activity, protein level, and relative mRNA level of the key anaplerotic enzyme pyruvate carboxylase (PC) were 80-90% lower in human pancreatic islets compared with islets of rats and mice and the rat insulinoma cell line INS-1 832/13.

Lower succinyl-CoA:3-ketoacid-CoA transferase (SCOT) and ATP citrate lyase in pancreatic islets of a rat model of type 2 diabetes: knockdown of SCOT inhibits insulin release in rat insulinoma cells.

Succinyl-CoA:3-ketoacid-CoA transferase (SCOT) is a mitochondrial enzyme that catalyzes the reversible transfer of coenzyme-A from acetoacetyl-CoA to succinate to form acetoacetate and succinyl-CoA. mRNAs of SCOT and ATP citrate lyase were decreased 55% and 58% and enzyme activities were decreased >70% in pancreatic islets of the GK rat, a model of type 2 diabetes. INS-1 832/13 cells were transfected with shRNAs targeting SCOT mRNA to generate cell lines with reduced SCOT activity.

Genetic association of the neuropilin-1 gene with type 1 diabetes in children: Neuropilin-1 expression in pancreatic islets.

Minor alleles of two SNPs in intron 9 of the NRP1 gene, which encodes neuropilin-1, were found to be associated with type 1 diabetes (T1D) in children. Neuropilin-1 peptides were confined to islets in human pancreas. This suggests neuropilins-1 could influence the development of some cases of T1D in children.

Chronic reduction of the cytosolic or mitochondrial NAD(P)-malic enzyme does not affect insulin secretion in a rat insulinoma cell line.

The cytosolic malic enzyme (ME1) has been suggested to augment insulin secretion via the malate-pyruvate and/or citrate-pyruvate shuttles, through the production of NADPH or other metabolites. We used selectable vectors expressing short hairpin RNA (shRNA) to stably decrease Me1 mRNA levels by 80-86% and ME1 enzyme activity by 78-86% with either of two shRNAs in the INS-1 832/13 insulinoma cell line. Contrary to published short term ME1 knockdown experiments, our long term targeted cells showed normal insulin secretion in response to glucose or to glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid.

Impaired anaplerosis and insulin secretion in insulinoma cells caused by small interfering RNA-mediated suppression of pyruvate carboxylase.

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.

Studies with leucine, beta-hydroxybutyrate and ATP citrate lyase-deficient beta cells support the acetoacetate pathway of insulin secretion.

We hypothesized that contrasting leucine with its non-metabolizable analog 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) might provide new information about metabolic pathways involved in insulin secretion. Both compounds stimulate insulin secretion by allosterically activating glutamate dehydrogenase, which enhances glutamate metabolism. However, we found that leucine was a stronger secretagogue in rat pancreatic islets and INS-1 cells.

Anaplerosis from glucose, alpha-ketoisocaproate, and pyruvate in pancreatic islets, INS-1 cells and liver mitochondria.

Methyl succinate (MS) and alpha-ketoisocaproate (KIC) when applied alone to cultured pancreatic islets or INS-1 832/13 cells do not stimulate insulin release. However, when the two metabolites are combined together they strongly stimulate insulin release. Studying the possible explanations for this complementarity has provided clues to the pathways involved in insulin secretion.

Acetoacetate and beta-hydroxybutyrate in combination with other metabolites release insulin from INS-1 cells and provide clues about pathways in insulin secretion.

Mitochondrial anaplerosis is important for insulin secretion, but only some of the products of anaplerosis are known. We discovered novel effects of mitochondrial metabolites on insulin release in INS-1 832/13 cells that suggested pathways to some of these products. Acetoacetate, beta-hydroxybutyrate, alpha-ketoisocaproate (KIC), and monomethyl succinate (MMS) alone did not stimulate insulin release.

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.

Perspective: emerging evidence for signaling roles of mitochondrial anaplerotic products in insulin secretion.

The importance of mitochondrial biosynthesis in stimulus secretion coupling in the insulin-producing beta-cell probably equals that of ATP production. In glucose-induced insulin secretion, the rate of pyruvate carboxylation is very high and correlates more strongly with the glucose concentration the beta-cell is exposed to (and thus with insulin release) than does pyruvate decarboxylation, which produces acetyl-CoA for metabolism in the citric acid cycle to produce ATP. The carboxylation pathway can increase the levels of citric acid cycle intermediates, and this indicates that anaplerosis, the net synthesis of cycle intermediates, is important for insulin secretion.

Citrate oscillates in liver and pancreatic beta cell mitochondria and in INS-1 insulinoma cells.

Oscillations in citric acid cycle intermediates have never been previously reported in any type of cell. Here we show that adding pyruvate to isolated mitochondria from liver, pancreatic islets, and INS-1 insulinoma cells or adding glucose to intact INS-1 cells causes sustained oscillations in citrate levels. Other citric acid cycle intermediates measured either did not oscillate or possibly oscillated with a low amplitude.

Sp/Krüppel-like transcription factors are essential for the expression of mitochondrial glycerol phosphate dehydrogenase promoter B.

The human mitochondrial glycerol phosphate dehydrogenase (hmGPD) is abundant in the normal pancreatic insulin cell and its level is lowered to 50% by high glucose and diabetes. Using Drosophila and INS-1 cells, we have analysed the hmGPD gene promoter B to characterize cis-elements and trans-acting factors that affect its regulation. We identified ten efficient Sp/Krüppel-like transcription-factor-binding sites in the promoter sequence.