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. RESULTS: With knockdown of both mitochondrial IDH's mRNA, enzyme activity and protein level, (but not with knockdown of only one mitochondrial IDH) glucose- and BCH (an allosteric activator of glutamate dehydrogenase)-plus-glutamine-stimulated insulin release were inhibited. Cellular levels of citrate, α-ketoglutarate, malate and ATP were altered in patterns consistent with blockage at the mitochondrial IDH reactions. We were able to generate only 50% knockdown of Idh1 mRNA in multiple cell lines (without inhibition of insulin release) possibly because greater knockdown of IDH1 was not compatible with cell line survival. CONCLUSIONS: The mitochondrial IDHs are redundant for insulin secretion. When both enzymes are severely knocked down, their low activities (possibly assisted by transport of IDH products and other metabolic intermediates from the cytosol into mitochondria) are sufficient for cell growth, but inadequate for insulin secretion when the requirement for intermediates is certainly more rapid. The results also indicate that IDH2 can support the citric acid cycle. GENERAL SIGNIFICANCE: As almost all mammalian cells possess substantial amounts of all three IDH enzymes, the biological principles suggested by these results are probably extrapolatable to many tissues.

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. In FAS knockdown cells glucose carbon incorporation into lipids and the levels of the subclasses of phospholipids and cholesterol ester species were lower by 20-30% without inhibition of glucose oxidation. These studies suggest that rapid lipid modification is essential for normal glucose-stimulated 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. Activity and protein of ATP citrate lyase, which uses anaplerotic products in the cytosol, were 60-75% lower in human islets than in rodent islets or the cell line. In line with the lower PC, the percentage of glucose-derived pyruvate that entered mitochondrial metabolism via carboxylation in human islets was only 20-30% that in rat islets. This suggests human islets depend less on pyruvate carboxylation than rodent models that were used to establish the role of PC in insulin secretion. Human islets possessed high levels of succinyl-CoA:3-ketoacid-CoA transferase, an enzyme that forms acetoacetate in the mitochondria, and acetoacetyl-CoA synthetase, which uses acetoacetate to form acyl-CoAs in the cytosol. Glucose-stimulated human islets released insulin similarly to rat islets but formed much more acetoacetate. ß-Hydroxybutyrate augmented insulin secretion in human islets. This information supports previous data that indicate beta cells can use a pathway involving succinyl-CoA:3-ketoacid-CoA transferase and acetoacetyl-CoA synthetase to synthesize and use acetoacetate and suggests human islets may use this pathway more than PC and citrate to form cytosolic acyl-CoAs.

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. We found no increase in the mRNAs and enzyme activities of the cytosolic isocitrate dehydrogenase or glucose-6-phosphate dehydrogenase, which also produce cytosolic NADPH. There was no compensatory induction of the mRNAs for the mitochondrial malic enzymes Me2 or Me3. Interferon pathway genes induced in preliminary small interfering RNA experiments were not induced in the long term shRNA experiments. We repeated our study with an improved vector containing Tol2 transposition sequences to produce a higher rate of stable transferents and shortened time to testing, but this did not alter the results. We similarly used stably expressed shRNA to reduce mitochondrial NAD(P)-malic enzyme (Me2) mRNA by up to 95%, with severely decreased ME2 protein and a 90% decrease in enzyme activity. Insulin release to glucose or glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid remained normal. The maintenance of robust insulin secretion after lowering expression of either one of these malic enzymes is consistent with the redundancy of pathways of pyruvate cycling and/or cytosolic NADPH production in insulinoma cells.

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. Two cell lines with >70% knockdown of SCOT activity showed >70% reduction in glucose- or methyl succinate-plus-beta-hydroxybutyrate-stimulated insulin release. Less inhibition of insulin release was observed with two cell lines with less knockdown of SCOT. Previous studies showed knockdown of ATP citrate lyase in INS-1 832/13 cells does not lower insulin release. The results further support work that suggests mitochondrial pathways involving SCOT which supply acetoacetate for export to the cytosol are important for insulin secretion.

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. This suggested that leucine's metabolism contributed to its insulinotropism. Indeed, we found that leucine increased acetoacetate and was metabolized to CO(2) in pancreatic islets and increased short chain acyl-CoAs (SC-CoAs) in INS-1 cells. We then used the leucine-BCH difference to study the hypothesis that acyl groups derived from secretagogue carbon can be transferred as acetoacetate, in addition to citrate, from mitochondria to the cytosol where they can be converted to SC-CoAs. Since BCH cannot form sufficient acetoacetate from glutamate, transport of any glutamate-derived acyl groups to the cytosol in BCH-stimulated cells must proceed mainly via citrate. In ATP citrate lyase-deficient INS-1 cells, which are unable to convert citrate into cytosolic acetyl-CoA, insulin release by BCH was decreased and adding beta-hydroxybutyrate or alpha-ketoisocaproate, which increases mitochondrial acetoacetate, normalized BCH-induced insulin release. This strengthens the concept that acetoacetate-transferred acyl carbon can be converted to cytosolic SC-CoAs to stimulate insulin secretion.

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. We show that the mutant subunit acts in a dominant manner to completely inhibit glycogen phosphorylase kinase enzyme activity and that this interferes with glycogenolysis causing increased levels of glycogen in human liver cells. It is known that even normal blood glucose levels physiologically inhibit glycogen phosphorylase to diminish glucose release from the liver when glycogenolysis is not needed. The patient's mother possessed the same mutant glycogen phosphorylase kinase subunit, but did not have diabetes or hepatomegaly. His father had childhood type 1 diabetes in poor glycemic control, but lacked the mutation and had neither hepatomegaly nor growth failure. This case proves that the effect of a mutant enzyme of glycogen metabolism can combine with hyperglycemia to directly hyperinhibit glycogen phosphorylase, in turn blocking glycogenolysis causing the massive liver in Mauriac disease.

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

Pancreatic ß-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 ß-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. We also used double targeting of the same Me gene to achieve even more severe reduction in Me1 and Me2 mRNAs and enzyme activities than we reported previously. Knockdown of ME3, but not ME1 or ME2 alone or together, inhibited insulin release stimulated by glucose, pyruvate or 2-aminobicyclo [2,2,1]heptane-2-carboxylic acid-plus-glutamine. The data suggest that ME3, far more than ME1 or ME2, is necessary for insulin release. Because ME3 enzyme activity is low in ß-cells, its role in insulin secretion may involve a function other than its ME catalytic activity.

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. These sites include four GC-boxes (CCCGCCC at -227, -68, -46 and GGGCGAG at -382), three GT-boxes (CCCCACCC at -350, CCCACACCC at -257, and CACCCGCCC at -48), and three CT/GA-boxes (TCCCTCCC at -262, GGGAGGGAG at -129, and GGGAGGAGGA at -107). Transfection of Drosophila SL2 cells, which lack Sp/Krüppel-like factors, with constructs encoding either Sp1, Sp3, Sp4 or erythroid Krüppel-like factor (EKLF) specifically activates the hmGPD promoter B up to 50-fold. Promoter activation requires the Sp1 activation and the DNA binding domains. Co-transfected EKLF was synergistic with either Sp1 or Sp3. On the other hand, the basic Krüppel-like factor (BKLF) inhibited Sp1-and EKLF-mediated promoter activation. Similarly, constructs encoding either GA-binding protein (GABP) or PU.1 inhibited Sp1-mediated promoter activation. Oligonucleotide 'decoys' with potential transcription factor binding sites decreased promoter activity in both INS-1 and Drosophila cells. Significant loss of Sp- and EKLF-mediated activation was observed with some internal as well as sequential 5' deletions of the promoter DNA. The level of Sp1 protein was reduced by 50% in INS-1 cells chronically exposed to a high concentration of glucose. The results demonstrate that Sp/Krüppel-like factors are essential for mGPD gene expression.

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.

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. MS increased carbon incorporation of KIC into acid-precipitable material and lipid in INS-1 cells. In isolated mitochondria, MS alone increased malate, but MS plus KIC increased citrate, alpha-ketoglutarate, and isocitrate. These data and the known pathways of their metabolism suggest that MS supplies the oxaloacetate component of citrate and KIC supplies the acetate component of citrate. Other citric acid cycle intermediates can be formed from citrate enabling anaplerosis to supply precursors for extramitochondrial pathways. In addition, KIC, glucose and pyruvate can be metabolized to acetoacetate. In an INS-1 cell line deficient in ATP citrate lyase, incorporation of carbon from pyruvate into acid-precipitable material and lipid was not lowered. This negative result is in agreement with our recent discovery that citrate is not the only carrier of acyl groups from the mitochondria to the cytosol in the beta cell and that acetoacetate can also transfer acyl carbon to the cytosol.

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. Lactate released very little insulin. When acetoacetate, beta-hydroxybutyrate, or KIC were combined with MMS, or either ketone body was combined with lactate, insulin release was stimulated 10-fold to 20-fold the controls (almost as much as with glucose). Pyruvate was a potent stimulus of insulin release. In rat pancreatic islets, beta-hydroxybutyrate potentiated MMS- and glucose-induced insulin release. The pathways of their metabolism suggest that, in addition to producing ATP, the ketone bodies and KIC supply the acetate component and MMS supplies the oxaloacetate component of citrate. In line with this, citrate was increased by beta-hydroxybutyrate plus MMS in INS-1 cells and by beta-hydroxybutyrate plus succinate in mitochondria. The two ketone bodies and KIC can also be metabolized to acetoacetyl-CoA and acetyl-CoA, which are precursors of other short-chain acyl-CoAs (SC-CoAs). Measurements of SC-CoAs by LC-MS/MS in INS-1 cells confirmed that KIC, beta-hydroxybutyrate, glucose, and pyruvate increased the levels of acetyl-CoA, acetoacetyl-CoA, succinyl-CoA, hydroxymethylglutaryl-CoA, and malonyl-CoA. MMS increased incorporation of (14)C from beta-hydroxybutyrate into citrate, acid-precipitable material, and lipids, suggesting that the two molecules complement one another to increase anaplerosis. The results suggest that, besides citrate, some of the products of anaplerosis are SC-CoAs, which may be precursors of molecules involved in insulin secretion.

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. 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 beta-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 14C into lipid from [U-14C]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.

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.

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. Increased cycle intermediates will alter mitochondrial processes, and, therefore, the synthesized intermediates must be exported from mitochondria to the cytosol (cataplerosis). This further suggests that these intermediates have roles in signaling insulin secretion. Although evidence is quite good that all physiological fuel secretagogues stimulate insulin secretion via anaplerosis, evidence is just emerging about the possible extramitochondrial roles of exported citric acid cycle intermediates. This article speculates on their potential roles as signaling molecules themselves and as exporters of equivalents of NADPH, acetyl-CoA and malonyl-CoA, as well as alpha-ketoglutarate as a substrate for hydroxylases. We also discuss the "succinate mechanism," which hypothesizes that insulin secretagogues produce both NADPH and mevalonate. Finally, we discuss the role of mitochondria in causing oscillations in beta-cell citrate levels. These parallel oscillations in ATP and NAD(P)H. Oscillations in beta-cell plasma membrane electrical potential, ATP/ADP and NAD(P)/NAD(P)H ratios, and glycolytic flux are known to correlate with pulsatile insulin release. Citrate oscillations might synchronize oscillations of individual mitochondria with one another and mitochondrial oscillations with oscillations in glycolysis and, therefore, with flux of pyruvate into mitochondria. Thus citrate oscillations may synchronize mitochondrial ATP production and anaplerosis with other cellular oscillations.

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. In INS-1 mitochondria citrate oscillations are in phase with NAD(P) oscillations, and in intact INS-1 cells citrate oscillations parallel oscillations in ATP, suggesting that these processes are co-regulated. Oscillations have been extensively studied in the pancreatic beta cell where oscillations in glycolysis, NAD(P)/NAD(P)H and ATP/ADP ratios, plasma membrane electrical activity, calcium levels, and insulin secretion have been well documented. Because the mitochondrion is the major site of ATP synthesis and NADH oxidation and the only site of citrate synthesis, mitochondria need to be synchronized for these factors to oscillate. In suspensions of mitochondria from various organs, most of the citrate is exported from the mitochondria. In addition, citrate inhibits its own synthesis. We propose that this enables citrate itself to act as one of the cellular messengers that synchronizes mitochondria. Furthermore, because citrate is a potent inhibitor of the glycolytic enzyme phosphofructokinase, the pacemaker of glycolytic oscillations, citrate may act as a metabolic link between mitochondria and glycolysis. Citrate oscillations may coordinate oscillations in mitochondrial energy production and anaplerosis with glycolytic oscillations, which in the beta cell are known to parallel oscillations in insulin secretion.