A Novel Intron-Encoded Neuropilin-1 Isoform in Pancreatic Islets Associated With Very Young Age of Onset of Type 1 Diabetes.

Net synthesis of pancreatic ß-cells peaks before 2 years of life. ß-Cell mass is set within the first 5 years of life. In-frame translational readthrough of the NRP1 gene exon 9 into intron 9 generates a truncated neuropilin-1 protein lacking downstream sequence necessary for binding VEGF that stimulates ß-cell replication. VEGF is critical for developing but not adult islet neogenesis. Herein we show that cells in human pancreatic islets containing the full-length neuropilin-1 possess insulin but cells that contain the truncated neuropilin-1 are devoid of insulin. Decreased insulin cells increases susceptibility to onset of type 1 diabetes at a younger age. We also show that the frequency of a genetic marker in NRP1 intron 9 is higher among patients with onset of type 1 diabetes before age 4 years (31.8%), including those with onset at 0.67-2.00 and 2-4 years, compared with that in patients with onset at 4-8 years, at 8-12 years, and after 16 years (16.1%) with frequency equal to that in subjects without diabetes (16.0%). Decreased insulin cells plus the genetic data are consistent with a low effect mechanism that alters the onset of type 1 diabetes to a very young age in some patients, thus supporting the endotype concept that type 1 diabetes is a heterogeneous disease.

Metformin's Therapeutic Efficacy in the Treatment of Diabetes Does Not Involve Inhibition of Mitochondrial Glycerol Phosphate Dehydrogenase.

Mitochondrial glycerol phosphate dehydrogenase (mGPD) is the rate-limiting enzyme of the glycerol phosphate redox shuttle. It was recently claimed that metformin, a first-line drug used for the treatment of type 2 diabetes, inhibits liver mGPD 30-50%, suppressing gluconeogenesis through a redox mechanism. Various factors cast doubt on this idea. Total-body knockout of mGPD in mice has adverse effects in several tissues where the mGPD level is high but has little or no effect in liver, where the mGPD level is the lowest of 10 tissues. Metformin has beneficial effects in humans in tissues with high levels of mGPD, such as pancreatic ß-cells, where the mGPD level is much higher than that in liver. Insulin secretion in mGPD knockout mouse ß-cells is normal because, like liver, ß-cells possess the malate aspartate redox shuttle whose redox action is redundant to the glycerol phosphate shuttle. For these and other reasons, we used four different enzyme assays to reassess whether metformin inhibited mGPD. Metformin did not inhibit mGPD in homogenates or mitochondria from insulin cells or liver cells. If metformin actually inhibited mGPD, adverse effects in tissues where the level of mGPD is much higher than that in the liver could prevent the use of metformin as a diabetes medicine.

Differential contribution of pyruvate carboxylation to anaplerosis and cataplerosis during non-gluconeogenic and gluconeogenic conditions in HepG2 cells.

Pyruvate carboxylase (PC) is an anaplerotic enzyme that supplies oxaloacetate to mitochondria enabling the maintenance of other metabolic intermediates consumed by cataplerosis. Using liquid chromatography mass spectrometry (LC-MS) to measure metabolic intermediates derived from uniformly labeled 13C6-glucose or [3-13C]l-lactate, we investigated the contribution of PC to anaplerosis and cataplerosis in the liver cell line HepG2. Suppression of PC expression by short hairpin RNA lowered incorporation of 13C glucose incorporation into tricarboxylic acid cycle intermediates, aspartate, glutamate and sugar derivatives, indicating impaired cataplerosis. The perturbation of these biosynthetic pathways is accompanied by a marked decrease of cell viability and proliferation. In contrast, under gluconeogenic conditions where the HepG2 cells use lactate as a carbon source, pyruvate carboxylation contributed very little to the maintenance of these metabolites. Suppression of PC did not affect the percent incorporation of 13C-labeled carbon from lactate into citrate, α-ketoglutarate, malate, succinate as well as aspartate and glutamate, suggesting that under gluconeogenic condition, PC does not support cataplerosis from lactate.

Characterization of Acyl-CoA synthetase isoforms in pancreatic beta cells: Gene silencing shows participation of ACSL3 and ACSL4 in insulin secretion.

Long-chain acyl-CoA synthetases (ACSLs) convert fatty acids to fatty acyl-CoAs to regulate various physiologic processes. We characterized the ACSL isoforms in a cell line of homogeneous rat beta cells (INS-1 832/13 cells) and human pancreatic islets. ACSL4 and ACSL3 proteins were present in the beta cells and human and rat pancreatic islets and concentrated in insulin secretory granules and less in mitochondria and negligible in other intracellular organelles. ACSL1 and ACSL6 proteins were not seen in INS-1 832/13 cells or pancreatic islets. ACSL5 protein was seen only in INS-1 832/13 cells. With shRNA-mediated gene silencing we developed stable ACSL knockdown cell lines from INS-1 832/13 cells. Glucose-stimulated insulin release was inhibited ∼50% with ACSL4 and ACSL3 knockdown and unaffected in cell lines with knockdown of ACSL5, ACLS6 and ACSL1. Lentivirus shRNA-mediated gene silencing of ACSL4 and ACSL3 in human pancreatic islets inhibited glucose-stimulated insulin release. ACSL4 and ACSL3 knockdown cells showed inhibition of ACSL enzyme activity more with arachidonate than with palmitate as a substrate, consistent with their preference for unsaturated fatty acids as substrates. ACSL4 knockdown changed the patterns of fatty acids in phosphatidylserines and phosphatidylethanolamines. The results show the involvement of ACLS4 and ACLS3 in 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.

Characterization of P4 ATPase Phospholipid Translocases (Flippases) in Human and Rat Pancreatic Beta Cells: THEIR GENE SILENCING INHIBITS INSULIN SECRETION.

The negative charge of phosphatidylserine in lipid bilayers of secretory vesicles and plasma membranes couples the domains of positively charged amino acids of secretory vesicle SNARE proteins with similar domains of plasma membrane SNARE proteins enhancing fusion of the two membranes to promote exocytosis of the vesicle contents of secretory cells. Our recent study of insulin secretory granules (ISG) (MacDonald, M. J., Ade, L., Ntambi, J. M., Ansari, I. H., and Stoker, S. W. (2015) Characterization of phospholipids in insulin secretory granules in pancreatic beta cells and their changes with glucose stimulation. J. Biol. Chem. 290, 11075-11092) suggested that phosphatidylserine and other phospholipids, such as phosphatidylethanolamine, in ISG could play important roles in docking and fusion of ISG to the plasma membrane in the pancreatic beta cell during insulin exocytosis. P4 ATPase flippases translocate primarily phosphatidylserine and, to a lesser extent, phosphatidylethanolamine across the lipid bilayers of intracellular vesicles and plasma membranes to the cytosolic leaflets of these membranes. CDC50A is a protein that forms a heterodimer with P4 ATPases to enhance their translocase catalytic activity. We found that the predominant P4 ATPases in pure pancreatic beta cells and human and rat pancreatic islets were ATP8B1, ATP8B2, and ATP9A. ATP8B1 and CDC50A were highly concentrated in ISG. ATP9A was concentrated in plasma membrane. Gene silencing of individual P4 ATPases and CDC50A inhibited glucose-stimulated insulin release in pure beta cells and in human pancreatic islets. This is the first characterization of P4 ATPases in beta cells. The results support roles for P4 ATPases in translocating phosphatidylserine to the cytosolic leaflets of ISG and the plasma membrane to facilitate the docking and fusion of ISG to the plasma membrane during insulin exocytosis.

Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis.

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.

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.

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.

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.

The role of rapid lipogenesis in insulin secretion: Insulin secretagogues acutely alter lipid composition of INS-1 832/13 cells.

Pancreatic beta cell mitochondria convert insulin secretagogues into products that support insulin exocytosis. We explored the idea that lipids are some of these products formed from acyl group transfer out of mitochondria to the cytosol, the site of lipid synthesis. There are two isoforms of acetyl-CoA carboxylase, the enzyme that forms malonyl-CoA from which C(2) units for lipid synthesis are formed. We found that ACC1, the isoform seen in lipogenic tissues, is the only isoform present in human and rat pancreatic islets and INS-1 832/13 cells. Inhibitors of ACC and fatty acid synthase inhibited insulin release in islets and INS-1 cells. Carbon from glucose and pyruvate were rapidly incorporated into many lipid classes in INS-1 cells. Glucose and other insulin secretagogues acutely increased many lipids with C14-C24 chains including individual cholesterol esters, phospholipids and fatty acids. Many phosphatidylcholines and phosphatidylserines were increased and many phosphatidylinositols and several phosphatidylethanolamines were decreased. The results suggest that lipid remodeling and rapid lipogenesis from secretagogue carbon support 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. 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.