The broad long-term objective is to obtain new information about the metabolic signals for insulin secretion. The immediate purpose of this project is to gain a better understanding of the role of mitochondrial biosynthetic pathways in supporting insulin secretion. Glucose, the most potent insulin secretagogue, and all other metabolizable secretagogues, stimulate insulin secretion via their metabolism in mitochondria. Our earlier work showed that one-half of glucose-derived pyruvate enters mitochondrial metabolism via carboxylation catalyzed by pyruvate carboxylase and one-half enters via decarboxylation. From the resulting oxaloacetate from carboxylation and acetyl-CoA from decarboxylation of pyruvate, any citric acid cycle intermediate can be synthesized. Recent work showed the presence of intra- and extra-mitochondrial enzymes, including succinyl- CoA: 3-ketoacid-CoA transferase (SCOT), that catalyze the synthesis and utilization of mitochondrial products. SCOT can form acetoacetate from all insulin secretagogues. Thus acetoacetate, in addition to citric acid cycle intermediates, can transfer carbon to the cytosol for the synthesis of short chain acyl-CoAs, lipids and other factors.
Aim 1 A is to study the pathways in mitochondria of formation of molecules that are exported to the cytosol for the synthesis of the compounds in the cytosol that support or signal insulin secretion.
Aim 1 B is to study the extramitochondrial utilization of these compounds. Cell lines with knocked down pyruvate carboxylase, SCOT, acetoacetyl-CoA synthetase, cytosolic malic enzyme, fatty acid synthase and other enzymes;as well as enzyme assays, including an assay for mitochondrial malic enzyme, will facilitate this work. Recent work has shown the beta cell is a lipogenic tissue. Lipids in cells and subcellular fractions are measured with gas chromatography. Mass spectrometry is used to measure short chain acyl-CoAs and to identify small molecules and lipids that change with secretagogue stimulation.
Aim 2 is to discern the relevance of mitochondrial biosynthesis to normal and abnormal insulin secretion in humans.
Aim 2 A will further study the surprising observation that pathways of anaplerosis that are very active in rodent beta cells are far less active in human islets. We have accumulated conclusive evidence that pyruvate carboxylase, the major enzyme of anaplerosis, is 90% lower in normal human islets than in islets of rodents and clonal beta cell lines. Thus, the human beta cell may be programmed to more heavily use alternative pathways to pyruvate carboxylase for mitochondrial synthesis. The regulation of pyruvate carboxylase expression and rate of pyruvate carboxylation in human islets will be studied.
Aim 2 B is to continue to study intra- and extra- mitochondrial metabolic enzymes that are decreased in islets of rodents and humans with type 2 diabetes and factors that might regulate their expression.

Public Health Relevance

Understanding the normal metabolic pathways of secretagogue-stimulated insulin secretion is essential for developing artificial beta cells (e.g. by altering normal non-insulin cells, such as liver cells, or developing insulin-secreting cells from stem cells), for the preservation of healthy pancreas or islets for use in the treatment of type 1 diabetes and also for understanding the impaired insulin secretion in type 2 diabetes.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Cellular Aspects of Diabetes and Obesity Study Section (CADO)
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Appel, Michael C
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University of Wisconsin Madison
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Ansari, Israr-Ul H; Longacre, Melissa J; Stoker, Scott W et al. (2017) Characterization of Acyl-CoA synthetase isoforms in pancreatic beta cells: Gene silencing shows participation of ACSL3 and ACSL4 in insulin secretion. Arch Biochem Biophys 618:32-43
El Azzouny, Mahmoud; Longacre, Melissa J; Ansari, Israr-Ul H et al. (2016) Knockdown of ATP citrate lyase in pancreatic beta cells does not inhibit insulin secretion or glucose flux and implicates the acetoacetate pathway in insulin secretion. Mol Metab 5:980-7
Ansari, Israr-ul H; Longacre, Melissa J; Paulusma, Coen C et al. (2015) Characterization of P4 ATPase Phospholipid Translocases (Flippases) in Human and Rat Pancreatic Beta Cells: THEIR GENE SILENCING INHIBITS INSULIN SECRETION. J Biol Chem 290:23110-23
MacDonald, Michael J; Ade, Lacmbouh; Ntambi, James M et al. (2015) Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation. J Biol Chem 290:11075-92
Hasan, Noaman M; Longacre, Melissa J; Stoker, Scott W et al. (2015) Mitochondrial malic enzyme 3 is important for insulin secretion in pancreatic ?-cells. Mol Endocrinol 29:396-410
Wutthisathapornchai, Apilak; Vongpipatana, Tuangtong; Muangsawat, Sureeporn et al. (2014) Multiple E-boxes in the distal promoter of the rat pyruvate carboxylase gene function as a glucose-responsive element. PLoS One 9:e102730
Madiraju, Anila K; Erion, Derek M; Rahimi, Yasmeen et al. (2014) Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase. Nature 510:542-6
MacDonald, Michael J; Hasan, Noaman M; Dobrzyn, Agnieszka et al. (2013) Knockdown of pyruvate carboxylase or fatty acid synthase lowers numerous lipids and glucose-stimulated insulin release in insulinoma cells. Arch Biochem Biophys 532:23-31
Thonpho, Ansaya; Rojvirat, Pinnara; Jitrapakdee, Sarawut et al. (2013) Characterization of the distal promoter of the human pyruvate carboxylase gene in pancreatic beta cells. PLoS One 8:e55139
MacDonald, M J; Langberg, E-C; Tibell, A et al. (2013) Identification of ATP synthase as a lipid peroxide protein adduct in pancreatic islets from humans with and without type 2 diabetes mellitus. J Clin Endocrinol Metab 98:E727-31

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