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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK028348-29
Application #
8301014
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Appel, Michael C
Project Start
1981-04-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
29
Fiscal Year
2012
Total Cost
$563,148
Indirect Cost
$183,924
Name
University of Wisconsin Madison
Department
Pediatrics
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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, Michael J; Brown, Laura J; Longacre, Melissa J et al. (2013) Knockdown of both mitochondrial isocitrate dehydrogenase enzymes in pancreatic beta cells inhibits insulin secretion. Biochim Biophys Acta 1830:5104-11
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
Macdonald, M J; Longacre, M J; Warner, T F et al. (2013) High level of ATP citrate lyase expression in human and rat pancreatic islets. Horm Metab Res 45:391-3
Hasan, N M; Longacre, M J; Stoker, S W et al. (2012) Sphingosine kinase 1 knockdown reduces insulin synthesis and secretion in a rat insulinoma cell line. Arch Biochem Biophys 518:23-30
MacDonald, Michael J; Longacre, Melissa J; Stoker, Scott W et al. (2011) 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. J Biol Chem 286:18383-96
Jitrapakdee, S; Wutthisathapornchai, A; Wallace, J C et al. (2010) Regulation of insulin secretion: role of mitochondrial signalling. Diabetologia 53:1019-32
MacDonald, M J; Longacre, M J; Langberg, E-C et al. (2009) Decreased levels of metabolic enzymes in pancreatic islets of patients with type 2 diabetes. Diabetologia 52:1087-91

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