A Role for ATP-Citrate Lyase, Malic Enzyme, and Pyruvate/Citrate Cycling in Glucose-induced Insulin Secretion

In pancreatic β-cells, metabolic coupling factors generated during glucose metabolism and pyruvate cycling through anaplerosis/cataplerosis processes contribute to the regulation of insulin secretion. Pyruvate/citrate cycling across the mitochondrial membrane leads to the production of malonyl-CoA and NADPH, two candidate coupling factors. To examine the implication of pyruvate/citrate cycling in glucose-induced insulin secretion (GIIS), different steps of the cycle were inhibited in INS 832/13 cells by pharmacological inhibitors and/or RNA interference (RNAi) technology: mitochondrial citrate export, ATP-citrate lyase (ACL), and cytosolic malic enzyme (ME1). The inhibitors of the di- and tri-carboxylate carriers, n-butylmalonate and 1,2,3-benzenetricarboxylate, respectively, reduced GIIS, indicating the importance of transmitochondrial transport of tri- and dicarboxylates in the action of glucose. To directly test the role of ACL and ME1 in GIIS, small hairpin RNA (shRNA) were used to selectively decrease ACL or ME1 expression in transfected INS 832/13 cells. shRNA-ACL reduced ACL protein levels by 67%, and this was accompanied by a reduction in GIIS. The amplification/KATP-independent pathway of GIIS was affected by RNAi knockdown of ACL. The ACL inhibitor radicicol also curtailed GIIS. shRNA-ME1 reduced ME1 activity by 62% and decreased GIIS. RNAi suppression of either ACL or ME1 did not affect glucose oxidation. However, because ACL is required for malonyl-CoA formation, inhibition of ACL expression by shRNA-ACL decreased glucose incorporation into palmitate and increased fatty acid oxidation in INS 832/13 cells. Taken together, the results underscore the importance of pyruvate/citrate cycling in pancreatic β-cell metabolic signaling and the regulation of GIIS.

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Probing the link between citrate and malonyl-CoA in perfused rat hearts

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00244.2002 ◽

2002 ◽

Vol 283 (4) ◽

pp. H1379-H1386 ◽

Cited By ~ 17

Author(s):

Myriame Poirier ◽

Geneviève Vincent ◽

Aneta E. Reszko ◽

Bertrand Bouchard ◽

Joanne K. Kelleher ◽

...

Keyword(s):

Carbon Sources ◽

Acid Oxidation ◽

Atp Citrate Lyase ◽

Acetyl Coa ◽

Rat Hearts ◽

Citrate Lyase ◽

Malonyl Coa ◽

Perfused Rat Hearts ◽

Citrate Release

Little is known about the sources of cytosolic acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of fatty acid oxidation in the heart. We tested the hypothesis that citrate provides acetyl-CoA for malonyl-CoA synthesis after its mitochondrial efflux and cleavage by cytosolic ATP-citrate lyase. We expanded on a previous study where we characterized citrate release from perfused rat hearts (Vincent G, Comte B, Poirier M, and Des Rosiers C. Citrate release by perfused rat hearts: a window on mitochondrial cataplerosis. Am J Physiol Endocrinol Metab 278: E846–E856, 2000). In the present study, we show that citrate release rates, ranging from 6 to 22 nmol/min, can support a net increase in malonyl-CoA concentrations induced by changes in substrate supply, at most 0.7 nmol/min. In experiments with [U-13C](lactate + pyruvate) and [1-13C]oleate, we show that the acetyl moiety of malonyl-CoA is derived from both pyruvate and long-chain fatty acids. This 13C-labeling of malonyl-CoA occurred without any changes in its concentration. Hydroxycitrate, an inhibitor of ATP-citrate lyase, prevents increases in malonyl-CoA concentrations and decreases its labeling from [U-13C](lactate + pyruvate). Our data support at least a partial role of citrate in the transfer from the mitochondria to cytosol of acetyl units for malonyl-CoA synthesis. In addition, they provide a dynamic picture of malonyl-CoA metabolism: even when the malonyl-CoA concentration remains constant, there appears to be a constant need to supply acetyl-CoA from various carbon sources, both carbohydrates and lipids, for malonyl-CoA synthesis.

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Cytosolic carnitine acetyltransferase as a source of cytosolic acetyl-CoA: a possible mechanism for regulation of cardiac energy metabolism

Biochemical Journal ◽

10.1042/bcj20170823 ◽

2018 ◽

Vol 475 (5) ◽

pp. 959-976 ◽

Cited By ~ 14

Author(s):

Tariq R. Altamimi ◽

Panakkezhum D. Thomas ◽

Ahmed M. Darwesh ◽

Natasha Fillmore ◽

Mohammad U. Mahmoud ◽

...

Keyword(s):

Energy Metabolism ◽

Glucose Oxidation ◽

Carnitine Acetyltransferase ◽

Atp Citrate Lyase ◽

Acetyl Coa ◽

Protein Levels ◽

Citrate Lyase ◽

Malonyl Coa ◽

Cardiac Energy Metabolism ◽

Cardiac Energy

The role of carnitine acetyltransferase (CrAT) in regulating cardiac energy metabolism is poorly understood. CrAT modulates mitochondrial acetyl-CoA/CoA (coenzyme A) ratios, thus regulating pyruvate dehydrogenase activity and glucose oxidation. Here, we propose that cardiac CrAT also provides cytosolic acetyl-CoA for the production of malonyl-CoA, a potent inhibitor of fatty acid oxidation. We show that in the murine cardiomyocyte cytosol, reverse CrAT activity (RCrAT, producing acetyl-CoA) is higher compared with the liver, which primarily uses ATP-citrate lyase to produce cytosolic acetyl-CoA for lipogenesis. The heart displayed a lower RCrAT Km for CoA compared with the liver. Furthermore, cytosolic RCrAT accounted for 4.6 ± 0.7% of total activity in heart tissue and 12.7 ± 0.2% in H9C2 cells, while highly purified heart cytosolic fractions showed significant CrAT protein levels. To investigate the relationship between CrAT and acetyl-CoA carboxylase (ACC), the cytosolic enzyme catalyzing malonyl-CoA production from acetyl-CoA, we studied ACC2-knockout mouse hearts which showed decreased CrAT protein levels and activity, associated with increased palmitate oxidation and acetyl-CoA/CoA ratio compared with controls. Conversely, feeding mice a high-fat diet for 10 weeks increased cardiac CrAT protein levels and activity, associated with a reduced acetyl-CoA/CoA ratio and glucose oxidation. These data support the presence of a cytosolic CrAT with a low Km for CoA, favoring the formation of cytosolic acetyl-CoA, providing an additional source to the classical ATP-citrate lyase pathway, and that there is an inverse relation between CrAT and the ratio of acetyl-CoA/CoA as evident in conditions affecting the regulation of cardiac energy metabolism.

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Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)42624-5 ◽

1992 ◽

Vol 267 (9) ◽

pp. 5802-5810 ◽

Cited By ~ 4

Author(s):

M Prentki ◽

S Vischer ◽

M.C. Glennon ◽

R Regazzi ◽

J.T. Deeney ◽

...

Keyword(s):

Insulin Secretion ◽

Metabolic Coupling ◽

Malonyl Coa ◽

Chain Acyl ◽

Coupling Factors ◽

Long Chain

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Malonyl-CoA regulation in skeletal muscle: its link to cell citrate and the glucose-fatty acid cycle

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1997.272.4.e641 ◽

1997 ◽

Vol 272 (4) ◽

pp. E641-E648 ◽

Cited By ~ 32

Author(s):

A. K. Saha ◽

D. Vavvas ◽

T. G. Kurowski ◽

A. Apazidis ◽

L. A. Witters ◽

...

Keyword(s):

Skeletal Muscle ◽

Fatty Acid ◽

Pancreatic Beta Cell ◽

Acid Oxidation ◽

Atp Citrate Lyase ◽

Acetyl Coa ◽

Acid Cycle ◽

Citrate Lyase ◽

Malonyl Coa ◽

Glucose Fatty Acid Cycle

Malonyl-CoA is an inhibitor of carnitine palmitoyltransferase I, the enzyme that controls the oxidation of fatty acids by regulating their transfer into the mitochondria. Despite this, knowledge of how malonyl-CoA levels are regulated in skeletal muscle, the major site of fatty acid oxidation, is limited. Two- to fivefold increases in malonyl-CoA occur in rat soleus muscles incubated with glucose or glucose plus insulin for 20 min [Saha, A. K., T. G. Kurowski, and N. B. Ruderman. Am. J. Physiol. 269 (Endocrinol. Metab. 32): E283-E289, 1995]. In addition, as reported here, acetoacetate in the presence of glucose increases malonyl-CoA levels in the incubated soleus. The increases in malonyl-CoA in all of these situations correlated closely with increases in the concentration of citrate (r2 = 0.64) and to an even greater extent the sum of citrate plus malate (r2 = 0.90), an antiporter for citrate efflux from the mitochondria. Where measured, no increase in the activity of acetyl-CoA carboxylase (ACC) was found. Inhibition of ATP citrate lyase with hydroxycitrate markedly diminished the increases in malonyl-CoA in these muscles, indicating that citrate was the major substrate for the malonyl-CoA precursor, cytosolic acetyl-CoA. Studies with enzyme purified by immunoprecipitation indicated that the observed increases in citrate could have also allosterically activated ACC. The results suggest that in the presence of glucose, insulin and acetoacetate acutely increase malonyl-CoA levels in the incubated soleus by increasing the cytosolic concentration of citrate. This novel mechanism could complement the glucose-fatty acid cycle in determining how muscle chooses its fuels. It could also provide a means by which glucose acutely modulates signal transduction in muscle and other cells (e.g., the pancreatic beta-cell) in which its metabolism is determined by substrate availability.

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Malonyl-CoA metabolism in cardiac myocytes and its relevance to the control of fatty acid oxidation

Biochemical Journal ◽

10.1042/bj2950061 ◽

1993 ◽

Vol 295 (1) ◽

pp. 61-66 ◽

Cited By ~ 133

Author(s):

M M Awan ◽

E D Saggerson

Keyword(s):

Fatty Acid ◽

Cardiac Myocytes ◽

Fatty Acid Synthase ◽

Acid Oxidation ◽

Fatty Acid Elongation ◽

Acetyl Coa Carboxylase ◽

Atp Citrate Lyase ◽

Rat Hearts ◽

Citrate Lyase ◽

Malonyl Coa

1. Viable myocytes were obtained from rat hearts. Oxidation of [1-14C]palmitate by these cells could be decreased by the addition of glucose (5 mM) or lactate (2 mM). In the presence of glucose, insulin decreased and adrenaline increased palmitate oxidation. 2. The myocytes contained activities of ATP citrate-lyase, acetyl-CoA carboxylase and the condensing enzyme of the fatty acid elongation system. No fatty acid synthase activity was demonstrable in myocytes. 3. In rat hearts perfused with 5 mM glucose, malonyl-CoA content was acutely raised by insulin. In the presence of glucose+insulin, perfusion with palmitate or adrenaline decreased the malonyl-CoA content. 4. It is concluded that malonyl-CoA can be synthesized within cardiac myocytes and that the level of this metabolite can be acutely regulated. This is likely to have consequences for the regulation of carnitine palmitoyltransferase in the heart.

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Coordinate regulation of the biosynthesis of ATP-citrate lyase and malic enzyme during adipocyte differentiation. Studies on 3T3-L1 cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)42920-6 ◽

1984 ◽

Vol 259 (8) ◽

pp. 4827-4832 ◽

Cited By ~ 9

Author(s):

L S Wise ◽

H S Sul ◽

C S Rubin

Keyword(s):

Malic Enzyme ◽

Adipocyte Differentiation ◽

Atp Citrate Lyase ◽

Coordinate Regulation ◽

Citrate Lyase

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ATP citrate-lyase and glycogen synthase kinase-3β in 3T3-L1 cells during differentiation into adipocytes

Biochemical Journal ◽

10.1042/bj3000477 ◽

1994 ◽

Vol 300 (2) ◽

pp. 477-482 ◽

Cited By ~ 23

Author(s):

W B Benjamin ◽

S N Pentyala ◽

J R Woodgett ◽

Y Hod ◽

D Marshak

Keyword(s):

Glycogen Synthase ◽

Protein Band ◽

Glycogen Synthase Kinase ◽

Fat Cells ◽

Apparent Mass ◽

Atp Citrate Lyase ◽

Protein Levels ◽

Citrate Lyase ◽

Sds Page ◽

Dependent Protein

ATP citrate-lyase (CL), acetyl-CoA carboxylase (ACC) and glycogen synthase kinase-3 beta (GSK-3 beta) levels were measured in cytosol from 3T3-L1 cells during differentiation from fibroblasts into fat-cells. Protein levels were estimated from immunoblots using specific antisera. Cytosol from confluent cells contain significant amounts of GSK-3 beta, which fell during differentiation of these cells into adipocytes. CL from confluent cells was found to be mostly in the form of a single protein band of apparent mass 110 kDa. Levels of CL and ACC increased during cell differentiation into adipocytes. During the first 3 days of differentiation, CL migration changed, and it was expressed as a complex of protein bands of apparent mass 110 kDa, 113 kDa and 115 kDa. At later stages of differentiation, when these cells had assumed the phenotype of fat-cells, they expressed CL mainly as protein bands of 110 and 113 kDa. When samples containing these bands were treated with alkaline phosphatase, the 113 kDa protein band collapsed into the 110 kDa species. This suggests that the slower-migrating species of CL is a higher-order phosphorylation state of the same protein. Furthermore, when purified CL, mostly expressed as the 110 kDa species, was phosphorylated with cyclic AMP-dependent protein kinase alone or together with GSK-3 and resolved by SDS/PAGE, the phosphorylated CL now migrated more slowly as the 113 kDa and 115 kDa forms. CL phosphorylation was hormone-regulated, since, in samples from fat-cells that had the complex two-band pattern, when cultured in medium without serum or hormones, CL migration reverted to a single band of 110 kDa, similar to confluent cells. Treatment of these ‘down-regulated’ cells with insulin rapidly induced substantial amounts of the 113 kDa species, with a concomitant decrease in the 110 kDa species.

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