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Roche, Pamela Corkey, Stphane Thumelin, Barbara E. Corkey and Marc Prentki
Am J Physiol Endocrinol Metab 277:521-528, 1999.
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P. O. Westermark, J. H. Kotaleski, A. Bjorklund, V. Grill and A. Lansner
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Lipid rather than glucose metabolism is implicated in
altered insulin secretion caused by oleate in INS-1 cells
LAURA SEGALL,1 NATHALIE LAMELOISE,2 FRANC OISE ASSIMACOPOULOS-JEANNET,2
ENRIQUE ROCHE,1 PAMELA CORKEY,3 STE PHANE THUMELIN,1 BARBARA E. CORKEY,3
AND MARC PRENTKI1
1Molecular Nutrition Unit, Department of Nutrition, University of Montreal, and the Centre de
Segall, Laura, Nathalie Lameloise, Francoise Assima- transfer experiments (13). In addition, their Lc-CoA or
copoulos-Jeannet, Enrique Roche, Pamela Corkey, Ste- complex lipid derivatives may act as coupling factors in
phane Thumelin, Barbara E. Corkey, and Marc Prentki. nutrient-stimulated insulin secretion (8, 31).
Lipid rather than glucose metabolism is implicated in altered Acute exposure of -cells to fatty acids potentiates
insulin secretion caused by oleate in INS-1 cells. Am. J.
Physiol. 277 (Endocrinol. Metab. 40): E521E528, 1999.A
glucose-induced insulin secretion by mechanisms that
apparently do not implicate KATP channels but may
explanation for the long-term action of fatty acids on protein pellets in 0.5 N NaOH. Malate was determined with
insulin secretion. the malate dehydrogenase (Boehringer) reaction (46). Citrate
As reviewed previously (34), the following sequence was measured by coupling the citrate lyase-malate dehydroge-
of events associated with predicted alterations in me- nase (Boehringer) reactions according to Williamson and
tabolite levels characterize a Randle cycle. Accelerated Corkey (46). Malonyl-CoA and glucose 6-phosphate were
extracted from cells with 10% trichloroacetic acid. After
fat oxidation results in exaggerated NADH and acetyl- centrifugation of precipitated proteins, cell extracts were
CoA production, thus causing PDH inhibition and brought to pH 56 by successive ether extractions. Samples
reduced glucose oxidation. Citrate also rises via a mass were lyophilized and stored at 70C. Malonyl-CoA was
effect in the mitochondrion and, after its export to the assayed with a radioactive method with fatty acid synthase
cytosolic compartment, allosterically inhibits 6-phospho- (27). Glucose 6-phosphate was measured by a fluorometric
fructo-1-kinase, thus causing reduced glycolytic flux method with glucose 6-phosphate dehydrogenase (6). ATP
and a rise in glucose 6-phosphate. To better understand and ADP were measured by a bioluminescence assay (40). For
how fatty acids increase insulin secretion at low glucose insulin secretion determinations, INS-1 cells were plated (105
and to determine whether altered glucose metabolism cells/well) into 24-well plates. They were cultured and incu-
via a Randle glucose-fatty acid cycle may account for bated as described previously for metabolite measurements.
the lack of glucose response in cells chronically exposed The insulin concentration in the medium was determined by
RIA with rat insulin as standard (2). Total cellular insulin
to FFA, we have carried out a comprehensive study of
content was measured after acid-ethanol (1.5% HCl, 75%
glucose and fatty acid metabolism in INS-1 -cells after ethanol) extraction. DNA was measured according to Labarca
long-term exposure to oleate. and Paigen (14).
metabolic coupling factor in secretion not only via ATP 4. Brun, T., F. Assimacopoulos-Jeannet, B. E. Corkey, and M.
production, for example by controlling sulfhydryl groups Prentki. Long chain fatty acids inhibit acetyl-CoA carboxylase
gene expression in the pancreatic cell line INS-1. Diabetes 46:
of transducing proteins in the -cell, it may be that a 393400, 1997.
rise in glucose from 5 to 25 mM cannot further enhance 5. Brun, T., E. Roche, F. Assimacopoulos-Jeannet, B. E. Cor-
secretion because the reduction state of pyridine nucle- key, K. H. Kim, and M. Prentki. Evidence for an anaplerotic
otides is already maximal at low (5 mM) glucose and malonyl-CoA pathway in pancreatic beta-cell nutrient signaling.
consequently secretion cannot be further enhanced. 2) Diabetes 45: 190198, 1996.
6. Brun, T., E. Roche, K. H. Kim, and M. Prentki. Glucose
The elevated TG content of the -cell is expected to
regulates acetyl-CoA carboxylase gene expression in a pancreatic
result, after lipolysis, in a rise in cytosolic FFA and beta-cell line (INS-1). J. Biol. Chem. 268: 1890518911, 1993.
Lc-CoA. FFA and Lc-CoA stimulate C-kinase enzymes 7. Civelek, V. N., J. T. Deeney, N. J. Shalosky, K. Tornheim,
(1, 28), which might be downregulated after long-term R. G. Hansford, M. Prentki, and B. E. Corkey. Regulation of
exposure to FFA with a resulting loss of a possible pancreatic beta-cell mitochondrial metabolism: influence of Ca2,
important component implicated in secretion. 3) Lc- substrate and ADP. Biochem. J. 318: 615621, 1996.
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NADH produced during glycolysis for beta-cell glucose signaling.
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are known to be elevated in FFA-treated -cells, are
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