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• W2049564955 abstract "Hyperlipidemia appears to play an integral role in loss of glucose-stimulated insulin secretion (GSIS) in type 2 diabetes. This impairment can be simulated in vitro by chronic culture of 832/13 insulinoma cells with high concentrations of free fatty acids, or by study of lipid-laden islets from Zucker diabetic fatty rats. Here we show that impaired GSIS is not a simple result of saturation of lipid storage pathways, as adenovirus-mediated overexpression of a cytosolically localized variant of malonyl-CoA decarboxylase in either cellular model results in dramatic lowering of cellular triglyceride stores but no improvement in GSIS. Instead, the glucose-induced increment in “pyruvate cycling” activity (pyruvate exchange with tricarboxylic acid cycle intermediates measured by 13C NMR), previously shown to play an important role in GSIS, is completely ablated in concert with profound suppression of GSIS in lipid-cultured 832/13 cells, whereas glucose oxidation is unaffected. Moreover, GSIS is partially restored in both lipid-cultured 832/13 cells and islets from Zucker diabetic fatty rats by addition of a membrane permeant ester of a pyruvate cycling intermediate (dimethyl malate). We conclude that chronic exposure of islet β-cells to fatty acids grossly alters a mitochondrial pathway of pyruvate metabolism that is important for normal GSIS. Hyperlipidemia appears to play an integral role in loss of glucose-stimulated insulin secretion (GSIS) in type 2 diabetes. This impairment can be simulated in vitro by chronic culture of 832/13 insulinoma cells with high concentrations of free fatty acids, or by study of lipid-laden islets from Zucker diabetic fatty rats. Here we show that impaired GSIS is not a simple result of saturation of lipid storage pathways, as adenovirus-mediated overexpression of a cytosolically localized variant of malonyl-CoA decarboxylase in either cellular model results in dramatic lowering of cellular triglyceride stores but no improvement in GSIS. Instead, the glucose-induced increment in “pyruvate cycling” activity (pyruvate exchange with tricarboxylic acid cycle intermediates measured by 13C NMR), previously shown to play an important role in GSIS, is completely ablated in concert with profound suppression of GSIS in lipid-cultured 832/13 cells, whereas glucose oxidation is unaffected. Moreover, GSIS is partially restored in both lipid-cultured 832/13 cells and islets from Zucker diabetic fatty rats by addition of a membrane permeant ester of a pyruvate cycling intermediate (dimethyl malate). We conclude that chronic exposure of islet β-cells to fatty acids grossly alters a mitochondrial pathway of pyruvate metabolism that is important for normal GSIS. A major contributing factor to the development of type 2 diabetes is inadequate insulin secretion to compensate for insulin resistance. A hallmark of this β-cell dysfunction is the impairment and eventual complete loss of glucose-stimulated insulin secretion (GSIS). 1The abbreviations used are: GSIS, glucose-stimulated insulin secretion; DMM, dimethyl malate; Glc, glucose, HBSS, HEPES-buffered saline; MCD, malonyl-CoA decarboxylase; TG, triglycerides; FFA, free fatty acids; ZDF, Zucker diabetic fatty; PC, pyruvate carboxylase; m.o.i., multiplicity of infection; pfu, plaque-forming units; PBS, phosphate-buffered saline. Hyperlipidemia, and the consequent accumulation of triglycerides (TG) and other lipid-derived intermediates in β-cells, is now well recognized as a variable that correlates with development of impaired insulin secretion (1Sako Y. Grill V.E. Endocrinology. 1990; 127: 1580-1589Crossref PubMed Scopus (347) Google Scholar, 2Lee Y. Hirose H. Ohneda M. Johnson J.H. McGarry J.D. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10878-10882Crossref PubMed Scopus (721) Google Scholar, 3Unger R.H. Diabetes. 1995; 44: 863-870Crossref PubMed Google Scholar, 4Prentki M. Corkey B.E. Diabetes. 1996; 45: 273-283Crossref PubMed Scopus (0) Google Scholar, 5McGarry J.D. Dobbins R.L. Diabetologia. 1999; 42: 128-138Crossref PubMed Scopus (498) Google Scholar, 6Poitout V. Robertson R.P. Endocrinology. 2002; 143: 339-342Crossref PubMed Scopus (550) Google Scholar). Furthermore, culture of pancreatic islets (3Unger R.H. Diabetes. 1995; 44: 863-870Crossref PubMed Google Scholar, 7Zhou Y.P. Grill V.E. J. Clin. Investig. 1994; 93: 870-876Crossref PubMed Scopus (631) Google Scholar, 8Milburn Jr., J.L. Hirose H. Lee Y.H. Nagasawa Y. Ogawa A. Ohneda M. Beltrandel Rio H. Newgard C.B. Johnson J.H. Unger R.H. J. Biol. Chem. 1995; 270: 1295-1299Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) or insulinoma cell lines (9Segall L. Lameloise N. Assimacopoulos-Jeannet F. Roche E. Corkey P. Thumelin S. Corkey B.E. Prentki M. Am. J. Physiol. 1999; 277: E521-E528PubMed Google Scholar) with elevated levels of free fatty acids in vitro results in loss of GSIS, and glucose sensing is also dramatically impaired in fat-laden islets from Zucker diabetic fatty (ZDF) rats (2Lee Y. Hirose H. Ohneda M. Johnson J.H. McGarry J.D. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10878-10882Crossref PubMed Scopus (721) Google Scholar, 3Unger R.H. Diabetes. 1995; 44: 863-870Crossref PubMed Google Scholar). However, a biochemical mechanism linking chronic exposure of islet cells to high levels of free fatty acids and impairment of GSIS has not emerged. To gain more insight into this important issue, two independent model systems were exploited. First, we have described recently (10Hohmeier H.E. Mulder H. Chen G. Henkel-Rieger R. Prentki M. Newgard C.B. Diabetes. 2000; 49: 424-430Crossref PubMed Scopus (716) Google Scholar) stable subclones of the rat insulinoma INS-1 cell line with robust GSIS, such as cell line 832/13. As shown here, chronic culture of these cells in 1 mm oleate/palmitate (2:1) causes profound impairment of GSIS. Second, islets from ZDF rats are both lipid-laden and poorly glucose-responsive (3Unger R.H. Diabetes. 1995; 44: 863-870Crossref PubMed Google Scholar). By using these model systems, two hypotheses about the mechanism of lipid-induced impairment of GSIS were tested. The first is that accumulation of lipid-derived metabolites caused by chronic exposure of β-cells to fatty acids plays a direct role in the functional impairment. To test this idea, we have employed a recombinant adenovirus encoding a variant, cytosolically localized form of malonyl-CoA decarboxylase (AdCMV-MCDΔ5) (11Mulder H. Lu D. Finley J.T. An J. Cohen J. Antinozzi P.A. McGarry J.D. Newgard C.B. J. Biol. Chem. 2001; 276: 6479-6484Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) to lower malonyl-CoA levels in lipid-laden cells. Application of this method caused a dramatic lowering of TG levels in both lipid-cultured 832/13 cells and in islets from ZDF rats but failed to improve GSIS. This led us to test a second hypothesis based on our recent discovery of a critical link between pyruvate carboxylase (PC)-mediated pyruvate exchange with tricarboxylic acid cycle intermediates (“pyruvate cycling”) and GSIS (12Lu D. Mulder H. Zhao P. Burgess S.C. Jensen M.V. Kamzolova S. Newgard C.B. Sherry A.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2708-2713Crossref PubMed Scopus (221) Google Scholar). This link between pyruvate cycling and GSIS was uncovered by NMR-based analysis of [U-13C]glucose metabolism in a set of variously glucose-responsive INS-1-derived cell lines. More precisely, pyruvate cycling refers either to the “pyruvate/malate cycle,” involving PC-catalyzed conversion of pyruvate to oxaloacetate, reduction of oxaloacetate to malate, and decarboxylation of malate to pyruvate via malic enzyme and/or to the “pyruvate/citrate cycle,” wherein the first and last steps are the same as in the pyruvate/malate cycle. Oxaloacetate formed in the PC reaction is converted to citrate, after which malate is regenerated via citrate lyase and cytosolic malate dehydrogenase (12Lu D. Mulder H. Zhao P. Burgess S.C. Jensen M.V. Kamzolova S. Newgard C.B. Sherry A.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2708-2713Crossref PubMed Scopus (221) Google Scholar). The NMR methods that we employ are not capable of distinguishing between these cycles but do discriminate total cycling activity relative to tricarboxylic acid cycle flux. In the current study, we demonstrate that the profound impairment of GSIS that occurs in response to chronic exposure of 832/13 cells to fatty acids is accompanied by complete ablation of the normal glucose-induced increment in pyruvate cycling, with no change in the rates of glucose oxidation at basal or stimulatory glucose. Furthermore, we demonstrate that addition of a membrane-permeant ester of a pyruvate cycling intermediate, dimethyl malate (DMM), restores a significant portion of GSIS in both fat-cultured 832/13 cells and fat-laden islets from ZDF rats. Thus, our data support a model for lipid-induced impairment of GSIS in which chronic exposure to elevated levels of fatty acids alters a mitochondrial pathway of pyruvate metabolism that is involved in GSIS. Materials were obtained from Sigma unless otherwise noted. Cell Culture—A clonal β-cell line, 832/13 (10Hohmeier H.E. Mulder H. Chen G. Henkel-Rieger R. Prentki M. Newgard C.B. Diabetes. 2000; 49: 424-430Crossref PubMed Scopus (716) Google Scholar), derived from INS-1 rat insulinoma cells (13Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Crossref PubMed Scopus (748) Google Scholar) by a transfection-selection strategy, was used in these studies. 832/13 cells were cultured in RPMI 1640 containing 11.1 mm d-glucose and supplemented with 10% fetal bovine serum, 10 mm HEPES, 2 mm glutamine, 1 mm sodium pyruvate, and 50 μm β-mercaptoethanol. Cells were cultured in 6- or 12-well plates at 37 °C in a humidified atmosphere containing 5% CO2, and media were changed routinely every 2nd day. For studies of lipid-induced impairment of GSIS, we prepared a 10 mm oleate/palmitate (2:1 molar ratio) stock solution complexed to 10% fat-free bovine serum albumin (8Milburn Jr., J.L. Hirose H. Lee Y.H. Nagasawa Y. Ogawa A. Ohneda M. Beltrandel Rio H. Newgard C.B. Johnson J.H. Unger R.H. J. Biol. Chem. 1995; 270: 1295-1299Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). This was added at a final concentration of 1 mm to cells at a state of 30–90% confluency in complete RPMI medium, dependent upon the planned duration of lipid exposure. Cells were then cultured for an additional 2–7 days as indicated in the figure legends or text. Experimental Animals—Lean wild type(+/+) male ZDF rats and obese homozygous (fa/fa) male ZDF rats were bred at the Veterans Affairs Medical Center, Dallas, TX. 11-Week-old male rats were used for the experiments. Rats were fed with standard chow (Harlan/Teklad 4% 7001; Madison, WI) ad libitum and had free access to water. Recombinant Adenoviruses—Malonyl-CoA decarboxylase (MCD) was overexpressed in the cytoplasmic compartment of 832/13 cells and islets by treatment with a recombinant adenovirus containing the cDNA encoding human MCD, modified by removal of its N-terminal mitochondrial localization sequence and its C-terminal peroxisomal targeting sequence (AdCMV-MCDΔ5 (11Mulder H. Lu D. Finley J.T. An J. Cohen J. Antinozzi P.A. McGarry J.D. Newgard C.B. J. Biol. Chem. 2001; 276: 6479-6484Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar)). As controls, other groups of cells were treated either with a virus encoding a catalytically inactive form of MCD (AdCMV-MCDmut (11Mulder H. Lu D. Finley J.T. An J. Cohen J. Antinozzi P.A. McGarry J.D. Newgard C.B. J. Biol. Chem. 2001; 276: 6479-6484Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 14Gao J. Waber L. Bennett M.J. Gibson K.M. Cohen J.C. J. Lipid Res. 1999; 40: 178-182Abstract Full Text Full Text PDF PubMed Google Scholar)) or the bacterial β-galactosidase gene (AdCMV-βGAL (15Herz J. Gerard R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2812-2816Crossref PubMed Scopus (504) Google Scholar)). Adenovirus Transduction of 832/13 Cells—832/13 cells at a confluency of ∼90% were treated with 10 or 20 m.o.i. (multiplicity of infection) of the various recombinant adenoviruses for 2 h. Cells were washed once in RPMI and cultured for an additional 24–72 h. Islet Isolation and Adenovirus Transduction—To overexpress MCD in islets of obese and diabetic rats, pancreases of 11-week-old male ZDF (fa/fa) rats were perfused for 1 h with 1 × 1012 plaque-forming units (pfu) of AdCMV-MCDΔ5, suspended in Krebs-Ringer bicarbonate buffer with 4.5% dextran T70, 1% bovine serum albumin, 5.6 mm glucose, and 5 mm each of sodium pyruvate, sodium glutamate, and sodium fumarate (16Wang M.Y. Koyama K. Shimabukuro M. Newgard C.B. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 714-718Crossref PubMed Scopus (90) Google Scholar). Pancreatic islets were then isolated by the method of Naber et al. (17Naber S.P. McDonald J.M. Jarett L. McDaniel M.L. Ludvigsen C.W. Lacy P.E. Diabetologia. 1980; 19: 439-444Crossref PubMed Scopus (55) Google Scholar) with modifications (8Milburn Jr., J.L. Hirose H. Lee Y.H. Nagasawa Y. Ogawa A. Ohneda M. Beltrandel Rio H. Newgard C.B. Johnson J.H. Unger R.H. J. Biol. Chem. 1995; 270: 1295-1299Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Isolated islets were either used immediately or were cultured for 48 h in RPMI 1640 with 8 mm glucose, supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 10 mm HEPES, 2 mm glutamine, 1 mm sodium pyruvate, and 50 μm β-mercaptoethanol. Malonyl-CoA Decarboxylase Activity Assay—MCD activity was determined as the rate of decarboxylation of malonyl-CoA to acetyl-CoA as described previously (18Antinozzi P.A. Segall L. Prentki M. McGarry J.D. Newgard C.B. J. Biol. Chem. 1998; 273: 16146-16154Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). In brief, the rate of acetyl-CoA formation was monitored by cleavage of its thioester bond by acetylcarnitine transferase over 5–10 min, a period when the rate of product accumulation was linear. Oil Red O Staining of Lipid Droplets—832/13 cells were cultured with or without 1 mm oleate/palmitate (2:1) for 6 days. Aliquots of cells cultured in the presence of fat were treated with 10 or 20 m.o.i. of AdCMV-MCDΔ5 virus, or were left untreated as indicated in the legend to Fig. 1. After viral treatment, cells were cultured for an additional 24 h in the presence of fatty acids. Following this period, media were removed, and cells were washed once with PBS and fixed with 2.5% glutaraldehyde (Fisher) for 15 min. Glutaraldehyde was removed, cells were washed with PBS, and then treated for 20 min with Oil Red O staining solution. After a wash with PBS, images of stained lipids were obtained using a Nikon phase contrast ELWD 0.3 microscope at high magnification. Triglyceride Content of 832/13 Cells or Islets—832/13 cells were cultured in the presence or absence of 1 mm oleate/palmitate (2:1) for 6 days, treated with the various recombinant adenoviruses, and harvested 24 h after transduction. Islets from lean or obese ZDF rats were cultured for 48 h prior to harvesting. Measurement of TG content was based on assay of glycerol produced by hydrolysis of neutral lipids in the presence of lipoprotein lipase. Islets or cells were centrifuged at 1200 rpm for 10 min at 4 °C, washed with PBS, and re-centrifuged. Lipids were extracted with chloroform/methanol (2:1) and the lower phase was evaporated under N2. The samples were resuspended in 50 μl of chloroform, and 10 μl was air-dried. The dry pellet was resuspended in 10 μl of Thesit (ICN). A standard curve (1–50 μg) was prepared using triolein diluted in chloroform/methanol 2:1, and 10 μl of sample or standard were assayed in duplicate by adding 200 μl of GPO Trinder Reagent and measurement of absorbance at 540 nm. Insulin Secretion Studies—832/13 cells were grown in the presence or absence of 1 mm oleate/palmitate (2:1) for the periods indicated in the figure legends. Cells were washed with HEPES-buffered saline (HBSS) with 114 mm NaCl, 4.7 mm KCl, 1.2 mm KH2PO4, 1.16 mm MgSO4, 20 mm HEPES, 2.5 mm CaCl2, and 25.5 mm NaHCO3, pH 7.2), containing 0.2% bovine serum albumin, followed by a 2-h preincubation in the same buffer. Insulin secretion was then measured by static incubation of the cells for 2 h in HBSS containing either 3 or 12 mm glucose. DMM (Aldrich) was added at a final concentration of 10 mm in certain experiments during this second incubation. Insulin levels were determined by radioimmunoassay using the DPC Coat-A-Count kit (Los Angeles, CA), according to the manufacturer's recommendations, and expressed as nanograms of insulin per mg of cellular protein. For studies of insulin secretion from ZDF islets, cells were washed with PBS and preincubated in HBSS containing 3 mm glucose for 1 h. Insulin secretion was then measured with the same static incubation protocol as described for 832/13 cells, using two uniformly sized islets/condition in triplicate and exposed to 3 or 16.7 mm glucose. NMR Measurements—832/13 cells were cultured for 3 days in 15-cm Petri dishes in the presence or absence of 1 mm oleate/palmitate (2:1). Cells were washed once with PBS, preincubated in HBSS for 1 h, and then incubated with 3 or 12 mm [U-13C6]glucose (Cambridge Isotope Laboratories, Cambridge, MA) in HBSS. An incubation time of 4 h was chosen based on previous experiments showing that glutamate became highly enriched in 13C in these cells within this time, and isotopic steady state is reached by ≈3 h (12Lu D. Mulder H. Zhao P. Burgess S.C. Jensen M.V. Kamzolova S. Newgard C.B. Sherry A.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2708-2713Crossref PubMed Scopus (221) Google Scholar). After 4 h of incubation, the assay buffer was collected for determination of insulin levels, and cells were washed once with ice-cold PBS and extracted with 3.5% ice-cold perchloric acid. Extracts from three dishes were pooled and neutralized with KOH in an ice bath. The KClO4 precipitate was centrifuged, and the supernatant was decanted and lyophilized. The sample was then dissolved in 2H2O for mass isotopomer analysis of extracted glutamate by 13C NMR. Proton-decoupled 13C NMR spectra were recorded on a 600-MHz 14T Varian INOVA NMR spectrometer by using a 45° pulse and a 3-s repetition time in a 5-mm tunable broadband probe. The areas of the multiplets arising from 13C to 13C spin-spin coupling in the glutamate C2, C3, and C4 resonances were determined by using the line-fitting routine in the PC-based NMR program, NUTS (Acorn NMR, Fremont, CA). These multiplet areas were used to perform a 13C-isotopomer analysis with the program tcaCALC described previously (19Jeffrey F.M. Storey C.J. Sherry A.D. Malloy C.R. Am. J. Physiol. 1996; 271: E788-E799Crossref PubMed Google Scholar). The program was applied using the same model parameters as reported recently (12Lu D. Mulder H. Zhao P. Burgess S.C. Jensen M.V. Kamzolova S. Newgard C.B. Sherry A.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2708-2713Crossref PubMed Scopus (221) Google Scholar) to determine a metabolic profile for metabolism of [U-13C6]glucose in the tricarboxylic acid cycle. O2 Consumption and Conversion of Relative Fluxes to Absolute Fluxes—Oxygen consumption was measured at 37 °C using an Oxytherm electrode connected to an Oxygraph Measurement System (Hansatech Instruments Ltd., King's Lynn, Norfolk, UK). 832/13 cells were cultured in the presence or absence of 1 mm oleate/palmitate in 6-well plates for 3 days. Tissue culture medium was removed, and cells were washed and preincubated in HBSS containing 3 mm glucose. Two hours later, cells were switched to 2 ml of the same buffer containing either 3 or 12 mm glucose for another hour. Before collecting the cells, the measurement system was calibrated according to the manufacturer's instructions. For each measurement, cells from three wells were gently scraped free and pooled in a total volume of ∼350 μl, and 250 μl of the cell suspension was immediately added to the oxygen electrode chamber. Oxygen consumption rates were expressed as nanomoles of oxygen/mg of protein/min. 13C NMR isotopomer analysis (19Jeffrey F.M. Storey C.J. Sherry A.D. Malloy C.R. Am. J. Physiol. 1996; 271: E788-E799Crossref PubMed Google Scholar, 20Sherry A.D. Malloy C.R. Berliner L.J. Robitaille P.-M.L. In Vivo Carbon-13 NMR, Biological Magnetic Resonance. 15. Kluwer Academic Publishers/Plenum Publishing Corp., New York1998: 59-97Google Scholar, 21Malloy C.R. Sherry A.D. Jeffrey F.M. Am. J. Physiol. 1990; 259: H987-H995Crossref PubMed Google Scholar) provided a direct measure of the fraction of acetyl-CoA contributed by [U-13C6]glucose (commonly designated as FC3), the fraction of acetyl-CoA contributed by endogenous unlabeled substrates (by difference, FC0 = 1 - FC3), and pyruvate cycling flux relative to total tricarboxylic acid cycle flux. Given well established relationships between tricarboxylic acid cycle flux (NADH and FADH2 production) and O2 consumption, these NMR determined parameters were then converted into absolute flux values (22Malloy C.R. Jones J.G. Jeffrey F.M. Jessen M.E. Sherry A.D. Magn. Reson. Mater. Phys. Biol. Med. 1996; 4: 35-46Crossref Scopus (68) Google Scholar). Briefly, given that complete oxidation of 1 mol of acetyl-CoA consumes 2 mol of molecular oxygen (one cycle turn nets 4 reducing equivalents or 8 electrons), one can derive the proportionality factor (Ri = Qi/Ci) relating O2 consumption (Qi) to tricarboxylic acid cycle flux (Ci) for any given substrate. For example, glycolysis yields 2 triose units. Complete oxidation per triose unit produces 4 reducing equivalents in the tricarboxylic acid cycle and 2 additional reducing equivalents, 1 in glycolysis and the other at the level of pyruvate dehydrogenase, for a total of 6; hence, Ri = 3 for each triose unit of glucose. The Ri values for other common substrates have been tabulated elsewhere (22Malloy C.R. Jones J.G. Jeffrey F.M. Jessen M.E. Sherry A.D. Magn. Reson. Mater. Phys. Biol. Med. 1996; 4: 35-46Crossref Scopus (68) Google Scholar). Total O2 consumption by tissue may be defined as Qt = Q0 + Qglucose, where Q0 refers to O2 consumption from oxidation of endogenous triglycerides or fats and Qglucose to O2 consumption from oxidation of glucose. Similarly, total tricarboxylic acid cycle flux is defined as Ct = C0 + Cglucose, where C0 refers to tricarboxylic acid cycle flux due to oxidation of endogenous substrates and Cglucose to oxidation of glucose. Since the Ri factor for each substrate relates O2 consumption to tricarboxylic acid cycle flux, it follows that Qt = C0R0 + CglucoseRglucose. Given that the FCi variables are defined by the fraction any given substrate makes to total acetyl-CoA entering the tricarboxylic acid cycle, FC0 = C0/Ct and FC3 = C3/Ct (where FC0 and FC3 is the fraction of acetyl-CoA derived from fats or glucose, respectively). These relationships can be combined to yield Qt/Ct = FC0R0 + FC3Rglucose. Hence, if O2 consumption (Qt) can be determined as an absolute flux (using an oxygen electrode for instance), and the FCi values measured by 13C NMR and the Ri factor for each substrate are known, then tricarboxylic acid cycle flux (Ct) may be calculated. It should be noted that this equation applies only when anaplerosis can be ignored. However, anaplerosis was also determined as a fraction of tricarboxylic acid cycle flux (y), and separate proportionality factors exist for anaplerotic substrates (Ra) (Ra is 0 for carboxylation of exogenous pyruvate and 0.5 for carboxylation of pyruvate derived from glucose) (22Malloy C.R. Jones J.G. Jeffrey F.M. Jessen M.E. Sherry A.D. Magn. Reson. Mater. Phys. Biol. Med. 1996; 4: 35-46Crossref Scopus (68) Google Scholar). Thus, after measuring oxygen consumption, the fractional contribution of each substrate to acetyl-CoA and anaplerosis, Ct, is easily determined as shown in Equation 1. Qt/Ct=FC0R0+FC3Rglucose+yRα (Eq. 1) It follows then that absolute glucose oxidation is simply FC3Ct, and endogenous lipid oxidation is FC0Ct. Furthermore, since pyruvate cycling is determined as a fraction of Ct, it too can be expressed as an absolute flux. Let us then consider the example of the control cells incubated with 12 mm glucose. The complete isotopomer analysis of the NMR data indicated that acetyl-CoA was derived from endogenous lipids (FC0 = 0.19) or glucose (FC3 = 0.81) and that anaplerosis was high (y = 0.95, we assume from glucose-derived pyruvate). Oxygen consumption was determined by oxygen electrode to be 12.5 nmol/min/mg protein. One can then estimate the contribution from each term in Equation 1 to O2 consumption as follows: FC0 = 0.19, R0 for lipids is ∼2.8 (based on known R0 for palmitate), hence term 1 = 0.19 × 2.8 = 0.53; FC3 = 0.81, R3 for glucose is 3, hence term 2 = 0.81 × 3 = 2.4; y = 0.95, Ra = 0.5, hence term 3 = 3.2 × 0.5 = 0.48. Thus, the proportionality constant (Qt/Ct) relating total tricarboxylic acid cycle flux to total O2 consumption is 3.4, and tricarboxylic acid cycle flux is 12.5/3.4 = 3.7 nmol of acetyl-CoA/min/mg of protein; endogenous lipid oxidation is 0.19 × 3.7 = 0.70 nmol of acetyl-CoA/min/mg of protein; and glucose oxidation is 0.81 × 3.7 = 3.0 nmol of acetyl-CoA/min/mg of protein. Statistical Methods—Statistical analysis of the data was performed using the two-tailed Student's t test, assuming unequal variances. MCD Expression Reduces TG Content—Functional impairment of β-cells has been correlated with elevated TG stores in a number of different model systems. However, these studies have not addressed the issue of whether saturation of lipid storage pathways participates directly in loss of GSIS. To address this point, we used a recombinant adenovirus to express a modified form of malonyl-CoA decarboxylase (AdCMV-MCDΔ5) in 832/13 cells. MCDΔ5 is preferentially localized to the cytosol and completely blocks the glucose-induced rise in malonyl-CoA levels (11Mulder H. Lu D. Finley J.T. An J. Cohen J. Antinozzi P.A. McGarry J.D. Newgard C.B. J. Biol. Chem. 2001; 276: 6479-6484Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). 832/13 cells were cultured in the presence of 1 mm oleate/palmitate for 6 days and then treated with one of the following viruses: AdCMV-MCDΔ5, a control virus containing the bacterial β-galactosidase gene AdCMV-βGAL (15Herz J. Gerard R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2812-2816Crossref PubMed Scopus (504) Google Scholar), or a control virus containing a catalytically inactive form of MCD, AdCMV-MCDmut (11Mulder H. Lu D. Finley J.T. An J. Cohen J. Antinozzi P.A. McGarry J.D. Newgard C.B. J. Biol. Chem. 2001; 276: 6479-6484Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and cultured for an additional 24 h in the presence of lipids. Another group of 832/13 cells was simply cultured in the presence or absence of 1 mm oleate/palmitate for the total experimental period of 7 days, with no addition of viruses. Cells treated with AdCMV-MCDΔ5 had ∼5 times more MCD enzymatic activity than either control group (1.5 ± 0.2 versus 0.3 ± 0.1 μmol/min/mg protein, respectively). Treatment of lipid-cultured 832/13 cells with AdCMV-MCDΔ5 caused a dramatic decrease in Oil Red O staining of stored lipids (Fig. 1) and also lowered TG content to levels indistinguishable from those in cells cultured in the absence of exogenous fatty acids (from 332 ± 31 to 118 ± 15 ng/mg protein; Fig. 2). MCD Overexpression Fails to Reverse Lipid-induced Impairment of GSIS in 832/13 Cells or Islets from ZDF Rats—We next investigated the effect of these maneuvers on GSIS (Fig. 3). A culture of 832/13 cells in 1 mm oleate/palmitate for 7 days resulted in clear impairment of insulin secretion during stimulation with 12 mm glucose. When normalized to basal secretion at 3 mm glucose, GSIS fell from 10.6 ± 2.5-, 9.4 ± 2.3-, and 9.6 ± 1.7-fold in cells cultured in normal medium containing 11 mm glucose (without supplemental fatty acids) to 2.3 ± 0.4-, 2.4 ± 0.4-, and 2.7 ± 0.6-fold in cells cultured in 1 mm oleate/palmitate for 7 days, in the AdCMV-βGAL, AdCMV-MCDΔ5, and AdCMV-MCDmut-treated groups, respectively (Fig. 3). Thus, normalization of cellular TG levels by overexpression of MCDΔ5 did not prevent the lipid-induced impairment in GSIS. Consistent with our previous work, overexpression of MCDΔ5 did not affect GSIS in cells cultured in the absence of fat (11Mulder H. Lu D. Finley J.T. An J. Cohen J. Antinozzi P.A. McGarry J.D. Newgard C.B. J. Biol. Chem. 2001; 276: 6479-6484Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 18Antinozzi P.A. Segall L. Prentki M. McGarry J.D. Newgard C.B. J. Biol. Chem. 1998; 273: 16146-16154Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). We also performed experiments in which the three adenoviruses were delivered after 4 days of culture in fatty acids, followed by 3 more days of culture in the presence of the exogenous lipids and assay of GSIS at day 7 of culture. In this experimental design, AdCMV-MCDΔ5 treatment prevented the rise in TG content but again did not prevent the fat-induced impairment in GSIS (data not shown). Taken together, these findings suggest that factors other than TG overstorage are involved in the lipid-induced impairment of GSIS in 832/13 cells. One potential criticism of the studies just summarized is that chronic culture of an insulinoma cell line in elevated fatty acids may not be fully reflective of β-cell deterioration that occurs in vivo in animals or humans with type 2 diabetes and chronic hyperlipidemia. To investigate this further, we delivered the AdCMV-MCDΔ5 virus to lipid-laden islets from obese ZDF (fa/fa) rats by pancreas perfusion (16Wang M.Y. Koyama K. Shimabukuro M. Newgard C.B. Unger R.H. Proc. Nat" @default.
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