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• W2139272214 abstract "The aim of this study was to investigate the role of the cytosolic form of phosphoenolpyruvate carboxykinase (Pck1) in the development of insulin resistance. Previous studies have shown that the roles of Pck1 in white adipose tissue (WAT) in glyceroneogenesis and reesterification of free fatty acids (FFA) to generate triglyceride are vital for the prevention of diabetes. We hypothesized that insulin resistance develops when dysregulation of Pck1 occurs in the triglyceride/fatty acid cycle, which regulates lipid synthesis and transport between adipose tissue and the liver. We examined this by analyzing mice with a deletion of the PPARγ binding site in the promoter of Pck1 (PPARE−/−). This mutation reduced the fasting Pck1 mRNA expression in WAT in brown adipose tissue (BAT). To analyze insulin resistance, we performed hyperinsulinemic-euglycemic glucose clamp analyses. PPARE−/− mice were profoundly insulin resistant and had more FFA and glycerol released during the hyperinsulinemic-euglycemic clamp compared with wild-type mice (WT). Finally, we analyzed insulin secretion in isolated islets. We found a 2-fold increase in insulin secretion in the PPARE−/− mice at 16.7 mM glucose. Thus, the PPARE site in the Pck1 promoter is essential for maintenance of lipid metabolism and glucose homeostasis and disease prevention. The aim of this study was to investigate the role of the cytosolic form of phosphoenolpyruvate carboxykinase (Pck1) in the development of insulin resistance. Previous studies have shown that the roles of Pck1 in white adipose tissue (WAT) in glyceroneogenesis and reesterification of free fatty acids (FFA) to generate triglyceride are vital for the prevention of diabetes. We hypothesized that insulin resistance develops when dysregulation of Pck1 occurs in the triglyceride/fatty acid cycle, which regulates lipid synthesis and transport between adipose tissue and the liver. We examined this by analyzing mice with a deletion of the PPARγ binding site in the promoter of Pck1 (PPARE−/−). This mutation reduced the fasting Pck1 mRNA expression in WAT in brown adipose tissue (BAT). To analyze insulin resistance, we performed hyperinsulinemic-euglycemic glucose clamp analyses. PPARE−/− mice were profoundly insulin resistant and had more FFA and glycerol released during the hyperinsulinemic-euglycemic clamp compared with wild-type mice (WT). Finally, we analyzed insulin secretion in isolated islets. We found a 2-fold increase in insulin secretion in the PPARE−/− mice at 16.7 mM glucose. Thus, the PPARE site in the Pck1 promoter is essential for maintenance of lipid metabolism and glucose homeostasis and disease prevention. brown adipose tissue citrate synthase glucose infusion rate glucose tolerance test glycerol kinase intraperitoneally phosphoenolpyruvate carboxykinase succinate dehydrogenase uncoupling protein white adipose tissue wild type Obesity and type 2 diabetes are growing epidemics worldwide in adults and children. The incidence of type 2 diabetes increased by 10-fold from 1982 to 1994 and is expected to reach 366 million worldwide in 2030 (1Pinhas-Hamiel O. Dolan L.M. Daniels S.R. Standiford D. Khoury P.R. Zeitler P. Increased incidence of non-insulin-dependent diabetes mellitus among adolescents.J. Pediatr. 1996; 128: 608-615Abstract Full Text PDF PubMed Scopus (949) Google Scholar, 2Wild S. Roglic G. Green A. Sicree R. King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030.Diabetes Care. 2004; 27: 1047-1053Crossref PubMed Scopus (11978) Google Scholar). Therefore, it is important to determine the causes of this disease and develop preventive strategies as well as more effective treatments. One risk factor for the development of type 2 diabetes is elevated free fatty acids (FFA) in the blood. Elevated FFAs have been shown to induce insulin resistance in peripheral tissues by inhibiting insulin-stimulated glucose uptake and glycogen synthesis (3Randle P.J. Regulatory interactions between lipids and carbohydrates: the glucose fatty acid cycle after 35 years.Diabetes Metab. Rev. 1998; 14: 263-283Crossref PubMed Scopus (688) Google Scholar, 4Boden G. Role of fatty acids in the pathogenesis of insulin resistance and NIDDM.Diabetes. 1997; 46: 3-10Crossref PubMed Scopus (1442) Google Scholar, 5Boden G. Free fatty acids (FFA), a link between obesity and insulin resistance.Front. Biosci. 1998; 3: d169-d175Crossref PubMed Scopus (121) Google Scholar). In the fed state, triglycerides are synthesized using glycerol-3-phosphate generated from the oxidation of glucose. In the fasted state, however, insulin levels are lower and lipolysis occurs. A significant amount of FFA is reesterified to generate triglyceride in the fasted state in white adipose tissue (WAT) and liver. Since glucose is limited in the fasted state, the ability to generate glycerol-3-phosphate from glucose in adipose tissue is greatly reduced. Also, due to negligible levels of glycerol kinase (GyK) expression in adipose tissue, glycerol-3-phosphate cannot be generated from glycerol. Reshef et al. showed that gluconeogenic precursors, such as pyruvate, lactate, and alanine, are converted into the glycerol backbone of triglyceride by Pck1 and the glyceroneogenic pathway (6Reshef L. Hanson R.W. Ballard F.J. A possible physiological role for glyceroneogenesis in rat adipose tissue.J. Biol. Chem. 1970; 245: 5979-5984Abstract Full Text PDF PubMed Google Scholar) (7Ballard F.J. Hanson R.W. Leveille G.A. Phosphoenolpyruvate carboxykinase and the synthesis of glyceride-glycerol from pyruvate in adipose tissue.J. Biol. Chem. 1967; 242: 2746-2750Abstract Full Text PDF PubMed Google Scholar, 8Reshef L. Niv J. Shapiro B. Effect of propionate on lipogenesis in adipose tissue.J. Lipid Res. 1967; 8: 682-687Abstract Full Text PDF PubMed Google Scholar). In brown adipose tissue (BAT), glyceroneogenesis has been shown to generate as much as 81% of total glycerol-3-phosphate in rats. Hahn and Novak suggested that maintenance of triglyceride stores in the BAT is associated with activation of heat production. They proposed that FFA released from triglyceride were substrates for BAT mitochondria and increase the expression of uncoupling proteins (UCP) (9Hahn P. Novak M. Development of brown and white adipose tissue.J. Lipid Res. 1975; 16: 79-91Abstract Full Text PDF PubMed Google Scholar). Glyceroneogenesis has also been described for the liver during fasting (10Botion L.M. Brito M.N. Brito N.A. Brito S.R. Kettelhut I.C. Migliorini R.H. Glucose contribution to in vivo synthesis of glyceride-glycerol and fatty acids in rats adapted to a high-protein, carbohydrate-free diet.Metabolism. 1998; 47: 1217-1221Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 11Festuccia W.T. Kawashita N.H. Garofalo M.A. Moura M.A. Brito S.R. Kettelhut I.C. Migliorini R.H. Control of glyceroneogenic activity in rat brown adipose tissue.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2003; 285: R177-R182Crossref PubMed Scopus (25) Google Scholar). FFA released from adipose tissue is oxidized and converted to ketone bodies and carbon dioxide. However, a significant portion of the FFA (60%) is reesterified back to triglyceride and packaged into VLDL in the liver. VLDL can then be released for transport to muscle and adipose tissues (10Botion L.M. Brito M.N. Brito N.A. Brito S.R. Kettelhut I.C. Migliorini R.H. Glucose contribution to in vivo synthesis of glyceride-glycerol and fatty acids in rats adapted to a high-protein, carbohydrate-free diet.Metabolism. 1998; 47: 1217-1221Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 12Kalhan S.C. Mahajan S. Burkett E. Reshef L. Hanson R.W. Glyceroneogenesis and the source of glycerol for hepatic triacylglycerol synthesis in humans.J. Biol. Chem. 2001; 276: 12928-12931Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) (13Hanson R.W. Reshef L. Glyceroneogenesis revisited.Biochimie. 2003; 85: 1199-1205Crossref PubMed Scopus (121) Google Scholar). Therefore, the liver plays a central role in the triglyceride/fatty acid cycle, and the pathway of glyceroneogenesis is essential for regulation of the FFA flux in the triglyceride/fatty acid cycle (14Jensen M.D. Ekberg K. Landau B.R. Lipid metabolism during fasting.Am. J. Physiol. Endocrinol. Metab. 2001; 281: E789-E793Crossref PubMed Google Scholar). The aim of this study was to examine the role of Pck1 in the regulation of the triglyceride/fatty acid cycle in the liver and WAT. To accomplish this, we analyzed PPARE−/− mice (15Olswang Y. Cohen H. Papo O. Cassuto H. Croniger C.M. Hakimi P. Tilghman S.M. Hanson R.W. Reshef L. A mutation in the peroxisome proliferator-activated receptor gamma-binding site in the gene for the cytosolic form of phosphoenolpyruvate carboxykinase reduces adipose tissue size and fat content in mice.Proc. Natl. Acad. Sci. USA. 2002; 99: 625-630Crossref PubMed Scopus (118) Google Scholar) that have 0.2% Pck1 mRNA expression of WT in WAT, 20% Pck1 mRNA expression of WT in BAT, and altered regulation of Pck1 mRNA expression in the liver. Mice with reduced Pck1 mRNA expression in WAT were generated as previously described (15Olswang Y. Cohen H. Papo O. Cassuto H. Croniger C.M. Hakimi P. Tilghman S.M. Hanson R.W. Reshef L. A mutation in the peroxisome proliferator-activated receptor gamma-binding site in the gene for the cytosolic form of phosphoenolpyruvate carboxykinase reduces adipose tissue size and fat content in mice.Proc. Natl. Acad. Sci. USA. 2002; 99: 625-630Crossref PubMed Scopus (118) Google Scholar). Briefly, E14 embryonic stem (ES) cells (129/OlaHsd) were manipulated to generate male chimeric mice, and these mice were mated with C57BL/6J female mice, resulting in heterozygous offspring. The mice were intercrossed (brother × sister matings) for over 20 generations to make a stable isogenic strain. Mice containing the wild-type promoter sequence were designated wild-type mice (WT) and were used as controls throughout the study. Mice with a disrupted PPARγ binding site in the Pck1 promoter were identified as homozygous (PPARE−/−) for the mutation. We screened the PPARE−/− mice by polymerase chain reaction (PCR) analysis using primers spanning the PPARE site within the Pck1 promoter. Primer sequences were as follows: forward primer 5′-AGC CAC TTC TTC TGT ACC-3′ and reverse primer 5′-GTA AGC TTT GTT CTG ACA GG-3′. The XhoI restriction endonuclease was used to digest the PCR products and to identify the mutant allele. The mice were housed in microisolator cages and maintained on a 12-h light-dark cycle. Mice had free access to water and were fed a normal mouse diet (LabDiet, St. Louis, MO; Diet #5P76). The approximate composition of the diet was 1.08 kJ protein, 0.58 kJ fat, and 14.33 kJ carbohydrate. The diet consisted of 4100 kJ × kg−1 gross energy (16Mardones P. Strobel P. Miranda S. Leighton F. Quinones V. Amigo L. Rozowski J. Krieger M. Rigotti A. Alpha-tocopherol metabolism is abnormal in scavenger receptor class B type I (SR-BI)-deficient mice.J. Nutr. 2002; 132: 443-449Crossref PubMed Scopus (156) Google Scholar). All experimental protocols were approved by the Case Western Reserve University Institutional Animal Care and Use Committee (IACUC). Total RNA was prepared from 30 mg of liver with the RNeasy Mini kit (Qiagen, Valencia, CA) and from 100 mg of epididymal WAT and BAT with the RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA). We synthesized single-strand cDNA from 2 µg of total RNA with random hexamer primers and MMTV reverse transcriptase (Ambion, Austin, TX) and amplified the cDNAs using the SYBR Green PCR Core reagent mix (Applied Biosystems, Foster City, CA). Real-time quantitative reverse transcription PCR (qRT-PCR) was performed in a Chromo4 Cycler (MJ Research), and the relative amounts of mRNA were determined using a linear regression from the standard curve derivative maximum method using the Opticon Monitor 3 software (MJ Research) as previously described (17Millward C.A. Heaney J.D. Sinasac D.S. Chu E.C. Bederman I.R. Gilge D.A. Previs S.F. Croniger C.M. Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta are protected against diet-induced obesity.Diabetes. 2007; 56: 161-167Crossref PubMed Scopus (79) Google Scholar). The observed mRNA expression levels were normalized to the 18S rRNA levels. The utilized primer sequences (IDT, Coralville, IA) are presented in supplementary Table I). Male mice were deprived of food overnight, and then heart punctures were performed under anesthesia (tribromoethyl alcohol at 249 mg × kg−1) to collect blood. We generated plasma using Microtainer plasma separator tubes (Becton Dickinson, Franklin Lakes, NJ). Veterinary Diagnostic Services (Marshfield Laboratories, Marshfield, WI) measured the levels of cholesterol, β-hydroxybutyrate, FFA, and triglycerides with an automated analyzer (Roche Modular Autoanalyzer). We assayed the liver triglycerides using the Triglyceride Glycerol Phosphate Oxidase (GPO) reagent as previously described (18Millward C.A. Burrage L.C. Shao H. Sinasac D.S. Kawasoe J.H. Hill-Baskin A.E. Ernest S.R. Gornicka A. Hsieh C.W. Pisano S. et al.Genetic factors for resistance to diet-induced obesity and associated metabolic traits on mouse chromosome 17.Mamm. Genome. 2009; 20: 71-82Crossref PubMed Scopus (34) Google Scholar) (Pointe Scientific, Lincoln Park, MI). Plasma insulin concentrations were measured with an Ultrasensitive Mouse Insulin ELISA according to the manufacturer's instructions (Mercodia, Winston Salem, NC). Male mice were deprived of food overnight for 18 h, and 2 g glucose × kg−1 of body weight was injected intraperitoneally (IP) into the mice. Blood was collected from the tail vein, and plasma insulin concentrations were measured at 0 and 30 min after glucose injection. Plasma insulin concentrations were measured as described above. Glucose tolerance tests (GTT) were performed on a separate group of male mice. Briefly, we starved the mice (8 weeks old) for 18 h, subsequently injected IP 2 g glucose × kg−1 of body weight, collected blood from the tail vein, and measured glucose levels at 0, 15, 30, 60, and 120 min using an UltraTouch Glucose Meter®. Clamp studies were performed at the Vanderbilt University Mouse Metabolic Phenotyping Core (MMPC) in Nashville, Tennessee. The clamp studies were conducted in WT and PPARE−/− mice at 12–14 weeks of age. The surgical procedures utilized for implanting chronic jugular vein and carotid artery catheters have been previously described (19Halseth A.E. Bracy D.P. Wasserman D.H. Overexpression of hexokinase II increases insulinand exercise-stimulated muscle glucose uptake in vivo.Am. J. Physiol. 1999; 276: E70-E77PubMed Google Scholar, 20Niswender K.D. Shiota M. Postic C. Cherrington A.D. Magnuson M.A. Effects of increased glucokinase gene copy number on glucose homeostasis and hepatic glucose metabolism.J. Biol. Chem. 1997; 272: 22570-22575Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Mice were allowed to recover from surgery for ≥5 days and were studied when body weight was restored to within 10% of the presurgery weight. The clamp was performed in mice fasted for 5 h. A continuous (0.05 μCi × min−1) infusion of HPLC-purified (21Balak K.J. Keith R.H. Felder M.R. Genetic and developmental regulation of mouse liver alcohol dehydrogenase.J. Biol. Chem. 1982; 257: 15000-15007Abstract Full Text PDF PubMed Google Scholar) 3H-glucose was initiated 120 min before the clamp. At t = 0 min, the basal glucose concentration and specific activity were determined, after which a constant infusion of regular human insulin (2.5 mU × kg−1 × min−1) and red blood cells (3 μl/min) was initiated. The 3H-glucose infusion rate was increased (0.1 μCi/min) to minimize changes in glucose specific activity. Glucose (20%) was infused at a variable rate to maintain euglycemia. Glucose levels were tested every 10 min from the carotid artery catheter. At t = 80, 90, 100, and 120 min, the plasma glucose specific activity was assessed. At t = 120 min, a 12-μCi bolus of 2-deoxyglucose (DG) was administered (22Bagby G.J. Lang C.H. Skrepnik N. Spitzer J.J. Attenuation of glucose metabolic changes resulting from TNF-alpha administration by adrenergic blockade.Am. J. Physiol. 1992; 262: R628-R635Crossref PubMed Google Scholar). Blood (30 μl per mouse) was sampled at different times (t = 122, 125, 130, 135, and 145 min) to determine the arterial blood glucose and plasma DG concentrations (22Bagby G.J. Lang C.H. Skrepnik N. Spitzer J.J. Attenuation of glucose metabolic changes resulting from TNF-alpha administration by adrenergic blockade.Am. J. Physiol. 1992; 262: R628-R635Crossref PubMed Google Scholar). We also analyzed the concentrations of plasma FFA and glycerol under clamp conditions using the FFA and free glycerol kits (Zen-Bio Inc., Research Triangle Park, NC) following the manufacturer's directions. At t = 145 min, a blood sample (100 μl) was obtained, and the mice were anesthetized with an infusion of sodium pentobarbital. Tissues (gastrocnemius, superficial vastus lateralis, and soleus muscles, as well as brain, epididymal fat, and liver) were removed, immediately frozen in liquid nitrogen, and stored at −70°C until further analysis. The rate of whole body glucose appearance (Ra) was calculated as the ratio of the 3H-glucose infusion rate (dpm × kg−1 × min−1) and plasma glucose specific activity (dpm/mg). The rate of endogenous glucose appearance during the hyperinsulinemic clamp was calculated as the difference between Ra and the exogenous glucose infusion rate. We isolated RNA from the liver (100 mg) and measured Pck1 expression as described above. To observe the effects of the PPARE mutation in the Pck1 promoter on insulin signaling, insulin (1 mU × kg−1 body weight) was administered to anesthetize mice by injection into the portal vein. Liver, skeletal muscle, and fat biopsies were removed before (basal) and 5 min after the insulin injection. We snap-froze the tissues for analysis by Western blot analysis. Islets were isolated by the Islet Procurement Core Facility at Vanderbilt University (Nashville, TN) as previously described (23Brissova M. Blaha M. Spear C. Nicholson W. Radhika A. Shiota M. Charron M.J. Wright C.V. Powers A.C. Reduced PDX-1 expression impairs islet response to insulin resistance and worsens glucose homeostasis.Am. J. Physiol. Endocrinol. Metab. 2005; 288: E707-E714Crossref PubMed Scopus (81) Google Scholar, 24Brissova M. Nicholson W.E. Shiota M. Powers A.C. Assessment of insulin secretion in the mouse.Methods Mol. Med. 2003; 83: 23-45PubMed Google Scholar, 25Brissova M. Fowler M. Wiebe P. Shostak A. Shiota M. Radhika A. Lin P.C. Gannon M. Powers A.C. Intraislet endothelial cells contribute to revascularization of transplanted pancreatic islets.Diabetes. 2004; 53: 1318-1325Crossref PubMed Scopus (200) Google Scholar). The islets were isolated from WT and PPARE−/− mice by dissection of the splenic portion of the pancreas followed by digestion with collagenase P (Roche Molecular Biochemicals) (26Stefan Y. Meda P. Neufeld M. Orci L. Stimulation of insulin secretion reveals heterogeneity of pancreatic B cells in vivo.J. Clin. Invest. 1987; 80: 175-183Crossref PubMed Scopus (129) Google Scholar). After a purification procedure, islets were rinsed three times with 12 ml of RPMI-1640 media containing 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 5 mM glucose and then cultured in 10-cm untreated plates overnight at 37°C. As islets from WT and PPARE−/− mice varied in diameter, we hand-picked islets under a stereomicroscope and matched them by size. This common microscopic analysis is based on individual islet sizing, calculation of the frequency distribution, and conversion into an islet equivalent (IEQ), which is the volume of a spherical islet with a diameter of 150 mm (27Lembert N. Wesche J. Petersen P. Doser M. Becker H.D. Ammon H.P. Areal density measurement is a convenient method for the determination of porcine islet equivalents without counting and sizing individual islets.Cell Transplant. 2003; 12: 33-41Crossref PubMed Scopus (35) Google Scholar). On the following day, 20 IEQs were incubated in 2 ml of RPMI-1640 media with 5 mM or 16.7 mM glucose. At the end of a static incubation, islets were collected in 1.5-ml tubes and washed three times with 1 ml of 1× PBS, and insulin was extracted in 0.2 ml of acid alcohol for 48 h at 4°C. The media from the static incubation was harvested in 15-ml conical tubes and centrifuged at 2000 rpm. Islet insulin extracts and the static incubation media were stored at −80°C until insulin measurement by radioimmunoassay. The results are expressed as ng of insulin secreted per 100 IEQs for 30 min. The total islet insulin concentration (% content) was calculated as ng of insulin in the media per ng of insulin isolated from the islets at the end of 30 min. Proteins were isolated from the liver, kidney, WAT, BAT and skeletal muscle from PPARE−/− and WT mice. A piece of tissue was homogenized in 15 ml/g of tissue of ice-cold homogenizing buffer consisting of 20 mM Tris-HCl, pH 7.6, 0.1 mM EDTA, 0.5 mM EGTA, 1.0% Triton-X, 250 mM sucrose, and 50 μl/5 ml protease inhibitor mixture as described previously (28Croniger C.M. Millward C. Yang J. Kawai Y. Arinze I.J. Liu S. Harada-Shiba M. Chakravarty K. Friedman J.E. Poli V. et al.Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta have an attenuated response to cAMP and impaired carbohydrate metabolism.J. Biol. Chem. 2001; 276: 629-638Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The concentration of protein was measured with the Bio-Rad protein assay reagent using BSA as a standard. We sonicated the protein samples for 20 s, and 10–20 μg of protein was diluted in Laemmli sample buffer (100 mM Tris-HCl, pH 6.8, 20% β-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, and 20% glycerol) and separated by 10% SDS-PAGE. For analysis of PCK1, 0.8 mg of liver and kidney proteins and 10 mg of WAT and BAT proteins were loaded. For AKT phosphorylation analysis, 15 µg of liver, WAT, and skeletal proteins were loaded on 10% SDS-PAGE gels. Proteins were electrophoretically transferred to Immobilon® polyvinylidene difluoride membranes, and gels were stained with Coomassie stain (45% methanol, 10% acetic acid, and 2.5% Coomassie Blue R250). The polyvinylidene difluoride membrane was incubated with antibody to PCK1 (1.5 μg/ml) diluted 1:1000 in blocking buffer for 4 h at 22°C, followed by extensive washing with Tris-buffered saline (150 mM NaCl, 10 mM Tris, and 0.05% Tween 20). The primary PCK1 antibody was a generous gift from Richard W. Hanson (Case Western Reserve University). The blots were incubated with 1:10,000 secondary antibody linked to horseradish peroxidase (Santa Cruz Biotechechnology, Inc., Santa Cruz, CA) in 10 ml of blocking buffer for 1 h at 22°C and washed again. Normalization for loading differences was done with an HSC70 antibody diluted at 1:16,000 (Santa Cruz Biotechnology) and anti-mouse IgG-HRP conjugated secondary antibody at 1:10,000 (Santa Cruz Biotechnology). Immunoreactive proteins were detected using the Super-Signal Chemiluminescent Substrate® Kit, and the density of the immunoreactive bands was measured by scanning densitometry using the UN-Scan-IT software (Silk Scientific, Inc., Orem, UT). For portal vein injection of insulin studies, the polyvinylidene difluoride membrane was incubated with antibodies to phosphorylated AKT-Ser473, phosphorylated AKT-Thr308, and total AKT (Cell Signaling Technology, Beverly, MA) diluted in blocking buffer for 4 h at 22°C. The blots were washed and incubated with secondary antibody, and immunoreactive proteins were detected as described above. Wild-type and PPARE−/− male mice were sacrificed, and epididymal fat pads were removed and weighed. Adipose cells were isolated from gonadal fat pads (2–3 mice per experiment) by collagenase digestion (1 mg/ml, collagenase CLS Type I, Worthington Biochemical Corp., Lakewood, NJ). Cells were incubated at 37°C with constant shaking in Krebs-Ringer-Phosphate (KRP) buffer (50 mM HEPES, pH 7.4, 128 mM NaCl, 4.7 mM KCL, 1.25 mM CaCl2, 1.25 mM MgSO4, 10 mM sodium phosphate and 2.0 mM pyruvic acid) and 2.5% BSA (BSA)-FFA free and in the absence (basal) or presence of insulin (100 nM) or epinephrine (10 μM). Concentrations of FFA released from the adipocytes were measured using a commercial kit (Zen-Bio Inc., Research Triangle Park, NC) following the manufacturer's directions. Total cell protein was prepared from adipocytes using a commercial lysate butter (Promega, Madison, WI). The protein concentration was measured with the Bradford assay (Bio-Rad Protein Assay) using BSA as a standard. GyK activity was measured as previously described (29Hibuse T. Maeda N. Funahashi T. Yamamoto K. Nagasawa A. Mizunoya W. Kishida K. Inoue K. Kuriyama H. Nakamura T. et al.Aquaporin 7 deficiency is associated with development of obesity through activation of adipose glycerol kinase.Proc. Natl. Acad. Sci. USA. 2005; 102: 10993-10998Crossref PubMed Scopus (236) Google Scholar, 30Guan H.P. Li Y. Jensen M.V. Newgard C.B. Steppan C.M. Lazar M.A. A futile metabolic cycle activated in adipocytes by antidiabetic agents.Nat. Med. 2002; 8: 1122-1128Crossref PubMed Scopus (332) Google Scholar). Briefly, WAT, BAT, or liver was homogenized in extraction buffer (50 mM HEPES, pH 7.8, 40 mM KCl, 11 mM MgCl2, 1 mM EDTA, and 1 mM DTT) on ice. The samples were centrifuged at 15,000 g for 15 min at 4°C, and 10 µg of protein was used for the enzymatic assay. The protein samples were incubated with 50 µl of assay buffer (50 mM Tris-HCl, pH 7.2, 5 mM ATP, 10 mM MgCl2, 100 mM KCl, 2.5 mM DTT, 4 mM glycerol, and 500 µmol/l 3H-glycerol) for 90 min at 37°C. The reaction was terminated with 100 µl stop solution (ethanol:methanol, 97:3). Equal amounts of samples (20 µl) were spotted onto DE81 Whatman filters (Whatman). The filters were air-dried and washed in water overnight. Radioactivity on the filters was measured by liquid scintillation counting. BAT (20 mg) was homogenized in 1 ml of MSM-EDTA buffer (220 mM mannitol, 70 mM sucrose, 5 mM Mops, and 2 mM EDTA, pH 7.4) supplemented with 1 mg cholate/1 mg wet wt and 1 µl/ml mammalian protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) (31Krahenbuhl S. Chang M. Brass E.P. Hoppel C.L. Decreased activities of ubiquinol:ferricytochrome c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobalamin[c-lactam]-induced methylmalonic aciduria.J. Biol. Chem. 1991; 266: 20998-21003Abstract Full Text PDF PubMed Google Scholar). Mitochondrial content was assessed by measuring the succinate dehydrogenase (SCD) and citrate synthase (CS) activities by established spectrophotometric methods (31Krahenbuhl S. Chang M. Brass E.P. Hoppel C.L. Decreased activities of ubiquinol:ferricytochrome c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobalamin[c-lactam]-induced methylmalonic aciduria.J. Biol. Chem. 1991; 266: 20998-21003Abstract Full Text PDF PubMed Google Scholar). Activities were normalized to the tissue wet weight (mg) and protein (µg/µl) and expressed as nmol/min/mg tissue or nmol/min/total protein. In male PPARE−/− and WT mice, ATP tissue concentrations were measured in BAT and WAT. Samples were dissected from mice under anesthesia and flash-frozen. ATP tissue concentrations were analyzed enzymatically from 25–30 mg of tissue as previously described (32Puchowicz M.A. Xu K. Magness D. Miller C. Lust W.D. Kern T.S. LaManna J.C. Comparison of glucose influx and blood flow in retina and brain of diabetic rats.J. Cereb. Blood Flow Metab. 2004; 24: 449-457Crossref PubMed Scopus (38) Google Scholar) and reported as nmol/mg wet tissue weight. The Mouse Metabolic Phenotyping Center (MMPC) of Case Western Reserve University (Cleveland, OH) measured the triglyceride content and newly synthesized triglyceride levels. To enrich the body water with ∼2% 2H, MMPC administered an IP injection of labeled water (20 µl × g−1 of body weight of 9 g/l NaCl in 99% atomic percentage excess 2H2O) into adult male mice, and the mice were returned to their cages and maintained on 5% 2H-labeled drinking water for 5 days. The mice were killed, and blood and tissue samples were collected and flash-frozen in liquid nitrogen. The samples were stored at −80°C until analysis. MMPC measured triglyceride concentrations and de novo lipogenesis as previously described (33Bederman I.R. Foy S. Chandramouli V. Alexander J.C. Previs S.F. Triglyceride synthesis in epididymal adipose tissue: contribution of glucose and non-glucose carbon sources.J. Biol. Chem. 2009; 284: 6101-6108Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Briefly, triglyceride from tissues was isolated, and labeled glycerol and palmitate were analyzed after derivatization by mass spectrometry. The 2H-labeled triglyceride covalently linked to glycerol measures the amount of newly synthesized triglyceride, and the 2H-labeled triglyceride covalently attached to palmitate indicates the amount of new palmitate. In mice given 2H2O for 5 days, the contribution of de novo lipogenesis to the pool of triglyceride and palmitate was calculated using the following equation: % newly made palmitate = [total 2H-labeled palmitate × (2H-labeled body water × n)]−1 × 100 where n is the number of exchangeable hydrogens, which is assumed to 22 (34Lee W.N. Bassilian S. Guo Z. Schoeller D. Edmond J. Bergner E.A. Byerley L.O. Measurement of fractional lipid synthesis using deuterated water (2H2O) and mass isotopomer analysis.Am. J. Physiol. 1994; 266: E372-E383Crossref PubMed Google Scholar, 35Diraison F. Pachiaudi C. Beylot M. Measuring lipogenesis" @default.
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• W2139272214 title "Phosphoenolpyruvate carboxykinase (Pck1) helps regulate the triglyceride/fatty acid cycle and development of insulin resistance in mice" @default.
• W2139272214 cites W1498897377 @default.
• W2139272214 cites W1508641641 @default.
• W2139272214 cites W1537134958 @default.
• W2139272214 cites W1583122397 @default.
• W2139272214 cites W1673990805 @default.
• W2139272214 cites W1834811765 @default.
• W2139272214 cites W1924042451 @default.
• W2139272214 cites W1959770930 @default.
• W2139272214 cites W1968059884 @default.
• W2139272214 cites W1977464316 @default.
• W2139272214 cites W1981100453 @default.
• W2139272214 cites W1998726695 @default.
• W2139272214 cites W2009411683 @default.
• W2139272214 cites W2013657154 @default.
• W2139272214 cites W2018768852 @default.
• W2139272214 cites W2020267609 @default.
• W2139272214 cites W2023565873 @default.
• W2139272214 cites W2026784736 @default.
• W2139272214 cites W2027117869 @default.
• W2139272214 cites W2029934114 @default.
• W2139272214 cites W2043106623 @default.
• W2139272214 cites W2047399305 @default.
• W2139272214 cites W2055733112 @default.
• W2139272214 cites W2056125864 @default.
• W2139272214 cites W2058355180 @default.
• W2139272214 cites W2067532199 @default.
• W2139272214 cites W2077097398 @default.
• W2139272214 cites W2083278430 @default.
• W2139272214 cites W2088858977 @default.
• W2139272214 cites W2113667153 @default.
• W2139272214 cites W2120282127 @default.
• W2139272214 cites W2122998674 @default.
• W2139272214 cites W2126806931 @default.
• W2139272214 cites W2133208346 @default.
• W2139272214 cites W2133544685 @default.
• W2139272214 cites W2135027079 @default.
• W2139272214 cites W2141470945 @default.
• W2139272214 cites W2146845495 @default.
• W2139272214 cites W2149937631 @default.
• W2139272214 cites W2156812020 @default.
• W2139272214 cites W2165426462 @default.
• W2139272214 cites W2171302838 @default.
• W2139272214 cites W2338808163 @default.
• W2139272214 cites W2437913713 @default.
• W2139272214 cites W4231918589 @default.
• W2139272214 cites W4246612992 @default.
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