Advertisement

A pilot study of the effects of pioglitazone and rosiglitazone on de novo lipogenesis in type 2 diabetes

Open AccessPublished:July 18, 2008DOI:https://doi.org/10.1194/jlr.M800165-JLR200
      Treatment of type 2 diabetes mellitus (T2DM) patients with pioglitazone results in a more favorable lipid profile, and perhaps more favorable cardiac outcomes, than treatment with rosiglitazone. Pioglitazone treatment increases VLDL-triacylglycerol clearance, but the role of de novo lipogenesis (DNL) has not been explored, and no direct comparison has been made between the thiazolidinediones (TZDs). Twelve subjects with T2DM and hypertriacylglyceridemia were randomized to either rosiglitazone or pioglitazone treatment. Stable isotope infusion studies were performed at baseline and after 20 weeks of treatment. Both treatments reduced glucose and HbA1c concentrations equally. Pioglitazone treatment resulted in a 40% reduction in hepatic DNL (P < 0.01) and in a 25% reduction in hepatic glucose production (P < 0.05), while rosiglitazone did not significantly change either parameter, although comparisons of changes between treatments were not significantly different. These pilot results indicate that pioglitazone reduces hepatic DNL while rosiglitazone does not. Larger follow-up studies are required to confirm differential effects of these agents definitively. The reduction in DNL may underlie altered assembly or atherogenicity of lipoprotein particles and may reflect PPARα or other non-PPARγ actions on the liver by pioglitazone. These differences might help explain previously reported differences in lipid profiles and cardiovascular disease outcomes for rosiglitazone and pioglitazone.
      Sources of triacylglycerols (TAG) assembled in the liver include plasma nonesterified fatty acids (NEFA), dietary fats, and fatty acids newly synthesized through the pathway of de novo lipogenesis (DNL). Hepatic TAG are either secreted immediately as very low density lipoprotein (VLDL)-TAG or stored in the cytosol and secreted after a delay (
      • Vedala A.
      • Wang W.
      • Neese R.A.
      • Christiansen M.P.
      • Hellerstein M.K.
      Delayed secretory pathway contributions to VLDL-triglycerides from plasma NEFA, diet, and de novo lipogenesis in humans.
      ). Although the majority of the fatty acids in VLDL-TAG derive from plasma NEFA, rates of DNL vary markedly in different settings, suggesting that this pathway may play important metabolic roles. DNL increases by 50–200% in the fasted state in obese (
      • Diraison F.
      • Dusserre E.
      • Vidal H.
      • Sothier M.
      • Beylot M.
      Increased hepatic lipogenesis but decreased expression of lipogenic gene in adipose tissue in human obesity.
      ), insulin-resistant humans (
      • Schwarz J.M.
      • Neese R.
      • Shackleton C.H.L.
      • Hellerstein M.K.
      De novo lipogenesis (DNL) during fasting and oral fructose in lean and obese hyperinsulinemic subjects. [Abstract].
      ) and in subjects with hypertriacylglyceridemia (
      • Vedala A.
      • Wang W.
      • Neese R.A.
      • Christiansen M.P.
      • Hellerstein M.K.
      Delayed secretory pathway contributions to VLDL-triglycerides from plasma NEFA, diet, and de novo lipogenesis in humans.
      ), with or without T2DM. DNL also contributes a significant portion (∼25%) of the TAG in liver and VLDL of patients with nonalcoholic fatty liver disease (
      • Donnelly K.L.
      • Smith C.I.
      • Schwarzenberg S.J.
      • Jessurun J.
      • Boldt M.D.
      • Parks E.J.
      Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease.
      ). Perhaps most importantly, the intrahepatic metabolic milieu associated with high activity of DNL (e.g., increased availability of malonyl-CoA and glycolytic metabolites, including α-glycerol-phosphate) promotes re-esterification rather than oxidation of incoming NEFA by the liver. Accordingly, the synthesis and secretion of TAG by the liver in subjects with type 2 diabetes mellitus (T2DM) may play a role in dyslipidemia as well as in nonalcoholic fatty liver disease. Reducing hepatic DNL is therefore a potential target for reducing hepatic fat accumulation and VLDL-TAG secretion in T2DM.
      The peroxisome proliferator-activated receptor (PPAR)-γ agonists, pioglitazone and rosiglitazone, are widely used for treatment of T2DM. These thiazolidinediones (TZDs) lower glucose concentrations by improving insulin sensitivity (
      • Goldberg R.B.
      • Kendall D.M.
      • Deeg M.A.
      • Buse J.B.
      • Zagar A.J.
      • Pinaire J.A.
      • Tan M.H.
      • Khan M.A.
      • Perez A.T.
      • Jacober S.J.
      A comparison of lipid and glycemic effects of pioglitazone and rosiglitazone in patients with type 2 diabetes and dyslipidemia.
      ,
      • Tran M.T.
      • Navar M.D.
      • Davidson M.B.
      Comparison of the glycemic effects of rosiglitazone and pioglitazone in triple oral therapy in type 2 diabetes.
      ,
      • Boyle P.J.
      • King A.B.
      • Olansky L.
      • Marchetti A.
      • Lau H.
      • Magar R.
      • Martin J.
      Effects of pioglitazone and rosiglitazone on blood lipid levels and glycemic control in patients with type 2 diabetes mellitus: a retrospective review of randomly selected medical records.
      ). Meta-analyses and prospective trials (
      • Erdmann E.
      • Dormandy J.A.
      • Charbonnel B.
      • Massi-Benedetti M.
      • Moules I.K.
      • Skene A.M.
      The effect of pioglitazone on recurrent myocardial infarction in 2,445 patients with type 2 diabetes and previous myocardial infarction: results from the PROactive (PROactive 05) Study.
      ,
      • Home P.D.
      • Pocock S.J.
      • Beck-Nielsen H.
      • Gomis R.
      • Hanefeld M.
      • Jones N.P.
      • Komajda M.
      • McMurray J.J.
      Rosiglitazone evaluated for cardiovascular outcomes—an interim analysis.
      ,
      • Nissen S.E.
      • Wolski K.
      Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes.
      ) have raised the possibility of different cardiovascular outcomes, however, for these two TZDs. Rosiglitazone has been suggested to cause adverse (
      • Nissen S.E.
      • Wolski K.
      Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes.
      ), or at best neutral (
      • Home P.D.
      • Pocock S.J.
      • Beck-Nielsen H.
      • Gomis R.
      • Hanefeld M.
      • Jones N.P.
      • Komajda M.
      • McMurray J.J.
      Rosiglitazone evaluated for cardiovascular outcomes—an interim analysis.
      ), effects on cardiovascular disease outcomes, while pioglitazone has been shown to reduce adverse events in T2DM patients with a prior myocardial infarction (
      • Erdmann E.
      • Dormandy J.A.
      • Charbonnel B.
      • Massi-Benedetti M.
      • Moules I.K.
      • Skene A.M.
      The effect of pioglitazone on recurrent myocardial infarction in 2,445 patients with type 2 diabetes and previous myocardial infarction: results from the PROactive (PROactive 05) Study.
      ).
      It would be helpful in evaluating the clinical outcomes data to establish whether underlying metabolic or biochemical differences exist between TZDs (i.e., whether differential biochemical actions can be identified that may explain the clinical observations). Different effects of pioglitazone and rosiglitazone have been observed on plasma lipids, with pioglitazone treatment resulting in a more favorable lipid profile than rosiglitazone treatment (
      • Goldberg R.B.
      • Kendall D.M.
      • Deeg M.A.
      • Buse J.B.
      • Zagar A.J.
      • Pinaire J.A.
      • Tan M.H.
      • Khan M.A.
      • Perez A.T.
      • Jacober S.J.
      A comparison of lipid and glycemic effects of pioglitazone and rosiglitazone in patients with type 2 diabetes and dyslipidemia.
      ,
      • Chiquette E.
      • Ramirez G.
      • Defronzo R.
      A meta-analysis comparing the effect of thiazolidinediones on cardiovascular risk factors.
      ). A randomized, prospective study comparing the effects of pioglitazone and rosiglitazone monotherapy in patients with T2DM, for example, showed a 12% reduction in plasma TAG concentrations with pioglitazone compared with a 15% increase with rosiglitazone in the face of identical reductions in HbA1c values (
      • Goldberg R.B.
      • Kendall D.M.
      • Deeg M.A.
      • Buse J.B.
      • Zagar A.J.
      • Pinaire J.A.
      • Tan M.H.
      • Khan M.A.
      • Perez A.T.
      • Jacober S.J.
      A comparison of lipid and glycemic effects of pioglitazone and rosiglitazone in patients with type 2 diabetes and dyslipidemia.
      ). In contrast, a meta-analysis showed no change in TAG concentrations with rosiglitazone (
      • Chiquette E.
      • Ramirez G.
      • Defronzo R.
      A meta-analysis comparing the effect of thiazolidinediones on cardiovascular risk factors.
      ). Pioglitazone as compared with rosiglitazone treatment is associated with a greater increase in HDL cholesterol (14.9% vs. 7.8%), a smaller increase in LDL cholesterol concentrations (15.7% vs. 23.3%), and less increase in apolipoprotein B (0% vs. 10%) (
      • Goldberg R.B.
      • Kendall D.M.
      • Deeg M.A.
      • Buse J.B.
      • Zagar A.J.
      • Pinaire J.A.
      • Tan M.H.
      • Khan M.A.
      • Perez A.T.
      • Jacober S.J.
      A comparison of lipid and glycemic effects of pioglitazone and rosiglitazone in patients with type 2 diabetes and dyslipidemia.
      ). Rosiglitazone and pioglitazone have both been reported to reduce the release of fatty acids from adipocytes and decrease plasma NEFA concentrations (
      • Goldberg R.B.
      • Kendall D.M.
      • Deeg M.A.
      • Buse J.B.
      • Zagar A.J.
      • Pinaire J.A.
      • Tan M.H.
      • Khan M.A.
      • Perez A.T.
      • Jacober S.J.
      A comparison of lipid and glycemic effects of pioglitazone and rosiglitazone in patients with type 2 diabetes and dyslipidemia.
      ,
      • Miyazaki Y.
      • Glass L.
      • Triplitt C.
      • Matsuda M.
      • Cusi K.
      • Mahankali A.
      • Mahankali S.
      • Mandarino L.J.
      • DeFronzo R.A.
      Effect of rosiglitazone on glucose and non-esterified fatty acid metabolism in type II diabetic patients.
      ), thereby reducing NEFA flux to the liver. Pioglitazone has also been shown to increase the fractional clearance rate of plasma VLDL-TAG (
      • Nagashima K.
      • Lopez C.
      • Donovan D.
      • Ngai C.
      • Fontanez N.
      • Bensadoun A.
      • Fruchart-Najib J.
      • Holleran S.
      • Cohn J.S.
      • Ramakrishnan R.
      • et al.
      Effects of the PPARgamma agonist pioglitazone on lipoprotein metabolism in patients with type 2 diabetes mellitus.
      ), although the effects of these two TZDs on intrahepatic lipid metabolism, especially as it relates to the disposition of incoming NEFA and synthesis of VLDL-TAG, have not yet been reported in humans with T2DM. Differential effects of pioglitazone and rosiglitazone on DNL, for example, might influence hepatic packaging, secretion, and the ultimate atherogenicity of VLDL particles.
      The present pilot study was designed to investigate the effects of rosiglitazone and pioglitazone on hepatic lipid and glucose metabolism in T2DM, with an emphasis on hepatic DNL.

      MATERIALS AND METHODS

      Subjects

      Twelve subjects (five males and seven females) with T2DM and hypertriacylglyceridemia were recruited and gave written informed consent. Subjects were studied at the General Clinical Research Center, at San Francisco General Hospital, University of California-San Francisco (UCSF), or at the University of New Mexico Health Science Center. The protocol was approved by the ethical committees of both institutions (Committee on Human Research at UCSF and the Human Research Review Committee of the University of New Mexico Health Sciences Center). Subjects were eligible to take part in the study if T2DM was not adequately controlled [HbA1c > 7.5% or fasting glucose > 10 mmol/l (180 mg/dl)] with metformin alone or metformin in combination with a sulfonylurea, and if hypertriacylglyceridemia was present [fasting TAG concentrations between 1.7 mmol/l (150 mg/dl) and 4.5 mmol/l (400 mg/dl)]. Exclusion criteria were pregnancy, alanine aminotransferase ALT > 1.5 times the upper limit of normal, serum creatinine > 1.4 mg/dl, congestive heart failure, history of coronary artery, and pulmonary or neurologic disease. Subjects were also excluded if they had been treated with insulin or had started treatment with a statin or any fibric acid derivative within 2 months of the beginning of the study.

      Study design

      This was an open-label, randomized study. Subjects were randomized to pioglitazone or rosiglitazone treatments for 20 weeks. Pioglitazone was started at a dose of 15–30 mg QD for 4 weeks followed by 45 mg QD for 16 weeks. Rosiglitazone was started at 4 mg QD for 4 weeks followed 4 mg twice daily for the remaining 16 weeks of the study. Stable isotope infusion studies were performed at baseline and after completion of the 20-week intervention trial to evaluate glucose and lipid metabolism.

      Stable isotope infusion protocol

      Stable isotope labeled metabolites were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). The infusion protocol is shown in Fig. 1. On day 1 of each infusion protocol, subjects received a standardized American Diabetes Association meal at 5:00 PM. After 6:00 PM, no calories or caffeine were taken until the end of the infusion (12:00 PM on day 2). Noncaloric beverages were allowed. Sodium [1-13C1]acetate (10 mg/min) and [1,2,3,4-13C4]palmitate (0.008 mg/kg body weight/min) were infused from 12:00 AM until 12:00 PM on day 2. A bolus of [6,6-2H2]glucose (4.8 mg/kg body weight) was given at 8:00 AM on day 2 followed by a constant infusion (0.08 mg/kg body weight/min) until 12:00 PM the same day. Indirect calorimetry was performed using a Deltatreck unit with a ventilated hood (Sensor Medics, Yorba Linda, CA) at 8:00 AM on day 2 and was completed 1 h later. Blood samples were drawn as shown (Fig. 1). Immediately after collection, one aliquot of plasma was transferred to heptane/isopropanol (30:70, v/v) for plasma NEFA analysis. The remaining plasma was stored at −80°C for further analyses.
      Figure thumbnail gr1
      Fig. 1Stable isotope infusion and blood drawing protocol.

      Analyses

      Plasma TAG, total cholesterol, LDL-cholesterol, HDL-cholesterol, HbA1C, aspartate aminotransferase (AST), and ALT concentrations were analyzed by standard clinical laboratory techniques. Plasma insulin concentrations were determined by radioimmunoassay (Linco, St. Charles, MO). Plasma glucose concentrations were analyzed with a Yellow Springs Instrument (Yellow Springs Instruments Co., Yellow Springs, OH).

      NEFA and lipoprotein isolation and analysis

      VLDL particles were isolated from plasma by sequential ultracentrifugation (
      • Vedala A.
      • Wang W.
      • Neese R.A.
      • Christiansen M.P.
      • Hellerstein M.K.
      Delayed secretory pathway contributions to VLDL-triglycerides from plasma NEFA, diet, and de novo lipogenesis in humans.
      ). Briefly, two 30 min spins (40,000 rpm at 10°C) were performed to isolate lipoproteins with a Svedberg flotation rate (Sf) > 400 (chylomicron-like particles) followed by an 18 h spin (40,000 rpm at 10°C) to isolate VLDL particles (Sf 20–400). Total lipids from VLDL particles and plasma samples were extracted with chloroform:methanol (2:1, v/v) and were subjected to thin layer chromatography to separate VLDL-TAG and plasma NEFAs from other lipid fractions. VLDL-TAG fatty acids and plasma NEFAs were then trans-esterified to fatty acid-methyl esters for gas chromatography mass-spectrometry (GC-MS) analysis. It should be noted that separation of buoyant VLDL particles in lipoprotein remnants, to the extent that these are removed with the chylomicron-like fraction, may alter DNL values (
      • Donnelly K.L.
      • Smith C.I.
      • Schwarzenberg S.J.
      • Jessurun J.
      • Boldt M.D.
      • Parks E.J.
      Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease.
      ).

      Mass spectrometry

      GC-MS was performed using a model 5973 instrument (Agilent Technologies, Palo Alto, CA) with a DB-17 or VB-17 column to measure 13C1- enrichment in VLDL-TAG-palmitate and plasma enrichment of [1,2,3,4-13C4]palmitate. Electron impact ionization was used, monitoring mass/charge ratio (m/z) 270–272 and 274, representing the parent M0, M1, M2, and M4 isotopomers. Plasma [1,2,3,4-13C4]palmitate enrichments were quantified by comparisons with standard curves of [1,2,3,4-13C4]palmitate. The relative concentrations of fatty acids in plasma were quantified by GC with flame ionization detection, as described previously (
      • Vedala A.
      • Wang W.
      • Neese R.A.
      • Christiansen M.P.
      • Hellerstein M.K.
      Delayed secretory pathway contributions to VLDL-triglycerides from plasma NEFA, diet, and de novo lipogenesis in humans.
      ). Hepatic glucose production (HGP) was determined based on tracer dilution with [6,6-2H2]glucose infusion by GC-MS, as previously described (
      • Argoud G.M.
      • Schade D.S.
      • Eaton R.P.
      Underestimation of hepatic glucose production by radioactive and stable tracers.
      ).

      Calculations

      Fractional DNL: the fraction of VLDL-TAG-palmitate synthesized by the DNL pathway was calculated using mass isotopomer distribution analysis (MIDA) (
      • Hellerstein M.K.
      • Neese R.A.
      Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations.
      ,
      • Siler S.Q.
      • Neese R.A.
      • Hellerstein M.K.
      De novo lipogenesis, lipid kinetics, and whole-body lipid balances in humans after acute alcohol consumption.
      ). Briefly, the isotopic enrichment of the precursor for lipogenesis (hepatic cytosolic acetyl-CoA) was calculated from the ratio of excess double-labeled to excess single-labeled species (EM2/EM1) in VLDL-TAG-palmitate, based on combinatorial probabilities. Fractional DNL was determined by the standard precursor-product relationship as described previously (
      • Hellerstein M.K.
      • Neese R.A.
      Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations.
      ). Fractional DNL at steady state was calculated as the mean of DNL values at 10 and 12 h after the start of the sodium [1-13C1]acetate infusion.
      The rates of appearance (Ra) of plasma NEFA and plasma glucose were calculated by the isotope dilution technique (
      • Wolfe R.R.
      Radioactive and Stable Isotope Tracers in Biomedicine.
      ). Ra (mg/kg body weight per min) = isotope infusion rate (mg/kg body weight per min)/metabolite enrichment, where metabolite enrichment is molar excess fraction. For Ra NEFA, Ra palmitate was first calculated and was then divided by the % of plasma NEFA that is palmitate. The average of the results from samples drawn at 6:00 AM, 8:00 AM, 10:00 AM, and 12:00 AM was used for the NEFA enrichment and 11:00 AM, 11:30 AM, 11:45 AM, and 12:00 PM for glucose enrichments. Under fasting conditions, Ra glucose is taken to represent HGP.
      Total fatty acid oxidation rate: whole-body fat oxidation was calculated by indirect calorimetry using the following equation: whole-body fat oxidation (g/min) = {[1.67 × VO2 (l/min)] − [1.67 × VCO2 (l/min)]} − [1.92 × urinary N (g/min)] (
      • Frayn K.N.
      Calculation of substrate oxidation rates in vivo from gaseous exchange.
      ). Urinary N was assumed to be 150 mg/kg body weight/day (
      • Wolfe R.R.
      • Chinkes D.L.
      Isotope Tracers in Metabolic Research. Principles and Practice of Kinetic Analysis.
      ). Total fatty acid oxidation was also expressed on a molar basis by converting whole-body fat oxidation to its molecular equivalent using 287 g/mol as the average molecular weight of a fatty acid.
      Extra-adipose tissue re-esterification of NEFA was calculated as the difference between Ra NEFA and whole-body fatty acid oxidation

      Statistical analysis

      Statistical analyses were performed for three different comparisons: 1) Baseline parameters between the two treatment groups were compared by unpaired Student's t-test. 2) Within each treatment group, the pre- and post-treatment results were compared using repeated-measures ANOVA (ANOVA) with Bonferroni correction. 3) Unpaired Student's t-tests were used to establish if the changes seen with pioglitazone were significantly different from changes seen with rosiglitazone.

      RESULTS

      Subject characteristics and metabolic parameters

      Subject characteristics and metabolic parameters at baseline and post-treatment are shown in Table 1. There were no significant differences between the rosiglitazone and pioglitazone treatment groups at baseline with respect to age, weight, body mass index, or metabolic parameters. In the pioglitazone treatment group, two subjects were taking metformin alone and four subjects were taking metformin in combination with sulfonylurea. In the rosiglitazone group, three subjects were on metformin alone and three subjects were on metformin in combination with sulfonylurea. In all cases, the dose of metformin and/or sulfonylurea was at least 50% of the maximum recommended dose. Five subjects in the rosiglitazone treatment group and one subject in the pioglitazone group were on stable regimens of statins.
      TABLE 1Subject characteristics of the rosiglitazone and pioglitazone treatment groups
      RosiglitazonePioglitazone
      ParametersPretreatmentPost-treatmentChangePretreatmentPost-treatmentChange
      Subjects (male/female)6 (2/4)6 (3/3)
      Age (years)56 ± 653 ± 8
      Weight (kg)86.8 ± 26.789.8 ± 27.13.0 ± 3.498.5 ± 36.6103.4 ± 40.4
      P < 0.05 significantly different from pretreatment by ANOVA. HbA1C data from one subject was not measured in the pioglitazone group.
      4.9 ± 4.2
      BMI (kg/m2)31.3 ± 5.432.3 ± 5.41.1 ± 1.436.9 ± 12.538.7 ± 13.8
      P < 0.05 significantly different from pretreatment by ANOVA. HbA1C data from one subject was not measured in the pioglitazone group.
      1.8 ± 1.5
      Fasting glucose (mmol/l)8.9 ± 5.56.4 ± 3.5
      P < 0.05 significantly different from pretreatment by ANOVA. HbA1C data from one subject was not measured in the pioglitazone group.
      −2.5 ± 2.28.8 ± 2.57.3 ± 3.5
      P < 0.05 significantly different from pretreatment by ANOVA. HbA1C data from one subject was not measured in the pioglitazone group.
      −1.5 ± 1.2
      Fasting insulin (pmol/l)90 ± 2675 ± 18−15 ± 2292 ± 6183 ± 26−9 ± 46
      Fasting HbA1C (%)8.2 ± 1.16.9 ± 1.1
      P < 0.05 significantly different from pretreatment by ANOVA. HbA1C data from one subject was not measured in the pioglitazone group.
      −1.3 ± 0.88.1 ± 0.67.0 ± 1.0
      P < 0.05 significantly different from pretreatment by ANOVA. HbA1C data from one subject was not measured in the pioglitazone group.
      −1.1 ± 0.6
      Fasting TAG (mmol/l)2.44 ± 0.532.82 ± 1.110.38 ± 1.132.53 ± 0.922.19 ± 1.80−0.34 ± 0.81
      Fasting NEFA (mmol/l)0.61 ± 0.360.50 ± 0.15−0.11 ± 0.260.75 ± 0.320.51 ± 0.04−0.23 ± 0.30
      Fasting total cholesterol (mmol/l)4.24 ± 1.445.21 ± 1.610.98 ± 1.244.04 ± 1.043.99 ± 0.59−0.05 ± 1.07
      Fasting LDL-cholesterol (mmol/l)2.27 ± 1.313.03 ± 1.180.76 ± 1.051.94 ± 0.912.13 ± 0.570.19 ± 1.20
      Fasting HDL-cholesterol (mmol/l)0.86 ± 0.260.89 ± 0.150.04 ± 0.190.94 ± 0.200.86 ± 0.11−0.08 ± 0.11
      Fasting AST (U/l)27 ± 627 ± 90 ± 424 ± 822 ± 5−2 ± 5
      Fasting ALT (U/l)36 ± 1030 ± 10−6 ± 632 ± 1028 ± 7−4 ± 6
      AST, aspartate aminotransferase; BMI, body mass index; NEFA, nonesterified fatty acids; TAG, triacylglycerols. Data are mean ± SD.
      a P < 0.05 significantly different from pretreatment by ANOVA. HbA1C data from one subject was not measured in the pioglitazone group.
      All six subjects in the pioglitazone group and five out of six subjects in the rosiglitazone group gained weight after 20 weeks of treatment, although the weight increase was statistically significant for the pioglitazone group only (P < 0.05). Similar results were seen for changes in body mass index after treatment [P < 0.05 for pioglitazone treatment but not significant (NS) for rosiglitazone treatment].
      Fasting plasma glucose and HbA1C concentrations decreased significantly (P < 0.05) with both treatments. The reductions in mean insulin concentrations with treatment were not significant in either group.
      Changes in plasma lipids with TZD treatment were not significant, although the trends were consistent with published results (
      • Goldberg R.B.
      • Kendall D.M.
      • Deeg M.A.
      • Buse J.B.
      • Zagar A.J.
      • Pinaire J.A.
      • Tan M.H.
      • Khan M.A.
      • Perez A.T.
      • Jacober S.J.
      A comparison of lipid and glycemic effects of pioglitazone and rosiglitazone in patients with type 2 diabetes and dyslipidemia.
      ,
      • Boyle P.J.
      • King A.B.
      • Olansky L.
      • Marchetti A.
      • Lau H.
      • Magar R.
      • Martin J.
      Effects of pioglitazone and rosiglitazone on blood lipid levels and glycemic control in patients with type 2 diabetes mellitus: a retrospective review of randomly selected medical records.
      ,
      • Chiquette E.
      • Ramirez G.
      • Defronzo R.
      A meta-analysis comparing the effect of thiazolidinediones on cardiovascular risk factors.
      ). Mean fasting TAG concentrations tended to increase with rosiglitazone treatment and decrease with pioglitazone treatment, mean fasting total cholesterol tended to increase with rosiglitazone treatment but not with pioglitazone treatment, and the increase in LDL cholesterol concentrations was greater for rosiglitazone treatment compared with pioglitazone treatment. HDL cholesterol concentrations did not change with either treatment. Changes with treatment were not significantly different between groups for any of these parameters.

      Hepatic glucose production

      At baseline, mean hepatic glucose production (HGP) was 2.75 ± 0.65 and 2.46 ± 0.42 mg/kg body weight per min for the rosiglitazone and the pioglitazone groups, respectively (NS). Pioglitazone treatment resulted in a significant reduction in HGP (1.91 ± 0.43 mg/kg body weight per min, P < 0.05), while rosiglitazone treatment did not (2.32 ± 0.57 mg/kg body weight per min, P = 0.2). The change with treatment was not significantly different between the two groups by unpaired t-test (−0.57 ± 0.42 mg/kg body weight per min for pioglitazone vs. −0.43 ± 0.60 mg/kg body weight per min for rosiglitazone, NS).

      DNL contribution to VLDL-TAG-palmitate

      The time course of hepatic DNL during a constant infusion of [1-13C1]acetate is shown in Fig. 2A, B. Fractional DNL (%) represents the fraction of plasma VLDL-TAG-palmitate derived from the DNL pathway (
      • Hellerstein M.K.
      De novo lipogenesis in humans: metabolic and regulatory aspects.
      ). Prior to treatment, fractional DNL was not significantly different between groups. Pioglitazone treatment significantly reduced fractional DNL at steady-state, from 6.6 ± 0.7% at baseline to 4.2 ± 0.6% (P < 0.01), while rosiglitazone treatment did not significantly alter fractional DNL, from baseline 6.9 ± 1.4%, to post-treatment 5.5 ± 1.4% (P = 0.5). The change with treatment was not significantly different between the two groups by unpaired t-test (−2.4 ± 1.2% for pioglitazone vs. −1.4 ± 4.5% for rosiglitazone, NS).
      Figure thumbnail gr2
      Fig. 2The effect of 20 weeks of rosiglitazone and pioglitazone treatment on hepatic de novo lipogenesis (DNL). The time course of fractional DNL is shown during a constant infusion of [1-13C1]acetate before and after rosiglitazone treatment (A) and pioglitazone treatment (B). * P < 0.05, ** P < 0.01, *** P < 0.001 statistically significant from pretreatment by ANOVA.

      Ra NEFA, whole-body fat oxidation, and extra-adipose tissue re-esterification of plasma NEFA

      Neither rosiglitazone nor pioglitazone treatment resulted in statistically significant changes of Ra NEFA, whole-body fat oxidation or extra-adipose tissue re-esterification rates (Table 2).
      TABLE 2Effect of rosiglitazone and pioglitazone treatment on nonesterified fatty acid (NEFA) fluxes
      RosiglitazonePioglitazone
      ParametersPretreatmentPost-treatmentnPretreatmentPost-treatmentn
      Ra NEFA6.3 ± 2.76.1 ± 2.467.7 ± 3.46.9 ± 3.06
      Whole-body fatty acid oxidation3.7 ± 1.83.5 ± 1.743.3 ± 1.22.7 ± 1.44
      Extra-adipose tissue re-esterification of NEFA2.5 ± 2.71.9 ± 1.643.7 ± 3.03.4 ± 1.64
      All data are expressed as μmol/kg body weight per minute. Values are mean ± SD.

      DISCUSSION

      The central questions addressed in this pilot study were whether TZDs reduce hepatic DNL in T2DM patients and whether pioglitazone and rosiglitazone have different metabolic effects, particularly on the liver, in addition to the previously established differences on lipoprotein profiles (
      • Goldberg R.B.
      • Kendall D.M.
      • Deeg M.A.
      • Buse J.B.
      • Zagar A.J.
      • Pinaire J.A.
      • Tan M.H.
      • Khan M.A.
      • Perez A.T.
      • Jacober S.J.
      A comparison of lipid and glycemic effects of pioglitazone and rosiglitazone in patients with type 2 diabetes and dyslipidemia.
      ,
      • Boyle P.J.
      • King A.B.
      • Olansky L.
      • Marchetti A.
      • Lau H.
      • Magar R.
      • Martin J.
      Effects of pioglitazone and rosiglitazone on blood lipid levels and glycemic control in patients with type 2 diabetes mellitus: a retrospective review of randomly selected medical records.
      ,
      • Chiquette E.
      • Ramirez G.
      • Defronzo R.
      A meta-analysis comparing the effect of thiazolidinediones on cardiovascular risk factors.
      ). Different effects on the regulation of hepatic metabolic pathways may be relevant to the differences in cardiovascular disease outcomes that have been reported for these two TZDs (
      • Erdmann E.
      • Dormandy J.A.
      • Charbonnel B.
      • Massi-Benedetti M.
      • Moules I.K.
      • Skene A.M.
      The effect of pioglitazone on recurrent myocardial infarction in 2,445 patients with type 2 diabetes and previous myocardial infarction: results from the PROactive (PROactive 05) Study.
      ,
      • Home P.D.
      • Pocock S.J.
      • Beck-Nielsen H.
      • Gomis R.
      • Hanefeld M.
      • Jones N.P.
      • Komajda M.
      • McMurray J.J.
      Rosiglitazone evaluated for cardiovascular outcomes—an interim analysis.
      ,
      • Nissen S.E.
      • Wolski K.
      Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes.
      ). Using sensitive in vivo stable isotope-mass spectrometric techniques, we identified differences in effects on hepatic lipid and glucose intermediary metabolism for pioglitazone and rosiglitazone. Treatment with these TZDs improved plasma glucose and HbA1C concentrations equally, as shown previously (
      • Goldberg R.B.
      • Kendall D.M.
      • Deeg M.A.
      • Buse J.B.
      • Zagar A.J.
      • Pinaire J.A.
      • Tan M.H.
      • Khan M.A.
      • Perez A.T.
      • Jacober S.J.
      A comparison of lipid and glycemic effects of pioglitazone and rosiglitazone in patients with type 2 diabetes and dyslipidemia.
      ,
      • Boyle P.J.
      • King A.B.
      • Olansky L.
      • Marchetti A.
      • Lau H.
      • Magar R.
      • Martin J.
      Effects of pioglitazone and rosiglitazone on blood lipid levels and glycemic control in patients with type 2 diabetes mellitus: a retrospective review of randomly selected medical records.
      ,
      • Chiquette E.
      • Ramirez G.
      • Defronzo R.
      A meta-analysis comparing the effect of thiazolidinediones on cardiovascular risk factors.
      ), but the hepatic metabolic response to the two TZDs was not the same. The most interesting and novel finding here is that hepatic DNL was significantly reduced (by 40%) by pioglitazone, whereas no significant change was seen with rosiglitazone treatment. The change in fractional DNL before and after pioglitazone treatment was not significantly different from the change in fractional DNL with rosiglitazone treatment, however, perhaps because of the small group sizes in this pilot study. Power calculations show that, with the same means, standard deviations and effects as were observed here, one would need at least 41 subjects per group to detect a statistically significant difference between pioglitazone and rosiglitazone treatment. The DNL values reported in the present study represent newly synthesized fatty acids in VLDL-TAG and not newly synthesized fatty acids retained in the liver. Longer labeling protocols might include a contribution from the cytosolic delay pool and might result in greater % DNL values than seen in this study.
      The DNL pathway contributes a minority of the nonessential fatty acids present in TAG secreted from the liver (
      • Hellerstein M.K.
      De novo lipogenesis in humans: metabolic and regulatory aspects.
      ,
      • Hellerstein M.K.
      • Benowitz N.L.
      • Neese R.A.
      • Schwartz J.M.
      • Hoh R.
      • Jacob 3rd, P.
      • Hsieh J.
      • Faix D.
      Effects of cigarette smoking and its cessation on lipid metabolism and energy expenditure in heavy smokers.
      ) but represents an integrated signal of hepatic carbohydrate availability and a key regulatory pathway in the liver. Intermediary metabolites generated from the metabolism of glucose or glycogen, such as pyruvate, oxaloacetate, acetyl-CoA, and citrate, increase the generation of malonyl-CoA by acetyl-CoA carboxylase, the first metabolic step in DNL, and stimulate flux through the DNL pathway. Malonyl-CoA is also the primary regulator of fatty acid transport into mitochondria for β-oxidation, by modulating the activity of carnitine palmitoyl transferase 1 (
      • McGarry J.D.
      • Leatherman G.F.
      • Foster D.W.
      Carnitine palmitoyltransferase I. The site of inhibition of hepatic fatty acid oxidation by malonyl-CoA.
      ). Accordingly, states of increased fatty acid synthesis in a tissue are typically associated with reduced fatty acid oxidation, and, in the liver, a milieu of activated DNL results in increased cytosolic re-esterification of NEFA, rather than mitochondrial oxidation (
      • McGarry J.D.
      • Leatherman G.F.
      • Foster D.W.
      Carnitine palmitoyltransferase I. The site of inhibition of hepatic fatty acid oxidation by malonyl-CoA.
      ). The result is partitioning of NEFA into TAG synthesis, which must either be secreted as VLDL-TAG or accumulate as hepatic fat droplets. Moreover, newly synthesized fatty acids exert intrahepatic signaling actions on the assembly and secretion of VLDL particles, and may have specific actions to regulate the assembly and atherogenicity of plasma lipoprotein particles (
      • Gibbons G.F.
      • Bartlett S.M.
      • Sparks C.E.
      • Sparks J.D.
      Extracellular fatty acids are not utilized directly for the synthesis of very-low-density lipoprotein in primary cultures of rat hepatocytes.
      ). Agents that reduce hepatic DNL are therefore expected to alter the partitioning of incoming NEFA toward oxidation rather than TAG synthesis and VLDL assembly, and are being evaluated as a therapeutic target in dyslipidemia and insulin resistance (
      • Savage D.B.
      • Choi C.S.
      • Samuel V.T.
      • Liu Z.X.
      • Zhang D.
      • Wang A.
      • Zhang X.M.
      • Cline G.W.
      • Yu X.X.
      • Geisler J.G.
      • et al.
      Reversal of diet-induced hepatic steatosis and hepatic insulin resistance by antisense oligonucleotide inhibitors of acetyl-CoA carboxylases 1 and 2.
      ).
      The effect of treatment with specific TZDs on hepatic DNL in humans with T2DM were difficult to predict a priori. Although TZDs are classically recognized to stimulate DNL in adipocytes, their effects on hepatic lipogenesis have been uncertain. Activation of PPARγ by TZDs in animal models is associated with reduced hepatic expression of DNL enzymes (
      • Ota T.
      • Takamura T.
      • Kurita S.
      • Matsuzawa N.
      • Kita Y.
      • Uno M.
      • Akahori H.
      • Misu H.
      • Sakurai M.
      • Zen Y.
      • et al.
      Insulin resistance accelerates a dietary rat model of nonalcoholic steatohepatitis.
      ) and increased expression of fatty acid oxidation enzymes (
      • Edvardsson U.
      • Bergstrom M.
      • Alexandersson M.
      • Bamberg K.
      • Ljung B.
      • Dahllof B.
      Rosiglitazone (BRL49653), a PPARgamma-selective agonist, causes peroxisome proliferator-like liver effects in obese mice.
      ). On the other hand, PPARγ gene expression is upregulated in liver in models of obesity and diabetes (
      • Memon R.A.
      • Tecott L.H.
      • Nonogaki K.
      • Beigneux A.
      • Moser A.H.
      • Grunfeld C.
      • Feingold K.R.
      Up-regulation of peroxisome proliferator-activated receptors (PPAR-alpha) and PPAR-gamma messenger ribonucleic acid expression in the liver in murine obesity: troglitazone induces expression of PPAR-gamma-responsive adipose tissue-specific genes in the liver of obese diabetic mice.
      ,
      • Vidal-Puig A.
      • Jimenez-Linan M.
      • Lowell B.B.
      • Hamann A.
      • Hu E.
      • Spiegelman B.
      • Flier J.S.
      • Moller D.E.
      Regulation of PPAR gamma gene expression by nutrition and obesity in rodents.
      ), and induction of hepatic PPARγ2 gene expression by high-fat feeding or other nutritional interventions has been linked to stimulation of hepatic DNL and development of hepatic steatosis (
      • Zhang Y.L.
      • Hernandez-Ono A.
      • Siri P.
      • Weisberg S.
      • Conlon D.
      • Graham M.J.
      • Crooke R.M.
      • Huang L.S.
      • Ginsberg H.N.
      Aberrant hepatic expression of PPARgamma2 stimulates hepatic lipogenesis in a mouse model of obesity, insulin resistance, dyslipidemia, and hepatic steatosis.
      ,
      • Schadinger S.E.
      • Bucher N.L.
      • Schreiber B.M.
      • Farmer S.R.
      PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes.
      ,
      • Gavrilova O.
      • Haluzik M.
      • Matsusue K.
      • Cutson J.J.
      • Johnson L.
      • Dietz K.R.
      • Nicol C.J.
      • Vinson C.
      • Gonzalez F.J.
      • Reitman M.L.
      Liver peroxisome proliferator-activated receptor gamma contributes to hepatic steatosis, triglyceride clearance, and regulation of body fat mass.
      ). Moreover, the effects of TZDs are further complicated by the possibility of cross-reactivity with PPARα (
      • Sakamoto J.
      • Kimura H.
      • Moriyama S.
      • Odaka H.
      • Momose Y.
      • Sugiyama Y.
      • Sawada H.
      Activation of human peroxisome proliferator-activated receptor (PPAR) subtypes by pioglitazone.
      ), in that PPARα agonists are well established to increase hepatic fatty acid oxidation pathways and reduce hepatic TAG synthesis (
      • Kim H.
      • Haluzik M.
      • Asghar Z.
      • Yau D.
      • Joseph J.W.
      • Fernandez A.M.
      • Reitman M.L.
      • Yakar S.
      • Stannard B.
      • Heron-Milhavet L.
      • et al.
      Peroxisome proliferator-activated receptor-alpha agonist treatment in a transgenic model of type 2 diabetes reverses the lipotoxic state and improves glucose homeostasis.
      ). Pioglitazone may directly bind to PPARα (
      • Sakamoto J.
      • Kimura H.
      • Moriyama S.
      • Odaka H.
      • Momose Y.
      • Sugiyama Y.
      • Sawada H.
      Activation of human peroxisome proliferator-activated receptor (PPAR) subtypes by pioglitazone.
      ); moreover, activation of genes related to PPARα appears to occur with pioglitazone binding to PPARγ, so that increased α-like effects may be driven through this indirect mechanism (
      • Bogacka I.
      • Xie H.
      • Bray G.A.
      • Smith S.R.
      Pioglitazone induces mitochondrial biogenesis in human subcutaneous adipose tissue in vivo.
      ).
      In this context, the preliminary finding that pioglitazone but not rosiglitazone reduces hepatic DNL in T2DM patients becomes potentially important. Even though we observed no differences in plasma NEFA flux or delivery of NEFA to the liver, a reduction in DNL could alter partitioning of incoming NEFA and influence the assembly and secretion of apoB-containing particles, in addition to directly contributing fewer new fatty acids to hepatic TAG (
      • Vedala A.
      • Wang W.
      • Neese R.A.
      • Christiansen M.P.
      • Hellerstein M.K.
      Delayed secretory pathway contributions to VLDL-triglycerides from plasma NEFA, diet, and de novo lipogenesis in humans.
      ). Pioglitazone has also been shown to alter the clearance of VLDL-TAG in people with T2DM (
      • Nagashima K.
      • Lopez C.
      • Donovan D.
      • Ngai C.
      • Fontanez N.
      • Bensadoun A.
      • Fruchart-Najib J.
      • Holleran S.
      • Cohn J.S.
      • Ramakrishnan R.
      • et al.
      Effects of the PPARgamma agonist pioglitazone on lipoprotein metabolism in patients with type 2 diabetes mellitus.
      ), in addition to these effects on hepatic fatty acid metabolism.
      Moreover, although fasting blood glucose concentrations and HbA1C levels were not different in the two TZD treatment arms, fasting glucose production by the liver was significantly reduced by pioglitazone, but not by rosiglitazone. Previous studies have reported a decrease in fasting HGP with pioglitazone (
      • Gastaldelli A.
      • Miyazaki Y.
      • Mahankali A.
      • Berria R.
      • Pettiti M.
      • Buzzigoli E.
      • Ferrannini E.
      • DeFronzo R.A.
      The effect of pioglitazone on the liver: role of adiponectin.
      ) and rosiglitazone (
      • Miyazaki Y.
      • Glass L.
      • Triplitt C.
      • Matsuda M.
      • Cusi K.
      • Mahankali A.
      • Mahankali S.
      • Mandarino L.J.
      • DeFronzo R.A.
      Effect of rosiglitazone on glucose and non-esterified fatty acid metabolism in type II diabetic patients.
      ,
      • Gastaldelli A.
      • Miyazaki Y.
      • Pettiti M.
      • Santini E.
      • Ciociaro D.
      • Defronzo R.A.
      • Ferrannini E.
      The effect of rosiglitazone on the liver: decreased gluconeogenesis in patients with type 2 diabetes.
      ) treatment in T2DM. In the present study, the findings for HGP complement the findings for hepatic lipid synthesis and might suggest greater activity of pioglitazone on liver metabolism in humans. Again, these metabolic actions may reflect greater functional PPARα cross-reactivity for pioglitazone, as has been reported previously (
      • Sakamoto J.
      • Kimura H.
      • Moriyama S.
      • Odaka H.
      • Momose Y.
      • Sugiyama Y.
      • Sawada H.
      Activation of human peroxisome proliferator-activated receptor (PPAR) subtypes by pioglitazone.
      ), compared with rosiglitazone, at least in the liver of people with T2DM.
      In summary, our pilot results indicate that pioglitazone reduces hepatic DNL while rosiglitazone does not in T2DM patients in the absence of differences in glycemic control. The metabolic implications of reduced hepatic DNL and glucose production are not certain but could, in principle, contribute to differences in plasma lipoprotein profiles or atherogenicity. At a minimum, these results support the notion that pioglitazone and rosiglitazone, though both in the TZD class of PPARγ activators, cannot be considered the same drug. These findings may be germane to the recent debate concerning cardiovascular outcomes of rosiglitazone and pioglitazone treatment in T2DM (
      • Erdmann E.
      • Dormandy J.A.
      • Charbonnel B.
      • Massi-Benedetti M.
      • Moules I.K.
      • Skene A.M.
      The effect of pioglitazone on recurrent myocardial infarction in 2,445 patients with type 2 diabetes and previous myocardial infarction: results from the PROactive (PROactive 05) Study.
      ,
      • Nissen S.E.
      • Wolski K.
      Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes.
      ,
      • Psaty B.M.
      • Furberg C.D.
      The record on rosiglitazone and the risk of myocardial infarction.
      ).

      Acknowledgments

      The authors gratefully acknowledge Drina Boban, Alan Bostrom, Erica Cheng, Kris Prado, and Ellen Tsang for their contributions to this study and the nurses and dietician of the GCRC staff at San Francisco General Hospital and the New Mexico Health Science Center for their help.

      References

        • Vedala A.
        • Wang W.
        • Neese R.A.
        • Christiansen M.P.
        • Hellerstein M.K.
        Delayed secretory pathway contributions to VLDL-triglycerides from plasma NEFA, diet, and de novo lipogenesis in humans.
        J. Lipid Res. 2006; 47: 2562-2574
        • Diraison F.
        • Dusserre E.
        • Vidal H.
        • Sothier M.
        • Beylot M.
        Increased hepatic lipogenesis but decreased expression of lipogenic gene in adipose tissue in human obesity.
        Am. J. Physiol. Endocrinol. Metab. 2002; 282: E46-E51
        • Schwarz J.M.
        • Neese R.
        • Shackleton C.H.L.
        • Hellerstein M.K.
        De novo lipogenesis (DNL) during fasting and oral fructose in lean and obese hyperinsulinemic subjects. [Abstract].
        Diabetes. 1993; 42: 39A
        • Donnelly K.L.
        • Smith C.I.
        • Schwarzenberg S.J.
        • Jessurun J.
        • Boldt M.D.
        • Parks E.J.
        Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease.
        J. Clin. Invest. 2005; 115: 1343-1351
        • Goldberg R.B.
        • Kendall D.M.
        • Deeg M.A.
        • Buse J.B.
        • Zagar A.J.
        • Pinaire J.A.
        • Tan M.H.
        • Khan M.A.
        • Perez A.T.
        • Jacober S.J.
        A comparison of lipid and glycemic effects of pioglitazone and rosiglitazone in patients with type 2 diabetes and dyslipidemia.
        Diabetes Care. 2005; 28: 1547-1554
        • Tran M.T.
        • Navar M.D.
        • Davidson M.B.
        Comparison of the glycemic effects of rosiglitazone and pioglitazone in triple oral therapy in type 2 diabetes.
        Diabetes Care. 2006; 29: 1395-1396
        • Boyle P.J.
        • King A.B.
        • Olansky L.
        • Marchetti A.
        • Lau H.
        • Magar R.
        • Martin J.
        Effects of pioglitazone and rosiglitazone on blood lipid levels and glycemic control in patients with type 2 diabetes mellitus: a retrospective review of randomly selected medical records.
        Clin. Ther. 2002; 24: 378-396
        • Erdmann E.
        • Dormandy J.A.
        • Charbonnel B.
        • Massi-Benedetti M.
        • Moules I.K.
        • Skene A.M.
        The effect of pioglitazone on recurrent myocardial infarction in 2,445 patients with type 2 diabetes and previous myocardial infarction: results from the PROactive (PROactive 05) Study.
        J. Am. Coll. Cardiol. 2007; 49: 1772-1780
        • Home P.D.
        • Pocock S.J.
        • Beck-Nielsen H.
        • Gomis R.
        • Hanefeld M.
        • Jones N.P.
        • Komajda M.
        • McMurray J.J.
        Rosiglitazone evaluated for cardiovascular outcomes—an interim analysis.
        N. Engl. J. Med. 2007; 357: 28-38
        • Nissen S.E.
        • Wolski K.
        Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes.
        N. Engl. J. Med. 2007; 356: 2457-2471
        • Chiquette E.
        • Ramirez G.
        • Defronzo R.
        A meta-analysis comparing the effect of thiazolidinediones on cardiovascular risk factors.
        Arch. Intern. Med. 2004; 164: 2097-2104
        • Miyazaki Y.
        • Glass L.
        • Triplitt C.
        • Matsuda M.
        • Cusi K.
        • Mahankali A.
        • Mahankali S.
        • Mandarino L.J.
        • DeFronzo R.A.
        Effect of rosiglitazone on glucose and non-esterified fatty acid metabolism in type II diabetic patients.
        Diabetologia. 2001; 44: 2210-2219
        • Nagashima K.
        • Lopez C.
        • Donovan D.
        • Ngai C.
        • Fontanez N.
        • Bensadoun A.
        • Fruchart-Najib J.
        • Holleran S.
        • Cohn J.S.
        • Ramakrishnan R.
        • et al.
        Effects of the PPARgamma agonist pioglitazone on lipoprotein metabolism in patients with type 2 diabetes mellitus.
        J. Clin. Invest. 2005; 115: 1323-1332
        • Argoud G.M.
        • Schade D.S.
        • Eaton R.P.
        Underestimation of hepatic glucose production by radioactive and stable tracers.
        Am. J. Physiol. 1987; 252: E606-E615
        • Hellerstein M.K.
        • Neese R.A.
        Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations.
        Am. J. Physiol. 1999; 276: E1146-E1170
        • Siler S.Q.
        • Neese R.A.
        • Hellerstein M.K.
        De novo lipogenesis, lipid kinetics, and whole-body lipid balances in humans after acute alcohol consumption.
        Am. J. Clin. Nutr. 1999; 70: 928-936
        • Wolfe R.R.
        Radioactive and Stable Isotope Tracers in Biomedicine.
        John Wiley & Sons, Inc., New York, NY1992
        • Frayn K.N.
        Calculation of substrate oxidation rates in vivo from gaseous exchange.
        J. Appl. Physiol. 1983; 55: 628-634
        • Wolfe R.R.
        • Chinkes D.L.
        Isotope Tracers in Metabolic Research. Principles and Practice of Kinetic Analysis.
        2nd edition. John Wiley & Sons, Inc., New York, NY2005
        • Hellerstein M.K.
        De novo lipogenesis in humans: metabolic and regulatory aspects.
        Eur. J. Clin. Nutr. 1999; 53: S53-S65
        • Hellerstein M.K.
        • Benowitz N.L.
        • Neese R.A.
        • Schwartz J.M.
        • Hoh R.
        • Jacob 3rd, P.
        • Hsieh J.
        • Faix D.
        Effects of cigarette smoking and its cessation on lipid metabolism and energy expenditure in heavy smokers.
        J. Clin. Invest. 1994; 93: 265-272
        • McGarry J.D.
        • Leatherman G.F.
        • Foster D.W.
        Carnitine palmitoyltransferase I. The site of inhibition of hepatic fatty acid oxidation by malonyl-CoA.
        J. Biol. Chem. 1978; 253: 4128-4136
        • Gibbons G.F.
        • Bartlett S.M.
        • Sparks C.E.
        • Sparks J.D.
        Extracellular fatty acids are not utilized directly for the synthesis of very-low-density lipoprotein in primary cultures of rat hepatocytes.
        Biochem. J. 1992; 287: 749-753
        • Savage D.B.
        • Choi C.S.
        • Samuel V.T.
        • Liu Z.X.
        • Zhang D.
        • Wang A.
        • Zhang X.M.
        • Cline G.W.
        • Yu X.X.
        • Geisler J.G.
        • et al.
        Reversal of diet-induced hepatic steatosis and hepatic insulin resistance by antisense oligonucleotide inhibitors of acetyl-CoA carboxylases 1 and 2.
        J. Clin. Invest. 2006; 116: 817-824
        • Ota T.
        • Takamura T.
        • Kurita S.
        • Matsuzawa N.
        • Kita Y.
        • Uno M.
        • Akahori H.
        • Misu H.
        • Sakurai M.
        • Zen Y.
        • et al.
        Insulin resistance accelerates a dietary rat model of nonalcoholic steatohepatitis.
        Gastroenterology. 2007; 132: 282-293
        • Edvardsson U.
        • Bergstrom M.
        • Alexandersson M.
        • Bamberg K.
        • Ljung B.
        • Dahllof B.
        Rosiglitazone (BRL49653), a PPARgamma-selective agonist, causes peroxisome proliferator-like liver effects in obese mice.
        J. Lipid Res. 1999; 40: 1177-1184
        • Memon R.A.
        • Tecott L.H.
        • Nonogaki K.
        • Beigneux A.
        • Moser A.H.
        • Grunfeld C.
        • Feingold K.R.
        Up-regulation of peroxisome proliferator-activated receptors (PPAR-alpha) and PPAR-gamma messenger ribonucleic acid expression in the liver in murine obesity: troglitazone induces expression of PPAR-gamma-responsive adipose tissue-specific genes in the liver of obese diabetic mice.
        Endocrinology. 2000; 141: 4021-4031
        • Vidal-Puig A.
        • Jimenez-Linan M.
        • Lowell B.B.
        • Hamann A.
        • Hu E.
        • Spiegelman B.
        • Flier J.S.
        • Moller D.E.
        Regulation of PPAR gamma gene expression by nutrition and obesity in rodents.
        J. Clin. Invest. 1996; 97: 2553-2561
        • Zhang Y.L.
        • Hernandez-Ono A.
        • Siri P.
        • Weisberg S.
        • Conlon D.
        • Graham M.J.
        • Crooke R.M.
        • Huang L.S.
        • Ginsberg H.N.
        Aberrant hepatic expression of PPARgamma2 stimulates hepatic lipogenesis in a mouse model of obesity, insulin resistance, dyslipidemia, and hepatic steatosis.
        J. Biol. Chem. 2006; 281: 37603-37615
        • Schadinger S.E.
        • Bucher N.L.
        • Schreiber B.M.
        • Farmer S.R.
        PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes.
        Am. J. Physiol. Endocrinol. Metab. 2005; 288: E1195-E1205
        • Gavrilova O.
        • Haluzik M.
        • Matsusue K.
        • Cutson J.J.
        • Johnson L.
        • Dietz K.R.
        • Nicol C.J.
        • Vinson C.
        • Gonzalez F.J.
        • Reitman M.L.
        Liver peroxisome proliferator-activated receptor gamma contributes to hepatic steatosis, triglyceride clearance, and regulation of body fat mass.
        J. Biol. Chem. 2003; 278: 34268-34276
        • Sakamoto J.
        • Kimura H.
        • Moriyama S.
        • Odaka H.
        • Momose Y.
        • Sugiyama Y.
        • Sawada H.
        Activation of human peroxisome proliferator-activated receptor (PPAR) subtypes by pioglitazone.
        Biochem. Biophys. Res. Commun. 2000; 278: 704-711
        • Kim H.
        • Haluzik M.
        • Asghar Z.
        • Yau D.
        • Joseph J.W.
        • Fernandez A.M.
        • Reitman M.L.
        • Yakar S.
        • Stannard B.
        • Heron-Milhavet L.
        • et al.
        Peroxisome proliferator-activated receptor-alpha agonist treatment in a transgenic model of type 2 diabetes reverses the lipotoxic state and improves glucose homeostasis.
        Diabetes. 2003; 52: 1770-1778
        • Bogacka I.
        • Xie H.
        • Bray G.A.
        • Smith S.R.
        Pioglitazone induces mitochondrial biogenesis in human subcutaneous adipose tissue in vivo.
        Diabetes. 2005; 54: 1392-1399
        • Gastaldelli A.
        • Miyazaki Y.
        • Mahankali A.
        • Berria R.
        • Pettiti M.
        • Buzzigoli E.
        • Ferrannini E.
        • DeFronzo R.A.
        The effect of pioglitazone on the liver: role of adiponectin.
        Diabetes Care. 2006; 29: 2275-2281
        • Gastaldelli A.
        • Miyazaki Y.
        • Pettiti M.
        • Santini E.
        • Ciociaro D.
        • Defronzo R.A.
        • Ferrannini E.
        The effect of rosiglitazone on the liver: decreased gluconeogenesis in patients with type 2 diabetes.
        J. Clin. Endocrinol. Metab. 2006; 91: 806-812
        • Psaty B.M.
        • Furberg C.D.
        The record on rosiglitazone and the risk of myocardial infarction.
        N. Engl. J. Med. 2007; 357: 67-69