1. Rapid in vivo Effects of Pioglitazone on Adipose Tissue Gene Expression and Insulin Action in Humans with Type 2 Diabetes Mellitus(T2DM) P. Kishore, W.J. Li, C. Weaver, J. Tonelli, P. Scherer, E. Goodman and M. Hawkins Albert Einstein College of Medicine, Bronx, NY Thiazolidinediones (TZD’s) improve insulin action in skeletal muscle, despite a paucity of PPAR- receptors. Possible mechanisms include changes in circulating free fatty acids (FFA) and/or adipokines, eg. tumor necrosis factor- (TNF-), adiponectin, resistin, and interleukins. We had shown that pioglitazone (PIO) increases whole-body insulin sensitivity and adiponectin levels in T2DM after only 21 days. Thus, we examined its impact on the expression of key adipokines at this early time point, prior to confounding effects on FFA and glucose levels. Nine T2DM subjects (7M/2F, age 47  3yr, HbA1C 10.8  0.7%, BMI 32.9  1.9 kg/m2) received either PIO (45 mg) and placebo (PL) for 21 days (random order, double-blinded). Glucose uptake (GU; [3-3H]- glucose) was measured during 6h euglycemic (5mM) stepped insulin (0-4h =50 U/ml; 4-6h = 200 U/ml) clamp studies. Subcutaneous abdominal fat was biopsied at t=6h. 21 days PIO increased GU at physiologic hyperinsulinemia (PIO=4.70.7 vs. PL=3.20.4 mg/kg.min, p<0.05) and at higher insulin levels (PIO=11.00.7 vs. PL=9.50.5 mg/kg.min, p<0.05). The table shows the effects of PIO vs. PL on adipose gene expression (real-time rt-PCR) and circulating adipokines (sandwich ELISA). Gene expression data was corrected for both -actin and GAPDH, using plasmid standard curves to calculate copy number. Thus, 21 days PIO improved insulin action in T2DM with marked increases in adiponectin (both gene expression and plasma levels)as shown here, yet minimal or no effects on leptin, TNF- or IL- 6. Hence, of the potential mechanisms examined, increased adiponectin is most likely to contribute to the effects of TZDs on insulin action. Abstract Thiazolidinediones: Apparent Paradoxes Promote adipocyte differentiation and weight gain, yet improve whole-body insulin action in type 2 diabetes mellitus (T2DM) Enhance insulin action in skeletal muscle, despite a low abundance of PPAR-g receptors in that tissue While TZDs are known to exert acute effects on insulin action in rodents, they have not been studied for less than 12 weeks in humans Experimental Question Can the improved insulin action observed following 21 days’ therapy with pioglitazone be attributed to changes in adipokines or FFA or both? Possible Mechanisms Changes in circulating FFA Changes in adipokines TNF Adiponectin Resistin Interleukins i.e IL-6, IL-1 Resistin Adipose Tissue Muscle Liver FFA PPARγ + FA storage +/- Adipokines: Leptin TNF-a IL-1/6 Adiponectin - Gluconeogenesis Whole body effects of TZD’s Insulin sensitization Glucose lowering Triglyceride lowering Introduction Effect of Pioglitazone on gene expression in subcutaneous adipose tiisue n=9 (7 males, 2 females) subjects with type 2 diabetes mellitus: mean age 47± 3 years HbA1C 10.8 ± 0.7% BMI 32.9 ± 1.9 kg/m2 Received 45mg of Pioglitazone(PIO) or Placebo(PL) in a double blind, random fashion 6 hour euglycemic-hyperinsulinemic “stepped clamp” following overnight insulinization Subcutaneous adipose tissue biopsies performed at end of the clamp Methods Variable Glucose Infusion Plasma glucose (mg/dl) Basal Insulin Glucose Tracer Somatostatin, GH, Glucagon Plasma insulin (µU/mL) 100 20 0 300 400 0 Low Insulin High Insulin 50 10 0 150 200 0 60120180240300360 Time (min) Placebo Pioglitazone GP (mg/kg/min) 0 0.5 1.0 1.5 2.0 2.5 * GP (Low insulin) GU (High insulin) * GU (mg/kg/min) 0 2 4 6 8 10 12 Time 0 Time 300-360 Placebo Pioglitazone 0 FFA (uM/ml) 100 200 Effect of 21 Days Pioglitazone on Glucose Production (GP) and Glucose Uptake (GU) at Low Insulin Infusion Rates Effect of 21 Days of Pioglitazone on Plasma Free Fatty Acid Levels in T2DM subjects Results Experimental Question 2 Are changes in plasma adipokines accompanied by changes in gene expression in muscle and adipose tissue? Gene expression by quantitative, ‘real-time’ reverse transcriptase polymerase chain reaction Specific protocol for Roche LightCycler instrument (see schematic figure) Fluorescent detection of double-stranded DNA by SYBRGreen dye Reaction conditions: 40 cycles, denaturation at 95◦C for 2 sec, annealing for 59◦C for 5 secs, elongation at 74 for 12 sec Pioglitazone therapy for only 21 days improved both hepatic and peripheral insulin action in patients with T2DM The improved insulin sensitivity was accompanied by marked increases in plasma adiponectin However, this short duration of pioglitazone therapy did not affect plasma levels of free fatty acids or interleukin-6, and had only small effects on leptin and TNF-alpha Effect of Pioglitazone on Fasting Plasma Adipponectin Levels in T2DM Subjects 0 10 20 *P<0.01 Fasting * High Insulin * Placebo Pio PlaceboPioglitazone Leptin (ng/ml) TNF-alpha (pg/ml) IL-6 (pg/m ) 14  6 257  51 62  15 12  5* 217  50* 53  8 *P<0.05 **p<0.01 Adiponectin (mcg/ml) 6.1  0.8 12.4  1.7** Effect of Pioglitazone on Plasma Levels of “Adipokines” Summary 1 Schematic of the LightCycler instrument for real-time rt-PCR Adiponectin Placebo Beta-actin PIO Beta-actin Placebo Adiponectin PIO Representative exponential portions of LightCycler curves comparing adipocyte Adiponectin and ß-actin gene expression in one subject with Pioglitazone (PIO) and placebo administration 21 days of Pioglitazone administration is associatedwith marked increases in Adiponectin gene expression There was a minimal or no effect of gene expression of leptin, IL-6 and TNF-alpha Hence, of the potential mechanisms examined, increased adiponectin is most likely to contribute to the effects of TZD’s on insulin action Given the time course and magnitude of effect, it is likely that increases in plasma levels of the fat-derived peptide adiponectin may contribute importantly to the beneficial effects of TZD’s on insulin action in people with type 2 diabetes mellitus Pio vs Placebo: Fold change Adiponectin Leptin IL-6 8.7  2.7* 0.7  1.3 1.1  5.2 *P<0.05 TNF-  0.9  1.3 Gene Conclusions Methods: rt-PCR Effect of Pioglitazone on Gene Expression in Adipose Tissue Summary (rt-PCR studies)