Antidiabetic Effects of IGFBP2, a Leptin-Regulated Gene Kristina Hedbacker, Kıvanç Birsoy, Robert W. Wysocki, Esra Asilmaz, Rexford S. Ahima, I. Sadaf Farooqi, Jeffrey M. Friedman  Cell Metabolism  Volume 11, Issue 1, Pages 11-22 (January 2010) DOI: 10.1016/j.cmet.2009.11.007 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Low-Dose Leptin Treatment of ob/ob Mice Corrects Blood Glucose and Hyperinsulinemia Independently of Body Weight (A) Mice receiving 12 day leptin treatment; percent change in body weight during treatment. Arrows show days 4 and 8 of treatment. Mice were fasted for 6 hr prior to receiving anesthesia and blood collection at days 0, 4, 8, and 12. Dose of leptin is as indicated in (A). For each group, n ≥ 4. Dotted line indicates food-restricted animals (see Figures 3A–3D). (B) Food intake in grams each day during treatment. Dotted line indicates food-restricted animals (see Figures 3A–3D). (C–E) Plasma leptin (ng/ml), blood glucose (mg/dl), and plasma insulin (ng/ml) at day 12 of leptin treatment. Blood glucose and plasma insulin for food-restricted animals can be found in Figures 3C and 3D. Error bars show SEM. ∗p < 0.05, ∗∗p < 0.01. Cell Metabolism 2010 11, 11-22DOI: (10.1016/j.cmet.2009.11.007) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Leptin Regulation of IGFBP2 (A) Relative IGFBP2 mRNA expression in liver samples from ob/ob mice leptin treated for 12 days as indicated. (B) Plasma IGFBP2 levels in ob/ob mice during treatment with leptin at the indicated concentrations. (C) Plasma leptin (left y axis) and plasma IGFBP2 (right y axis) in mice as follows: WT, wild-type; WT + leptin, wild-type after 8 days of 1 μg/hr leptin; DIO, diet-induced obese (high-fat diet for 15 weeks); STZ, streptozotocin diabetic; ob/ob mice; ob/ob + leptin, ob/ob after 12 days of 100 ng/hr leptin treatment; ob/ob food restr., ob/ob after 12 days of food restriction to 0.5 g/day (insulin levels one-tenth of free-feeding: data not shown); Ay, agouti; Srebp-1c. Grey bars indicate IGFBP2. Black bars indicate leptin levels in same animals. Error bars show standard error. ∗p < 0.05, ∗∗p < 0.01. Cell Metabolism 2010 11, 11-22DOI: (10.1016/j.cmet.2009.11.007) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 IGFBP2 Treatment Corrects Hyperglycemia, Hyperinsulinemia, and Hepatic Steatosis in ob/ob Diabetic Mice (A–F) ob/ob mice treated with Ad-control (blue) or Ad-IGFBP2 (red) or untreated and pair-fed to Ad-IGFBP2. N ≥ 4. Body weight and food intake in grams are shown in (A) and (B). Food intake is average for 24 hr. Mice were injected with adenovirus on day 0. Arrow indicates 18 hr fast (for GTT); x axis indicates day of experiment. Dotted line shows body weight and food intake of mice pair-fed to the IGFBP2-treated mice. Plasma glucose and plasma insulin in treated, control, and pair-fed mice are shown in (C) and (D). Milligrams triglycerides per gram liver tissue in ob/ob control, ob/ob + IGFBP2, ob/ob + 12 days 100 ng/hr leptin, and ob/ob + 12 days of 25 ng/hr leptin are shown in (E). H&E-stained liver paraffin sections of treated and control mice are shown in (F) (10× and 40× as indicated, ∗p < 0.05, ∗∗p < 0.01). Error bars show standard error. Cell Metabolism 2010 11, 11-22DOI: (10.1016/j.cmet.2009.11.007) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 IGFBP2 Treatment Improves Glucose Metabolism and Insulin Sensitivity in Diabetic Mice (A) Glucose tolerance test (GTT) in ob/ob mice receiving IGFBP2 or empty adenovirus. (B) Relative IGFBP2 mRNA expression in frozen liver samples from ob/ob mice ∼3 weeks after Ad-IGFBP2 or Ad-control infection (fatty acid synthase [FAS], PEPCK, and G6Pase). (C) Glucose infusion rate (GIR) in mg/kg/min necessary to keep hyperinsulinemic mice euglycemic during clamp studies of ob/ob mice ± IGFBP2. (D) Rd (whole-body glucose disappearance) in mg/kg/min. (E) Hepatic glucose production (HGP) in mg/kg/min. (F) Percent suppression of HGP in response to hyperinsulinemic clamp. In all panels: red, IGFBP2-treated ob/ob mice; blue, control-treated ob/ob mice. ∗p < 0.05, ∗∗p < 0.01, n = 7. Error bars show standard error. Cell Metabolism 2010 11, 11-22DOI: (10.1016/j.cmet.2009.11.007) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Comparison of Effect of IGFBP2 Treatment on Blood Glucose, Insulin, Daily Food Intake, Body Weight, and Glucose Tolerance in Wild-Type, ob/ob, Ay, STZ, and DIO Mice (A–H) Red, Ad-IGFBP2-treated; blue, Ad-control-treated. ∗p < 0.05, ∗∗p < 0.01. Error bars show standard error. WT, wild-type; Ay, Agouti; DIO, diet-induced obese (15 weeks on high-fat diet); STZ, streptozotocin-induced type 1 diabetic mice. Blood glucose and insulin were taken on day 5 or 6 postinfection. Daily food intake is the average of days 4–14 postinfection. Change in weight is the total change in weight from days 4–14 postinfection. Cell Metabolism 2010 11, 11-22DOI: (10.1016/j.cmet.2009.11.007) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 IGFBP2 Levels in Leptin-Deficient Patients (A) Serum IGFBP2 in leptin-deficient and age- and weight-matched controls. (B) Serum IGFBP2 in three leptin-deficient patients before (light gray) and 6 months after (dark gray) low-dose leptin treatment. Error bars show standard error. Cell Metabolism 2010 11, 11-22DOI: (10.1016/j.cmet.2009.11.007) Copyright © 2010 Elsevier Inc. Terms and Conditions