Leptin administration enhances islet transplant performance in diabetic mice

Abstract

Islet transplantation is an effective method to obtain long-term glycemic control for patients with type 1 diabetes, yet its widespread use is limited by an inadequate supply of donor islets. The hormone leptin has profound glucose-lowering and insulin-sensitizing action in type 1 diabetic rodent models. We hypothesized that leptin administration could reduce the dose of transplanted islets required to achieve metabolic control in a mouse model of type 1 diabetes. We first performed a leptin dose-response study in C57Bl/6 mice with streptozotocin (STZ)-induced diabetes to determine a leptin dose insufficient to reverse hyperglycemia. Subsequently, we compared the ability of suboptimal islet transplants of 50 or 125 syngeneic islets to achieve glycemic control in STZ-induced diabetic C57Bl/6 mice treated with or without this dose of leptin. The dose-response study revealed that leptin reverses STZ-induced diabetes in a dose-dependent manner. Supraphysiological leptin levels were necessary to restore euglycemia but simultaneously increased risk of hypoglycemia, and also lost efficacy after 12 days of administration. In contrast, 1 µg/day leptin only modestly reduced blood glucose but maintained efficacy throughout the study duration. We then administered 1 µg/day leptin to diabetic mice that underwent transplantation of 50 or 125 islets. Although these islet doses were insufficient to ameliorate hyperglycemia alone, coadministration of leptin with islet transplantation robustly improved control of glucose and lipid metabolism, without increasing circulating insulin levels. This study reveals that low-dose leptin administration can reduce the number of transplanted islets required to achieve metabolic control in STZ-induced diabetic mice.

FIG. 1.

Leptin reverses STZ-induced diabetes in a dose-dependent manner. Mice were treated with STZ and 6 days later received implantation of osmotic pumps (day 0, vertical broken line) delivering doses of either leptin ranging from 1 to 10 µg/day (STZ and 1, 3, 5, or 10 µg/day) or vehicle only (STZ-vehicle). Nondiabetic mice served as controls. Fasting blood glucose (A) and change in body weight (B) relative to day −1. AUC analysis of blood glucose levels from day 5 to day 12 is presented (A). AUC analysis of body weight change from day 5 to day 12 revealed no significant differences and is not shown. C: The 4-h fasted plasma insulin levels on day 25. Limit of detection is shown by broken horizontal line. D: The 4-h fasted plasma leptin levels on days 12 and 25. Four of five STZ-vehicle mice had leptin levels below the detection limit (indicated by broken line) on both days, and this group was not included in statistical analyses. Statistical analyses were performed by two-way ANOVA with Tukey post hoc test. E: The 4-h fasted plasma IGFBP2 levels on day 12. Data are presented as mean ± SEM, n = 4–5. *P < 0.05. †P < 0.05 vs. nondiabetic controls. ‡P < 0.05 vs. STZ-vehicle controls.

FIG. 2.

Leptin improves glucose tolerance in STZ-diabetic mice in a dose-dependent manner. OGTTs were performed in nondiabetic controls and STZ-induced diabetic mice receiving different doses of leptin (STZ and 1, 3, 5, or 10 µg/day) or vehicle only (STZ-vehicle) after a 6-h fast on day 19 after surgery. Mice were gavaged with 2 g/kg glucose at time 0. Time course of blood glucose tracking (A) and AUC from 0–90 min (B). Four of five mice treated with 1 µg/day and all STZ-vehicle–treated mice had blood glucose above the detection limit (33.3 mmol/L) at one or more time points in which case values of 33.3 mmol/L were assigned. These groups were not included in statistical analyses. Two mice treated with STZ and 5 µg/day (STZ-5 µg/day) and one mouse treated with STZ and 3 µg/day (STZ-3 µg/day) had blood glucose <2.9 mmol/L at 90 min and were rescued with exogenous glucose. Data from these mice are omitted from the 120-min time point; n = 5 for 0–90 min and n = 3–5 for 120 min. Data are presented as mean ± SEM. NS, not significant.

FIG. 3.

Leptin administration enhances the efficacy of islet transplantation for treatment of STZ-induced diabetes. Mice were treated with STZ on day −6 and subsequently received transplants of 50, 125, or 300 islets or sham surgery (day 0) and simultaneous osmotic pump implants delivering 1 µg/day leptin or vehicle for 6 weeks. A: The 4-h fasted blood glucose levels. B: Blood glucose data from days 5 to 43 analyzed by AUC. C: The 4-h fasted HbA_1c levels in whole blood are presented as percents and mmol/mol equivalents are in parentheses. The 4-h fasted body weight gain normalized to day −1 (D) and net AUC from days 5 to 43 (E). F: The 4-h fasted plasma leptin measured from cardiac puncture samples collected 6 weeks after surgery. Data are presented as mean ± SEM (n = 3–5). *P < 0.05. †P < 0.05 vs. nondiabetic controls. ‡P < 0.05 vs. sham-vehicle controls. NS, not significant.

FIG. 4.

Leptin cotherapy does not alter circulating insulin levels or β-cell recovery. A: The 4-h fasted plasma insulin levels were measured 6 weeks after transplant in all groups by ultrasensitive insulin ELISA. Limit of detection (0.019 ng/mL) is shown as horizontal broken line (n = 3–5). B: Immunofluorescent quantifications of β-cell and α-cell areas were performed in nondiabetic controls and STZ-induced diabetic mice treated with 125 islets (125 islets-vehicle), with 125 islets and leptin (125 islets-leptin), or left untreated (sham-vehicle) (n = 3–4). Data are presented as mean ± SEM. †P < 0.05 vs. nondiabetic controls. ‡P < 0.05 vs. sham-vehicle controls. C: Representative images of pancreata from nondiabetic and sham-vehicle groups costained for insulin (red), glucagon (green), and DAPI (white) (n = 3–4). Scale bars represent 1,000 µm (left top, left bottom) and 100 µm (right top, right bottom). NS, not significant.

FIG. 5.

Leptin and islet cotherapy improves glucose tolerance. A: OGTT on day 20 after a 6-h fast in mice transplanted with islets with or without leptin and untreated diabetic (sham-vehicle) mice, leptin-treated diabetic (sham-leptin) mice, and nondiabetic controls. B: Because some mice had blood glucose levels over the limit of detection after gavage, blood was collected at 10 and 20 min for plasma glucose measurement. C: Plasma insulin levels after gavage. The limit of detection (0.188 ng/mL) is indicated by a broken horizontal line. Only nondiabetic and 300 islets-vehicle groups had detectable plasma insulin levels at any given time. The remainder of mice were assigned values of 0.188 ng/mL. Data are presented as mean ± SEM (n = 3–5). *P < 0.05. †P < 0.05 vs. nondiabetic controls. ‡P < 0.05 vs. sham-vehicle controls. NS, not significant.

FIG. 6.

Low-dose leptin administration normalizes lipid levels in STZ-diabetic mice. The 4-h fasted plasma triglycerides (A), FFA (B), and β-hydroxybutyrate (C) were measured in mice transplanted with islets with or without leptin and in untreated diabetic (sham-vehicle) mice, leptin-treated diabetic (sham-leptin) mice, and nondiabetic controls on day 12 after surgery. Data are presented as mean ± SEM (n = 3–5). *P < 0.05. †P < 0.05 vs. nondiabetic controls. ‡P < 0.05 vs. STZ-vehicle controls.