A GIP receptor agonist exhibits beta-cell anti-apoptotic actions in rat models of diabetes resulting in improved beta-cell function and glycemic control

Abstract

Aims: The gastrointestinal hormone GIP promotes pancreatic islet function and exerts pro-survival actions on cultured beta-cells. However, GIP also promotes lipogenesis, thus potentially restricting its therapeutic use. The current studies evaluated the effects of a truncated GIP analog, D-Ala(2)-GIP(1-30) (D-GIP(1-30)), on glucose homeostasis and beta-cell mass in rat models of diabetes.

Materials and methods: The insulinotropic and pro-survival potency of D-GIP(1-30) was evaluated in perfused pancreas preparations and cultured INS-1 beta-cells, respectively, and receptor selectivity evaluated using wild type and GIP receptor knockout mice. Effects of D-GIP(1-30) on beta-cell function and glucose homeostasis, in vivo, were determined using Lean Zucker rats, obese Vancouver diabetic fatty rats, streptozotocin treated rats, and obese Zucker diabetic fatty rats, with effects on beta-cell mass determined in histological studies of pancreatic tissue. Lipogenic effects of D-GIP(1-30) were evaluated on cultured 3T3-L1 adipocytes.

Results: Acutely, D-GIP(1-30) improved glucose tolerance and insulin secretion. Chronic treatment with D-GIP(1-30) reduced levels of islet pro-apoptotic proteins in Vancouver diabetic fatty rats and preserved beta-cell mass in streptozotocin treated rats and Zucker diabetic fatty rats, resulting in improved insulin responses and glycemic control in each animal model, with no change in body weight. In in vitro studies, D-GIP(1-30) exhibited equivalent potency to GIP(1-42) on beta-cell function and survival, but greatly reduced action on lipoprotein lipase activity in 3T3-L1 adipocytes.

Conclusions: These findings demonstrate that truncated forms of GIP exhibit potent anti-diabetic actions, without pro-obesity effects, and that the C-terminus contributes to the lipogenic actions of GIP.

Figure 1. A DPP-IV resistant GIP analog (D-GIP_1–30) demonstrates equivalent islet actions to GIP_1–42.

A, OGTTs were performed on fasted Lean (n = 3) and obese VDF (n = 6) rats and blood glucose levels measured. Mean ± SEM; ***, p<0.001 vs Lean rats. B, Insulin levels were determined from blood samples collected in A. Mean ± SEM with significance as indicated. C, Pancreas perfusions with 16.7 mM glucose + D-GIP_1–30 or GIP_1–42 (0–1 nM) were performed on Lean and obese VDF rats and insulin levels determined in perfusate. Mean ± SEM (n = 3). D, i.p. glucose tolerance tests (IPGTT) were performed on fasted Lean (n = 3) and obese VDF (n = 4) rats that received the glucose immediately following s.c. injections with PBS or D-GIP_1–30 (8 nmol/kg BW) and blood glucose levels measured. Mean ± SEM; **, p<0.01, ***, p<0.001 vs Lean controls; #, p<0.05 vs VDF controls. E, Insulin levels were determined from blood samples collected in D. Mean ± SEM; ***, p<0.001 vs Lean controls; #, p<0.05, ###, p<0.001 vs VDF controls. F, IPGTTs were performed on fasted wild type (GIPR^+/+) mice and GIPR knockout (GIPR^−/−) mice that received the glucose immediately following s.c. injections with PBS or D-GIP_1–30 (8 nmol/kg BW), and blood glucose levels measured. Mean ± SEM (n = 3); *, p<0.05 vs GIPR^+/+ mice treated with PBS. G, Islets from GIPR^+/+ and GIPR^−/− mice were incubated for 2 h in 3 or 11 mM glucose ± 10 nM D-GIP_1–30 and secreted insulin levels determined. Mean ± SEM (n = 3); significance as indicated. H, INS-1 cells were treated without or with 100 nM staurosporine + increasing concentrations of D-GIP_1–30 or GIP_1–42 (0–100 nM) for 6 h and cell death determined. Mean ± SEM (n = 4); ###, p<0.001 vs control (no staurosporine); ***, p<0.001 vs staurosporine alone. In the upper right is the calculated EC₅₀ value for GIP_1–42 and D-GIP_1–30.

Figure 2. D-GIP_1–30 partially protects β-cells in streptozotocin (STZ) treated rats.

A, Glucose levels were monitored 2 days prior to (day −2) and 4 days (day 4) following an i.p. injection of STZ (35 mg/kg BW; on day 0) to Lean rats treated twice daily with PBS or D-GIP_1–30 (8 nmol/kg BW) from day −2 to day 1 as well as in untreated Lean rats. B, On day 5, OGTTs were performed on rats described in A and blood glucose levels measured. C, Insulin levels were determined from blood samples collected in B. For A–C, Mean ± SEM (n = 4); *, p<0.05, **, p<0.01, ***, p<0.001 vs rats treated with STZ and PBS. D, Representative images of pancreases collected on day 6 stained with hematoxylin & eosin or with insulin (green), glucagon (red) and DAPI (blue); scale bar = 100 µm. E, Mean ± SEM of -cell (insulin positive) area relative to pancreas area (n = 4; 4 sections per animal); significance as shown.

Figure 3. D-GIP_1–30 improves islet function and diminishes islet pro-apoptotic protein levels in VDF rats.

A, OGTTs were performed on obese VDF rats ∼24 h prior to and ∼48 h following 10 days of twice daily treatment with PBS or D-GIP_1–30 (8 nmol/kg BW) and blood glucose levels measured. Mean ± SEM (n = 6); **, p<0.01 vs same VDF rats prior to treatment. B, Integrated glucose profile for OGTTs described in A. Mean ± SEM (n = 6); significant differences as shown. C, Insulin levels were determined from blood samples collected in A. Mean ± SEM (n = 6); *, p<0.05 vs same VDF rats prior to treatment. B, Integrated acute insulin response (from 0 to 30 minutes) for insulin profiles described in C. Mean ± SEM (n = 6); significant differences as shown. E, Islets were isolated from VDF rats and age matched Lean rats ∼24 h following OGTTs and Western analysis performed on cell lysates with indicated antibodies. F, For quantification, protein levels were normalized to beta-actin and expressed relative to Lean controls. Mean ± SEM (n = 3); $, p<0.05 vs Lean; #, p<0.05 vs VDF controls.

Figure 4. D-GIP_1–30 improves glycemic control in ZDF rats.

A, Schematic depicting the treatment protocol in which Lean or obese ZDF rats (starting at 6 weeks of age) were monitored every 3 days from day −6 to day 18 and treated twice daily with PBS or D-GIP_1–30 (8 nmol/kg BW) from day 0 to day 18. B–E, Routine monitoring involved measurements of blood glucose (B), body weight (C), food intake (D), and water intake (E). Mean ± SEM (n = 6); *, p<0.05, **, p<0.01, ***, p<0.001 vs ZDF rats treated with PBS. F, On day 18, blood glucose levels were determined every 3 h over a 24 h period. Mean ± SEM (n = 6); **, p<0.01, ***, p<0.001 vs ZDF rats treated with PBS.

Figure 5. D-GIP_1–30 improves β-cell function and mass and glucose tolerance in ZDF rats.

A, OGTTs were performed on fasted Lean and ZDF rats (described in figure 4) ∼48 h following the last day of treatment and blood glucose levels were measured. Mean ± SEM (n = 6); **, p<0.01, ***, p<0.001 vs ZDF rats treated with PBS. B, Insulin levels were determined from blood samples collected in A. Mean ± SEM (n = 6); **, p<0.01 vs ZDF rats treated with PBS. C, Integrated acute insulin response (from 0 to 30 minutes) for profiles described in B was plotted with respect to HOMA S_I. Mean ± SEM (n = 6). D, Representative images of pancreases collected ∼24 h following OGTTs. Pancreases were stained with hematoxylin & eosin or with insulin (green), glucagon (red) and DAPI (blue); scale bar = 100 µm. E, Mean ± SEM of β-cell (insulin positive) area relative to pancreas area (n = 3; 4 sections per animal); significance as shown. E, Mean percent ± SEM of β-cells undergoing apoptosis as determined via TUNEL positive nuclei (n = 6); significance as shown. F, Mean percent ± SEM of -cells undergoing proliferation as determined via PCNA positive nuclei (n = 6).

Figure 6. Cultured 3T3-L1 adipocytes differentially respond to D-GIP_1–30 and GIP_1–42.

3T3-L1 adipocytes were serum starved in 3 mM glucose DMEM containing 0.1% BSA overnight and then treated for 24 h with increasing concentrations (0–1000 nM) of GIP_1–42, D-GIP_1–42, GIP_1–30, or D-GIP_1–30 in the presence of 1 nM insulin and then LPL activity determined. Mean ± SEM (n = 7); significance as shown.