Beneficial effects of growth hormone-releasing hormone agonists on rat INS-1 cells and on streptozotocin-induced NOD/SCID mice

Abstract

Agonists of growth hormone-releasing hormone (GHRH) have been previously reported to promote growth, function, and engraftment of islet cells following transplantation. Here we evaluated recently synthesized GHRH agonists on the proliferation and biological functions of rat pancreatic β-cell line (INS-1) and islets. In vitro treatment of INS-1 cells with GHRH agonists increased cell proliferation, the expression of cellular insulin, insulin-like growth factor-1 (IGF1), and GHRH receptor, and also stimulated insulin secretion in response to glucose challenge. Exposure of INS-1 cells to GHRH agonists, MR-356 and MR-409, induced activation of ERK and AKT pathways. Agonist MR-409 also significantly increased the levels of cellular cAMP and the phosphorylation of cAMP response element binding protein (CREB) in INS-1 cells. Treatment of rat islets with agonist, MR-409 significantly increased cell proliferation, islet size, and the expression of insulin. In vivo daily s.c. administration of 10 μg MR-409 for 3 wk dramatically reduced the severity of streptozotocin (STZ)-induced diabetes in nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice. The maximal therapeutic benefits with respect to the efficiency of engraftment, ability to reach normoglycemia, gain in body weight, response to high glucose challenge, and induction of higher levels of serum insulin and IGF1 were observed when diabetic mice were transplanted with rat islets preconditioned with GHRH agonist, MR-409, and received additional treatment with MR-409 posttransplantation. This study provides an improved approach to the therapeutic use of GHRH agonists in the treatment of diabetes mellitus.

Keywords: GHRH agonists; INS-1; diabetes; signaling pathways; transplantation.

Fig. 1.

Effect of GHRH agonists on INS-1 cell viability. INS-1 cells were treated with either MR agonists or JI-36 at concentrations indicated. Total numbers of viable cells were measured at 72 h of treatment using CellTiter 96 aqueous one solution cell proliferation assay and calculated as relative percentage of the control.

Fig. S1.

Effect of GHRH agonists on INS-1 cell viability and proliferation. (A) INS-1 cells were treated by MR agonists and JI-36 at concentrations indicated for 24 h. Total numbers of proliferating cells were measured using the BrdU incorporation assay. Values are shown as percentages of control. (B) INS-1 cells were treated with MR-356 at indicated concentrations in the presence or absence of 2 μM GHRH antagonist, MIA-602. Relative percentages were calculated by comparing the number of viable cells in the treatment to that in the control. Data were averaged from at least three individual tests done in quadruplicate for each test.

Fig. 2.

Effect of GHRH agonists on expression of insulin, IGF1, GHRH receptor, VEGF, and glucose-stimulated insulin secretion. INS-1 cells were exposed to 500 nM GHRH agonists or respective control medium for 48 h. The expression levels of messenger RNA for (A) insulin, (B) IGF1, and (C) GHRH receptor, are shown as percentages of the control. In addition, the protein levels of (A) intracellular insulin, (B) secreted IGF1, and (C) GHRH receptor, are also shown in parallel. A representative Western blot image for GHRH receptor is shown. (D) Insulin secretion in response to high glucose (17 mM) stimulation; *P < 0.05. (E) Elevated VEGF secretion from MR-409-treated INS-1 cells. All data were calculated from three individual experiments.

Fig. 3.

Stimulatory effect of GHRH agonists on ERK, AKT, and cAMP/CREB pathways. (A) Stimulation of p-ERK/ERK and p-AKT/AKT after a 30-min treatment with MR-356 and MR-409. (B) Inhibition of MR-409-induced cell proliferation by PD98059 (PD, 25 µM) or Wortmannin (Wt, 0.5 µM). Data shown are a representative experiment done in quintuplicate in each treatment; *P < 0.05, **P < 0.01, ***P < 0.001. (C) Stimulation of cellular cAMP in MR-409-treated INS-1 cells. (D) Elevated pCREB/CREB in MR-409-treated INS-1 cells. Data were averaged from individual experiments. A representative Western blot image is shown.

Fig. S2.

Effect of GHRH agonists on the proliferation of rat islets. (A) Proliferation of rat islet cells after 24 h of treatment with MR-356 or MR-409. (B) Relative expression levels of insulin in rat islets treated with 1 μM MR-356 or MR-409 for 48 h. (C and D) Comparison of total IPN and IEQ/IPN in rat islets treated with either 1 μM MR409 or vehicle for 48 h; T-0, islets before culture. **P < 0.01.

Fig. S3.

Effect of GHRH agonist MR-409 on survival and blood glucose levels of NOD/SCID mice. (A) Cumulative survival of the diabetic animals in control (C, ●) and MR-409-treated group (T, ○). (B) Blood glucose (average ± SEM) of the diabetic mice treated by either control (C, ●) or MR-409 (T, ○). Animals in group T received 10 μg of MR-409 per day s.c. from day 0 to day 21.

Fig. 4.

Effect of GHRH agonist MR-409 in the transplanted NOD/SCID mice. (A) Blood glucose follow-up (average ± SEM) in the diabetic mice after transplantation. Control (C, ●); M (○); and M + T (▼). Transplantation was conducted at day 0, animals in the group M + T were injected s.c. with 10 μg/day of MR-409 from day 2 to day 24. The horizontal line represents the average blood glucose level in nondiabetic mice (147.3 ± 7.6 mg/dL, n = 25). (B) Average weekly blood glucose, the data represent the average glucose of three measurements per week (mean ± SEM). Open box, control; light gray box, group M, and gray box, group M + T. −D1, 1 d before transplantation. (C) The IPGTT performed at 2 wk after transplantation.

Fig. 5.

Serum levels of insulin and IGF1 in transplanted NOD/SCID mice. The serum levels of (A) insulin and (B) IGF1 were analyzed after 3 wk of treatment. B-STZ, before STZ induction, n = 12); BT, diabetic mice before transplantation (n = 8); C, control (n = 7), M (n = 5), M + T (n = 7); *P < 0.05, **P < 0.01, ***P < 0.001. (C) Immunohistochemistry analysis of insulin expression. Representative immunostained tissue sections of islet-bearing kidneys retrieved from the animals in M and M + T groups. (Scale bar, 100 μm.)