Treatment with rHuEPO protects against hyperglycemia in the MLDS model of diabetes. (A) 6–8-wk-old C57BL/6 mice were injected with rHuEPO or saline three times per week for 4 wk starting on the first day of STZ treatment. Blood glucose levels were measured at the indicated time points (n = 15 per treatment group). (B) Fasting blood glucose levels were measured after 4 wk of rHuEPO or saline treatment (n = 10 per treatment group). (C) Insulin tolerance (insulin sensitivity) was measured after 4 wk of treatment (n = 6 per treatment group). *, P < 0.05. Results represent means ± SE.

rHuEPO-treated mice have enhanced β-cell mass. (A) β-cell area per pancreatic area was determined on insulin-immunostained sections (n = 11 per treatment group). (B) β-cell proliferation was assessed on Ki67/insulin-coimmunostained sections (n = 11 per treatment group). (C) TUNEL-positive apoptotic β cells were determined on the day after the last STZ injection (n = 3 per treatment group). (D and E) Islet vasculature was assessed on factor VIII (D)– and CD31 (E)-immunostained pancreatic sections (n = 5 per treatment group). Mice were sacrificed after 4 wk of treatment. (F) bcl-xL and vegf mRNA levels in isolated islets were assessed by quantitative real-time PCR after 1 wk of STZ and either rHuEPO or saline treatments (n = 3 per treatment group). The expression level of each unknown was normalized by the internal control 18S. Each sample was run in triplicate, and each experiment included three nontemplate control wells. Similar results were also found from the normalization by GAPDH and actin (not depicted). (G) Intact islet architecture in both rHuEPO- and saline-treated mice as assessed by insulin (red) and glucagon (green) coimmunostaining (n = 3 per treatment group). Mice were sacrificed after 4 wk of their respective treatments. *, P < 0.05. Results represent means ± SE. Bars: (A) 200 µm; (D) 20 µm; (G) 40 µm.

Treatment with rHuEPO reverses type 2 diabetes in the db/db mouse. (A) Random blood glucose levels were measured weekly (n = 10 per treatment group). (B) Fasting blood glucose levels were measured after 4 wk of treatment (n = 10 per treatment group). (C) Insulin sensitivity as assessed by insulin tolerance tests performed after 4 wk of treatment (n = 10 per treatment group). (D) In vivo glucose-stimulated insulin secretion was performed after 4 wk of rHuEPO or saline treatment (n = 9 per treatment group). *, P < 0.05. Results represent means ± SE.

Effect of rHuEPO treatment on the islets of db/db mice. (A) β-cell area per pancreatic area was calculated on insulin-immunostained pancreatic sections (n = 10 per treatment group). (B and C) The percentage of Ki67-positive proliferating β cells (B) and TUNEL-positive apoptotic β cells (C) per islet was assessed by immunohistochemistry (n = 10 per treatment group). (D and E) Islet vasculature was assessed by factor VIII (D) and CD31 (E) immunostaining on pancreatic sections (n = 5 per treatment group). Mice were sacrificed after 4 wk of treatment. (F) bcl-xL and vegf mRNA levels in isolated islets after 1 wk of rHuEPO or saline treatment were assessed by quantitative real-time PCR (n = 3 per treatment group). The expression level of each unknown was normalized by the internal control 18S. Each sample was run in triplicate, and each experiment included three nontemplate control wells. Similar results were also found from the normalization by GAPDH and actin (not depicted). (G) Insulin (red) and glucagon (green) coimmunostaining was performed to determine islet architecture (n = 3 per treatment group). Mice were sacrificed after 4 wk of treatment. *, P < 0.05. Results represent means ± SE. Bars: (A) 200 µm; (D and G) 40 µm.

Effect of rHuEPO administration on islet signaling and gene transcription. (A) Quantitative real-time PCR results of bcl-xL, c-myc, c-kit, and vegf mRNA levels in islets isolated after 1 wk of rHuEPO treatment (n = 3 per treatment group). The expression level of each unknown was normalized by the internal control 18S. Each sample was run in triplicate, and each experiment included three nontemplate control wells. Similar results were also found from the normalization by other internal controls, including GAPDH and actin (not depicted). (B) Western blots of islet protein lysates were performed after 1 wk of rHuEPO or saline administration to assess phospho (p)-JAK2, phospho-STAT5, and Bcl-xL expression levels as well as phospho-AKT, PDX-1, and GLUT2 levels (n = 3 per treatment group). *, P < 0.05. Results represent means ± SE.

EPO-R is required for rHuEPO-mediated diabetes protection against STZ treatment. (A) Blood glucose levels were measured on a weekly basis throughout the treatment period (n = 8 per treatment group). (B and C) Insulin-immunostained pancreatic sections were performed to assess β-cell area per pancreatic area (n = 8 per treatment group). (D) Ki67 immunostaining on pancreatic sections was performed to assess the percentage of Ki67-positive β cells per islet (n = 8 per treatment group). (E and F) Islet vasculature was assessed by factor VIII immunostaining on pancreatic sections (n = 5 per treatment group). Mice were sacrificed 4 wk after their respective treatments. *, P < 0.05. Results represent means ± SE. Bars: (C) 200 µm; (F) 20 µm.

Role for EPO-R in EPO-mediated gene transcription in islets. Quantitative real-time PCR was performed to measure expression levels of bcl-xl, c-kit, c-myc, and vegf in isolated islets after 1 wk of rHuEPO or saline treatment (n = 3 per genotype). The expression level of each unknown was normalized by the internal control 18S. Each sample was run in triplicate, and each experiment included three nontemplate control wells. Similar results were also found from the normalization by GAPDH and actin (not depicted). *, P < 0.05. Results represent means ± SE.