A non-erythropoietic peptide derivative of erythropoietin decreases susceptibility to diet-induced insulin resistance in mice

Abstract

Background and purpose: The haematopoietic activity of erythropoietin (EPO) is mediated by the classic EPO receptor (EpoR) homodimer, whereas tissue-protective effects are mediated by a heterocomplex between EpoR and the β-common receptor (βcR). Here, we investigated the effects of a novel, selective ligand of this heterocomplex - pyroglutamate helix B surface peptide (pHBSP) - in mice fed a diet enriched in sugars and saturated fats.

Experimental approach: Male C57BL/6J mice were fed a high-fat high-sucrose diet (HFHS) for 22 weeks. pHBSP (30 μg·kg(-1) s.c.) was administered for the last 11 weeks. Biochemical assays, histopathological and immunohistochemical examinations and Western blotting were performed on serum and target organs (liver, kidney and skeletal muscle).

Key results: Mice fed with HFHS diet exhibited insulin resistance, hyperlipidaemia, hepatic lipid accumulation and kidney dysfunction. In gastrocnemius muscle, HFHS impaired the insulin signalling pathway and reduced membrane translocation of glucose transporter type 4 and glycogen content. Treatment with pHBSP ameliorated renal function, reduced hepatic lipid deposition, and normalized serum glucose and lipid profiles. These effects were associated with an improvement in insulin sensitivity and glucose uptake in skeletal muscle. Diet-induced overproduction of the myokines IL-6 and fibroblast growth factor-21 were attenuated by pHBSP and, most importantly, pHBSP markedly enhanced mitochondrial biogenesis in skeletal muscle.

Conclusions and implications: Chronic treatment of mice with an EPO derivative, devoid of haematopoietic effects, improved metabolic abnormalities induced by a high-fat high-sucrose diet, by affecting several levels of the insulin signalling and inflammatory cascades within skeletal muscle, while enhancing mitochondrial biogenesis.

Figure 1

HFHS diet and chronic administration of pHBSP (30 μg·kg⁻¹, s.c.) on serum insulin (A), blood glucose (B) and oral glucose tolerance (C). Values are mean ± SEM of six animals per group. ⋆P < 0.05 versus control.

Figure 2

Effects of HFHS diet and pHBSP administration on kidney structure and function. Representative sections of kidneys (A) from mice maintained on the control diet or the HFHS diet and treated with pHBSP (30 μg·kg⁻¹, s.c.). Kidneys from mice fed with a standard laboratory chow showed a normal appearance in the presence or absence of drug treatment. The HFHS diet produced an intense vacuolar degeneration of the S1 and S2 portions of the proximal convoluted tubules. These alterations were significantly attenuated by pHBSP administration. (Arrows point to S1 and S2 portions of the proximal convoluted tubules; asterisks indicate tubular cells with vacuolar degeneration.) Urinary ACR (B) and BUN (C) levels were measured in mice exposed to the control diet or the HFHS diet in the absence or presence of pHBSP (30 μg·kg⁻¹, s.c.). Values are mean ± SEM of six animals per group. ⋆P < 0.05 versus control; •P < 0.05 versus HFHS.

Figure 3

Effects of HFHS diet and pHBSP administration on liver lipid accumulation. (A) Representative photomicrographs (×40 magnification) of Oil Red O staining on liver sections from mice maintained on the control diet or the HFHS diet and treated with pHBSP (30 μg·kg⁻¹, s.c.). Arrows point hepatocytes with lipid droplets. (B) TG content in mouse liver. Data are mean ± SEM of six animals per group. ⋆P < 0.05 versus control; •P < 0.05 versus HFHS.

Figure 4

Alterations in insulin signalling pathway in the skeletal muscle of mice maintained on HFHS diet in the absence or presence of pHBSP. Total protein expression of IRS-1 (A), Akt (B) and GSK-3β (C) as well as their related phosphorylated forms were analysed by Western blot on the gastrocnemius homogenates obtained from mice exposed to the control diet or the HFHS diet and treated with pHBSP (30 μg·kg⁻¹, s.c.). Protein expression is measured as relative optical density (O.D.), corrected for the corresponding tubulin contents and normalized to the control band. The data are means ± SEM of three separate experiments. ⋆P < 0.05 versus control; •P < 0.05 versus HFHS.

Figure 5

Effects of dietary manipulation and pHBSP treatment on GLUT-4 localization (A, original magnification: 400×) and expression (B), AS160 total protein expression and Thr⁶⁴² phosphorylation (C), and glycogen content (D) in the skeletal muscle from mice exposed to the control diet or the HFHS diet and treated with pHBSP (30 μg·kg⁻¹, s.c.). Protein expression is measured as relative optical density (O.D.), corrected for the corresponding tubulin and normalized using the related control band. Data are means ± SEM of three separate experiments for Western blot analysis and six to eight animals per group for skeletal muscle glycogen content. ⋆P < 0.05 versus control; •P < 0.05 versus HFHS.

Figure 6

Effects of pHBSP on local and systemic levels of IL-6 and FGF-21 in mice fed with HFHS diet. IL-6 and FGF-21 concentrations were analysed by elisa in gastrocnemius homogenates (A and B, respectively) and serum (C and D, respectively) of mice maintained on the control or the HFHS diet and treated with pHBSP (30 μg·kg⁻¹, s.c.). Data are means ± SEM of six to eight animals per group. ⋆P < 0.05 versus control; •P < 0.05 versus HFHS.

Figure 7

Effects of pHBSP treatment on markers of mitochondrial production/biogenesis in the gastrocnemius of mice fed with a HFHS diet. COI (A), PGC1-α (B), mtTFA (C) and NRF-1 (D) protein expression was analysed by Western blot on the gastrocnemius homogenates obtained from mice maintained on the control or the HFHS diet and treated with pHBSP (30 μg·kg⁻¹, s.c.). Protein expression is measured as relative optical density (O.D.), corrected for the corresponding tubulin contents and normalized using the related control band. The data are means ± SEM of three separate experiments. ⋆P < 0.05 versus control; •P < 0.05 versus HFHS.

Figure 8

Heterodimerization between EpoR and βcR in the mouse skeletal muscle. Co-immunoprecipitation analysis illustrating the heteromeric complex consisting of EpoR and βcR in gastrocnemius homogenates obtained from mice maintained on the control or the HFHS diet and treated with pHBSP (30 μg·kg⁻¹, s.c.). Lysate from skeletal muscles was subjected to immunoprecipitation (IP) with anti-EpoR antibody and then exposed to immunoblot (IB) analysis with either anti-EpoR (left panel) or anti-βcR antibodies (right panel). The results, representative of two independent experiments, show interaction between the two receptors both under control conditions and after dietary manipulation and/or pHBSP treatment.