AMPK is involved in mediation of erythropoietin influence on metabolic activity and reactive oxygen species production in white adipocytes

Abstract

Erythropoietin, discovered for its indispensable role during erythropoiesis, has been used in therapy for selected red blood cell disorders in erythropoietin-deficient patients. The biological activities of erythropoietin have been found in animal models to extend to non-erythroid tissues due to the expression of erythropoietin receptor. We previously demonstrated that erythropoietin promotes metabolic activity and white adipocytes browning to increase mitochondrial function and energy expenditure via peroxisome proliferator-activated receptor alpha and Sirtuin1. Here we report that AMP-activated protein kinase was activated by erythropoietin possibly via Ca(2+)/calmodulin-dependent protein kinase kinase in adipocytes as well as in white adipose tissue from diet induced obese mice. Erythropoietin increased cellular nicotinamide adenine dinucleotide via increased AMP-activated protein kinase activity, possibly leading to Sirtuin1 activation. AMP-activated protein kinase knock down reduced erythropoietin mediated increase in cellular oxidative function including the increased oxygen consumption rate, fatty acid utilization and induction of key metabolic genes. Under hypoxia, adipocytes were found to generate more reactive oxygen species, and erythropoietin reduced the reactive oxygen species and increased antioxidant gene expression, suggesting that erythropoietin may provide protection from oxidative stress in adipocytes. Erythropoietin also reversed increased nicotinamide adenine dinucleotide by hypoxia via increased AMP-activated protein kinase. Additionally, AMP-activated protein kinase is found to be involved in erythropoietin stimulated increase in oxygen consumption rate, fatty acid oxidation and mitochondrial gene expression. AMP-activated protein kinase knock down impaired erythropoietin stimulated increases in antioxidant gene expression. Collectively, our findings identify the AMP-activated protein kinase involvement in erythropoietin signaling in regulating adipocyte cellular redox status and metabolic activity.

Keywords: AMP-activated protein kinase; Adipocytes; Erythropoietin; Oxidative metabolism; Reactive oxygen species.

Figure 1. EPO increases AMPK activity in vitro and in vivo

(A–B) Western blotting to determine total AMPKα (t-AMPK) and phosphorylated AMPKα protein (p-AMPKα) levels in 3T3-L1 adipocytes treated without (PBS) and with EPO (5U/mL) at times indicated (A) and in S-WAT and V-WAT from DIO mice with EPO treatment for 5 weeks (B) (n=4). One-way ANOVA was used in B and other statistics were performed using Student’s t-test, and bar graphs are mean±s.e.m. In vitro data are means of three independent experiments. *P bold> 0.05; **P < 0.01

Figure 2. EPO regulated NAD⁺/NADH ratio and PGC-1α deacetylation was inhibited by AMPK inhibitor

(A) NAD⁺/NADH ratio was determined in 3T3-L1 adipocytes exposed to EPO at indicated dosage in the absence or presence of AMPK inhibitor Compound C at indicated dosage. (B) PGC-1α deacetylation was determined in 3T3-L1 adipocytes with PGC-1α overexpression exposed to EPO (5U/ml) in the absence or presence of AMPK inhibitor Compound C at indicated dosage. (C) Total ACC (t-ACC) and phosphorylated ACC protein levels were determined in 3T3-L1 adipocytes without or with EPO treatment at indicated dosage in the absence or presence of AMPK inhibitor Compound C at indicated dosage. One-way ANOVA was used in A, B and C. And the bar graphs are mean±s.e.m. The data are means of three independent experiments. *P < 0.05; **P < 0.01

Figure 3. EPO regulated AMPK activity is mediated by CaMKK

(A) Total AMPKα and p-AMPKα protein levels were determined in adipocytes without or with EPO treatment in the absence or presence of CaMKK inhibitor STO-609 or siRNA knock-down of LKB1 (LKB1-KD), Ctrl (no siRNA) and negative control siRNA (NC-siRNA) were used as control for LKB1 knock down. (B) The expression of mitochondrial genes was determined in adipocytes without or with EPO treatment in the absence or presence of CaMKK inhibitor STO-609. (C) OCR was determined in adipocytes without or with EPO treatment (5U/ml) in the absence or presence of CaMKK inhibitor STO-609. One-way ANOVA was used in A, B and C. And the bar graphs are mean±s.e.m. The data are means of three independent experiments. *P < 0.05.

Figure 4. EPO regulates ROS production and expression of Antioxidant gene

(A) Total AMPKα (t-AMPKα) and p-AMPKα protein levels were determined in the adipocyte under normoxia (21%O₂) and hypoxia (2%O₂) without or with EPO treatment. (B) NAD⁺/NADH ratio, NAD⁺ and NADH level were shown in 3T3-L1 adipocytes under normoxia and hypoxia without or with EPO treatment (5U/ml). (C) 3T3-L1 adipocytes were incubated with CellRox Green for 30 min after being incubated for 4 hours in normoxia or hypoxia without or with EPO treatment (5U/ml). The fluorescence was measured. Results are presented as the fold of stimulation compared with control. (D) Antioxidant gene expression level in 3T3-L1 adipocytes under normoxia and hypoxia without or with EPO treatment (5U/ml). (E) Western blotting shows SOD, Gpx and Catalase protein levels in in 3T3-L1 adipocytes under normoxia and hypoxia without or with EPO treatment (5U/ml). (F) SOD, Gpx and Catalase protein levels were determined in white fat tissue from the normal chow (NC) and high fat diet (HFD) induced obese mice with EPO treatment for 5 weeks. One-way ANOVA was used in A–D. And the bar graphs are mean±s.e.m. In vitro data are means of three independent experiments. *P < 0.05; **P < 0.01.

Figure 5. AMPK mediates EPO effects in adipocytes

(A) Total AMPKα protein level was determined in 3T3-L1 adipocyte with AMPKα knock down (KD). Ctrl (no siRNA) and negative control siRNA (NC-siRNA) were used as control for AMPKα knock down. (B–D) OCR (B) of basal and with presence of oligomycin (OM) and carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP), the fatty acid oxidation (C) of basal and with the presence of palmitate (PALM) and expression of mitochondrial genes (D) were determined in adipocytes with knock down of AMPKα without (open bar; control) and with EPO treatment (5U/ml; closed bar). (E) Antioxidant gene expression was determined in 3T3-L1 adipocytes with knock down of AMPKα without (open bar; control) and with EPO treatment (5U/ml; closed bar). One-way ANOVA was used in B–E. And the bar graphs are mean±s.e.m. The data are means of three independent experiments.*P < 0.05; **P < 0.01

Figure 6. The crosstalk between EPO and important energy sensors

EPO regulated AMPK activity is involved in adipocyte energy metabolism, mitochondrial function regulation and protection from oxidative stress. EPO regulated AMPK activity may, at least in part, regulate Sirt1 activity via modulating NAD⁺/NADH ratio, which contributes to energy metabolism and mitochondrial function in adipocytes. As the downstream target of Sirt1 and AMPK, PGC-1α may also be directly regulated by EPO or via regulating Sirt1 and AMPK activity to promote adipocyte oxidative metabolism, mitochondrial biogenesis and protection from oxidative stress.