Doxorubicin caused severe hyperglycaemia and insulin resistance, mediated by inhibition in AMPk signalling in skeletal muscle

Abstract

Background: Cancer is considered the second leading cause of death in the world, and for the treatment of this disease, pharmacological intervention strategies are frequently based on chemotherapy. Doxorubicin (DOX) is one of the most widely used chemotherapeutic agents in clinical practice for treating a number of solid tumours. The treatment with DOX mimics some effects of cancer cachexia, such as anorexia, asthenia, decreases in fat and skeletal muscle mass and fatigue. We observed that treatment with DOX increased the systemic insulin resistance and caused a massive increase in glucose levels in serum. Skeletal muscle is a major tissue responsible for glucose uptake, and the positive role of AMPk protein (AMP-activated protein kinase) in GLUT-4 (Glucose Transporter type 4) translocation, is well established. With this, our aim was to assess the insulin sensitivity after treatment with DOX and involvement of AMPk signalling in skeletal muscle in this process.

Methods: We used Wistar rats which received a single dose of doxorubicin (DOX group) or saline (CT group) intraperitoneally at a dose of 15 mg/kg b.w. The expression of proteins involved in insulin sensitivity, glucose uptake, inflammation, and activity of electron transport chain was assessed in extensor digitorum longus muscle, as well as the histological evaluation. In vitro assays were performed in L6 myocytes to assess glucose uptake after treatment with DOX. Agonist of AMPk [5-aminoimidazole-4-carboxamide (AICAR)] and the antioxidant n-acetyl cysteine were used in L6 cells to evaluate its effect on glucose uptake and cell viability.

Results: The animals showed a significant insulin resistance, hyperglycaemia, and hyperinsulinemia. A decrease in the expression of AMKP and GLUT-4 was observed in the extensor digitorum longus muscle. Also in L6 cells, DOX leads to a decrease in glucose uptake, which is reversed with AICAR.

Conclusions: DOX leads to conditions similar to cachexia, with severe glucose intolerance both in vivo and in vitro. The decrease of AMPk activity of the protein is modulated negatively with DOX, and treatment with agonist of AMPk (AICAR) has proved to be a possible therapeutic target, which is able to recover glucose sensitivity in skeletal muscle.

Keywords: AMPk; Anthracycline; Chemotherapy; Doxorubicin; Glucose intolerance; Hyperglycaemia; Skeletal muscle.

Figure 1

Effect of doxorubicin on muscular atrophy and the electron transport chain activity. (A) Ratio muscle extensor digitorum longus/tibia. (B) Cross‐sectional area in extensor digitorum longus muscle. (C) Histological slices of the extensor digitorum longus muscle stained with H & E. (D) Ratio corticosterone/testosterone. (E) Corticosterone levels. (F) Testosterone levels. The groups were compared by Test T. P < 0.05 was considered statistically significant. * P < 0.5, ** P < 0.01, *** P < 0.001. n = 4–10.

Figure 2

Doxorubicin leads to impaired systemic insulin sensitivity. (A) Homeostatic model assessment of insulin resistance. (B) Curve of the insulin tolerance test C. Kitt. (D)Gene expression involved in glucose metabolism. (E/F) protein expression involved in glucose metabolism in extensor digitorum longus muscle. (G/H) AMPk and GLUT‐4 protein expression in culture L6 myocytes treated with doxorubicin. The groups were compared by Test T. P < 0.05 was considered statistically significant. * P < 0.5, ** P < 0.01, *** P < 0.001. n = 3–8.

Figure 3

Doxorubicin decrease glucose uptake in L6 cells, which is recovered with chronic treatment with AICAR. (A) The L6 cells were differentiated and submitted to the glucose uptake assay, with (plus sign) and without (minus sign) insulin stimulation (100 nM), after 48 h the treatment with doxorubicin (100 nM). (B–D). Effect of acute and chronic treatment with AICAR on the phosphorylation of AMPK in myocytes. (E) 2‐Deoxy‐[C14]‐D‐glucose uptake in L6 cells. This cells were subjected to treatment with doxorubicin, associated with insulin, and submitted to acute (1 h) or chronic (48 h) AICAR (2 mmol) treatment; The results were relativized by control group without insulin stimulation. For the glucose uptake assay the groups were compared using ANOVA two‐way test with Bonferroni post‐test to compare the groups. P < 0.05 was considered statistically significant. * P < 0.5, ** P < 0.01, *** P < 0.001. n = 4–6.

Figure 4

Mitochondrial complex 1 and 3 activity. The groups were compared by Test T. P < 0.05 was considered statistically significant. * P < 0.5. n = 4–6.