Effects of Mineralocorticoid Receptor Blockade on Angiogenesis and Glucose Uptake in a High‐Fat Fed Mouse Model with Hindlimb Ischemia

Abstract

Increased plasma aldosterone levels and mineralocorticoid receptor (MR) activation contribute to vascular damage in atherosclerosis. Peripheral arterial disease (PAD), a likely manifestation of a chronic atherosclerosis process, is characterized by reduced arterial perfusion of the lower extremities and decreased skeletal muscle insulin sensitivity. Given these observations, the objective of the current study was to elucidate the effect of MR inhibition on the development of PAD in a high‐fat fed mouse hindlimb ischemia model.MethodsIn mice fed the high fat diet we evaluated post‐ligation recovery of blood perfusion and angiogenesis as well as glucose uptake and further analyzed the expression and phosphorylation levels of selected proteins in skeletal muscle cells.ResultsDoppler imaging and immunofluorescence staining indicated that MR blockade in high fat fed mice impaired the post‐ligation recovery of blood perfusion and reduced ischemia‐induced angiogenesis in the ischemic hindlimb (Fig. 1). Data from PET/CT scanning further suggested that MR blockade increased glucose uptake in ischemic hindlimb muscles (Fig. 2). Quantitative real‐time PCR showed that MR blockade decreased mRNA expression of inflammatory markers including MCP‐1, IL‐6 and TNFa while increasing GLUT‐4, IRa and IRS‐1 mRNA in the ischemic hindlimb muscle. In vitro experiments, in cultured skeletal muscle cells, showed that MR inhibition attenuated an aldosterone‐induced decrease of GLUT‐4, Akt phosphorylation and total IRS‐1 protein.ConclusionsThe results demonstrate that MR blockade impairs post‐ligation recovery of blood perfusion and angiogenesis in high‐fat fed mice subject to hindlimb ischemia, while increasing glucose uptake into the ischemic muscle via upregulation of GLUT‐4, in part, through IRS‐1 and Akt signaling pathways. These results support the view that in this model of ischemia combined with metabolic dysfunction, MR activation appears to exert differential effects on angiogenesis and glucose handling.Support or Funding InformationThis work was supported by research grants from the National Natural Science Foundation of China (81172050 and 81570263 to J. Wu).In mice fed a high‐fat diet and subjected to lower limb ischemia, mineralocorticoid receptor inhibition with spironolactone attenuates subsequent recovery of blood perfusion.(A) Representative laser Doppler images acquired from the lower extremities of the mice scanned immediately before and after artery ligation (Pre/Post) and on the 3th, 7th, 14th days after surgery. (B) Group blood flow recovery (ischemic/non‐ischemic lower limbs) data for n = 4–6 mice/group, *P<0.05.Figure 1[18F]FDG distribution in tissues of high‐fat fed mice as measured by small animal PET/CT imaging.(A) Representative whole body PET/CT images of mice. Each row displays transverse, coronal and sagittal sections. (B) Representative images of skeletal muscles from each experimental animal group. Rows displays the respective CT and PET images as well as the PET image co‐registered with CT image, in coronal or transverse sections. White arrows point to ischemicc hindlimb.Figure 2