Fig. 7.

Knockdown of CAMKK1 in MSC-ex increases hepatic lipid accumulation and injury in HFD-fed mice.

(A) C57BL/6 mice were placed on a HFD (40%) and administered 10 mg/kg of MSC-ex^shCtr or MSC-ex^shCAMKK1 i.v. from the 10th week to the 14th week of HFD feeding. As a control, the same volume of PBS was injected. (B) Immunoblotting analyses of CAMKK1 proteins in mice and quantification of the results. (C) Representative immunofluorescence confocal microscopy images of CD63 (green) and CAMKK1 (red) colocalisation; nuclei were labelled with DAPI (blue). Scale bars, 20 μm. (D) Representative images of gross liver appearance for mice fed a normal chow diet (normal group) or HFD for 10 weeks and then injected with PBS, MSC-ex^shCtr, or MSC-ex^shCAMKK1 for 4 weeks. (E) Representative images of H&E (top; scale bars, 50 μm) and Oil Red O (bottom; Scale bars, 20 μm) staining of liver sections. (F) Liver contents of total TG and TC, n = 10 per group. (G) Body weight changes of mice. (H) Changes in the liver indexes of mice (liver index = liver wet weight/body weight). (I) i.p. ITTs evaluated individual insulin tolerance by injecting insulin at a dose of 0.75 IU/kg body weight; blood glucose levels were detected at 0, 30, 60, 90, and 120 min and compared with that at 0 min. Individual glucose tolerance was assessed by IPGTT; fasted mice were administered 1 g of glucose/kg body weight by i.p. injection, and blood glucose levels were determined at 0, 30, 60, 90, and 120 min. (J) Fasting glycaemia (after 16-h fast) and serum insulin levels, n = 10 per group. (K and L) Serum TG and TC (K), and AST and ALT (L) levels, n = 10 per group. (M) Immunoblotting analyses of AMPK signalling proteins in livers and quantification. Data are represented as the mean ± SEM. Statistical analyses by a one-way ANOVA (F–M). ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 vs. HFD group; ^#p <0.05, ^##p <0.01, ^###p <0.001 vs. MSC-ex^shCtr (10 mg/kg) group. ALT, alanine transaminase; AMPK, AMP-activated protein kinase; AST, aspartate transaminase; CAMKK1, calcium/calmodulin-dependent protein kinase 1; CPT-1A, carnitine palmitoyltransferase 1A; FASn, fatty acid synthase; HFD, high-fat diet; IPGTT, i.p. glucose tolerance test; IRS1, insulin receptor substrate 1; ITT, insulin tolerance test; MSC-ex, MSC-derived exosomes; MSC, mesenchymal stem cell; p-AMPK, phosphorylated AMPK; p-IRS1, phosphorylated IRS1; PPARα, peroxisome proliferator-activated receptor alpha; SREBP-1C, sterol regulatory element-binding protein-1C; TC, total cholesterol; TG, triglycerides.