Role of sphingolipid mediator ceramide in obesity and renal injury in mice fed a high-fat diet

Abstract

The present study tested a hypothesis that excess accumulation of sphingolipid, ceramide, its metabolites, or a combination contributes to the development of obesity and associated kidney damage. Liquid chromatography/mass spectrometry analysis demonstrated that C57BL/6J mice on the high-fat diet (HFD) had significantly increased plasma total ceramide levels compared with animals fed a low-fat diet (LFD). Treatment of mice with the acid sphingomyelinase (ASMase) inhibitor amitriptyline significantly attenuated the HFD-induced plasma ceramide levels. Corresponding to increase in plasma ceramide, the HFD significantly increased the body weight gain, plasma leptin concentration, urinary total protein and albumin excretion, glomerular damage index, and adipose tissue ASMase activity compared with the LFD-fed mice. These HFD-induced changes were also significantly attenuated by treatment of mice with amitriptyline. In addition, the decline of plasma glucose concentration after an intraperitoneal injection of insulin (0.15 U/kg b.wt.) was more sustained in mice on the HFD with amitriptyline than on the HFD alone. Intraperitoneal injection of glucose (3 g/kg b.wt.) resulted in a slow increase followed by a rapid decrease in the plasma glucose concentration in LFD and HFD plus amitriptyline-treated mice, but such blood glucose response was not observed in HFD-fed mice. Immunofluorescence analysis demonstrated a decrease in the podocin and an increase in the desmin in the glomeruli of HFD-fed mice compared with the LFD and HFD plus amitriptyline-treated mice. In conclusion, our results reveal a pivotal role for ceramide biosynthesis in obesity, metabolic syndrome, and associated kidney damage.

Fig. 1.

Effects of the low-fat and high-fat diet on body weight with or without amitriptyline treatment. A, values are means ± S.E. (n = 10–12/group) of body weight in LFD- and HFD-fed C57BL/6J mice with or without Ami treatment. *, P < 0.05, significant difference compared with the values from mice receiving the LFD; #, P < 0.05, significant difference compared with the values from mice receiving the HFD.

Fig. 2.

Plasma total ceramide concentrations, delta body weight, adipocyte size, and arterial blood pressure in C57BL/6J mice on low-fat or high-fat diet with or without amitriptyline treatment. Data are arithmetic means ± S.E. (n = 4–12/group) of plasma total ceramide concentrations (A), delta body weight (B), adipocyte size (original magnification, 100×) (C), and mean arterial blood pressure (D) in LFD- or HFD-fed C57BL/6J mice with or without amitriptyline treatment. *, P < 0.05, significant difference compared with the values from mice receiving the LFD. #, P < 0.05, significant difference compared with the values from mice receiving the HFD. Scale bar, 50 μm.

Fig. 3.

Plasma glucose concentrations after intraperitoneal glucose or insulin injection in C57BL/6J mice on low-fat or high-fat diet with or without amitriptyline treatment. Values are arithmetic means ± S.E. (n = 6/group) of plasma glucose concentrations after intraperitoneal injection of glucose (3 g/kg b.wt.; A) or insulin (0.15 U/kg b.wt.; B) in LFD- or HFD-fed C57BL/6J mice with or without amitriptyline treatment. *, P < 0.05, significant difference compared with the values from mice receiving the LFD; #, P < 0.05, significant difference compared with the values from mice receiving the HFD.

Fig. 4.

Morphological features of the glomeruli in low-fat or high-fat diet treatment in C57BL/6J mice on low-fat or high-fat diet with or without amitriptyline treatment. A, photomicrographs show typical glomerular structure (original magnification, 400×) in LFD or HFD treatment with or without amitriptyline treatment. B, summarized data of GDI by semiquantitation of scores in four different groups of mice (n = 6/group). For each kidney section, 50 glomeruli were randomly chosen for the calculation of GDI. C, urinary total protein excretion. D, urinary albumin excretion (n = 6–10/group). *, P < 0.05, significant difference compared with the values from mice receiving the LFD; #, P < 0.05, significant difference compared with the values from mice receiving the HFD. Scale bar, 50 μm.

Fig. 5.

Immunofluorescent staining of desmin and podocin from C57BL/6J mice on low-fat or high-fat diet with or without amitriptyline treatment. A, typical images of desmin staining in glomeruli from C57BL/6J mice on the LFD or HFD with or without amitriptyline treatment (n = 4/group). B, typical images of podocin staining in glomeruli from C57BL/6J mice on the LFD or HFD with or without amitriptyline treatment (n = 4/group). Scale bar, 20 μm.

Fig. 6.

Plasma leptin concentrations. Values are arithmetic means ± S.E.M. (n = 6/group) of plasma leptin concentrations in LFD- and HFD-fed C57BL/6J mice with or without amitriptyline treatment. *, P < 0.05, significant difference compared with the values from mice receiving the LFD. #, P < 0.05, significant difference compared with the values from mice receiving the HFD.

Fig. 7.

Effect of the low-fat diet and high-fat diet on adipose tissue ASMase mRNA expression and ASMase activity in C57BL/6J mice on low-fat or high-fat diet with or without amitriptyline treatment. Values are arithmetic means ± S.E.M. (n = 6/group) of ASMase mRNA expression (A) and ASMase activity (B) in LFD- and HFD-fed C57BL/6J mice with or without amitriptyline treatment. *, P < 0.05, significant difference compared with the values from mice receiving the LFD. #, P < 0.05, significant difference compared with the values from mice receiving the HFD.