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The present study tested a hypothesis that excess accumulation of sphingolipid, ceramide and/or its metabolites contribute to the development of obesity and associated kidney damage. LC/MS spectroscopy analysis demonstrated that C57BL/6J mice on the high fat diet (HFD) had significantly increased plasma total ceramide levels compared to the low fat diet (LFD)-fed animals. Treatment of mice with an 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 to 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 following an intraperitoneal injection of insulin (0.15 U/kg B.W.) was more sustained in mice on the HFD with amitriptyline than on the HFD alone. Intraperitoneal injection of glucose (3 g/kg B.W.) 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 to 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. This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on June 11, 2010 as DOI: 10.1124/jpet.110.168815 at A PE T Jornals on M arch 0, 2017 jpet.asjournals.org D ow nladed from