Ceramides are bioactive sphingolipids, which are composed of sphingoid bases carrying acyl chains of various lengths. Ceramides are synthesized by a family of six ceramide synthases (CerS) in mammals, which produce ceramides with different N-linked acyl chains. Increased ceramide levels are known to contribute to the development of obesity and insulin resistance. Recently, it has been demonstrated that the ceramide acylation pattern is of particular importance for an organism to maintain energy homeostasis. However, which of the CerS family members are involved in this process is not yet completely known. Using newly developed CerS5 knock-out mice, we show here that CerS5 is essential to maintain cellular C 16:0 sphingolipid pools in lung, spleen, muscle, liver, and white adipose tissue. Glycerophospholipid levels in CerS5-deficient mice were not altered. We found a strong impact of CerS5-dependent ceramide synthesis in white adipose tissue after high fat diet feeding. In skeletal muscle, liver, and spleen, C 16:0 -ceramide levels were altered independent of feeding conditions. The loss of CerS5 is associated with reduced weight gain and improved systemic health, including maintenance of glucose homeostasis and reduced white adipose tissue inflammation after high fat diet challenge. Our findings indicate that reduction of endogenous C 16:0 -ceramide by genetic inhibition of CerS5 is sufficient to ameliorate obesity and its comorbidities.Ceramide synthases are sphingosine N-acyltransferases and represent an important metabolic hub in the ceramide synthesis pathway (Fig. 1A). They acylate sphingoid bases with fatty acid acyl chains of different length and saturation ( Fig. 1B) (1-6), thereby creating ceramides with diverse biological properties (7-9). The ceramide synthase enzyme family contains six members (CerS1-6) 6 in mammals (1, 9, 10). The individual enzymes differ in their substrate specificity and show different expression patterns (Fig. 1A) (1, 11). CerS1 is specifically expressed in muscle and neurons and has strong substrate specificity toward C 18:0 -CoA (1, 4, 11), whereas CerS2 and CerS4 are broadly expressed with specificity toward very long chain C 20: 0 -26:0 CoAs and C 18:0 -C 22:0 CoAs, respectively (1, 11). CerS3 is highly expressed in the epidermis and testis showing a substrate specificity for ultra-long chain CoAs (2, 10). CerS6 is expressed in most tissues at low levels and shows substrate specificity toward long chain C 14:0 -16:0 -CoAs (Fig. 1B) (1, 11, 12). CerS5 expression has also been studied at the mRNA level and is expressed in most tissues at low levels (11) and has a specificity toward the long chain CoAs C 14:0 -18:0 (1).Most murine CerS family members have also been characterized using knock-out (KO) mouse models. CerS1-deficient mice show behavioral abnormalities and Purkinje cell loss (4, 13), whereas CerS2 knock-out mice develop hepatocarcinomas and show myelination defects (3,5,14). CerS3 KO mice are lethal shortly after birth due to skin barrier disruption (2), and it was s...
