The role of ceramide synthases in pancreatic beta cell demise

Background:

Increased ceramide biosynthesis has been linked to pancreatic beta cell demise during the development of diabetes. Ceramides are generated by six different enzymes (CerS1 – 6), which attach fatty acids with a specific chain length to a sphingoid backbone and therefore have different biochemical properties. We aim to define the CerS responsible for diabetogenic beta cell dysfunction and analyse the molecular mechanism.

Methods:

CerS2, CerS5 and CerS6, which are the most prominent CerS in human and mouse islets, were knocked down and knocked out in the rat insulinoma cell line Ins1E using RNA interference and CrispR/Cas9 technology. We performed semi-quantitative real time PCR (qPCR), insulin secretion assays, as well as westernblotting (detection of cleaved caspase 3) after knockdown and treatment with diabetogenic stressors (Palmitate and Tunicamycin).

Results:

Knockdown of CerS2 decreases the expression of Pdx1 as well as Ins2, markers for beta cell identity, and increases cell death after ER stress induction. In contrast, ER stress dependent beta cell death is decreased after CerS6 knockdown, but increased after CerS5 knockdown. Lipid analyses of CrispR/Cas9 KO cells indicate that CerS5 is dominant over CerS6 in terms of C16 ceramide generation.

Conclusion:

We demonstrate that CerS are involved in maintenance of beta cell identity marker expression and CerS2, CerS5 and CerS6 differentially regulate beta cell survival in a stress dependent manner. Our results also indicate that CerS5 and CerS6 inversely affect the cellular response to diabetogenic ER stress, although both are thought to generate C16 ceramides.