Inhibition of central de novo ceramide synthesis restores insulin signaling in hypothalamus and enhances β-cell function of obese Zucker rats

Objectives: Hypothalamic lipotoxicity has been shown to induce central insulin resistance and dysregulation of glucose homeostasis; nevertheless, elucidation of the regulatory mechanisms remains incomplete. Here, we aimed to determine the role of de novo ceramide synthesis in hypothalamus on the onset of central insulin resistance and the dysregulation of glucose homeostasis induced by obesity. Methods: Hypothalamic GT1-7 neuronal cells were treated with palmitate. De novo ceramide synthesis was inhibited either by pharmacological (myriocin) or molecular (si-Serine Palmitoyl Transferase 2, siSPT2) approaches. Obese Zucker rats (OZR) were intracerebroventricularly infused with myriocin to inhibit de novo ceramide synthesis. Insulin resistance was determined by quantification of Akt phosphorylation. Ceramide levels were quantified either by a radioactive kinase assay or by mass spectrometry analysis. Glucose homeostasis were evaluated in myriocin-treated OZR. Basal and glucose-stimulated parasympathetic tonus was recorded in OZR. Insulin secretion from islets and β-cell mass was also determined. Results: We show that palmitate impaired insulin signaling and increased ceramide levels in hypothalamic neuronal GT1-7 cells. In addition, the use of deuterated palmitic acid demonstrated that palmitate activated several enzymes of the de novo ceramide synthesis pathway in hypothalamic cells. Importantly, myriocin and siSPT2 treatment restored insulin signaling in palmitate-treated GT1-7 cells. Protein kinase C (PKC) inhibitor or a dominant-negative PKCζ also counteracted palmitate-induced insulin resistance. Interestingly, attenuating the increase in levels of hypothalamic ceramides with intracerebroventricular infusion of myriocin in OZR improved their hypothalamic insulin-sensitivity. Importantly, central myriocin treatment partially restored glucose tolerance in OZR. This latter effect is related to the restoration of glucose-stimulated insulin secretion and an increase in β-cell mass of OZR. Electrophysiological recordings also showed an improvement of glucose-stimulated parasympathetic nerve activity in OZR centrally treated with myriocin. Conclusion: Our results highlight a key role of hypothalamic de novo ceramide synthesis in central insulin resistance installation and glucose homeostasis dysregulation associated with obesity. Keywords: Ceramide, Lipotoxicity, Hypotalamus, Insulin resistance, Insulin secretio