Serine palmitoyltransferase-mediated de novo sphingolipid biosynthesis is required for normal insulin production and glucose tolerance

Abstract

Aims/hypothesis: The importance for normal insulin secretion of ceramide synthesis is unclear. De novo ceramide synthesis requires serine palmitoyl transferase, SPT2, encoded by Sptl2.

Methods: We generated β-cell-selective Sptl2 null mice by crossing animals with floxed alleles to mice expressing Cre recombinase from the Ins1 locus. Metabolic phenotyping, transcriptomic, functional analyses and histology were performed using standard approaches.

Results: Islets from Sptlc2 ^ΔInsl mice displayed marked alterations in ceramide and sphingomyelin levels: ceramide content: p=0.016 and p=0.109; sphingomyelin content: p=0.016 and p=0.004 in Sptlc2 ^ΔInsl vs Sptlc2 ^CTL mice under regular and high fat diet, respectively, despite compensatory increases in the expression of enzymes in the salvage and sphingomyelinase pathways. Correspondingly, profound abnormalities were observed in glucose-regulated insulin secretion and glucose tolerance in vivo, both on a regular chow and high fat diet. These changes were associated with a drastic (~80%) lowering in β-cell numbers, and a more minor increase in delta cell numbers. They were also preserved in animals maintained on a ketogenic diet, consistent with a cell autonomous effect on the β-cell. Despite normal glucose-regulated intracellular calcium dynamics and insulin secretion, marked transcriptomic changes were observed in Sptlc2 ^ΔInsl mouse islets, with affected GO terms including lysosome organisation and regulation of autophagy. Consistent with roles for compromised SPT2 function in diseased β-cells, Sptl2 expression in Balbc and DBA2J mouse islets was lowered by a high fat-diet. Moreover, SPTLC2 mRNA tended to be lower, and SPTLC1 mRNA was significantly decreased, in islets from human subjects with type 2 diabetes versus normoglycemic individuals.

Conclusions: Preserved de novo ceramide synthesis is required to maintain normal β-cell mass and thus insulin secretion in mice. Therapeutic approaches which seek to target this process systemically using pharmacological SPT2 inhibitors should thus be treated with caution.

Keywords: Beta cell mass; ceramides; diabetes; sphingomyelin.

Figure 1:. Sptlc2 deficiency in β-cell modulate sphingolipids balance in pancreatic islet

Schematic representation of the recombination of the floxed alleles of Sptlc2 using an Ins1-driven Cre recombinase (A). Gene expression of Sptlc1, Sptlc2 and Exon1 of Sptlc2 measured by qPCR in isolated islets of Sptlc2^CTL and Sptlc2^ΔIns1 mice (B). Quantification of sphingolipids, ceramides and sphingomyelin in islets of regular chow (CD) and high fat (60% HFD) diet -fed mice (C-D). Analysis was performed in 14-week-old mice either on CD or HFD, starting at 8 weeks. Error bars represent SEM, n=4-8 mice per group, *p<0,05, **p<0,01, ***p<0,001 with Kruskal-Wallis or Mann and Whitney test.

Figure 2:. Impaired glucose induced insulin secretion in Sptlc2^ΔIns1 mice regardless of the diet.

Body composition (A, B). Morning fed glycemia of animals was followed from 5 to 8 weeks of age, under chow (C) or high fat diet initiated at 5 weeks of age (D). Oral glucose (2g/kg) tolerance test: glycaemia (E, F, H, I) and insulin secretion (G, J). Insulin (0.5 ui/kg) tolerance test for CD mice (K, L). The insulin dose was 0.75 iu/kg for HFD mice (M, N). Fasted metabolic analysis (free fatty acids, Leptin, MCP-1 (O-Q). Analysis was performed in 13 weeks old mice (except for the fed glycemia experiment) either on CD or HFD from 8 weeks of age. Error bars represent SEM, n=4-8 mice per group, *p<0,05, **p<0,01, ***p<0,001 with Kruskal-Wallis or Mann and Whitney test.

Figure 3:. Sptlc2 deletion in the β-cell induces a drastic reduction in the number of pancreatic islets

Pancreatic histology analysis for total area (A). Pancreatic histology analysis for insulin staining: evaluating total islets area (B), islets number (C-D) and β-cell area (E-G). Pancreatic histology analysis for glucagon (H-J) and somatostatin staining (K-M).

Figure 4:. Preserved insulin secretory functionality despite β-cell-selective Sptlc2 deficiency.

Glucose-stimulated insulin secretion (GSIS) of isolated islets from 14-week-old mice under CD or HF was measured under low glucose (2.8mM) or high glucose (16.7mM) (A-C). Total insulin content (B-D). Measurement of intracellular free Ca2+ (E-F), ATP/ADP (G-H) and connectivity (I-K) assessed by imaging analysis under low glucose (3mM), high glucose (17mM) and KCl stimulation. Error bars represent SEM, n=3-5 mice per group, *p<0,05, **p<0,01, ***p<0,001 with Kruskal-Wallis or Mann and Whitney test.

Figure 5:. Alteration of membrane and autophagy function implied by transcriptomic analysis of islets from Sptlc2^ΔIns1 mice

RNA seq analysis was performed using islets isolated from Sptlc2^ΔIns1 or Sptlc2^CTL mice under chow (CD) and High fat (HFD) diet. Volcano plot of gene expression in CD (A) and HFD (B). Gene Ontology (GO) analysis of the most enriched terms in Sptlc2^ΔIns1 compared to Sptlc2^CTL mice under CD (C) and HFD (D). Heatmap representing gene expression of enriched GO Terms: GO:0010506 Regulation of autophagy (E) n=3-4 mice per group, mice were aged of 14 weeks

Figure 6:. Reduced Sptlc1 and Sptlc2 expression induced by high fat diet in islets of BABLC and DBA2J mice.

Sptlc2 and Sptlc1 expression in RNA seq analysis of isolated islets of 1292S (A, G), AJ (B, H), AKR (C, I), BALBC (D, J), C57Bl6 (E, K) and DBA2J (F, L) mice under chow diet (CD) and High fat diet (HFD) at different time point. n=6 male mice per group, mice aged of eight weeks. Sptlc2 (M) and Sptlc1 (N) expression in RNA seq analysis of isolated islets from pancreatectomized human non diabetic (ND) living with type 2 diabetes (T2D), diabetes secondary to pancreatic disease (T3cD) or glucose intolerant patient (IGT).