Vitamin K-dependent carboxylation regulates Ca2+ flux and adaptation to metabolic stress in ß cells.

Vitamin K is a micronutrient necessary for γ-carboxylation of glutamic acids. This post-translational modification occurs in the endoplasmic reticulum (ER) and affects secreted proteins. Recent clinical studies implicate vitamin K in the pathophysiology of diabetes, but the underlying molecular mechanism remains unknown. Here, we show that mouse ß cells lacking γ-carboxylation fail to adapt their insulin secretion in the context of age-related insulin resistance or diet-induced ß cell stress. In human islets, γ-carboxylase expression positively correlates with improved insulin secretion in response to glucose. We identify endoplasmic reticulum Gla protein (ERGP) as a γ-carboxylated ER-resident Ca2+-binding protein expressed in ß cells. Mechanistically, γ-carboxylation of ERGP protects cells against Ca2+ overfilling by diminishing STIM1 and Orai1 interaction and restraining store-operated Ca2+ entry. These results reveal a critical role of vitamin K-dependent carboxylation in regulation of Ca2+ flux in ß cells and in their capacity to adapt to metabolic stress.

Single-Cell RNA Sequencing Reveals a Role for Reactive Oxygen Species and Peroxiredoxins in Fatty Acid-Induced Rat ß-Cell Proliferation.

The functional mass of insulin-secreting pancreatic ß-cells expands to maintain glucose homeostasis in the face of nutrient excess, in part via replication of existing ß-cells. Type 2 diabetes appears when these compensatory mechanisms fail. Nutrients including glucose and fatty acids are important contributors to the ß-cell compensatory response, but their underlying mechanisms of action remain poorly understood. We investigated the transcriptional mechanisms of ß-cell proliferation in response to fatty acids. Isolated rat islets were exposed to 16.7 mmol/L glucose with or without 0.5 mmol/L oleate (C18:1) or palmitate (C16:0) for 48 h. The islet transcriptome was assessed by single-cell RNA sequencing. ß-Cell proliferation was measured by flow cytometry. Unsupervised clustering of pooled ß-cells identified different subclusters, including proliferating ß-cells. ß-Cell proliferation increased in response to oleate but not palmitate. Both fatty acids enhanced the expression of genes involved in energy metabolism and mitochondrial activity. Comparison of proliferating versus nonproliferating ß-cells and pseudotime ordering suggested the involvement of reactive oxygen species (ROS) and peroxiredoxin signaling. Accordingly, N-acetyl cysteine and the peroxiredoxin inhibitor conoidin A both blocked oleate-induced ß-cell proliferation. Our study reveals a key role for ROS signaling through peroxiredoxin activation in oleate-induced ß-cell proliferation.

Very-Long-Chain Unsaturated Sphingolipids Mediate Oleate-Induced Rat ß-Cell Proliferation.

Fatty acid (FA) signaling contributes to ß-cell mass expansion in response to nutrient excess, but the underlying mechanisms are poorly understood. In the presence of elevated glucose, FA metabolism is shifted toward synthesis of complex lipids, including sphingolipids. Here, we tested the hypothesis that sphingolipids are involved in the ß-cell proliferative response to FA. Isolated rat islets were exposed to FA and 16.7 mmol/L glucose for 48-72 h, and the contribution of the de novo sphingolipid synthesis pathway was tested using the serine palmitoyltransferase inhibitor myriocin, the sphingosine kinase (SphK) inhibitor SKI II, or knockdown of SphK, fatty acid elongase 1 (ELOVL1) and acyl-CoA-binding protein (ACBP). Rats were infused with glucose and the lipid emulsion ClinOleic and received SKI II by gavage. ß-Cell proliferation was assessed by immunochemistry or flow cytometry. Sphingolipids were analyzed by liquid chromatography-tandem mass spectrometry. Among the FAs tested, only oleate increased ß-cell proliferation. Myriocin, SKI II, and SphK knockdown all decreased oleate-induced ß-cell proliferation. Oleate exposure did not increase the total amount of sphingolipids but led to a specific rise in 24:1 species. Knockdown of ACBP or ELOVL1 inhibited oleate-induced ß-cell proliferation. We conclude that unsaturated very-long-chain sphingolipids produced from the available C24:1 acyl-CoA pool mediate oleate-induced ß-cell proliferation in rats.