Beta-Cell Injury in Ncb5or-null Mice is Exacerbated by Consumption of a High-Fat Diet.

NADH-cytochrome b5 oxidoreductase (Ncb5or) in endoplasmic reticulum (ER) is involved in fatty acid metabolism, and Ncb5or(-/-) mice fed standard chow (SC) are insulin-sensitive but weigh less than wild type (WT) littermates. Ncb5or(-/-) mice develop hyperglycemia at about age 7 weeks due to ß-cell dysfunction and loss associated with saturated fatty acid accumulation and manifestations of ER and oxidative stress. Here we report that when Ncb5or(-/-) mice born to heterozygous mothers fed a high fat (HF) diet continue to ingest HF, they weigh as much as SC-fed WT at age 5 weeks. By age 7 weeks, diabetes mellitus develops in all HF-fed vs. 68% of SC-fed Ncb5or(-/-) mice. Islet ß-cell content in age 5-week Ncb5or(-/-) mice fed HF for 7 days is lower (53%) than for those fed SC (63%), and both are lower than for WT (75%, SC, vs. 69%, HF). Islet transcript levels for markers of mitochondrial biogenesis (PGC-1α) and ER stress (ATF6α) are higher in Ncb5or(-/-) than WT mice but not significantly affected by diet. Consuming a HF diet exacerbates Ncb5or(-/-) ß-cell accumulation of intracellular saturated fatty acids and increases the frequency of ER distention from 11% (SC) to 47% (HF), thus accelerates ß-cell injury in Ncb5or(-/-) mice.

Development of diabetes in lean Ncb5or-null mice is associated with manifestations of endoplasmic reticulum and oxidative stress in beta cells.

NADH-cytochrome b5 oxidoreductase (Ncb5or) is an endoplasmic reticulum (ER)-associated redox enzyme involved in fatty acid metabolism, and phenotypic abnormalities of Ncb5or(-/-) mice include diabetes and lipoatrophy. These mice are lean and insulin-sensitive but become hyperglycemic at age 7 weeks as a result of ß-cell dysfunction and loss. Here we examine early cellular and molecular events associated with manifestations of ß-cell defects in Ncb5or(-/-) mice. We observe lower islet ß-cell content in pancreata at age 4 weeks and prominent ER distention in ß-cells by age 5 weeks. Ultrastructural changes progress rapidly in severity from age 5 to 6 weeks, and their frequency rises from 10% of ß-cells at 5 weeks to 33% at 6 weeks. These changes correlate temporally with the onset of diabetes. ER stress responses and lipid load in Ncb5or(-/-) ß-cells were assessed with isolated islets from mice at age 5 weeks. Expression levels of the stress marker protein Grp78/BiP and of phosphorylated eIF2α protein were found to be reduced, although their transcript levels did not decline. This pattern stands in contrast to the canonical unfolded protein response. Ncb5or(-/-) ß-cells also accumulated higher intracellular levels of palmitate and other free fatty acids and exhibited greater reactive oxygen species production than wild-type cells. An alloxan-susceptible genetic background was found to confer accelerated onset of diabetes in Ncb5or(-/-) mice. These findings provide the first direct evidence that manifestations of diabetes in lean Ncb5or(-/-) mice involve saturated free fatty acid overload of ß-cells and ER and oxidative stress responses.

Ncb5or deficiency increases fatty acid catabolism and oxidative stress.

The endoplasmic reticulum-associated NADH cytochrome b(5) oxidoreductase (Ncb5or) is widely distributed in animal tissues. Ncb5or(-/-) mice develop diabetes at age 7 weeks and have increased susceptibility to the diabetogenic oxidant streptozotocin. Ncb5or deficiency also results in lipoatrophy and increased hepatocyte sensitivity to cytotoxic effects of saturated fatty acids. Here we investigate the mechanisms of these phenomena in prediabetic Ncb5or(-/-) mice and find that, despite increased rates of fatty acid uptake and synthesis and higher stearoyl-CoA desaturase (SCD) expression, Ncb5or(-/-) liver accumulates less triacylglycerol (TAG) than wild type (WT). Increased fatty acid catabolism and oxidative stress are evident in Ncb5or(-/-) hepatocytes and reflect increased mitochondrial content, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) expression, fatty acid oxidation rates, oxidative stress response gene expression, and oxidized glutathione content. Ncb5or(-/-) hepatocytes readily incorporate exogenous fatty acids into TAG but accumulate more free fatty acids (FFA) and have greater palmitate-induced oxidative stress responses and cell death than WT, all of which are alleviated by co-incubation with oleate via TAG channeling. A high fat diet rich in palmitate and oleate stimulates both lipogenesis and fatty acid catabolism in Ncb5or(-/-) liver, resulting in TAG levels similar to WT but increased intracellular FFA accumulation. Hepatic SCD-specific activity is lower in Ncb5or(-/-) than in WT mice, although Ncb5or(-/-) liver has a greater increase in Scd1 mRNA and protein levels. Together, these findings suggest that increased FFA accumulation and catabolism and oxidative stress are major consequences of Ncb5or deficiency in liver.