Loss of Ncb5or Results in Impaired Fatty Acid Desaturation, Lipoatrophy, and Diabetes

Abstract

Targeted ablation of the novel flavoheme reductase Ncb5or knock-out (KO) results in progressive loss of pancreatic ␤-cells and white adipose tissue over time. Lipoatrophy persisted in KO animals in which the confounding metabolic effects of diabetes were eliminated by islet transplantation (transplanted knockout (TKO)). Lipid profiles in livers prepared from TKO animals were markedly deficient in triglycerides and diacylglycerides. Despite enhanced expression of stearoyl-Co-A desaturase-1, levels of palmitoleic and oleic acids (⌬9 fatty acid desaturation) were decreased in TKO relative to wild type controls. Treatment of KO hepatocytes with palmitic acid reduced cell viability and increased apoptosis, a response blunted by coincubation with oleic acid. The results presented here support the hypothesis that Ncb5or supplies electrons for fatty acid desaturation, offer new insight into the regulation of a crucial step in fatty acid biosynthesis, and provide a plausible explanation for both the diabetic and the lipoatrophic phenotype in Ncb5or ؊/؊ mice.The most prevalent and life-threatening metabolic disorders, type 2 diabetes and obesity, arise from the complex interplay of a large number of genetic determinants. Characterizing polygenic disorders is challenging, requiring insight into plausible candidate genes and analysis of genomic data on numerous kindred. Mouse models offer an attractive independent source of information on genes important in pancreatic ␤-cell and adipocyte viability and homeostasis.We have been investigating a novel flavoheme reductase that plays an important role in pancreatic ␤-cell viability and maintenance of white adipose tissue. Ncb5or (alternatively called b5ϩb5R and cyb5r4) is a single 58-kDa polypeptide composed of two well characterized domains tethered by a unique 90-residue hinge (1). The 130-residue N-terminal domain bears strong homology to classic microsomal cytochrome b 5 , a sixcoordinate heme protein. At the C terminus, there is a 300-residue domain with strong homology to classic microsomal cytochrome b 5 reductase, a flavoprotein. Ncb5or is of special interest because it is highly conserved in a wide range of animals from mammals to flies and worms and because there is no other example in the animal kingdom of such a two-domain protein.Ncb5or is expressed in a wide variety of organs, tissues, and cell lines and localized in the endoplasmic reticulum (2). Because of its high negative redox potential, Ncb5or would be expected to function primarily as an electron donor.Mice with a global knock-out of Ncb5or have normal embryonic and fetal development and normal viability at birth with no gross anatomic abnormalities (3). However, after the first month of life, Ncb5or Ϫ/Ϫ mice develop diabetes because of the loss of pancreatic ␤-cells. In addition, there is progressive atrophy of white adipose tissue (WAT).3 The diabetic and lipoatrophic phenotype of the Ncb5or Ϫ/Ϫ mouse raises the question of how this novel reductase impacts ␤-cell and adipocyte viability and function."Class...

The flavoheme reductase Ncb5or protects cells against endoplasmic reticulum stress-induced lipotoxicity

“…Wildtype cells treated for 12 h with tunicamycin (1 g/ml) were used acids. Although it was not technically feasible to carry out in vitro experiments on isolated pancreatic ␤ -cells, we did show that the viability of isolated Ncb5or Ϫ / Ϫ hepatocytes was markedly impaired and apoptosis enhanced by incubation with palmitate but not with oleate or a mixture of palmitate and oleate ( 7 ). There is growing evidence that in a number of cell types ( 8,9 ), including pancreatic ␤ -cells ( 10 ), the ER is a target of lipotoxicity induced by palmitate.…”

“…We previously reported that primary hepatocytes from Ncb5or Ϫ / Ϫ (KO) mice had impaired viability and enhanced apoptosis, analyzed by TUNEL, following 1 h and 4 h exposure to 1.0 mM palmitate but not to 1.0 mM oleate or an equimolar mixture of the two fatty acids ( 7 ). With a greatly improved method for cell isolation, we are now able to obtain cells at much higher yields and improved viability, allowing longer incubation times.…”

“…Mouse hepatocytes were prepared from 3-to 4-week-old wildtype and Ncb5or Ϫ / Ϫ male and female animals by the two-step perfusion procedure using Liver Perfusion Medium (Invitrogen) and Liver Digestion Medium (Invitrogen) separately as described previously ( 7 ). After isolation, hepatocytes were cultured in Williams' medium E (Invitrogen) containing penicillin/streptomycin and glutamine for 12 h before any treatment.…”

NAD(P)H Cytochrome b5 Oxidoreductase Deficiency in Leishmania major Results in Impaired Linoleate Synthesis Followed by Increased Oxidative Stress and Cell Death

“…Ncb5or deficiency in hepatocytes results in an impaired ⌬9 desaturation pathway, and saturated FA induced lipotoxicity is observed (12). Ncb5or knock-out mice have usual embryonic and fetal growth, and normal viability at birth with no gross anatomic abnormalities (31).…”

“…Ncb5or is an endoplasmic reticulum-bound protein that has been implicated in lipid metabolism and diabetes (11,12). Ncb5or contains two redox domains; the amino-terminal portion is highly homologous to classical microsomal cytochrome b 5 , and the carboxyl-terminal portion of the protein is highly similar to cytochrome b 5 reductase domain (10).…”