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.

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

NCB5OR is a novel flavoheme reductase with a cytochrome b5-like domain at the N-terminus and a cytochrome b5 reductase-like domain at the C terminus. Ncb5or knock-out mice develop insulin deficient diabetes and loss of white adipose tissue. Ncb5or(-/-) mice have impairment of Delta9 fatty acid desaturation with elevated ratios of palmitate to palmitoleate and stearate to oleate. In this study we assess the role of the endoplasmic reticulum (ER) stress response in mediating lipotoxicity in Ncb5or(-/-) mice. The ER stress response was assessed by induction of BiP, ATF3, ATF6, XBP-1, and C/EBP homologous protein (CHOP). Exposure to palmitate, but not oleate or mixtures of oleate and palmitate induced these markers of ER stress to a much greater extent in Ncb5or(-/-) hepatocytes than in wild-type cells. In contrast, Ncb5or(-/-) and Ncb5or(+/+) hepatocytes were equally sensitive to ER stress imposed by increasing concentrations of tunicamycin. In order to assess the role of ER stress in vivo, we prepared mice that lack both NCB5OR and CHOP, a proapoptotic transcription factor important in the ER stress response. Onset of hyperglycemia in the Chop(-/-);Ncb5or(-/-) mice was delayed two weeks beyond that observed in Chop(+/+);Ncb5or(-/-) mice. Taken together these results suggest that ER stress plays a critical role in palmitate-induced lipotoxicity both in vitro and in vivo.

Living without Oxygen: Anoxia-Responsive Gene Expression and Regulation.

Many species of marine mollusks demonstrate exceptional capacities for long term survival without oxygen. Analysis of gene expression under anoxic conditions, including the subsequent translational responses, allows examination of the functional mechanisms that support and regulate natural anaerobiosis and permit noninjurious transitions between aerobic and anoxic states. Identification of stress-specific gene expression can provide important insights into the metabolic adaptations that are needed for anoxia tolerance, with potential applications to anoxia-intolerant systems. Various methods are available to do this, including high throughput microarray screening and construction and screening of cDNA libraries. Anoxia-responsive genes have been identified in mollusks; some have known functions in other organisms but were not previously linked with anoxia survival. In other cases, completely novel anoxia-responsive genes have been discovered, some that show known motifs or domains that hint at function. Selected genes are expressed at different times over an anoxia-recovery time course with their transcription and translation being actively regulated to ensure protein expression at the optimal time. An examination of transcript status over the course of anoxia exposure and subsequent aerobic recovery identifies genes, and the proteins that they encode, that enhance cell survival under oxygen-limited conditions. Analysis of data generated from non-mainstream model systems allows for insight into the response by cells to anoxia stress.

Loss of Ncb5or results in impaired fatty acid desaturation, lipoatrophy, and diabetes.

Targeted ablation of the novel flavoheme reductase Ncb5or knock-out (KO) results in progressive loss of pancreatic beta-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 (Delta9 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 co-incubation 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.

Constructing and screening a cDNA library. Methods for identification and characterization of novel genes expressed under conditions of environmental stress.

Many organisms provide excellent models for studying disease states or for exploring the molecular adaptations that allow cells and organisms to cope with or survive different stresses. The construction of a cDNA library and subsequent screening for genes of interest allows researchers to select for genes that are likely to play key roles in the regulation or response to the condition or stress of interest, those that may not be expressed (or exist) in other systems. Determination of the open reading frame(s) of novel genes, and extensive analysis of the proteins they encode, can open up new avenues of research and promote intelligent design of downstream projects.

Identification of a granulin-like transcript expressed during anoxic exposure and translated during aerobic recovery in a marine gastropod.

A novel transcript encoding a cysteine-rich granulin-like peptide (l-grn) was identified in the hepatopancreas of the marine intertidal gastropod, Littorina littorea, an anoxia-tolerant species. Experimental exposure of snails to anoxia induced a gradual accumulation of l-grn transcripts over time, with expression regulated in vitro through elements responsive to second messengers of protein kinases A, C and G. Translation of this transcript was analyzed by examining l-grn association with ribosomes during normoxia, anoxia, and aerobic recovery. Transcripts of l-grn were associated with polysomes during normoxia, moved into the monosome fractions under anoxia, but shifted back to the polysomal fractions during aerobic recovery. Western blotting confirmed this with a granulin-like protein detected under normoxic conditions, but not during anoxia exposure. A significant increase in the precursor protein and peptide (L-GRN) was observed during the aerobic recovery period. The accumulation of l-grn transcripts during anoxic exposure and subsequent translation following the return to aerobic conditions may be a response to oxidant damage that occurs during re-oxygenation. Overall, the data show that the l-grn gene is anoxia-responsive in this species and may have pro-survival functions during the recovery period.

Arrest of transcription following anoxic exposure in a marine mollusc.

The intertidal marine snail, Littorina littorea, is an anoxia tolerant species that endures long-term oxygen deprivation using a suite of compensatory metabolic adaptations that includes overall metabolic rate depression. Nuclear run-off assays were used to quantify the relative rates of mRNA transcription in nuclear extracts from hepatopancreas of aerobic and anoxic snails. Total [(32)P]-UTP incorporation into RNA by nuclei from 48 h anoxic snails ranged from 42 to 50% of that observed for nuclei from normoxic snails. When this data is transformed with respect to incubation time, the rate of [(32)P]-UTP incorporation by nuclei from 48 h anoxic snails showed a decrease of 68% as compared with the normoxic level. Examination of selected expressed sequence tags also showed an overall decrease in mRNA transcription levels in samples derived from anoxic nuclei as compared with normoxic nuclei. Control of ribosomal translation was also examined by assessing the levels of the eukaryotic initiation factors eIF-2alpha and eIF-4E and the eukaryotic elongation factor-1gamma (eEF-1gamma). Levels of eIF-4E and eEF-1gamma did not change between aerobic and anoxic states, but the amount of phosphorylated inactive eIF-2alpha rose strongly under anoxic conditions indicating that control of this factor is key to suppressing protein translation in anoxic snails. Since gene transcription is an ATP expensive process in cells, suppression of transcription to minimum levels provides substantial energy savings for the hepatopancreas, and the organism as a whole, under anoxic conditions.

The reductase NCB5OR is responsive to the redox status in beta-cells and is not involved in the ER stress response.

The novel reductase NCB5OR (NADPH cytochrome b5 oxidoreductase) resides in the ER (endoplasmic reticulum) and may protect cells against ER stress. Levels of BiP (immunoglobulin heavy-chain-binding protein), CHOP (CCAAT/enhancer-binding protein homologous protein) and XBP-1 (X-box-binding protein-1) did not differ in WT (wild-type) and KO (Ncb5or-null) tissues or MEFs (mouse embryonic fibroblasts), and XBP-1 remained unspliced. MEFs treated with inducers of ER stress demonstrated no change in Ncb5or expression and expression of ER-stress-induced genes was not enhanced. Induction of ER stress in beta-cell lines did not change Ncb5or expression or promoter activity. Transfection with Ncb5or-specific siRNA (small interfering RNA) yielded similar results. Microarray analysis of mRNA from islets and liver of WT and KO animals revealed no significant changes in ER-stress-response genes. Induction of oxidative stress in betaTC3 cells did not alter Ncb5or mRNA levels or promoter activity. However, KO islets were more sensitive to streptozotocin when compared with WT islets. MEFs incubated with nitric oxide donors showed no difference in cell viability or levels of nitrite produced. No significant differences in mRNA expression of antioxidant enzymes were observed when comparing WT and KO tissues; however, microarray analysis of islets indicated slightly enhanced expression of some antioxidant enzymes in the KO islets. Short-term tBHQ (t-butylhydroquinone) treatment increased Ncb5or promoter activity, although longer incubation times yielded a dose-dependent decrease in activity. This response appears to be due to a consensus ARE (antioxidant-response element) present in the Ncb5or promoter. In summary, NCB5OR does not appear to be involved in ER stress, although it may be involved in maintaining or regulating the redox status in beta-cells.

Promoter characterization and transcriptional regulation of Ncb5or, a novel reductase necessary for pancreatic beta-cell maintenance.

Ncb5or is a ubiquitously expressed gene required for beta-cell survival in mice. Examination of mouse tissues demonstrated high levels of expression in the pancreas, heart and kidney. A transcription start site was identified 149 bp upstream from the start codon and transient expression analysis in betaTC3 cells indicated the presence of a core promoter located within 348 bp upstream of this site. Deletion of Region C (-216/-157) resulted in a significant decrease in promoter activity and specific nucleotides located in a region designated C2 were demonstrated to be critical for complex binding. Deletion of Region D (-60/-33), which contains multiple consensus Sp1 sites, resulted in an additional loss of promoter activity. The data presented here identify and characterize the previously unknown promoter of Ncb5or, a reductase critical for beta-cell survival.

Analysis of signal transduction pathways during anoxia exposure in a marine snail: a role for p38 MAP kinase and downstream signaling cascades.

The responses of members of the three main MAPK families (ERK, JNK/SAPK, p38 MAPK), as well as selected peripheral pathways, were examined in hepatopancreas of the marine periwinkle, Littorina littorea, to determine if anoxia exposure influenced the total protein content or the phosphorylation status of any key components. The content of active phospho-p38 MAPK was 2-fold higher in hepatopancreas from anoxic snails relative to controls. A 1.7-fold increase in the amount of phospho-Hsp27 and a 1.3-fold increase in phospho-CREB correlated well with the changes in p38 MAPK phosphorylation. Activation of these factors via p38 MAPK may be vital to the reorganization of metabolic responses to anoxia in hepatopancreas. No changes in components of the JNK/SAPK and ERK pathways occurred and transcription factors involved in lipid metabolism did not appear to be affected by anoxia. The present analysis of a variety of signaling pathways has implicated the p38 MAPK pathway as a key anoxia-responsive signal transduction pathway in L. littorea.

Variation in NCB5OR: studies of relationships to type 2 diabetes, maturity-onset diabetes of the young, and gestational diabetes mellitus.

Recent data show that homozygous Ncb5or(-/-) knock-out mice present with an early-onset nonautoimmune diabetes phenotype. Furthermore, genome-wide scans have reported linkage to the chromosome 6q14.2 region close to the human NCB5OR. We therefore considered NCB5OR to be a biological and positional candidate gene and examined the coding region of NCB5OR in 120 type 2 diabetic patients and 63 patients with maturity-onset diabetes of the young using denaturing high-performance liquid chromatography. We identified a total of 22 novel nucleotide variants. Three variants [IVS5+7del(CT), Gln187Arg, and His223Arg] were genotyped in a case-control design comprising 1,246 subjects (717 type 2 diabetic patients and 529 subjects with normal glucose tolerance). In addition, four rare variants were investigated for cosegregation with diabetes in multiplex type 2 diabetic families. The IVS5+7del(CT) variant was associated with common late-onset type 2 diabetes; however, we failed to relate this variant to any diabetes-related quantitative traits among the 529 control subjects. Thus, variation in the coding region of NCB5OR is not a major contributor in the pathogenesis of nonautoimmune diabetes.

Absence of a reductase, NCB5OR, causes insulin-deficient diabetes.

NCB5OR is a highly conserved NAD(P)H reductase that contains a cytochrome b5-like domain at the N terminus and a cytochrome b5 reductase-like domain at the C terminus. The enzyme is located in the endoplasmic reticulum (ER) and is widely expressed in organs and tissues. Targeted inactivation of this gene in mice has no impact on embryonic or fetal viability. At 4 weeks of age, Ncb5or-/- mice have normal blood glucose levels but impaired glucose tolerance. Isolated Ncb5or-/- islets have markedly impaired glucose- or arginine-stimulated insulin secretion. By 7 weeks of age, these mice develop severe hyperglycemia with markedly decreased serum insulin levels and nearly normal insulin tolerance. As the animals age, there is a progressive loss of beta cells in pancreatic islets, but there is no loss of alpha, delta, or PP cells. Electron microscopy reveals degranulation of beta cells and hypertrophic and hyperplastic mitochondria, some of which contain electron dense inclusions. Four-week-old Ncb5or-/- mice have enhanced sensitivity to the diabetogenic agent streptozotocin. NCB5OR appears to play a critical role in protecting pancreatic beta cells against oxidant stress.

NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum.

The NAD(P)H cytochrome b5 oxidoreductase, Ncb5or (previously named b5+b5R), is widely expressed in human tissues and broadly distributed among the animal kingdom. NCB5OR is the first example of an animal flavohemoprotein containing cytochrome b5 and chrome b5 reductase cytodomains. We initially reported human NCB5OR to be a 487-residue soluble protein that reduces cytochrome c, methemoglobin, ferricyanide, and molecular oxygen in vitro. Bioinformatic analysis of genomic sequences suggested the presence of an upstream start codon. We confirm that endogenous NCB5OR indeed has additional NH2-terminal residues. By performing fractionation of subcellular organelles and confocal microscopy, we show that NCB5OR colocalizes with calreticulin, a marker for endoplasmic reticulum. Recombinant NCB5OR is soluble and has stoichiometric amounts of heme and flavin adenine dinucleotide. Resonance Raman spectroscopy of NCB5OR presents typical signatures of a six-coordinate low-spin heme similar to those found in other cytochrome b5 proteins. Kinetic measurements showed that full-length and truncated NCB5OR reduce cytochrome c actively in vitro. However, both full-length and truncated NCB5OR produce superoxide from oxygen with slow turnover rates: kcat = approximately 0.05 and approximately 1 s(-1), respectively. The redox potential at the heme center of NCB5OR is -108 mV, as determined by potentiometric titrations. Taken together, these data suggest that endogenous NCB5OR is a soluble NAD(P)H reductase preferentially reducing substrate(s) rather than transferring electrons to molecular oxygen and therefore not an NAD(P)H oxidase for superoxide production. The subcellular localization and redox properties of NCB5OR provide important insights into the biology of NCB5OR and the phenotype of the Ncb5or-null mouse.

Anoxia-induced transcriptional upregulation of sarp-19: cloning and characterization of a novel EF-hand containing gene expressed in hepatopancreas of Littorina littorea.

Many marine molluscs have well-developed biochemical adaptations that allow them to live without oxygen for long periods of time, but very little is currently known about the molecular biology underlying these processes. Differential screening of a cDNA library derived from the hepatopancreas of the marine snail Littorina littorea revealed a novel anoxia-induced gene, sarp-19 (snail anoxia-responsive protein, 19 kDa). Examination of the sarp-19 transcript revealed an open reading frame that encoded a protein of 168 amino acids containing an N-terminal signal sequence and two putative EF-hand domains. Expression analysis of transcript levels established that sarp-19 accumulated over a time course of anoxia exposure, reaching a maximum 5.6-fold increase after 96 h compared with aerobic controls. However, transcript levels were reduced by 50% within 1 h when aerobic conditions were reestablished. Nuclear runoff assays confirmed transcriptional upregulation of sarp-19 during anoxia exposure, and organ explant experiments showed that the gene was also responsive to anoxia exposure in vitro. sarp-19 transcripts were also elevated in response to freezing, suggesting that the protein may have a role in the physiological responses of this intertidal snail to both aerial exposure and winter freezing. Hepatopancreas explants treated with a calcium ionophore showed increased levels of the sarp-19 transcript, suggesting a possible feedback mechanism regulated by levels of intracellular calcium. Expression was also responsive to tissue incubation with cyclic GMP and phorbol 12-myristate 13-acetate but was not affected by cyclic AMP, implicating involvement of protein kinases G and C but not protein kinase A in the expression of sarp-19. The SARP-19 protein may play a role in calcium-activated signaling during anoxia exposure in L. littorea.

Accumulation and translation of ferritin heavy chain transcripts following anoxia exposure in a marine invertebrate.

Differential screening of a Littorina littorea (the common periwinkle) cDNA library identified ferritin heavy chain as an anoxia-induced gene in hepatopancreas. Northern blots showed that ferritin heavy chain transcript levels were elevated twofold during anoxia exposure, although nuclear run-off assays demonstrated that ferritin heavy chain mRNAs were not transcriptionally upregulated during anoxia. Polysome analysis indicated that existing ferritin transcripts were actively translated during the anoxic period. This result was confirmed via western blotting, which demonstrated a twofold increase in ferritin heavy chain protein levels during anoxia, with a subsequent decrease to control levels during normoxic recovery. Organ culture experiments using hepatopancreas slices demonstrated a >50% increase in ferritin heavy chain transcript levels in vitro under conditions of anoxia and freezing, as well as after incubation with the second messenger cGMP. Taken together, these results suggest that ferritin heavy chain is actively regulated during anoxia exposure in the marine snail, L. littorea.

Reversible suppression of protein synthesis in concert with polysome disaggregation during anoxia exposure in Littorina littorea.

Many marine invertebrates can live without oxygen for long periods of time, a capacity that is facilitated by the ability to suppress metabolic rate in anoxia to a value that is typically less than 10% of the normal aerobic rate. The present study demonstrates that a reduction in the rate of protein synthesis is one factor in the overall anoxia-induced metabolic suppression in the marine snail, Littorina littorea. The rate of [3H]leucine incorporation into newly translated protein in hepatopancreas isolated from 48 h anoxic snails was determined to be 49% relative to normoxic controls. However, protein concentration in hepatopancreas did not change during anoxia, suggesting a coordinated suppression of net protein turnover. Analysis of hepatopancreas samples from snails exposed to 24-72 h anoxia showed a gradual disaggregation of polysomes into monosomes. A re-aggregation of monosomes into polysomes was observed after 3 h of aerobic recovery. Analysis of fractions from the ribosome profile using radiolabeled probe to detect alpha-tubulin transcripts confirmed a general decrease in protein translation during anoxia exposure (transcript association with polysomes decreased) with a reversal during aerobic recovery. Western blotting of hepatopancreas samples from normoxic, 24 h anoxic, and 1 h aerobic recovered snails demonstrated that eIF-2alpha is substantially phosphorylated during anoxia exposure and dephosphorylated during normoxia and aerobic recovery, suggesting a decrease in translation initiation during anoxia exposure. These results suggest that metabolic suppression during anoxia exposure in L. littorea involves a decrease in protein translation.

Characterization of a novel gene up-regulated during anoxia exposure in the marine snail, Littorina littorea.

Gene expression was investigated during anoxia exposure in the marine snail, Littorina littorea. Differential screening of a cDNA library made from hepatopancreas of anoxic L. littorea yielded a 525 bp clone coding for the novel gene kvn. The deduced amino acid sequence of the KVN protein contained 99 amino acid residues with a predicted molecular weight of 12 kDa and showed an N-terminal secretory signal. Analysis of hepatopancreas samples over a time course of anoxia exposure showed a maximum increase in transcript levels of 5.8-fold after 48 h relative to normoxic animals, with a subsequent decrease in transcript levels during normoxic recovery. Nuclear run-off assays confirmed the observed transcriptional up-regulation of kvn during anoxia. Organ culture experiments were performed to determine a possible pathway of up-regulation of kvn, with data indicating a putative role for cGMP in signal transduction. Profiles of ribosome distribution in polysomes versus monosomes revealed a reduction in the polysome peak during anoxia and a shift in the position of kvn transcripts to association with the lower density polysome/higher density monosome region. The data suggest that the kvn transcript is both transcribed and translated during anoxia, indicating a possible significant role for the KVN protein in the survival of anoxia by L. littorea.