Characterization of metabolic phenotypes and distinctive genes in mice with low-weight gain.

Being overweight exacerbates various metabolic diseases, necessitating the identification of target molecules for obesity control. In the current study, we investigated common physiological features related to metabolism in mice with low weight gain: (1) G protein-coupled receptor, family C, group 5, member B-knockout; (2) gastric inhibitory polypeptide receptor-knockout; and (3) Iroquois-related homeobox 3-knockout. Moreover, we explored genes involved in metabolism by analyzing differentially expressed genes (DEGs) between low-weight gain mice and the respective wild-type control mice. The common characteristics of the low-weight gain mice were low inguinal white adipose tissue (iWAT) and liver weight despite similar food intake along with lower blood leptin levels and high energy expenditure. The DEGs of iWAT, epididymal (gonadal) WAT, brown adipose tissue, muscle, liver, hypothalamus, and hippocampus common to these low-weight gain mice were designated as candidate genes associated with metabolism. One such gene tetraspanin 7 (Tspan7) from the iWAT was validated using knockout and overexpressing mouse models. Mice with low Tspan7 expression gained more weight, while those with high Tspan7 expression gained less weight, confirming the involvement of the Tspan7 gene in weight regulation. Collectively, these findings suggest that the candidate gene list generated in this study contains potential target molecules for obesity regulation. Further validation and additional data from low-weight gain mice will aid in understanding the molecular mechanisms associated with obesity.

Gut microbial carbohydrate metabolism contributes to insulin resistance.

Insulin resistance is the primary pathophysiology underlying metabolic syndrome and type 2 diabetes1,2. Previous metagenomic studies have described the characteristics of gut microbiota and their roles in metabolizing major nutrients in insulin resistance3-9. In particular, carbohydrate metabolism of commensals has been proposed to contribute up to 10% of the host's overall energy extraction10, thereby playing a role in the pathogenesis of obesity and prediabetes3,4,6. Nevertheless, the underlying mechanism remains unclear. Here we investigate this relationship using a comprehensive multi-omics strategy in humans. We combine unbiased faecal metabolomics with metagenomics, host metabolomics and transcriptomics data to profile the involvement of the microbiome in insulin resistance. These data reveal that faecal carbohydrates, particularly host-accessible monosaccharides, are increased in individuals with insulin resistance and are associated with microbial carbohydrate metabolisms and host inflammatory cytokines. We identify gut bacteria associated with insulin resistance and insulin sensitivity that show a distinct pattern of carbohydrate metabolism, and demonstrate that insulin-sensitivity-associated bacteria ameliorate host phenotypes of insulin resistance in a mouse model. Our study, which provides a comprehensive view of the host-microorganism relationships in insulin resistance, reveals the impact of carbohydrate metabolism by microbiota, suggesting a potential therapeutic target for ameliorating insulin resistance.

Exploring body weight-influencing gut microbiota by elucidating the association with diet and host gene expression.

We aimed to identify gut microbiota that influences body weight by elucidating the association with diets and host genes. Germ-free (GF) mice with and without fecal microbiota transplant (FMT) were fed a normal, high-carbohydrate, or high-fat diet. FMT mice exhibited greater total body weight; adipose tissue and liver weights; blood glucose, insulin, and total cholesterol levels; and oil droplet size than the GF mice, regardless of diet. However, the extent of weight gain and metabolic parameter levels associated with gut microbiota depended on the nutrients ingested. For example, a disaccharide- or polysaccharide-rich diet caused more weight gain than a monosaccharide-rich diet. An unsaturated fatty acid-rich diet had a greater microbial insulin-increasing effect than a saturated fatty acid-rich diet. Perhaps the difference in microbial metabolites produced from substances taken up by the host created metabolic differences. Therefore, we analyzed such dietary influences on gut microbiota, differentially expressed genes between GF and FMT mice, and metabolic factors, including body weight. The results revealed a correlation between increased weight gain, a fat-rich diet, increased Ruminococcaceae abundance, and decreased claudin 22 gene expression. These findings suggest that weight regulation might be possible through the manipulation of the gut microbiota metabolism using the host's diet.

Metabolic differences and differentially expressed genes between C57BL/6J and C57BL/6N mice substrains.

C57BL/6J (B6J) and C57BL/6N (B6N) mice are the most frequently used substrains in C57BL/6 (B6) inbred mice, serving as physiological models for in vivo studies and as background strains to build transgenic mice. However, the differences in metabolic phenotypes between B6J and B6N mice are not coherent, and genotypic differences in metabolically important tissues have not been well studied. The phenotypic differences between B6J and B6N substrains have often been attributed to the role of the nicotinamide nucleotide transhydrogenase (Nnt) gene, whereby B6J has a spontaneous missense mutation of Nnt. Nevertheless, phenotypic differences between the two cannot be explained by Nnt mutations alone, especially in metabolic traits. Therefore, we aimed to investigate the genetic cause of the phenotypic differences between B6J and B6N mice. Determining consistent genetic differences across multiple tissues involved in metabolic traits such as subcutaneous and visceral white adipose tissues, brown adipose tissue, skeletal muscle, liver, hypothalamus, and hippocampus, may help explain phenotypic differences in metabolism between the two substrains. We report candidate genes along with comparative data on body weight, tissue weight, blood components involved in metabolism, and energy balance of B6J and B6N mice. Insulin degrading enzyme, adenylosuccinate synthase 2, and ectonucleotide triphosphate diphosphohydrolase 4 were highly expressed in B6J mice compared with those in B6N mice, and Nnt, WD repeat and FYVE domain containing 1, and dynein light chain Tctex-type 1 were less expressed in B6J mice compared with those in B6N mice in all seven tissues. Considering the extremely wide use of both substrains and their critical importance in generating transgenic and knock-out models, these findings guide future research across several interrelated fields.

Death-effector domain-containing protein DEDD is an inhibitor of mitotic Cdk1/cyclin B1.

Accumulating evidence has shown that many molecules, including some cyclin-dependent kinases (Cdks) and cyclins, as well as the death-effector domain (DED)-containing FADD, function for both apoptosis and cell cycle. Here we identified that DEDD, which also possesses the DED domain, acts as a novel inhibitor of the mitotic Cdk1/cyclin B1 complex. DEDD associates with mitotic Cdk1/cyclin B1 complexes via direct binding to cyclin B1 and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (DEDD-/-) embryonic fibroblasts is increased compared with that in DEDD+/+ cells, which results in accelerated mitotic progression, thus exhibiting a shortened G2/M stage. Interestingly, DEDD-/- cells also demonstrated decreased G1 duration, which perhaps enhanced the overall reduction in rRNA amounts and cell volume, primarily caused by the rapid termination of rRNA synthesis before cell division. Likewise, DEDD-/- mice show decreased body and organ weights relative to DEDD+/+ mice. Thus, DEDD is an impeder of cell mitosis, and its absence critically influences cell and body size via modulation of rRNA synthesis.

Interactions between E2F1 and SirT1 regulate apoptotic response to DNA damage.

The nicotinamide adenine dinucleotide (NAD)-dependent deacetylase Sir2 (silent information regulator 2) regulates gene silencing in yeast and promotes lifespan extension during caloric restriction. The mammalian homologue of Sir2 (SirT1) regulates p53, NF-kappaB and Forkhead transcription factors, and is implicated in stress response. This report shows that the cell-cycle and apoptosis regulator E2F1 induces SirT1 expression at the transcriptional level. Furthermore, SirT1 binds to E2F1 and inhibits E2F1 activities, forming a negative feedback loop. Knockdown of SirT1 by small interference RNA (siRNA) increases E2F1 transcriptional and apoptotic functions. DNA damage by etoposide causes E2F1-dependent induction of SirT1 expression and knockdown of SirT1 increases sensitivity to etoposide. These results reveal a mutual regulation between E2F1 and SirT1 that affects cellular sensitivity to DNA damage.

Phosphatidylinositol 3-kinase-dependent suppression of the human inducible nitric-oxide synthase promoter is mediated by FKHRL1.

The synthesis of nitric oxide by inducible nitric-oxide synthase (iNOS) plays an important role in the innate immune response by promoting microbial killing and cell damage. In response to inflammatory cytokines and bacterial products, the human iNOS (hiNOS) gene undergoes rapid transcriptional activation via binding of stimulatory transcription factors (e.g. AP-1 and NF-kappaB) to its 5'-flanking region. However, maximal hiNOS promoter induction was suppressed via an unknown phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. We hypothesized that inhibition of the transcription factor FKHRL1 by the PI3K/protein kinase B pathway attenuates hiNOS promoter induction by bacterial lipopolysaccharide and interferon-gamma (LPS/IFN-gamma). Human lung epithelial adenocarcinoma (A549) cells were transiently transfected with an 8.3-kb hiNOS promoter luciferase reporter construct. Co-expression of dominant-negative protein kinase B potentiated LPS/IFN-gamma-stimulated hiNOS promoter activity. In response to LPS/IFN-gamma, FKHRL1 was phosphorylated in a PI3K- and time-dependent fashion. Co-expression of constitutively active FKHRL1 increased hiNOS promoter activity and mRNA levels. Dominant-negative siRNA expression showed that FKHRL1 was necessary for the inhibitory effects of PI3K on hiNOS induction. The same effect was observed upon mutation of a consensus FKHRL1-binding site in the hiNOS promoter. By gel-shift analysis, the corresponding oligonucleotide probe bound endogenous FKHRL1 in an LPS/IFN-gamma- and PI3K-sensitive fashion. Regulation of the hiNOS promoter by FKHRL1 represents a potentially important molecular mechanism by which the PI3K pathway might suppress pro-inflammatory and proapoptotic responses to cytokines and bacterial products.

The mammalian longevity-associated gene product p66shc regulates mitochondrial metabolism.

Previous studies have determined that mice with a homozygous deletion in the adapter protein p66(shc) have an extended life span and that cells derived from these mice exhibit lower levels of reactive oxygen species. Here we demonstrate that a fraction of p66(shc) localizes to the mitochondria and that p66(shc-/-) fibroblasts have altered mitochondrial energetics. In particular, despite similar cytochrome content, under basal conditions, the oxygen consumption of spontaneously immortalized p66(shc-/-) mouse embryonic fibroblasts were lower than similarly maintained wild type cells. Differences in oxygen consumption were particularly evident under chemically uncoupled conditions, demonstrating that p66(shc-/-) cells have a reduction in both their resting and maximal oxidative capacity. We further demonstrate that reconstitution of p66(shc) expression in p66(shc-/-) cells increases oxygen consumption. The observed defect in oxidative capacity seen in p66(shc-/-) cells is partially offset by augmented levels of aerobic glycolysis. This metabolic switch is manifested by p66(shc-/-) cells exhibiting an increase in lactate production and a stricter requirement for extracellular glucose in order to maintain intracellular ATP levels. In addition, using an in vivo NADH photobleaching technique, we demonstrate that mitochondrial NADH metabolism is reduced in p66(shc-/-) cells. These results demonstrate that p66(shc) regulates mitochondrial oxidative capacity and suggest that p66(shc) may extend life span by repartitioning metabolic energy conversion away from oxidative and toward glycolytic pathways.

Phosphorylation of p66Shc and forkhead proteins mediates Abeta toxicity.

Excessive accumulation of amyloid beta-peptide (Abeta) plays an early and critical role in synapse and neuronal loss in Alzheimer's Disease (AD). Increased oxidative stress is one of the mechanisms whereby Abeta induces neuronal death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66Shc, we investigated the role of p66Shc in Abeta toxicity. Treatment of cells and primary neuronal cultures with Abeta caused apoptotic death and induced p66Shc phosphorylation at Ser36. Ectopic expression of a dominant-negative SEK1 mutant or chemical JNK inhibition reduced Abeta-induced JNK activation and p66Shc phosphorylation (Ser36), suggesting that JNK phosphorylates p66Shc. Abeta induced the phosphorylation and hence inactivation of forkhead transcription factors in a p66Shc-dependent manner. Ectopic expression of p66ShcS36A or antioxidant treatment protected cells against Abeta-induced death and reduced forkhead phosphorylation, suggesting that p66Shc phosphorylation critically influences the redox regulation of forkhead proteins and underlies Abeta toxicity. These findings underscore the potential usefulness of JNK, p66Shc, and forkhead proteins as therapeutic targets for AD.

Mitochondria, oxidants, and aging.

The free radical theory of aging postulates that the production of intracellular reactive oxygen species is the major determinant of life span. Numerous cell culture, invertebrate, and mammalian models exist that lend support to this half-century-old hypothesis. Here we review the evidence that both supports and conflicts with the free radical theory and examine the growing link between mitochondrial metabolism, oxidant formation, and the biology of aging.

SIRT1 functionally interacts with the metabolic regulator and transcriptional coactivator PGC-1{alpha}.

In lower organisms, increased expression of the NAD-dependent deacetylase Sir2 augments lifespan. The mechanism through which this life extension is mediated remains incompletely understood. Here we have examined the cellular effects of overexpression of SIRT1, the closest mammalian ortholog of Sir2. In PC12 cells, increased expression of the NAD-dependent deacetylase SIRT1 reduces cellular oxygen consumption by approximately 25%. We further demonstrate that SIRT1 expression can alter the transcriptional activity of the mitochondrial biogenesis coactivator PGC-1alpha. In addition, SIRT1 and PGC-1alpha directly interact and can be co-immunoprecipitated as a molecular complex. A single amino acid mutation in the putative ADP-ribosyltransferase domain of SIRT1 inhibits the interaction of SIRT1 with PGC-1alpha but does not effect the interaction of SIRT1 with either p53 or Foxo3a. We further show that PGC-1alpha is acetylated in vivo. This acetylation is augmented by treatment with the SIRT1 inhibitor nicotinamide or by expression of the transcriptional coactivator p300. Finally we demonstrate that SIRT1 catalyzes PGC-1alpha deacetylation both in vitro and in vivo. These results provide a direct link between the sirtuins, a family of proteins linked to lifespan determination and PGC-1alpha, a coactivator that regulates cellular metabolism.

Nutrient availability regulates SIRT1 through a forkhead-dependent pathway.

Nutrient availability regulates life-span in a wide range of organisms. We demonstrate that in mammalian cells, acute nutrient withdrawal simultaneously augments expression of the SIRT1 deacetylase and activates the Forkhead transcription factor Foxo3a. Knockdown of Foxo3a expression inhibited the starvation-induced increase in SIRT1 expression. Stimulation of SIRT1 transcription by Foxo3a was mediated through two p53 binding sites present in the SIRT1 promoter, and a nutrient-sensitive physical interaction was observed between Foxo3a and p53. SIRT1 expression was not induced in starved p53-deficient mice. Thus, in mammalian cells, p53, Foxo3a, and SIRT1, three proteins separately implicated in aging, constitute a nutrient-sensing pathway.

Identification of a specific molecular repressor of the peroxisome proliferator-activated receptor gamma Coactivator-1 alpha (PGC-1alpha).

The nuclear co-activator PGC-1alpha is a pivotal regulator of numerous pathways controlling both metabolism and overall energy homeostasis. Inappropriate increases in PGC-1alpha activity have been linked to a number of pathological conditions including heart failure and diabetes mellitus. Previous studies (Puigserver, P., Adelmant, G., Wu, Z., Fan, M., Xu, J., O'Malley, B., and Spiegelman, B. M. (1999) Science 286, 1368-1371) have demonstrated an inhibitory domain within PGC-1alpha that limits transcriptional activity. Using this inhibitory domain in a yeast two-hybrid screen, we demonstrate that PGC-1alpha directly associates with the orphan nuclear receptor estrogen-related receptor-alpha (ERR-alpha). The binding of ERR-alpha to PGC-1alpha requires the C-terminal AF2 domain of ERR-alpha. PGC-1alpha and ERR-alpha have a similar pattern of expression in human tissues, with both being present predominantly in organs with high metabolic needs such as skeletal muscle and kidney. Similarly, we show that in mice physiological stimuli such as fasting coordinately induces PGC-1alpha and ERR-alpha transcription. We also demonstrate that under normal conditions PGC-1alpha is located within discrete nuclear speckles, whereas the expression of ERR-alpha results in PGC-1alpha redistributing uniformly throughout the nucleoplasm. Finally, we show that the expression of ERR-alpha can dramatically and specifically repress PGC-1alpha transcriptional activity. These results suggest a novel mechanism of transcriptional control wherein ERR-alpha can function as a specific molecular repressor of PGC-1alpha activity. In addition, our results suggest that other co-activators might also have specific repressors, thereby identifying another layer of combinatorial complexity in transcriptional regulation.

Redox regulation of forkhead proteins through a p66shc-dependent signaling pathway.

Genetic determinants of longevity include the forkhead-related transcription factor DAF-16 in the worm Caenorhabditis elegans and the p66shc locus in mice. We demonstrate that p66shc regulates intracellular oxidant levels in mammalian cells and that hydrogen peroxide can negatively regulate forkhead activity. In p66shc-/- cells, the activity of the mammalian forkhead homolog FKHRL1 is increased and redox-dependent forkhead inactivation is reduced. In addition, expression of FKHRL1 results in an increase in both hydrogen peroxide scavenging and oxidative stress resistance. These results demonstrate an important functional relation between three distinct elements linked to aging: forkhead proteins, p66shc, and intracellular oxidants.