The trillions of microbes that colonize our adult intestines function collectively as a metabolic organ that communicates with, and complements, our own human metabolic apparatus. Given the worldwide epidemic in obesity, there is interest in how interactions between human and microbial metabolomes may affect our energy balance. Here we report that, in contrast to mice with a gut microbiota, germ-free (GF) animals are protected against the obesity that develops after consuming a Western-style, high-fat, sugar-rich diet. Their persistently lean phenotype is associated with increased skeletal muscle and liver levels of phosphorylated AMP-activated protein kinase (AMPK) and its downstream targets involved in fatty acid oxidation (acetylCoA carboxylase; carnitinepalmitoyltransferase). Moreover, GF knockout mice lacking fasting-induced adipose factor (Fiaf), a circulating lipoprotein lipase inhibitor whose expression is normally selectively suppressed in the gut epithelium by the microbiota, are not protected from diet-induced obesity. Although GF Fiaf؊/؊ animals exhibit similar levels of phosphorylated AMPK as their wild-type littermates in liver and gastrocnemius muscle, they have reduced expression of genes encoding the peroxisomal proliferatoractivated receptor coactivator (Pgc-1␣) and enzymes involved in fatty acid oxidation. Thus, GF animals are protected from dietinduced obesity by two complementary but independent mechanisms that result in increased fatty acid metabolism: (i) elevated levels of Fiaf, which induces Pgc-1␣; and (ii) increased AMPK activity. Together, these findings support the notion that the gut microbiota can influence both sides of the energy balance equation, and underscore the importance of considering our metabolome in a supraorganismal context. AMP-activated protein kinase ͉ fasting-induced adipose factor ͉ fatty acid metabolism ͉ gut microbiota ͉ symbiosis

The distal human intestine harbors trillions of microbes that allow us to extract calories from otherwise indigestible dietary polysaccharides. The products of polysaccharide fermentation include shortchain fatty acids that are ligands for Gpr41, a G protein-coupled receptor expressed by a subset of enteroendocrine cells in the gut epithelium. To examine the contribution of Gpr41 to energy balance, we compared Gpr41؊/؊ and Gpr41؉/؉ mice that were either conventionally-raised with a complete gut microbiota or were reared germ-free and then cocolonized as young adults with two prominent members of the human distal gut microbial community: the saccharolytic bacterium, Bacteroides thetaiotaomicron and the methanogenic archaeon, Methanobrevibacter smithii. Both conventionallyraised and gnotobiotic Gpr41؊/؊ mice colonized with the model fermentative community are significantly leaner and weigh less than their WT (؉/؉) littermates, despite similar levels of chow consumption. These differences are not evident when germ-free WT and germ-free Gpr41 knockout animals are compared. Functional genomic, biochemical, and physiologic studies of germ-free and cocolonized Gpr41؊/؊ and ؉/؉ littermates disclosed that Gpr41-deficiency is associated with reduced expression of PYY, an enteroendocrine cell-derived hormone that normally inhibits gut motility, increased intestinal transit rate, and reduced harvest of energy (short-chain fatty acids) from the diet. These results reveal that Gpr41 is a regulator of host energy balance through effects that are dependent upon the gut microbiota.host-microbial interactions ͉ energy balance ͉ enteroendocrine cells ͉ nutrient sensing ͉ polysaccharide fermentation O ur ability to effectively digest food reflects the combined activities of enzymes encoded in our primate genome and in the genomes of the trillions of microbes that reside in our distal guts. This microbial community, or microbiota, affects both sides of the energy-balance equation, influencing both the harvest of calories and the activity of host genes involved in the metabolism and storage of absorbed energy (e.g., ref.

The human gut is home to trillions of microbes, thousands of bacterial phylotypes, as well as hydrogen-consuming methanogenic archaea. Studies in gnotobiotic mice indicate that Methanobrevibacter smithii, the dominant archaeon in the human gut ecosystem, affects the specificity and efficiency of bacterial digestion of dietary polysaccharides, thereby influencing host calorie harvest and adiposity. Metagenomic studies of the gut microbial communities of genetically obese mice and their lean littermates have shown that the former contain an enhanced representation of genes involved in polysaccharide degradation, possess more archaea, and exhibit a greater capacity to promote adiposity when transplanted into germ-free recipients. These findings have led to the hypothesis that M. smithii may be a therapeutic target for reducing energy harvest in obese humans. To explore this possibility, we have sequenced its 1,853,160-bp genome and compared it to other human gut-associated M. smithii strains and other Archaea. We have also examined M. smithii's transcriptome and metabolome in gnotobiotic mice that do or do not harbor Bacteroides thetaiotaomicron, a prominent saccharolytic bacterial member of our gut microbiota. Our results indicate that M. smithii is well equipped to persist in the distal intestine through (i) production of surface glycans resembling those found in the gut mucosa, (ii) regulated expression of adhesin-like proteins, (iii) consumption of a variety of fermentation products produced by saccharolytic bacteria, and (iv) effective competition for nitrogenous nutrient pools. These findings provide a framework for designing strategies to change the representation and/or properties of M. smithii in the human gut microbiota.archaeal-bacterial mutualism ͉ comparative microbial genomics ͉ functional genomics and metabolomics ͉ gnotobiotic mice ͉ human gut microbiota T he human gut microbiota is dominated by two divisions of bacteria, the Bacteroidetes and the Firmicutes, which together encompass Ͼ90% of all phylogenetic types (phylotypes). Archaea are also represented, most prominently by a methanogenic Euryarchaeote, Methanobrevibacter smithii, which comprises up to 10% of all anaerobes in the colons of healthy adults (1, 2).Complex dietary polysaccharides (fiber) and proteins are digested by enzymes encoded by genes in the microbial community's collective genome (microbiome), but not in our human genome (3, 4). Bacterial fermentation of polysaccharides yields short chain fatty acids (SCFAs) (principally acetate, propionate, and butyrate), other organic acids (e.g., formate), alcohols (e.g., methanol and ethanol), and gases [e.g., hydrogen (H 2 ) and carbon dioxide (CO 2 )]. Host absorption of SCFAs provides up to 10% of daily caloric intake, although this value varies depending on the glycan content of the diet (5). Archaeal methanogenesis improves the efficiency of polysaccharide fermentation in animal gut ''bioreactors'' by preventing the buildup of H 2 and other reaction end products (6).Several recent obs...

Yeast deprived of nutrients exhibit a marked life span extension that requires the activity of the NAD ؉ -dependent histone deacetylase, Sir2p. Here we show that increased dosage of NPT1, encoding a nicotinate phosphoribosyltransferase critical for the NAD ؉ salvage pathway, increases Sir2-dependent silencing, stabilizes the rDNA locus, and extends yeast replicative life span by up to 60%. Both NPT1 and SIR2 provide resistance against heat shock, demonstrating that these genes act in a more general manner to promote cell survival. We show that Npt1 and a previously uncharacterized salvage pathway enzyme, Nma2, are both concentrated in the nucleus, indicating that a significant amount of NAD ؉ is regenerated in this organelle. Additional copies of the salvage pathway genes, PNC1, NMA1, and NMA2, increase telomeric and rDNA silencing, implying that multiple steps affect the rate of the pathway. Although SIR2-dependent processes are enhanced by additional NPT1, steady-state NAD ؉ levels and NAD ؉ /NADH ratios remain unaltered. This finding suggests that yeast life span extension may be facilitated by an increase in the availability of NAD ؉ to Sir2, although not through a simple increase in steady-state levels. We propose a model in which increased flux through the NAD ؉ salvage pathway is responsible for the Sir2-dependent extension of life span.

A relationship between life span and cellular glucose metabolism has been inferred from genetic manipulations and caloric restriction of model organisms. In this report, we have used the Snf1p glucose-sensing pathway of Saccharomyces cerevisiae to explore the genetic and biochemical linkages between glucose metabolism and aging. Snf1p is a serine/threonine kinase that regulates cellular responses to glucose deprivation. Loss of Snf4p, an activator of Snf1p, extends generational life span whereas loss of Sip2p, a presumed repressor of the kinase, causes an accelerated aging phenotype. An annotated data base of global age-associated changes in gene expression in isogenic wild-type, sip2⌬, and snf4⌬ strains was generated from DNA microarray studies. The transcriptional responses suggested that gluconeogenesis and glucose storage increase as wild-type cells age, that this metabolic evolution is exaggerated in rapidly aging sip2⌬ cells, and that it is attenuated in longerlived snf4⌬ cells. To test this hypothesis directly, we applied microanalytic biochemical methods to generation-matched cells from each strain and measured the activities of enzymes and concentrations of metabolites in the gluconeogenic, glycolytic, and glyoxylate pathways, as well as glycogen, ATP, and NAD ؉ . The sensitivity of the assays allowed comprehensive biochemical profiling to be performed using aliquots of the same cell populations employed for the transcriptional profiling. The results provided additional evidence that aging in S. cerevisiae is associated with a shift away from glycolysis and toward gluconeogenesis and energy storage. They also disclosed that this shift is forestalled by two manipulations that extend life span, caloric restriction and genetic attenuation of the normal age-associated increase in Snf1p activity. Together, these findings indicate that Snf1p activation is not only a marker of aging but also a candidate mediator, because a shift toward energy storage over expenditure could impact myriad aspects of cellular maintenance and repair. Genetic studies in model organisms imply that changes in glucose and energy metabolism can alter life span (1-4), although there has been little direct biochemical analysis of this hypothesis in aging cells (5). Saccharomyces cerevisiae is an attractive model for studying how glucose and energy metabolism are linked to aging. Age-associated alterations in energy metabolism can be analyzed more readily in a unicellular eukaryote than in a multicellular organism with diverse

Bacteroides thetaiotaomicron is a prominent member of our normal adult intestinal microbial community and a useful model for studying the foundations of human-bacterial mutualism in our densely populated distal gut microbiota. A central question is how members of this microbiota sense nutrients and implement an appropriate metabolic response. B. thetaiotaomicron contains a large number of glycoside hydrolases not represented in our own proteome, plus a markedly expanded collection of hybrid twocomponent system (HTCS) proteins that incorporate all domains found in classical two-component environmental sensors into one polypeptide. To understand the role of HTCS in nutrient sensing, we used B. thetaiotaomicron GeneChips to characterize their expression in gnotobiotic mice consuming polysaccharide-rich or -deficient diets. One HTCS, BT3172, was selected for further analysis because it is induced in vivo by polysaccharides, and its absence reduces B. thetaiotaomicron fitness in polysaccharide-rich diet-fed mice. Functional genomic and biochemical analyses of WT and BT3172-deficient strains in vivo and in vitro disclosed that ␣-mannosides induce BT3172 expression, which in turn induces expression of secreted ␣-mannosidases. Yeast two-hybrid screens revealed that the cytoplasmic portion of BT3172's sensor domain serves as a scaffold for recruiting glucose-6-phosphate isomerase and dehydrogenase. These interactions are a unique feature of BT3172 and specific for the cytoplasmic face of its sensor domain. Loss of BT3172 reduces glycolytic pathway activity in vitro and in vivo. Thus, this HTCS functions as a metabolic reaction center, coupling nutrient sensing to dynamic regulation of monosaccharide metabolism. An expanded repertoire of HTCS proteins with diversified sensor domains may be one reason for B. thetaiotaomicron's success in our intestinal ecosystem.Bacteroides thetaiotaomicron ͉ glycoside hydrolases ͉ gut microbial ecology ͉ metabolic regulation ͉ signal transduction

Gas exchange between the environment and leaf interior is regulated by stomatal aperture. Stomatal opening results from guard cell swelling which is caused by increased osmotica in the cell. Most of the change in osmotic potential can be accounted for by K+ influx (2, 6). Potassium uptake is balanced by 18,19) and organic anion synthesis (1,14 The principle used for starch determination has been described by Lust et al. (9). Details of the initial portions of the assay are given below.Step 1. Guard cell contents were extracted in 20 nl of 0.2 N KOH containing 100 mm ethanol using the "oil well technique" (8). Incubation was for 20 min at 80 C. Starch standards were carried through all steps of the assay.Step 2. One hundred nl of specific step reagent was added to the oil well droplet. The reagent was 100 mm sodium acetate (70 mm acid, 30 mm base) and 200 Ag/ml amyloglucosidase (from Rhizopus, EC 3.2.1.3). Incubation was for 60 min at 30 C. This step was terminated by heating to 80 C for 20 min. Glucose standards were incorporated into some aliquots of this step and were carried through the remaining steps of the assay.Step 3. Fifty nl of glucose reagent was added to the reaction droplet. The reagent was 360 mm Tris-HCl (50 mm acid, 310 mM base), 3.6 mm MgCl2, 1 mM ATP, 0.1 mm NADP+, 3.6 mm DTT, 0.2 ,ug/ml glucose-6-P dehydrogenase (from yeast, EC 1.1.1.49) and 3.6 ,ug/ml hexokinase (from yeast, EC 2.7.1.1). Incubation was for 30 min at 24 C.Step 4. One pl of 0.1 N NaOH was added. The droplets were heated to 80 C for 20 min.Step 5. The NADP in a 0.5-,lI aliquot was amplified by an enzymic cycling technique (8).Heating in 0.2 N ethanolic KOH was necessary to make amylaceous polymers susceptible to enzymic degradation. When wheat starch, corn starch, "soluble" potato starch, shellfish glycogen, and rabbit liver glycogen were heated in only 0.02 N NaOH, the end point of glucose release by Aspergillus amyloglucosidase was 34, 53, 55, 88, and 97%, respectively, of

To investigate the role of glycogen synthase in controlling glycogen accumulation, we generated three lines of transgenic mice in which the enzyme was overexpressed in skeletal muscle by using promoter-enhancer elements derived from the mouse muscle creatine kinase gene. In all three lines, expression was highest in muscles composed primarily of fast-twitch fibers, such as the gastrocnemius and anterior tibialis. In these muscles, glycogen synthase activity was increased by as much as 10-fold, with concomitant increases (up to 5-fold) in the glycogen content. The uridine diphosphoglucose concentrations were markedly decreased, consistent with the increase in glycogen synthase activity. Levels of glycogen phosphorylase in these muscles increased (up to 3-fold), whereas the amount of the insulin-sensitive glucose transporter 4 either remained unchanged or decreased. The observation that increasing glycogen synthase enhances glycogen accumulation supports the conclusion that the activation of glycogen synthase, as well as glucose transport, contributes to the accumulation of glycogen in response to insulin in skeletal muscle.