Jill K. Manchester

Mechanisms underlying the resistance to diet-induced obesity in germ-free mice

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; carnitine-palmitoyltransferase). 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 proliferator-activated receptor coactivator (Pgc-1α) and enzymes involved in fatty acid oxidation. Thus, GF animals are protected from diet-induced obesity by two complementary but independent mechanisms that result in increased fatty acid metabolism: (i) elevated levels of Fiaf, which induces Pgc-1α; 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.

Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41.

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 short-chain 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 conventionally-raised 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.

Genomic and metabolic adaptations of Methanobrevibacter smithii to the human gut

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. smithiis 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.

Enhanced Gluconeogenesis and Increased Energy Storage as Hallmarks of Aging in Saccharomyces cerevisiae

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 ofSaccharomyces 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,sip2Δ, and snf4Δ 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 agingsip2Δ cells, and that it is attenuated in longer-livedsnf4Δ 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.

Diversity of Metabolic Patterns in Human Brain Tumors: Enzymes of Energy Metabolism and Related Metabolites and Cofactors

Abstract: Biopsies from 15 human gliomas, five meningiomas, four Schwannomas, one medulloblastoma, and four normal brain areas were analyzed for 12 enzymes of energy metabolism and 12 related metabolites and cofactors. Samples, 0.01–0.25 μg dry weight, were dissected from freeze‐dried microtome sections to permit all the assays on a given specimen to be made, as far as possible, on nonnecrotic pure tumor tissue from the same region. Great diversity was found with regard to both enzyme activities and metabolite levels among individual tumors, but the following generalities can be made. Activities of hexokinase, phosphorylase, phosphofructokinase, glycerophosphate dehydrogenase, citrate synthase, and malate dehydrogenase levels were usually lower than in brain; glycogen synthase and glucose‐6‐phosphate dehydrogenase were usually higher; and the averages for pyruvate kinase, lactate dehydrogenase, 6‐phosphogluconate dehydrogenase, and β‐hydroxyacyl coenzyme A dehydrogenase were not greatly different from brain. Levels of eight of the 12 enzymes were distinctly lower among the Schwannomas than in the other two groups. Average levels of glucose‐6‐phosphate, lactate, pyruvate, and uridine diphosphoglucose were more than twice those of brain; 6‐phosphogluconate and citrate were about 70% higher than in brain; glucose, glycogen, glycerol‐1‐phosphate, and malate averages ranged from 104% to 127% of brain; and fructose‐1,6‐bisphosphate and glucose‐1,6‐bis‐phosphate levels were on the average 50% and 70% those of brain, respectively.

Administration of prostaglandin E1 after lung transplantation improves early graft function

Early graft dysfunction continues to be a major clinical problem after lung transplantation. The objective of this experiment was to determine whether continuous administration of prostaglandin E1 (PGE1) after lung transplantation has a beneficial effect on early graft function. Left lung allotransplantation was performed in 10 size-matched mongrel dogs (weight, 24.4 to 31.4 kg). Lung preservation consisted of a bolus injection of PGE1 (250 micrograms) into the pulmonary artery, followed by a pulmonary artery flush with 50 mL/kg of 4 degrees C modified Euro-Collins solution. The lungs were then stored at 1 degree C for 12 hours. Left lung transplantation was performed using standard technique. The right pulmonary artery and right bronchus were ligated prior to chest closure. Animals were placed in the supine position and ventilated for 6 hours with 100% oxygen at a rate of 20 breaths/min, a tidal volume of 550 mL, and a positive end-expiratory pressure of 5 cm H2O. Animals were randomly allocated to one of two groups. Group I animals (n = 6) received continuous PGE1 infusion from the onset of implantation. The dose was gradually increased and fixed when mean systemic pressure showed a 10% decrease (mean PGE1 dose, 31.7 +/- 6.9 ng.kg-1.min-1). Group II animals (n = 4) received no PGE1. After the 6-hour assessment period, arterial oxygen tension and alveolar-arterial oxygen pressure difference were preserved in group I compared with group II (group I versus group II: arterial oxygen tension, 255.8 +/- 37.6 mm Hg versus 64.7 +/- 7.9 mm Hg [p < 0.05]; alveolar-arterial oxygen pressure difference, 411.1 +/- 70.5 mm Hg versus 597.5 +/- 1.3 mm Hg [p < 0.05]).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of microgravity on metabolic enzymes of individual muscle fibers

Eleven enzymes were measured in individual fibers of soleus and tibialis anterior (TA) muscles from two flight and two control (synchronous) animals. There were five enzymes of glycogenolytic metabolism: phosphorylase, glucose‐6‐phosphate isomerase, glyccrol‐3‐phosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase (group GLY); five of oxidative metabolism: citrate synthase, malate dehydrogenase, β‐hydroxyacyl‐CoA dehydrogenase, 3‐ketoacid CoA‐transferase, and mitochondrial thiolase (group OX); and hexokinase, subserving both groups. Fiber size (dry weight per unit length) was reduced about 35% in both muscles. On a dry weight basis, hexokinase levels were increased 100% or more in flight fibers from both soleus and TA. Group OX enzymes increased 56‐193% in TA without significant change in soleus. Group GLY enzymes increased an average of 28% in soleus fibers but underwent, if anything, a modest decrease (20%) in TA fibers. These changes in composition of TA fibers were those anticipated for a conversion of about half of the originally predominant fast glycolytic fibers into fast oxidative glycolytic fibers. Calculation on the basis of fiber length, rather than dry weight, gave an estimate of absolute enzyme changes: hexokinase was still calculated to have increased in both soleus and TA fibers, but only by 50 and 25%, respectively. Three of the OX enzymes were, on this basis, unchanged in TA fibers, but 3‐ketoacid CoA‐transferase and thiolase had still nearly doubled, whereas TA GLY enzymes had fallen about 40%. In soleus fibers, absolute levels of OX enzymes had decreased an average of 25% and GLY enzymes were marginally decreased.—Manchester, J. K.; Chi, M. M.‐Y.; Norris, B.; Ferrier, B.; Krasnov, I.; Nemeth, P. M.; McDougal, D. B., Jr.; and Lowry, O. H. Effect of microgravity on metabolic enzymes of individual muscle fibers. FASEB J. 4: 55‐63; 1990.

Linkage between cellular communications, energy utilization, and proliferation in metastatic neuroendocrine cancers

To identify metabolic features that support the aggressive behavior of human neuroendocrine (NE) cancers, we examined metastatic prostate NE tumors and derived prostate NE cancer (PNEC) cell lines from a transgenic mouse model using a combination of magic angle spinning NMR spectroscopy, in silico predictions of biotransformations that observed metabolites may undergo, biochemical tests of these predictions, and electrophysiological/calcium imaging studies. Malignant NE cells undergo excitation and increased proliferation when their GABAA, glutamate, and/or glycine receptors are stimulated, use glutamate and GABA as substrates for NADH biosynthesis, and produce propylene glycol, a precursor of pyruvate derived from glycine that increases levels of circulating free fatty acids through extra-NE cell effects. Treatment of nude mice containing PNEC tumor xenografts with (i) amiloride, a diuretic that inhibits Abp1, an enzyme involved in NE cell GABA metabolism, (ii) carbidopa, an inhibitor of dopa decarboxylase which functions upstream of Abp1, plus (iii) flumazenil, a benzodiazepine antagonist that binds to GABAA receptors, leads to significant reductions in tumor growth. These findings may be generally applicable: GeneChip data sets from 471 human neoplasms revealed that components of GABA metabolic pathways, including ABP1, exhibit statistically significant increases in their expression in NE and non-NE cancers.

Measurement of 2-deoxyglucose and 2-deoxyglucose 6-phosphate in tissues☆

The enzymatic methods previously described for 2-deoxyglucose (DG) and 2-deoxyglucose 6-phosphate have been refined and adapted to measurements of brain samples ranging from 50 mg wet weight to less than a microgram dry weight. Procedures for preparing such samples for assay are described. Analytical properties of the enzymes employed are given together with means for overcoming their possible short comings. Emphasis is placed on information useful for employing DG to assess rapid changes in glucose metabolism.

The relationship between protein content and dry weight of guard cells and other single cell samples ofVicia faba L. leaflet

The specific activities of plant enzymes are usually expressed on a chlorophyll, dry weight or protein basis. Although the latter is usually preferred, chlorophyll or dry weight are also adequate when comparisons are within one cell, tissue or organ type. If the comparisons are between dissimilar samples, interpretations may be quite different depending on the basis for comparison. For example, NADP+-malic enzyme on a chlorophyll basis is 402-fold higher in leaf epidermis than in the remainder of the leaf, but only 6.2-fold higher on a protein basis (Willmer et al., 1973). Differences between specific activities on a protein or dry weight basis are certain to exist at the cell level because of large differences in cell wall thicknesses (Fig. 1). This is particularly interesting to us because guard cells (Fig. 1), our main research interest, have very thick cell walls compared to other leaf cells (Figs. 1A, B). Unfortunately, protein estimation on single cell samples is not possible if these same samples are to be used for enzyme assays. However, it is convenient and non-destructive to weigh them on a Lowry fibre balance (Lowry & Passonneau, 1972) prior to assay. This communication reports protein: dry weight conversion factors for several cell types in Vicia leaflet. In addition, we report a brief evaluation of the quantitative histochemical methodology used. In preliminary experiments, protein determinations (at the/~g level) were made on several commercially available protein sources, extracts of Vicia leaflets and extracts of Vicia guard cell protoplasts. Bovine serum albumin (BSA) was the standard in all protein assays. Protein extractions and protoplast isolation were carried out according to Outlaw et aI. (1981). Plants were grown as described by Outlaw & Manchester (1979). Protein sources were as indicated in Table 1. Analytical enzymes were from Boehringer; other biochemicals were from Sigma. Protein assays were by (a) the Folin method (Lowry et al., 1951) to which the other protein assays were compared; (b) Coomassie Blue binding method (Bradford, 1976); (c) glutamate measurement after acid hydrolysis (Butcher & Lowry, 1976); (d) aspartate measurement after acid hydrolysis (a variation of Butcher & Lowry, 1976); and (e) amino acid estimation by 0-phthaldialdehyde after acid hydrolysis (Butcher & Lowry, 1976). The aim of these preliminary experiments was to compare results by procedures (c), (d) and (e)