Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice

3 Фев 2014 | Author: | No comments yet »
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Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice


Here, we study the intricate relationship between gut microbiota and host cometabolic phenotypes associated with dietary-induced impaired glucose homeostasis and nonalcoholic fatty liver disease (NAFLD) in a mouse strain (129S6) known to be susceptible to these disease traits, using plasma and urine metabotyping, achieved by 1 H NMR spectroscopy. Multivariate statistical modeling of the spectra shows that the genetic predisposition of the 129S6 mouse to impaired glucose homeostasis and NAFLD is associated with disruptions of choline metabolism, i.e. low circulating levels of plasma phosphatidylcholine and high urinary excretion of methylamines (dimethylamine, trimethylamine, and trimethylamine- N -oxide), coprocessed by symbiotic gut microbiota and mammalian enzyme systems. Conversion of choline into methylamines by microbiota in strain 129S6 on a high-fat diet reduces the bioavailability of choline and mimics the effect of choline-deficient diets, causing NAFLD. These data also indicate that gut microbiota may play an active role in the development of insulin resistance.

Highly complex animals such as mammals can be considered as “superorganisms” with a karyome, a chondriome, and a microbiome (1 ), resulting from a coevolutionary symbiotic ecosystem of diverse intestinal microbiota interacting metabolically with the host (2 ). Recent molecular analyses of human microbiota 16s ribosomal DNA sequences revealed a majority of uncultivated or unknown species with a strong degree of interindividual diversity (3. 4 ). Also, some of the molecular foundations of beneficial symbiotic host–bacteria relationships in the gut were revealed by colonization of germ-free mice with known microbes and by comparisons of the genomes of members of the intestinal microbiota (5 ). For instance, Bacteroides thetaiotaomicron . a dominant member of normal distal intestinal microbiota, hydrolyzes otherwise indigestible dietary polysaccharides, thus supplying the host with 10–15% of calorific requirement (6 ). Gut Lactobacillus spp. are also responsible for a significant proportion of bile acid deconjugation, a process that efficiently reduces lipid absorption in the gut (7 ). Such symbiotic relationships are the result of coevolution and operate at the genome, proteome, and metabolome levels (6. 8 ).

Insulin resistance (IR) is central to a cluster of frequent and increasingly prevalent pathologies, including type 2 diabetes mellitus, central obesity, hypertension hepatic steatosis, and dyslipidemia (9 ). IR contributes to major causes of morbidity and mortality worldwide (10 ). Epidemiological and genetic studies in human and animal models have demonstrated the importance of both genetic and environmental factors in the etiology of IR (9 ): Dietary variation and intervention, in particular, have a strong influence on the development of IR. Nonalcoholic fatty liver disease (NAFLD), is the most frequent liver condition associated with IR (11 ). It is associated with hepatic IR and characterized by hepatic accumulation of triglycerides, or steatosis. Although the causes of human NAFLD are not understood, it has been shown in animal models that choline-deficient diets are associated with NAFLD (12 ).

The critical involvement of the gut microbiota in biological processes controlling host metabolic regulations (13 ), including those involved in insulin sensitivity and caloric recovery from the diet, is emerging from recent studies (14 ): Conventionalized animals have 40% more body fat than germ-free animals. Moreover, diet is known to modulate gut-microbial composition (15 ), and obesity correlates with variation in the distribution of Bacteroidetes and Firmicutes in mice (16 ). Hence, symbiotic bacterial contributions to IR and NAFLD should not be overlooked.

Novel approaches are emerging to measure and model metabolism in diverse compartments in interacting multicellular systems that also involve symbiotic microorganisms (2 ). Alongside functional genomic profiling methods such as transcriptomics and proteomics, metabonomics is a metabolic systems-biology approach that can be encapsulated as “understanding the metabolic responses of living systems to pathophysiological stimuli by using multivariate statistical analysis of biological NMR spectroscopic data” (17. 18 ). 1 H NMR spectroscopy of biofluids has long been established as a method for profiling abnormal biochemistry and, indeed, was applied to describe diabetic and hyperglyceridaemic phenotypes 20 years ago (19 ). We have recently applied metabonomics to characterize the intergenome interactions in mice with symbiotic gut microflora and parasitic Schistosoma mansoni infection in mice (20 ). We have also monitored the gut-microbial metabolite variation in urine from acclimatizing formerly germ-free rats (21 ).

In this study, we have tested the effects of dietary changes, i.e. switching from a 5% control low-fat diet (LFD) to a 40% high-fat diet (HFD), on plasma and urine metabolic 1 H NMR profiles in inbred mouse strain 129S6, documented for its susceptibility to IR or NAFLD (22 ), and in BALBc strain, which exhibits evidence of resistance to these phenotypes. We characterize here the metabolic profiles related to the cometabolome homeostatic variation (23 ) and show that microbial metabolism strongly contributes to a NAFLD metabotype, i.e. a quantitative combination of several metabolites, related to IR.


Overview of the Pathophysiological Effects of Fat-Feeding.

We present here background data primarily focused on glucose tolerance and glucose-induced insulin secretion in vivo as well as structural and biochemical markers of hepatic dysfunction.

Glucose Homeostasis and Insulin Secretion.

Fasting insulinemia was not significantly different between the strains on LFDs, but, in response to glucose, 129S6 mice secrete more insulin during the GTT than BALB/c mice (Fig. 1 B ). This apparent increased insulin-secretion capacity in 129S6 when compared with BALB/c does not result in improved glucose tolerance (Fig. 1 A ), suggesting a relative reduction of the biological action of insulin in 129S6 mice compared with BALB/c mice. In BALB/c mice, fat-feeding induces a strongly significant enhancement of glucose-stimulated insulin secretion when compared with the LFD-fed group (Fig. 1 B ), which may account for concomitant improved glucose tolerance by HFD in this strain (Fig. 1 A ). In contrast, in 129S6, insulin-secretion pattern and capacity are not significantly altered by HFD, indicating that glucose intolerance in HFD-fed 129S6 mice develops as the result of insulin resistance rather than insulin-secretion deficiency (Fig. 1 A and B ).

Plasma Lipid Profiles.

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To characterize dyslipidemia, we applied standard clinical chemistry protocols to compare the concentration of plasma lipids, including triglycerides (TG) and total, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) cholesterol, in the four mouse groups (A.T. unpublished work). On LFD, the two strains show similar levels of plasma lipids, and prolonged HFD feeding induced similar hypercholesterolemia in both strains (Fig. 4 A – D . which is published as supporting information on the PNAS web site).

Liver Histopathology and Dysfunction.

After prolonged high-fat feeding, 129S6 mice develop micro- and macrovesicular steatosis, as evidenced by the accumulation of fat droplets in the liver (Fig. 1 D ), whereas liver histology remained unchanged in BALB/c mice (Fig. 1 C ). To further characterize steatosis, we assayed hepatic TG (one of the major storage forms of lipids in liver) and circulating levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (markers of hepatic dysfunction). Liver TG are significantly increased (3.3-fold) in fat-fed 129S6 mice (Fig. 4 E ). The levels of both AST and ALT are higher in 129S6 compared with BALB/c mice on LFD. Fat-feeding induces a significant increase in ALT and AST in both strains that is more prominent in 129S6 than BALB/c mice (Fig. 1 F and G ).

Body Weight Follow-Up.

To characterize obesity, we monitored body weight at different ages. On LFDs, body weight is similar in 129S6 and BALB/c mice at 2, 3, and 5 months of age. In 129S6 mice, body weight is significantly increased after 3, 7, and 15 weeks of HFD when compared with age- and strain-matched LFD-fed mice, whereas it remains similar in LFD- and HFD-fed BALB/c mice (data not shown).

Overall, our observations provide confirmatory evidence of the strong susceptibility of 129S6 mice to NAFLD, impaired glucose tolerance, dyslipidemia, and obesity in response to fat-feeding as observed by Biddinger et al . (22 ) and provide evidence of resistance to these pathologies in BALB/c mice.

Metabolic Profiling by 1 H NMR Spectroscopy at 4 Months After HFD Induction.

The chronic effects of the HFD induction on urinary and plasma metabolic profiles at 4 months after induction for 129S6 and BALB/c mouse strains are illustrated in Fig. 2 and Table 1. As shown in Fig. 2 A . a typical 600-MHz 1 H NMR spectrum of one HFD-fed 129S6 mouse plasma characterizes common markers of insulin resistance: (CH 2 ) n and CH 3 resonances from components of lipoproteins, e.g. cholesterol esters, TG, and phospholipids. A peak from the N -methyl group from phosphatidylcholine (PC) can be observed. Low-molecular-mass metabolites, e.g. lactate, alanine, and glucose, are also present (Fig. 2 A ).

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