Carmen A. Argmann

Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha.

Diminished mitochondrial oxidative phosphorylation and aerobic capacity are associated with reduced longevity. We tested whether resveratrol (RSV), which is known to extend lifespan, impacts mitochondrial function and metabolic homeostasis. Treatment of mice with RSV significantly increased their aerobic capacity, as evidenced by their increased running time and consumption of oxygen in muscle fibers. RSVs effects were associated with an induction of genes for oxidative phosphorylation and mitochondrial biogenesis and were largely explained by an RSV-mediated decrease in PGC-1alpha acetylation and an increase in PGC-1alpha activity. This mechanism is consistent with RSV being a known activator of the protein deacetylase, SIRT1, and by the lack of effect of RSV in SIRT1(-/-) MEFs. Importantly, RSV treatment protected mice against diet-induced-obesity and insulin resistance. These pharmacological effects of RSV combined with the association of three Sirt1 SNPs and energy homeostasis in Finnish subjects implicates SIRT1 as a key regulator of energy and metabolic homeostasis.

A gene expression network model of type 2 diabetes links cell cycle regulation in islets with diabetes susceptibility

Insulin resistance is necessary but not sufficient for the development of type 2 diabetes. Diabetes results when pancreatic beta-cells fail to compensate for insulin resistance by increasing insulin production through an expansion of beta-cell mass or increased insulin secretion. Communication between insulin target tissues and beta-cells may initiate this compensatory response. Correlated changes in gene expression between tissues can provide evidence for such intercellular communication. We profiled gene expression in six tissues of mice from an obesity-induced diabetes-resistant and a diabetes-susceptible strain before and after the onset of diabetes. We studied the correlation structure of mRNA abundance and identified 105 co-expression gene modules. We provide an interactive gene network model showing the correlation structure between the expression modules within and among the six tissues. This resource also provides a searchable database of gene expression profiles for all genes in six tissues in lean and obese diabetes-resistant and diabetes-susceptible mice, at 4 and 10 wk of age. A cell cycle regulatory module in islets predicts diabetes susceptibility. The module predicts islet replication; we found a strong correlation between (2)H(2)O incorporation into islet DNA in vivo and the expression pattern of the cell cycle module. This pattern is highly correlated with that of several individual genes in insulin target tissues, including Igf2, which has been shown to promote beta-cell proliferation, suggesting that these genes may provide a link between insulin resistance and beta-cell proliferation.

The metabolic footprint of aging in mice

Aging is characterized by a general decline in cellular function, which ultimately will affect whole body homeostasis. Although DNA damage and oxidative stress all contribute to aging, metabolic dysfunction is a common hallmark of aging at least in invertebrates. Since a comprehensive overview of metabolic changes in otherwise healthy aging mammals is lacking, we here compared metabolic parameters of young and 2 year old mice. We systemically integrated in vivo phenotyping with gene expression, biochemical analysis, and metabolomics, thereby identifying a distinguishing metabolic footprint of aging. Among the affected pathways in both liver and muscle we found glucose and fatty acid metabolism, and redox homeostasis. These alterations translated in decreased long chain acylcarnitines and increased free fatty acid levels and a marked reduction in various amino acids in the plasma of aged mice. As such, these metabolites serve as biomarkers for aging and healthspan.

Compromised intestinal lipid absorption in mice with a liver-specific deficiency of liver receptor homolog 1

ABSTRACT Bile acids (BAs) are water-soluble end products from cholesterol metabolism and are essential for efficient absorption of dietary lipids. By using targeted somatic mutagenesis of the nuclear receptor liver receptor homolog 1 (LRH-1) in mouse hepatocytes, we demonstrate here that LRH-1 critically regulates the physicochemical properties of BAs. The absence of LRH-1 and subsequent deficiency of Cyp8b1 eliminate the production of cholic acid and its amino acid conjugate taurocholic acid and increase the relative amounts of less amphipathic BA species. Intriguingly, while the expression of Cyp8b1 is almost extinguished in the livers of mice that lack LRH-1, the expression of the rate-limiting enzyme of BA synthesis, i.e., Cyp7a1, remains unchanged. The profound remodeling of the BA composition significantly reduces the efficacy of intestinal absorption of lipids and reuptake of BAs and facilitates the removal of lipids from the body. Our studies unequivocally demonstrate a pivotal role for LRH-1 in determining the composition of BAs, which, in turn has major consequences on whole-body lipid homeostasis.

Systems proteomics of liver mitochondria function

Expanded proteomic analysis of metabolism Combined analysis of large data sets characterizing genes, transcripts, and proteins can elucidate biological functions and disease processes. Williams et al. report an exceptionally detailed characterization of mitochondrial function in a genetic reference panel of recombinant inbred mice. They measured the metabolic function of nearly 400 mice under various environmental conditions and collected detailed quantitative information from livers of the animals on over 25,000 transcripts. These data were integrated with quantitation of over 2500 proteins and nearly 1000 metabolites. Such analysis showed a frequent lack of correlation of transcript and protein abundance, enabled the identification of genomic variants of mitochondrial enzymes that caused inborn errors in metabolism, and revealed two genes that appear to function in cholesterol metabolism. Science, this issue p. 10.1126/science.aad0189 Advances in mass spectrometry yield insights into mitochondrial function. INTRODUCTION Over the past two decades, continuous improvements in “omics” technologies have driven an ever-greater capacity to define the relationships between genetics, molecular pathways, and overall phenotypes. Despite this progress, the majority of genetic factors influencing complex traits remain unknown. This is exemplified by mitochondrial supercomplex assembly, a critical component of the electron transport chain, which remains poorly characterized. Recent advances in mass spectrometry have expanded the scope and reliability of proteomics and metabolomics measurements. These tools are now capable of identifying thousands of factors driving diverse molecular pathways, their mechanisms, and consequent phenotypes and thus substantially contribute toward the understanding of complex systems. RATIONALE Genome-wide association studies (GWAS) have revealed many causal loci associated with specific phenotypes, yet the identification of such genetic variants has been generally insufficient to elucidate the molecular mechanisms linking these genetic variants with specific phenotypes. A multitude of control mechanisms differentially affect the cellular concentrations of different classes of biomolecules. Therefore, the identification of the causal mechanisms underlying complex trait variation requires quantitative and comprehensive measurements of multiple layers of data—principally of transcripts, proteins, and metabolites and the integration of the resulting data. Recent technological developments now support such multiple layers of measurements with a high degree of reproducibility across diverse sample or patient cohorts. In this study, we applied a multilayered approach to analyze metabolic phenotypes associated with mitochondrial metabolism. RESULTS We profiled metabolic fitness in 386 individuals from 80 cohorts of the BXD mouse genetic reference population across two environmental states. Specifically, this extensive phenotyping program included the analysis of metabolism, mitochondrial function, and cardiovascular function. To understand the variation in these phenotypes, we quantified multiple, detailed layers of systems-scale measurements in the livers of the entire population: the transcriptome (25,136 transcripts), proteome (2622 proteins), and metabolome (981 metabolites). Together with full genomic coverage of the BXDs, these layers provide a comprehensive view on overall variances induced by genetics and environment regarding metabolic activity and mitochondrial function in the BXDs. Among the 2600 transcript-protein pairs identified, 85% of observed quantitative trait loci uniquely influenced either the transcript or protein level. The transomic integration of molecular data established multiple causal links between genotype and phenotype that could not be characterized by any individual data set. Examples include the link between D2HGDH protein and the metabolite D-2-hydroxyglutarate, the BCKDHA protein mapping to the gene Bckdhb, the identification of two isoforms of ECI2, and mapping mitochondrial supercomplex assembly to the protein COX7A2L. These respective measured variants in these mitochondrial proteins were in turn associated with varied complex metabolic phenotypes, such as heart rate, cholesterol synthesis, and branched-chain amino acid metabolism. Of note, our transomics approach clarified the contested role of COX7A2L in mitochondrial supercomplex formation and identified and validated Echdc1 and Mmab as involved in the cholesterol pathway. CONCLUSION Overall, these findings indicate that data generated by next-generation proteomics and metabolomics techniques have reached a quality and scope to complement transcriptomics, genomics, and phenomics for transomic analyses of complex traits. Using mitochondria as a case in point, we show that the integrated analysis of these systems provides more insights into the emergence of the observed phenotypes than any layer can by itself, highlighting the complementarity of a multilayered approach. The increasing implementation of these omics technologies as complements, rather than as replacements, will together move us forward in the integrative analysis of complex traits. Model of the transomics analysis. A transomics approach was taken to analyze genetic and environmental variation in metabolic and mitochondrial phenotypes by measuring five distinct layers of biology in a diverse population of BXD mice. The combined analysis of all layers together provides additional information not yielded by any single omics approach. Recent improvements in quantitative proteomics approaches, including Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), permit reproducible large-scale protein measurements across diverse cohorts. Together with genomics, transcriptomics, and other technologies, transomic data sets can be generated that permit detailed analyses across broad molecular interaction networks. Here, we examine mitochondrial links to liver metabolism through the genome, transcriptome, proteome, and metabolome of 386 individuals in the BXD mouse reference population. Several links were validated between genetic variants toward transcripts, proteins, metabolites, and phenotypes. Among these, sequence variants in Cox7a2l alter its protein’s activity, which in turn leads to downstream differences in mitochondrial supercomplex formation. This data set demonstrates that the proteome can now be quantified comprehensively, serving as a key complement to transcriptomics, genomics, and metabolomics—a combination moving us forward in complex trait analysis.

Multilayered genetic and omics dissection of mitochondrial activity in a mouse reference population

The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome--a subset of the metabolome--and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes. Most prominently, Dhtkd1 was identified as a primary regulator of 2-aminoadipate, explaining variance in fasted glucose and diabetes status in both mice and humans. These integrated molecular profiles also allowed further characterization of complex pathways, particularly the mitochondrial unfolded protein response (UPR(mt)). UPR(mt) shows strikingly variant responses at the transcript and protein level that are remarkably conserved among C. elegans, mice, and humans. Overall, these examples demonstrate the value of an integrated multilayered omics approach to characterize complex metabolic phenotypes.

Evaluation of glucose homeostasis.

Obesity and dyslipidemia are often found in association with insulin resistance (IR). These components combined with hypertension characterize the most common endocrine disorder in humans, the metabolic syndrome. Thus, in addition to profiling body weight evolution and lipid metabolites, glucose tolerance (a reflection of IR) and insulin sensitivity should also be considered as part of any metabolic phenotyping protocol. The ability to measure IR and glucose tolerance is important not only in the quest to fully understand the pathogenesis of the metabolic syndrome in the mouse, but also to test the effects of potential interventions. This unit presents a variety of tests used for this purpose, including direct blood glucose measurements, insulin measurement by ELISA, the homeostatic model assessment, glucose tolerance and insulin sensitivity tests, and the euglycemic clamp.

Peroxisome proliferator-activated receptor gamma: the more the merrier?

The consequence of activating the nuclear hormone receptor, peroxisome proliferator‐activated receptor gamma (PPARγ), which coordinates adipocyte differentiation, validates the concept, ‘you are what you eat’. Excessive caloric intake leads to fat formation if the energy from these nutrients is not expended. However, this evolutionary adaptation to store energy in fat, which can be released under the form of fatty acids, potent PPARγ agonists, has become a disadvantage in todays affluent society as it results in numerous metabolic imbalances, collectively known as the metabolic syndrome. With the surge of human and genetic studies on PPARγ function, the limitations to the benefits of PPARγ signalling have been realized. It is now evident that the most effective strategy for resetting the balance of this thrifty gene is through its modulation rather than full activation, with the goal to improve glucose homeostasis while preventing adipogenesis. Finally, as more PPARγ targeted pathways are revealed such as bone homeostasis, atherosclerosis and longevity, it is most certain that the PPARγ thrifty gene hypothesis will evolve to incorporate these.

The Pro12Ala PPARγ2 Variant Determines Metabolism at the Gene-Environment Interface

The metabolic impact of the common peroxisome proliferator-activated receptor gamma isoform 2 (PPARgamma2) variant Pro12Ala in human populations has been widely debated. We demonstrate, using a Pro12Ala knockin model, that on chow diet, Ala/Ala mice are leaner, have improved insulin sensitivity and plasma lipid profiles, and have longer lifespans. Gene-environment interactions played a key role as high-fat feeding eliminated the beneficial effects of the Pro12Ala variant on adiposity, plasma lipids, and insulin sensitivity. The underlying molecular mechanisms involve changes in cofactor interaction and adiponectin signaling. Altogether, our results establish the Pro12Ala variant of Ppargamma2 as an important modulator in metabolic control that strongly depends on the metabolic context.

Peroxisomal L-bifunctional enzyme (Ehhadh) is essential for the production of medium-chain dicarboxylic acids

L-bifunctional enzyme (Ehhadh) is part of the classical peroxisomal fatty acid β-oxidation pathway. This pathway is highly inducible via peroxisome proliferator-activated receptor α (PPARα) activation. However, no specific substrates or functions for Ehhadh are known, and Ehhadh knockout (KO) mice display no appreciable changes in lipid metabolism. To investigate Ehhadh functions, we used a bioinformatics approach and found that Ehhadh expression covaries with genes involved in the tricarboxylic acid cycle and in mitochondrial and peroxisomal fatty acid oxidation. Based on these findings and the regulation of Ehhadhs expression by PPARα, we hypothesized that the phenotype of Ehhadh KO mice would become apparent after fasting. Ehhadh mice tolerated fasting well but displayed a marked deficiency in the fasting-induced production of the medium-chain dicarboxylic acids adipic and suberic acid and of the carnitine esters thereof. The decreased levels of adipic and suberic acid were not due to a deficient induction of ω-oxidation upon fasting, as Cyp4a10 protein levels increased in wild-type and Ehhadh KO mice.We conclude that Ehhadh is indispensable for the production of medium-chain dicarboxylic acids, providing an explanation for the coordinated induction of mitochondrial and peroxisomal oxidative pathways during fasting.