Iphigenia Tzameli

TLR4 links innate immunity and fatty acid–induced insulin resistance

TLR4 is the receptor for LPS and plays a critical role in innate immunity. Stimulation of TLR4 activates proinflammatory pathways and induces cytokine expression in a variety of cell types. Inflammatory pathways are activated in tissues of obese animals and humans and play an important role in obesity-associated insulin resistance. Here we show that nutritional fatty acids, whose circulating levels are often increased in obesity, activate TLR4 signaling in adipocytes and macrophages and that the capacity of fatty acids to induce inflammatory signaling in adipose cells or tissue and macrophages is blunted in the absence of TLR4. Moreover, mice lacking TLR4 are substantially protected from the ability of systemic lipid infusion to (a) suppress insulin signaling in muscle and (b) reduce insulin-mediated changes in systemic glucose metabolism. Finally, female C57BL/6 mice lacking TLR4 have increased obesity but are partially protected against high fat diet-induced insulin resistance, possibly due to reduced inflammatory gene expression in liver and fat. Taken together, these data suggest that TLR4 is a molecular link among nutrition, lipids, and inflammation and that the innate immune system participates in the regulation of energy balance and insulin resistance in response to changes in the nutritional environment.

Enhanced leptin sensitivity and attenuation of diet-induced obesity in mice with haploinsufficiency of Socs3

Leptin is an adipocyte-derived hormone that regulates energy balance and neuroendocrine function primarily by acting on specific hypothalamic pathways. Resistance to the weight reducing effects of leptin is a feature of most cases of human and rodent obesity, yet the molecular basis of leptin resistance is poorly understood. We have previously identified suppressor of cytokine signaling-3 (Socs3) as a leptin-induced negative regulator of leptin receptor signaling and potential mediator of leptin resistance. However, due to the non-viability of mice with targeted disruption of Socs3 (ref. 6), the importance of Socs3 in leptin action in vivo was unclear. To determine the functional significance of Socs3 in energy balance in vivo we undertook studies in mice with heterozygous Socs3 deficiency (Socs3+/−). We report here that Socs3+/− mice display greater leptin sensitivity than wild-type control mice: Socs3+/− mice show both enhanced weight loss and increased hypothalamic leptin receptor signaling in response to exogenous leptin administration. Furthermore, Socs3+/− mice are significantly protected against the development of diet-induced obesity and associated metabolic complications. The level of Socs3 expression is thus a critical determinant of leptin sensitivity and obesity susceptibility in vivo and this molecule is a potential target for therapeutic intervention.

The Xenobiotic Compound 1,4-Bis(2-(3,5-Dichloropyridyloxy))Benzene Is an Agonist Ligand for the Nuclear Receptor CAR

ABSTRACT A wide range of xenobiotic compounds are metabolized by cytochrome P450 (CYP) enzymes, and the genes that encode these enzymes are often induced in the presence of such compounds. Here, we show that the nuclear receptor CAR can recognize response elements present in the promoters of xenobiotic-responsive CYP genes, as well as other novel sites. CAR has previously been shown to be an apparently constitutive transactivator, and this constitutive activity is inhibited by androstanes acting as inverse agonists. As expected, the ability of CAR to transactivate the CYP promoter elements is blocked by the inhibitory inverse agonists. However, CAR transactivation is increased in the presence of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), the most potent known member of the phenobarbital-like class of CYP-inducing agents. Three independent lines of evidence demonstrate that TCPOBOP is an agonist ligand for CAR. The first is that TCPOBOP acts in a dose-dependent manner as a direct agonist to compete with the inhibitory effect of the inverse agonists. The second is that TCPOBOP acts directly to stimulate coactivator interaction with the CAR ligand binding domain, both in vitro and in vivo. The third is that mutations designed to block ligand binding block not only the inhibitory effect of the androstanes but also the stimulatory effect of TCPOBOP. Importantly, these mutations do not block the apparently constitutive transactivation by CAR, suggesting that this activity is truly ligand independent. Both its ability to target CYP genes and its activation by TCPOBOP demonstrate that CAR is a novel xenobiotic receptor that may contribute to the metabolic response to such compounds.

Regulated Production of a Peroxisome Proliferator-activated Receptor-γ Ligand during an Early Phase of Adipocyte Differentiation in 3T3-L1 Adipocytes

Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear hormone receptor that is critical for adipogenesis and insulin sensitivity. Ligands for PPARγ include some polyunsaturated fatty acids and prostanoids and the synthetic high affinity antidiabetic agents thiazolidinediones. However, the identity of a biologically relevant endogenous PPARγ ligand is unknown, and limited insight exists into the factors that may regulate production of endogenous PPARγ ligands during adipocyte development. To address this question, we created a line of 3T3-L1 preadipocytes that carry a β-galactosidase-based PPARγ ligand-sensing vector system. In this system, induction of adipogenesis resulted in elevated β-galactosidase activity that signifies activation of PPARγ via its ligand-binding domain (LBD) and suggests generation and/or accumulation of a ligand moiety. The putative endogenous ligand appeared early in adipogenesis in response to increases in cAMP, accumulated in the medium, and dissipated later in adipogenesis. Organically extracted and high pressure liquid chromatography-fractionated conditioned media from differentiating cells, but not from mature adipocytes, were enriched in this activity. One or more components within the organic extract activated PPARγ through interaction with its LBD, induced lipid accumulation in 3T3-L1 cells as efficiently as the differentiation mixture, and competed for binding of rosiglitazone to the LBD of PPARγ. The active species appears to be different from other PPARγ ligands identified previously. Our findings suggest that a novel biologically relevant PPARγ ligand is transiently produced in 3T3-L1 cells during adipogenesis.

Complex Role of the Vitamin D Receptor and Its Ligand in Adipogenesis in 3T3-L1 Cells

The vitamin D receptor (VDR) and its ligand 1,25-OH2-VD3 (calcitriol) play an essential role in mineral homeostasis in mammals. Interestingly, the VDR is expressed very early in adipogenesis in 3T3-L1 cells, suggesting that the VDR signaling pathway may play a role in adipocyte biology and function. Indeed, it has been known for a number of years that calcitriol is a potent inhibitor of adipogenesis in this model but with no clear mechanism identified. In this study, we have further defined the molecular mechanism by which the unliganded VDR and calcitriol-liganded VDR regulate adipogenesis. In the presence of calcitriol, the VDR blocks adipogenesis by down-regulating both C/EBPβ mRNA expression and C/EBPβ nuclear protein levels at a critical stage of differentiation. In addition, calcitriol allows for the up-regulation of the recently described C/EBPβ corerepressor, ETO, which would further inhibit the action of any remaining C/EBPβ, whose action is required for adipogenesis. In contrast, in the absence of calcitriol, the unliganded VDR appears necessary for lipid accumulation, since knock-down of the VDR using siRNA both delays and prevents this process. Taken together, these data support the notion that the intracellular concentrations of calcitriol can play an important role in either promoting or inhibiting adipogenesis via the VDR and the transcriptional pathways that it targets. Further examination of this hypothesis in vivo may shed new light on the biology of adipogenesis.

Role reversal: new insights from new ligands for the xenobiotic receptor CAR

In the classic model of nuclear receptor signaling, specific hormone binding results in the recruitment of coactivator proteins and transcriptional activation. Recent results with newly characterized nuclear receptors have expanded this model to include new types of ligands and novel transcriptional responses. Both inverse agonists and conventional agonist ligands have been identified for the xenobiotic receptor constitutive androstane receptor (CAR), a constitutive activator of transcription in the absence of ligands.

Decreased expression of peroxisome proliferator activated receptor γ in CFTR-/- mice

Some of the pathological manifestations of cystic fibrosis are in accordance with an impaired expression and/or activity of PPARγ. We hypothesized that PPARγ expression is altered in tissues lacking the normal cystic fibrosis transmembrane regulator protein (CFTR). PPARγ mRNA levels were measured in colonic mucosa, ileal mucosa, adipose tissue, lung, and liver from wild‐type and cftr−/− mice by quantitative RT‐PCR. PPARγ expression was decreased twofold in CFTR‐regulated tissues (colon, ileum, and lung) from cftr−/− mice compared to wild‐type littermates. In contrast, no differences were found in fat and liver. Immunohistochemical analysis of PPARγ in ileum and colon revealed a predominantly nuclear localization in wild‐type mucosal epithelial cells while tissues from cftr−/− mice showed a more diffuse, lower intensity labeling. A significant decrease in PPARγ expression was confirmed in nuclear extracts of colon mucosa by Western blot analysis. In addition, binding of the PPARγ/RXR heterodimer to an oligonucletotide containing a peroxisome proliferator responsive element (PPRE) was also decreased in colonic mucosa extracts from cftr−/− mice. Treatment of cftr−/− mice with the PPARγ ligand rosiglitazone restored both the nuclear localization and binding to DNA, but did not increase RNA levels. We conclude that PPARγ expression in cftr−/− mice is downregulated at the RNA and protein levels and its function diminished. These changes may be related to the loss of function of CFTR and may be relevant to the pathogenesis of metabolic abnormalities associated with cystic fibrosis in humans.

Decreased expression of peroxisome proliferator activated receptor gamma in cftr-/- mice.

Some of the pathological manifestations of cystic fibrosis are in accordance with an impaired expression and/or activity of PPARγ. We hypothesized that PPARγ expression is altered in tissues lacking the normal cystic fibrosis transmembrane regulator protein (CFTR). PPARγ mRNA levels were measured in colonic mucosa, ileal mucosa, adipose tissue, lung, and liver from wild‐type and cftr−/− mice by quantitative RT‐PCR. PPARγ expression was decreased twofold in CFTR‐regulated tissues (colon, ileum, and lung) from cftr−/− mice compared to wild‐type littermates. In contrast, no differences were found in fat and liver. Immunohistochemical analysis of PPARγ in ileum and colon revealed a predominantly nuclear localization in wild‐type mucosal epithelial cells while tissues from cftr−/− mice showed a more diffuse, lower intensity labeling. A significant decrease in PPARγ expression was confirmed in nuclear extracts of colon mucosa by Western blot analysis. In addition, binding of the PPARγ/RXR heterodimer to an oligonucletotide containing a peroxisome proliferator responsive element (PPRE) was also decreased in colonic mucosa extracts from cftr−/− mice. Treatment of cftr−/− mice with the PPARγ ligand rosiglitazone restored both the nuclear localization and binding to DNA, but did not increase RNA levels. We conclude that PPARγ expression in cftr−/− mice is downregulated at the RNA and protein levels and its function diminished. These changes may be related to the loss of function of CFTR and may be relevant to the pathogenesis of metabolic abnormalities associated with cystic fibrosis in humans.

Nicotinamide N-methyltransferase regulates hepatic nutrient metabolism through Sirt1 protein stabilization

Nicotinamide N-methyltransferase (Nnmt) methylates nicotinamide, a form of vitamin B3, to produce N1-methylnicotinamide (MNAM). Nnmt has emerged as a metabolic regulator in adipocytes, but its role in the liver, the tissue with the strongest Nnmt expression, is not known. In spite of its overall high expression, here we find that hepatic expression of Nnmt is highly variable and correlates with multiple metabolic parameters in mice and humans. Further, we find that suppression of hepatic Nnmt expression in vivo alters glucose and cholesterol metabolism and that the metabolic effects of Nnmt in the liver are mediated by its product MNAM. Supplementation of high-fat diet with MNAM decreases serum and liver cholesterol and liver triglycerides levels in mice. Mechanistically, increasing Nnmt expression or MNAM levels stabilizes sirtuin 1 protein, an effect that is required for their metabolic benefits. In summary, we describe here a novel regulatory pathway for vitamin B3 that could provide a new opportunity for metabolic disease therapy.

The evolving role of mitochondria in metabolism

Mitochondria, the cells powerhouses, produce up to 95% of a eukaryotic cells energy (ATP) through oxidative phosphorylation to fuel cellular activity. They are also highly dynamic organelles that constantly remodel and turn over. The number of mitochondria and/or activity change in response to a variety of physiological conditions such as exercise, nutrients, or with aging. Several of these properties and the regulatory mechanisms that are involved in optimal mitochondrial function were summarized in a recent Cell SnapShot (Figure 1Figure 1) that is reproduced here as a backdrop for the current discussion. In addition, hear the related podcast that discusses changes in mitochondrial dynamics in nutrient loaded pancreatic beta-cells.Figure 1Mitochondrial Quality ControlFactors and pathways involved in the maintenance of optimal mitochondrial function and some of the changes that take place in dysfunctional mitochondria. Reprinted from [1xSnapShot: Mitochondrial quality control. Green, D.R. and Van Houten, B. Cell. 2011; 147Abstract | Full Text PDF | Scopus (18)See all References][1].View Large Image | Download PowerPoint SlideThe physiological importance of mitochondria has been widely appreciated for a long time, as disorders of the mitochondrial respiratory chain are associated with a number of major diseases, and dysfunctional mitochondria have been linked to health problems ranging from cancer to neurodegeneration and type 2 diabetes mellitus (T2DM). Progress over the past few years has produced new insights that advance our understanding of the mitochondrial function in metabolism and the metabolic syndrome, and new ideas have emerged for strategies to develop therapeutic approaches.This special issue of Trends in Endocrinology and Metabolism is a collection of articles dedicated to the mitochondrion and focuses on some of the most recent advances related to metabolic diseases. Harper et al. eloquently review the transformation of cellular redox potential into ATP synthetic capacity, and discusse the regulation of mitochondrial reactive oxygen species (ROS) production by the uncoupling proteins (UCPs). UCPs cause proton leaks resulting in poor fuel conversion efficiency, and some UCPs control ROS production from the mitochondrial respiratory chain. ROS may trigger cell signaling, but at excessive levels it might also contribute to mitochondrial dysfunction and disease development. This observation highlights the profound physiological effects UPCs have on mitochondria, as they acutely modulate mitochondrial ROS emission to maintain optimal function in a normal cell. Murphy et al. expand on the topic of redox potential and explain how a transient elevation in ROS production has positive effects. Under conditions of excess nutrient supply and low ATP demand, increased ROS act as a feedback signal to slow substrate oxidation and to divert carbohydrates to storage as fat, helping to decrease overall ROS production and facilitate more efficient energy usage. However, a combination of excess nutrition and physical inactivity, an underlying driver for the metabolic syndrome, chronically overactivates these redox signaling pathways and may contribute to pathology. This mechanistic link raises the possibility that pharmacological approaches to target ROS production in mitochondria could have therapeutic benefit.An essential molecule in mitochondrial function is nicotinamide adenine dinucleotide (NAD). The next article by Imai et al. highlights the importance of preserving an optimal pool of mitochondrial NAD, a co-factor consumed by key cellular mediators such as the sirtuins and poly-ADP-ribose polymerases (PARPs). Not only is the maintenance of an optimal NAD/NADH ratio essential for mitochondrial function, but so is a balance between NAD production and consumption. This physiological juggling act involves biosynthetic, transport, and catabolic enzymes and is responsive to nutritional and environmental stimuli including diet and aging. Its physiological importance suggests that there may be options for nutriceutical-based interventions focused on NAD intermediates that could improve human health. The NAD-dependent protein deacetylases Sirtuins are molecular sensors of cellular energy balance and have been implicated in regulating metabolism, stress responses, and aging. Verdin et al. offer us excellent insights into the functions of the three mitochondrial sirtuins by taking us through their distinct deacylase activities in response to acetylation, malonylation, or succinylation of proteins. They propose a conceptual model in which changes in energy homeostasis or nutrient availability lead to changes in the levels of metabolites including acyl-coAs. Acyl-coAs influence mitochondrial function by catalyzing the acylation of metabolic enzymes thus potentially changing their function, and mitochondrial sirtuins act to remove these groups from mitochondrial proteins. This balance of forces might ultimately coordinate the network of metabolic fluxes in response to dynamic changes in the metabolic state. Another key function is mitochondrial biogenesis. Kelly et al. dissect the transcriptional circuitry, both nuclear and mitochondrial, that controls mitochondrial biogenesis and provide unique and current insights into the regulation of these genes with emphasis on the role of key signal transduction pathways. At the core of the discussion is the peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family of proteins. These transcriptional co-activators, when activated by AMPK or the sirtuin SIRT1, co-activate transcription factors involved in respiratory gene expression, which in turn enhance mitochondrial biogenesis and oxidative function and increase functional mitochondrial capacity. Developing strategies to modulate this pathway represents yet another angle for potential therapeutic intervention.Mitochondria are present in all cell types except erythrocytes, and have cell-specific functions as well as general roles. An emerging and very interesting topic is the role of mitochondria in white adipose tissue (WAT). Although the importance of mitochondria in brown adipose tissue homeostasis has been discussed extensively, our understanding of the relevance of mitochondria in WAT homeostasis is still relatively limited. Scherer et al. skillfully tackle this topic and discuss how mitochondria might play a crucial role in WAT homeostasis and systemic insulin sensitivity, through their ability to influence key biochemical processes central to the adipocyte, such as fatty acid esterification and lipogenesis. The potential regulatory role of WAT mitochondria on whole body physiology suggests that therapeutic interventions, such as ones that increase WAT mitochondrial biogenesis, could positively impact systemic metabolism. Another topic that has recently attracted considerable attention is impaired mitochondrial oxidative phosphorylation in skeletal muscle, and its implication in the pathogenesis of human insulin resistance (IR) and T2DM. As discussed by Schrauwen et al, diminished mitochondrial function, most likely secondary to the development of IR, may aggravate the condition. Thus, the improved mitochondrial capacity that can be achieved through exercise and calorie restriction, most likely contributes to the positive metabolic health effects of these lifestyle changes. In addition, activators of AMPK, SIRT1, or PGC-1α may improve diminished mitochondrial function and future research focused on optimizing exercise training interventions or finding small molecules and stimulators of muscle mitochondrial capacity could help improve the metabolic profile of IR subjects and T2DM patients. Maechler et al. continue the tissue-specific theme and discuss how mitochondrial dysfunction affects pancreatic beta cell function. The authors provide a broad overview of factors contributing to mitochondrial dysfunction and discuss how oxidative stress, gluco(lipo)toxicity, or rare mitochondrial DNA mutations lead to beta cell apoptosis. The good news is that since the majority of alterations originate from an environment of inappropriate dietary composition and overfeeding, simple life style modifications might help reverse mitochondrial dysfunction and hence retain beta cell function. Still, major challenges remain because most of the factors contributing to beta cell dysfunction remain at the candidate stage. A better understanding of these putative targets will help drive translational approaches. Last but not least, Rustin et al. describe potential therapies for correcting or managing inborn errors of oxidative metabolism. It is estimated that over a million individuals suffer from clinical diseases related to mtDNA mutations. Because disorders of the mitochondrial respiratory chain manifest themselves very differently and underlie a large number of clinical conditions, the field is challenged by the need for both broad based and disease specific therapies. We currently have the ability to slow disease progression for only a very limited number of mitochondrial diseases, but promising new approaches based on nutritional modulation and the use of chemicals that act on factors involved in diet-associated regulatory pathways such as SIRT1, AMPK, PGC1, will hopefully lead to meaningful clinical trials.Collectively, the articles in this special issue highlight the evolving role of mitochondria in metabolism and emphasize some of the most promising areas of research for future exploration. We greatly appreciate the contributions that all of the authors and reviewers made to this special issue, and we hope you will enjoy the way that the overall collection highlights various different areas of current interest. All articles will be freely available on the TEM website. We would welcome any comments or feedback that you have by email at tem@cell.com or via Twitter to @Trends_Endo_Met.