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1.  Global Transcript Profiles of Fat in Monozygotic Twins Discordant for BMI: Pathways behind Acquired Obesity  
PLoS Medicine  2008;5(3):e51.
The acquired component of complex traits is difficult to dissect in humans. Obesity represents such a trait, in which the metabolic and molecular consequences emerge from complex interactions of genes and environment. With the substantial morbidity associated with obesity, a deeper understanding of the concurrent metabolic changes is of considerable importance. The goal of this study was to investigate this important acquired component and expose obesity-induced changes in biological pathways in an identical genetic background.
Methods and Findings
We used a special study design of “clonal controls,” rare monozygotic twins discordant for obesity identified through a national registry of 2,453 young, healthy twin pairs. A total of 14 pairs were studied (eight male, six female; white), with a mean ± standard deviation (SD) age 25.8 ± 1.4 y and a body mass index (BMI) difference 5.2 ± 1.8 kg/m2. Sequence analyses of mitochondrial DNA (mtDNA) in subcutaneous fat and peripheral leukocytes revealed no aberrant heteroplasmy between the co-twins. However, mtDNA copy number was reduced by 47% in the obese co-twin's fat. In addition, novel pathway analyses of the adipose tissue transcription profiles exposed significant down-regulation of mitochondrial branched-chain amino acid (BCAA) catabolism (p < 0.0001). In line with this finding, serum levels of insulin secretion-enhancing BCAAs were increased in obese male co-twins (9% increase, p = 0.025). Lending clinical relevance to the findings, in both sexes the observed aberrations in mitochondrial amino acid metabolism pathways in fat correlated closely with liver fat accumulation, insulin resistance, and hyperinsulinemia, early aberrations of acquired obesity in these healthy young adults.
Our findings emphasize a substantial role of mitochondrial energy- and amino acid metabolism in obesity and development of insulin resistance.
Leena Peltonen and colleagues uncover the metabolic changes that result from obesity through an analysis of genetically identical twin pairs in which one was obese and the other was not.
Editors' Summary
Around the world, the proportion of people who are obese (people with an unhealthy amount of body fat) is increasing. In the US, for example, 1 adult in 7 was obese in the mid 1970s. That is, their body mass index (BMI)—their weight in kilograms divided by their height in meters squared—was more than 30. Nowadays, 1 US adult in 3 has a BMI this high and, by 2025, it is predicted that 1 in 2 will be obese. This obesity epidemic is being driven by lifestyle changes that encourage the over-consumption of energy-rich foods and discourage regular physical activity. The resultant energy imbalance leads to weight gain (the excess energy is stored as body fat or adipose tissue) and also triggers numerous metabolic changes, alterations in the chemical processes that convert food into the energy and various substances needed to support life. These obesity-related metabolic changes increase a person's risk of developing adverse health conditions such as diabetes, a condition in which dangerously high levels of sugar from food accumulate in the blood.
Why Was This Study Done?
The changes in human fat in obesity have not been completely understood, although the abnormal metabolism of adipose tissue is increasingly seen as playing a critical part in excessive weight gain. It has been very difficult to decipher which molecular and metabolic changes associated with obesity are the result of becoming obese, and which might contribute towards the acquisition of obesity in humans in the first place. To discover more about the influence of environment on obesity-induced metabolic changes, the researchers in this study have investigated these changes in pairs of genetically identical twins.
What Did the Researchers Do and Find?
The researchers recruited 14 pairs of genetically identical Finnish twins born between 1975 and 1979 who were “obesity discordant”—that is, one twin of each pair had a BMI of about 25 (not obese); the other had a BMI of about 30 (obese). The researchers took fat and blood samples from each twin, determined the insulin sensitivity of each, and measured the body composition and various fat stores of each. They found that the obese twins had more subcutaneous, intra-abdominal, and liver fat and were less insulin sensitive than the non-obese twins. Insulin sensitivity correlated with the amount of liver fat. Analysis of gene expression in the fat samples showed that 19 gene pathways (mainly inflammatory pathways) were expressed more strongly (up-regulated) in the obese twins than the non-obese twins, whereas seven pathways were down-regulated. The most highly down-regulated pathway was a mitochondrial pathway involved in amino acid breakdown, but mitochondrial energy metabolism pathways were also down-regulated. Finally, mitochondrial DNA copy number in fat was reduced in the obese twins by nearly half, a novel observation that could partly account for the obesity-induced metabolic defects of these individuals.
What Do These Findings Mean?
These and other findings identify several pathways that are involved in the development of obesity and insulin resistance. In particular, they suggest that changes in mitochondrial energy production pathways and in mitochondrial amino acid metabolism pathways could play important roles in the development of obesity and of insulin resistance and in the accumulation of liver fat even in young obese people. The study design involving identical twins has here produced some evidence for aberrations in molecules critical for acquired obesity. The results suggest that careful management of obesity by lifestyle changes has the potential to correct the obesity-related metabolic changes in fat that would otherwise lead to diabetes and other adverse health conditions in obese individuals. In addition, they suggest that the development of therapies designed to correct mitochondrial metabolism might help to reduce the illnesses associated with obesity.
Additional Information.
Please access these Web sites via the online version of this summary at
The MedlinePlus encyclopedia has pages on obesity and diabetes (in English and Spanish)
The US Centers for Disease Control and Prevention provides information on all aspects of obesity (in English and Spanish)
The UK National Health Service's health Web site (NHS Direct) provides information about obesity
The International Obesity Taskforce provides information about preventing obesity and on diabetes and obesity
The UK Foods Standards Agency and the United States Department of Agriculture provide online tools and useful advice about healthy eating for adults and children
Information is available for patients and carers from the US National Diabetes Information Clearinghouse on diabetes, including information on insulin resistance
PMCID: PMC2265758  PMID: 18336063
2.  Role of T Cells in Malnutrition and Obesity 
Nutritional status is critically important for immune cell function. While obesity is characterized by inflammation that promotes metabolic syndrome including cardiovascular disease and insulin resistance, malnutrition can result in immune cell defects and increased risk of mortality from infectious diseases. T cells play an important role in the immune adaptation to both obesity and malnutrition. T cells in obesity have been shown to have an early and critical role in inducing inflammation, accompanying the accumulation of inflammatory macrophages in obese adipose tissue, which are known to promote insulin resistance. How T cells are recruited to adipose tissue and activated in obesity is a topic of considerable interest. Conversely, T cell number is decreased in malnourished individuals, and T cells in the setting of malnutrition have decreased effector function and proliferative capacity. The adipokine leptin, which is secreted in proportion to adipocyte mass, may have a key role in mediating adipocyte-T cell interactions in both obesity and malnutrition, and has been shown to promote effector T cell function and metabolism while inhibiting regulatory T cell proliferation. Additionally, key molecular signals are involved in T cell metabolic adaptation during nutrient stress; among them, the metabolic regulator AMP kinase and the mammalian target of rapamycin have critical roles in regulating T cell number, function, and metabolism. In summary, understanding how T cell number and function are altered in obesity and malnutrition will lead to better understanding of and treatment for diseases where nutritional status determines clinical outcome.
PMCID: PMC4127479  PMID: 25157251
obesity; inflammation; T cells; malnutrition; leptin
3.  Effects of obesity on bone metabolism 
Obesity is traditionally viewed to be beneficial to bone health because of well-established positive effect of mechanical loading conferred by body weight on bone formation, despite being a risk factor for many other chronic health disorders. Although body mass has a positive effect on bone formation, whether the mass derived from an obesity condition or excessive fat accumulation is beneficial to bone remains controversial. The underline pathophysiological relationship between obesity and bone is complex and continues to be an active research area. Recent data from epidemiological and animal studies strongly support that fat accumulation is detrimental to bone mass. To our knowledge, obesity possibly affects bone metabolism through several mechanisms. Because both adipocytes and osteoblasts are derived from a common multipotential mesenchymal stem cell, obesity may increase adipocyte differentiation and fat accumulation while decrease osteoblast differentiation and bone formation. Obesity is associated with chronic inflammation. The increased circulating and tissue proinflammatory cytokines in obesity may promote osteoclast activity and bone resorption through modifying the receptor activator of NF-κB (RANK)/RANK ligand/osteoprotegerin pathway. Furthermore, the excessive secretion of leptin and/or decreased production of adiponectin by adipocytes in obesity may either directly affect bone formation or indirectly affect bone resorption through up-regulated proinflammatory cytokine production. Finally, high-fat intake may interfere with intestinal calcium absorption and therefore decrease calcium availability for bone formation. Unraveling the relationship between fat and bone metabolism at molecular level may help us to develop therapeutic agents to prevent or treat both obesity and osteoporosis.
Obesity, defined as having a body mass index ≥ 30 kg/m2, is a condition in which excessive body fat accumulates to a degree that adversely affects health [1]. The rates of obesity rates have doubled since 1980 [2] and as of 2007, 33% of men and 35% of women in the US are obese [3]. Obesity is positively associated to many chronic disorders such as hypertension, dyslipidemia, type 2 diabetes mellitus, coronary heart disease, and certain cancers [4-6]. It is estimated that the direct medical cost associated with obesity in the United States is ~$100 billion per year [7].
Bone mass and strength decrease during adulthood, especially in women after menopause [8]. These changes can culminate in osteoporosis, a disease characterized by low bone mass and microarchitectural deterioration resulting in increased bone fracture risk. It is estimated that there are about 10 million Americans over the age of 50 who have osteoporosis while another 34 million people are at risk of developing the disease [9]. In 2001, osteoporosis alone accounted for some $17 billion in direct annual healthcare expenditure.
Several lines of evidence suggest that obesity and bone metabolism are interrelated. First, both osteoblasts (bone forming cells) and adipocytes (energy storing cells) are derived from a common mesenchymal stem cell [10] and agents inhibiting adipogenesis stimulated osteoblast differentiation [11-13] and vice versa, those inhibiting osteoblastogenesis increased adipogenesis [14]. Second, decreased bone marrow osteoblastogenesis with aging is usually accompanied with increased marrow adipogenesis [15,16]. Third, chronic use of steroid hormone, such as glucocorticoid, results in obesity accompanied by rapid bone loss [17,18]. Fourth, both obesity and osteoporosis are associated with elevated oxidative stress and increased production of proinflammatory cytokines [19,20]. At present, the mechanisms for the effects of obesity on bone metabolism are not well defined and will be the focus of this review.
PMCID: PMC3141563  PMID: 21676245
bone; fat; obesity; osteoporosis; inflammation
4.  The endocannabinoid system links gut microbiota to adipogenesis 
We investigated several models of gut microbiota modulation: selective (prebiotics, probiotics, high-fat), drastic (antibiotics, germ-free mice) and mice bearing specific mutations of a key gene involved in the toll-like receptors (TLR) bacteria-host interaction (Myd88−/−). Here we report that gut microbiota modulates the intestinal endocannabinoid (eCB) system-tone, which in turn regulates gut permeability and plasma lipopolysaccharide (LPS) levels.The activation of the intestinal endocannabinoid system increases gut permeability which in turn enhances plasma LPS levels and inflammation in physiological and pathological conditions such as obesity and type 2 diabetes.The investigation of adipocyte differentiation and lipogenesis (both markers of adipogenesis) indicate that gut microbiota controls adipose tissue physiology through LPS-eCB system regulatory loops and may play a critical role in the adipose tissue plasticity during obesity.In vivo, ex vivo and in vitro studies indicate that LPS acts as a master switch on adipose tissue metabolism, by blocking the cannabinoid-driven adipogenesis.
Obesity and type II diabetes have reached epidemic proportions and are associated with a massive expansion of the adipose tissue. Recent data have shown that these metabolic disorders are characterised by low-grade inflammation of unknown molecular origin (Hotamisligil and Erbay, 2008; Shoelson and Goldfine, 2009); therefore, it is of the utmost importance to identify the link between inflammation and adipose tissue metabolism and plasticity. Among the latest important discoveries published in the field, two new concepts have driven this study. First, emerging data have shown that gut microbiota is involved in the control of energy homeostasis (Ley et al, 2005; Turnbaugh et al, 2006; Claus et al, 2008) Obesity is characterised by the massive expansion of adipose tissues and is associated with inflammation (Weisberg et al, 2003). It is possible that both this expansion and the associated inflammation are controlled by microbiota and lipopolysaccharide (LPS) (Cani et al, 2007a, 2008), a cell wall component of Gram-negative bacteria that is among the most potent inducers of inflammation (Cani et al, 2007a, 2007b, 2008; Cani and Delzenne, 2009). Second, obesity is also characterised by greater endocannabinoid (eCB) system tone (increased eCB plasma levels, altered expression of the cannabinoid receptor 1 (CB1 mRNA) and increased eCB levels in the adipose tissue) (Engeli et al, 2005; Bluher et al, 2006; Matias et al, 2006; Cote et al, 2007; D'Eon et al, 2008; Starowicz et al, 2008; Di Marzo et al, 2009; Izzo et al, 2009).
Several studies have suggested a close relationship between LPS, gut microbiota and the eCB system. Indeed, LPS controls the synthesis of eCB in macrophages, whereas macrophage infiltration in the adipose tissue occurring during obesity is an important factor in the development of the metabolic disorders (Weisberg et al, 2003). We have shown that macrophage infiltration is not only dependent on the activation of the receptor CD14 by LPS, but is also dependent on the gut microbiota composition and the gut barrier function (gut permeability) (Cani et al, 2007a, 2008). Moreover, LPS controls the synthesis of eCBs both in vivo (Hoareau et al, 2009) and in vitro (Di Marzo et al, 1999; Maccarrone et al, 2001) through mechanisms dependent of the LPS receptor signalling pathway (Liu et al, 2003). Thus, obesity is nowadays associated with changes in gut microbiota and a higher endocannabinoid system tone, both having a function in the disease's pathophysiology.
Given that the convergent molecular mechanisms that may affect these different supersystem activities and adiposity remain to be elucidated, we tested the hypothesis that the gut microbiota and the eCB system control gut permeability and adipogenesis, by a LPS-dependent mechanism, under both physiological and obesity-related conditions.
First, we found that high-fat diet-induced obese and diabetic animals exhibit threefold higher colonic CB1 mRNA, whereas no modification was observed in the small intestinal segment (jejunum). Moreover, selective modulation of gut microbiota using prebiotics (i.e. non-digestible compounds fermented by specific bacteria in the gut) (Gibson and Roberfroid, 1995) reduces by about one half this effect. Similarly, in genetically obese mice (ob/ob), prebiotic treatment decreases colonic CB1 mRNA and colonic eCB concentrations (AEA) (Figure 2A). In addition, we have observed a modulation of FAAH and MGL mRNA (Figure 2A). Furthermore, we have found that antibiotic treatment decreasing the number of gut bacteria content was associated with a strong reduction of the CB1 receptor levels in the colon of healthy mice.
Second, we show that the endocannabinoid system controls gut barrier function (in vivo and in vitro) and endotoxaemia. More precisely, we designed two in vivo experiments in obese and lean mice (Figure 2). In a first experiment, we blocked the CB1 receptor in obese mice with a specific and selective antagonist (SR141716A) and found that the blockade of the CB1 receptor reduces plasma LPS levels by a mechanism linked to the improvement of the gut barrier function (Figure 2C) as shown by the lower alteration of tight junctions proteins (zonula occludens-1 (ZO-1) and occludin) distribution and localisation, and independently of food intake behaviour (Figures 2D and 3). In a second set of experiments performed in lean wild-type mice, we mimicked the increased eCB system tone observed during obesity by chronic (4-week) infusion of a cannabinoid receptor agonist (HU-210) through mini-pumps implanted subcutaneously. We found that cannabinoid agonist administration significantly increased plasma LPS levels. Furthermore, increased plasma fluorescein isothiocyanate-dextran levels were observed after oral gavage (Figure 2F and G). These sets of in vivo experiments strongly suggest that an overactive eCB system increases gut permeability. Finally, in a cellular model of intestinal epithelial barrier (Caco-2 cells monolayer), we found that CB1 receptor antagonist normalised LPS and the cannabinoid receptors agonist HU-210-induced epithelial barrier alterations.
Third, we provide evidence that adipogenesis is under the control of the gut microbiota, through the modulation of the gut and adipose tissue endocannabinoid systems in both physiological and pathological conditions. We found that the higher eCB system tone (found in obesity or mimicked by eCB agonist) participates to the regulation of adipogenesis by directly acting on the adipose tissue, but also indirectly by increasing plasma LPS levels, which consequently impair adipogenesis and promote inflammatory states. Here, we found that both the specific modulation of the gut microbiota and the blockade of the CB1 receptor decrease plasma LPS levels and is associated with higher adipocyte differentiation and lipogenesis rate. One possible explanation for these surprising data could be as follows: plasma LPS levels might be under the control of CB1 in the intestine (gut barrier function); therefore, under particular pathophysiological conditions in vivo (e.g. obesity/type II diabetes), this could lead to higher circulating LPS levels. Furthermore, CB1 receptor blockade might paradoxically increase adipogenesis because of the ability of CB1 antagonist to reduce gut permeability and counteract the LPS-induced inhibitory effect on adipocyte differentiation and lipogenesis (i.e. a disinhibition mechanism). In summary, given that these treatments reduce gut permeability and, hence, plasma LPS levels and inflammatory tone, we hypothesised that LPS could act as a regulator in this process. This hypothesis was further supported in vitro and in vivo by the observation that cannabinoid-induced adipocyte differentiation and lipogenesis were directly altered (i.e. reduced) in the presence of physiological levels of LPS. In summary, because these treatments reduce gut permeability, hence, plasma LPS and inflammatory tone, we hypothesised that LPS acts as a regulator in this process. Altogether, our data provide the evidence that the consequences of obesity and gut microbiota dysregulation on gut permeability and metabolic endotoxaemia are clearly mediated by the eCB system, those observed on adiposity are likely the result of two systems interactions: LPS-dependent pathways activities and eCB system tone dysregulation (Figure 9).
Our results indicate that the endocannabinoid system tone and the plasma LPS levels have a critical function in the regulation of the adipose tissue plasticity. As obesity is commonly characterised by increased eCB system tone, higher plasma LPS levels, altered gut microbiota and impaired adipose tissue metabolism, it is likely that the increased eCB system tone found in obesity is caused by a failure or a vicious cycle within the pathways controlling the eCB system.
These findings show that two novel therapeutic targets in the treatment of obesity, the gut microbiota and the endocannabinoid system, are closely interconnected. They also provide evidence for the presence of a new integrative physiological axis between gut and adipose tissue regulated by LPS and endocannabinoids. Finally, we propose that the increased endotoxaemia and endocannabinoid system tone found in obesity might explain the altered adipose tissue metabolism.
Obesity is characterised by altered gut microbiota, low-grade inflammation and increased endocannabinoid (eCB) system tone; however, a clear connection between gut microbiota and eCB signalling has yet to be confirmed. Here, we report that gut microbiota modulate the intestinal eCB system tone, which in turn regulates gut permeability and plasma lipopolysaccharide (LPS) levels. The impact of the increased plasma LPS levels and eCB system tone found in obesity on adipose tissue metabolism (e.g. differentiation and lipogenesis) remains unknown. By interfering with the eCB system using CB1 agonist and antagonist in lean and obese mouse models, we found that the eCB system controls gut permeability and adipogenesis. We also show that LPS acts as a master switch to control adipose tissue metabolism both in vivo and ex vivo by blocking cannabinoid-driven adipogenesis. These data indicate that gut microbiota determine adipose tissue physiology through LPS-eCB system regulatory loops and may have critical functions in adipose tissue plasticity during obesity.
PMCID: PMC2925525  PMID: 20664638
adipose tissue; endocannabinoids; gut microbiota; lipopolysaccharide (LPS); obesity
5.  Effects of Leucine Supplementation and Serum Withdrawal on Branched-Chain Amino Acid Pathway Gene and Protein Expression in Mouse Adipocytes 
PLoS ONE  2014;9(7):e102615.
The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of the transcriptomic response of this pathway to variations in nutrient availability.
PMCID: PMC4106850  PMID: 25050624
6.  Association of Lipidome Remodeling in the Adipocyte Membrane with Acquired Obesity in Humans 
PLoS Biology  2011;9(6):e1000623.
The authors describe a new approach to studying cellular lipid profiles and propose a compensatory mechanism that may help maintain the normal membrane function of adipocytes in the context of obesity.
Identification of early mechanisms that may lead from obesity towards complications such as metabolic syndrome is of great interest. Here we performed lipidomic analyses of adipose tissue in twin pairs discordant for obesity but still metabolically compensated. In parallel we studied more evolved states of obesity by investigating a separated set of individuals considered to be morbidly obese. Despite lower dietary polyunsaturated fatty acid intake, the obese twin individuals had increased proportions of palmitoleic and arachidonic acids in their adipose tissue, including increased levels of ethanolamine plasmalogens containing arachidonic acid. Information gathered from these experimental groups was used for molecular dynamics simulations of lipid bilayers combined with dependency network analysis of combined clinical, lipidomics, and gene expression data. The simulations suggested that the observed lipid remodeling maintains the biophysical properties of lipid membranes, at the price, however, of increasing their vulnerability to inflammation. Conversely, in morbidly obese subjects, the proportion of plasmalogens containing arachidonic acid in the adipose tissue was markedly decreased. We also show by in vitro Elovl6 knockdown that the lipid network regulating the observed remodeling may be amenable to genetic modulation. Together, our novel approach suggests a physiological mechanism by which adaptation of adipocyte membranes to adipose tissue expansion associates with positive energy balance, potentially leading to higher vulnerability to inflammation in acquired obesity. Further studies will be needed to determine the cause of this effect.
Author Summary
Obesity is characterized by excess body fat, which is predominantly stored in the adipose tissue. When adipose tissue expands too much it stops storing lipid appropriately. The excess lipid accumulates in organs such as muscle, liver, and pancreas, causing metabolic disease. In this study, we aim to identify factors that cause adipose tissue to malfunction when it reaches its limit of expansion. We performed lipidomic analyses of human adipose tissue in twin pairs discordant for obesity—that is, one of the twins was lean and one was obese—but still metabolically healthy. We identified multiple changes in membrane phospholipids. Using computer modeling, we show that “lean” and “obese” membrane lipid compositions have the same physical properties despite their different compositions. We hypothesize that this represents allostasis—changes in lipid membrane composition in obesity occur to protect the physical properties of the membranes. However, protective changes cannot occur without a cost, and accordingly we demonstrate that switching to the “obese” lipid composition is associated with higher levels of adipose tissue inflammation. In a separate group of metabolically unhealthy obese individuals we investigated how the processes that regulate the “lean” and “obese” lipid profiles are changed. To determine how these lipid membrane changes are regulated we constructed an in silico network model that identified key control points and potential molecular players. We validated this network by performing genetic manipulations in cell models. Therapeutic targeting of this network may open new opportunities for the prevention or treatment of obesity-related metabolic complications.
PMCID: PMC3110175  PMID: 21666801
7.  Impact of Obesity on IL-12 Family Gene Expression in Insulin Responsive Tissues 
Biochimica et biophysica acta  2012;1832(1):11-19.
Mounting evidence has established a role for chronic inflammation in the development of obesity-induced insulin resistance, as genetic ablation of pro-inflammatory cytokines and chemokines elevated in obesity improves insulin signaling in vitro and in vivo. Recent evidence further highlights interleukin (IL)-12 family cytokines as prospective inflammatory mediators linking obesity to insulin resistance. In this study, we present empirical evidence demonstrating that IL-12 family related genes are expressed and regulated in insulin-responsive tissues under conditions of obesity. First, we report that respective mRNAs for each of the known members of this cytokine family are expressed within detectable ranges in WAT, skeletal muscle, liver and heart. Second, we show that these cytokines and their cognate receptors are divergently regulated with genetic obesity in a tissue-specific manner. Third, we demonstrate that select IL-12 family cytokines are regulated in WAT in a manner that is dependent on the developmental stage of obesity as well as the inflammatory progression associated with obesity. Fourth, we report that respective mRNAs for IL-12 cytokines and receptors are also expressed and divergently regulated in cultured adipocytes under conditions of inflammatory stress. To our knowledge, this report is the first study to systemically evaluated mRNA expression of all IL-12 family cytokines and receptors in any tissue under conditions of obesity highlighting select family members as potential mediators linking excess nutrient intake to metabolic diseases such as insulin resistance, diabetes and heart disease.
PMCID: PMC3545633  PMID: 22952004
obesity; adipose tissue; adipocyte; inflammation; cytokines
8.  SCS3 and YFT2 Link Transcription of Phospholipid Biosynthetic Genes to ER Stress and the UPR 
PLoS Genetics  2012;8(8):e1002890.
The ability to store nutrients in lipid droplets (LDs) is an ancient function that provides the primary source of metabolic energy during periods of nutrient insufficiency and between meals. The Fat storage-Inducing Transmembrane (FIT) proteins are conserved ER–resident proteins that facilitate fat storage by partitioning energy-rich triglycerides into LDs. FIT2, the ancient ortholog of the FIT gene family first identified in mammals has two homologs in Saccharomyces cerevisiae (SCS3 and YFT2) and other fungi of the Saccharomycotina lineage. Despite the coevolution of these genes for more than 170 million years and their divergence from higher eukaryotes, SCS3, YFT2, and the human FIT2 gene retain some common functions: expression of the yeast genes in a human embryonic kidney cell line promotes LD formation, and expression of human FIT2 in yeast rescues the inositol auxotrophy and chemical and genetic phenotypes of strains lacking SCS3. To better understand the function of SCS3 and YFT2, we investigated the chemical sensitivities of strains deleted for either or both genes and identified synthetic genetic interactions against the viable yeast gene-deletion collection. We show that SCS3 and YFT2 have shared and unique functions that connect major biosynthetic processes critical for cell growth. These include lipid metabolism, vesicular trafficking, transcription of phospholipid biosynthetic genes, and protein synthesis. The genetic data indicate that optimal strain fitness requires a balance between phospholipid synthesis and protein synthesis and that deletion of SCS3 and YFT2 impacts a regulatory mechanism that coordinates these processes. Part of this mechanism involves a role for SCS3 in communicating changes in the ER (e.g. due to low inositol) to Opi1-regulated transcription of phospholipid biosynthetic genes. We conclude that SCS3 and YFT2 are required for normal ER membrane biosynthesis in response to perturbations in lipid metabolism and ER stress.
Author Summary
The ability to form lipid droplets is a conserved property of eukaryotic cells that allows the storage of excess metabolic energy in a form that can be readily accessed. In adipose tissue, the storage of excess calories in lipid droplets normally protects other tissues from lipotoxicity and insulin resistance, but this protection is lost with chronic over-nutrition. The FAT storage-inducing transmembrane (FIT) proteins were recently identified as a conserved family of proteins that reside in the lipid bilayer of the endoplasmic reticulum and are implicated in lipid droplet formation. In this work we show that specific functions of the FIT proteins are conserved between yeast and humans and that SCS3 and YFT2, the yeast homologs of mammalian FIT2, are part of a large genetic interaction network connecting lipid metabolism, vesicle trafficking, transcription, and protein synthesis. From these interactions we determined that yeast strains lacking SCS3 and YFT2 are defective in their response to chronic ER stress and cannot induce the unfolded protein response pathway or transcription of phospholipid biosynthetic genes in low inositol. Our findings suggest that the mammalian FIT genes may play an important role in ER stress pathways, which are linked to obesity and type 2 diabetes.
PMCID: PMC3426550  PMID: 22927826
9.  Control of Metabolic Homeostasis by Stress Signaling Is Mediated by the Lipocalin NLaz 
PLoS Genetics  2009;5(4):e1000460.
Metabolic homeostasis in metazoans is regulated by endocrine control of insulin/IGF signaling (IIS) activity. Stress and inflammatory signaling pathways—such as Jun-N-terminal Kinase (JNK) signaling—repress IIS, curtailing anabolic processes to promote stress tolerance and extend lifespan. While this interaction constitutes an adaptive response that allows managing energy resources under stress conditions, excessive JNK activity in adipose tissue of vertebrates has been found to cause insulin resistance, promoting type II diabetes. Thus, the interaction between JNK and IIS has to be tightly regulated to ensure proper metabolic adaptation to environmental challenges. Here, we identify a new regulatory mechanism by which JNK influences metabolism systemically. We show that JNK signaling is required for metabolic homeostasis in flies and that this function is mediated by the Drosophila Lipocalin family member Neural Lazarillo (NLaz), a homologue of vertebrate Apolipoprotein D (ApoD) and Retinol Binding Protein 4 (RBP4). Lipocalins are emerging as central regulators of peripheral insulin sensitivity and have been implicated in metabolic diseases. NLaz is transcriptionally regulated by JNK signaling and is required for JNK-mediated stress and starvation tolerance. Loss of NLaz function reduces stress resistance and lifespan, while its over-expression represses growth, promotes stress tolerance and extends lifespan—phenotypes that are consistent with reduced IIS activity. Accordingly, we find that NLaz represses IIS activity in larvae and adult flies. Our results show that JNK-NLaz signaling antagonizes IIS and is critical for metabolic adaptation of the organism to environmental challenges. The JNK pathway and Lipocalins are structurally and functionally conserved, suggesting that similar interactions represent an evolutionarily conserved system for the control of metabolic homeostasis.
Author Summary
Metabolism of multicellular organisms has to adjust to environmental changes. Insulin signaling plays an important role in this regulation. Stress signals can repress Insulin signaling, curtailing growth to promote stress tolerance and extend lifespan. While this interaction allows managing energy resources under stress conditions, excessive JNK activity in adipose tissue of vertebrates has been found to promote type II diabetes. Thus, the interaction between stress and Insulin signaling has to be carefully regulated to ensure proper metabolic adaptation. Here, we identify a new regulatory mechanism by which stress signaling influences metabolism in fruitflies. We show that an evolutionarily conserved secreted protein, Neural Lazarillo (NLaz), is induced in response to stress signals, and that it is required for metabolic regulation. NLaz mutant animals are more sensitive to stress and show significant metabolic deficiencies. Similarly, increased expression of NLaz inhibits growth, but increases stress and starvation tolerance. We show that these functions are mediated by an interaction with the Insulin signaling pathway. Our results show that the regulation of NLaz by stress signals is critical for metabolic adaptation of the organism to environmental challenges. Both the Insulin and JNK signaling mechanisms analyzed here are evolutionarily conserved, suggesting that similar interactions control metabolic adaptation in vertebrates.
PMCID: PMC2667264  PMID: 19390610
10.  Molecular characterization of the evolution of phagosomes 
First large-scale comparative proteomics/phosphoproteomics study characterizing some of the key steps that contributed to the remodeling of phagosomes that occurred during evolution. Comparison of profiling analyses of isolated phagosomes from three distant organisms (Dictyostelium, Drosophila, and mouse) revealed a protein core that defines a potential ‘ancient' phagosome and a set of 50 proteins that emerged while adaptive immunity was already well established.Gene duplication events of mouse phagosome paralogs occurred mostly in Bilateria and Euteleostomi, coinciding with the emergence of innate and adaptive immunity, and thus, provided the functional innovations needed for the establishment of these two crucial evolutionary steps of the immune system.Phosphoproteomics of isolated phagosomes from the same three distant species indicate that the phagosome phosphoproteome has been extensively modified during evolution. Still, some phosphosites have been maintained for >1.2 billion years, and thus, highlight their particular significance in the regulation of key phagosomal functions.
Phagocytosis is the process by which multiple cell types internalize large particulate material from the external milieu. The functional properties of phagosomes are acquired through a complex maturation process, referred to as phagolysosome biogenesis. This pathway involves a series of rapid interactions with organelles of the endocytic apparatus, enabling the gradual transformation of newly formed phagosomes into phagolysosomes in which proteolytic degradation occurs. The degradative environment encountered in the phagosome lumen has enabled the use of phagocytosis as a predation mechanism for feeding (phagotrophy) in amoeba, whereas multicellular organisms utilize this process as a defense mechanism to kill microbes and, in jawed vertebrates (fish), initiate a sustained immune response.
High-throughput proteomics profiling of isolated phagosomes has been tremendously helpful for the molecular comprehension of this organelle. This approach is achieved by feeding low buoyancy latex beads to phagocytic cells, enabling the subsequent isolation of latex bead-containing phagosomes, away from all the other cell organelles, by a single-isopicnic centrifugation in sucrose gradient. In order to characterize some of the key steps that contributed to the remodeling of phagosomes during evolution, we isolated this organelle from three distant organisms: the amoeba Dictyostelium discoideum, the fruit fly Drosophila melanogaster, and mouse (Mus musculus) that use phagocytosis for different purposes, and performed detailed proteomics and phosphoproteomics analyses with unparallel protein coverage for this organelle (two- to four-fold enhancements in identified proteins).
In order to establish the origin of the mouse phagosome proteome, we performed comparative analyses among 39 taxa including plants/algea, unicellular organisms, fungi, and more complex animal multicellular organisms. These genomic comparisons indicated that a large proportion of the mouse phagosome proteome is of ancient origin (73.1% of the proteome is conserved in eukaryotic organisms) (Figure 2A). This stresses the fact that phagocytosis is a very ancient process, as shown by its possible involvement in the emergence of eukaryotic cells (eukaryogenesis). Indeed, we identified close to 300 phagosome mouse proteins also present on Drosophila and Dictyostelium phagosomes, defining a potential ‘ancient' core of proteins from which the immune functions of phagosomes likely evolved. Around 16.7% of the mouse phagosome proteins appeared in organisms that use phagocytosis for innate immunity (Bilateria to Chordata), whereas 10.2% appeared in Euteleostomi or Tetrapoda where phagosomes have an important function in linking the killing of microorganisms with the development of a specific sustained immune response following antigen recognition. The phagosome is made of molecules taken from a variety of sources within the cell, including the cytoplasm, the cytoskeleton and membrane organelles. Despite the evolution and diversification of these various cellular systems, the mammalian phagosome proteome is made preferentially of ancient proteins (Figure 2B). Comparison of functional annotation during evolution highlighted the emergence of specific phagosomal functions at various steps during evolution (Figure 2C). Some of these proteins and their point of origin during evolution are highlighted in Figure 2D. Strikingly, we identified in Tetrapods a set of 50 proteins that arose while adaptive immunity was already well established in teleosts (fish), indicating that the phagocytic system is still evolving.
Our study highlights the fact that the functional properties of phagosomes emerged by the remodeling of ancient molecules, the addition of novel components, and the duplication of existing proteins (paralogs) leading to the formation of molecular machines of mixed origin. Gene duplication is a process that contributed continuously to the complexification of the mouse proteome during evolution. In sharp contrast, paralog analysis indicated that the phagosome proteome was mainly reorganized through two periods of gene duplication, in Bilateria and Euteleostomi, coinciding with the emergence of adaptive immunity (in jawed fish), and innate immunity (at the split between Metazoa and Bilateria). These results strongly suggest that selective constraints may have favored the maintenance of phagosome paralogs to ensure the establishment of novel functions associated with this organelle at these two crucial evolutionary steps of the immune system.
The emergence of genes associated to the MHC locus in mammals that appeared originally in the genome of jawed fishes, contributed to the development of complex molecular mechanisms linking innate (our immune system that defends the host from infection in a non-specific manner) and adaptive immunity (the part of the immune system triggered specifically after antigen recognition). Several of the genes of this locus encode proteins known to have important functions in antigen presentation, such as subunits of the immunoproteasome (LMP2 and LMP7), MHC class I and class II molecules, as well as tapasin and the transporter associated with antigen processing (TAP1 and TAP2), involved in the transport and loading of peptides on MHC class I molecules (Figure 6). In addition to their ability to present peptides on MHC class II molecules, phagosomes of vertebrates have been shown to be competent for the presentation of exogenous peptides on MHC class I molecules, a process referred to as cross-presentation. From a functional point of view, the involvement of phagosomes in antigen cross-presentation is the outcome of the successful integration of a wide range of multimolecular components that emerged throughout evolution (Figure 6). The trimming of exogenous proteins into small peptides that can be loaded on MHC class I molecules is inherited from the phagotrophic properties of unicellular organisms, where internalized bacteria are degraded into basic molecules and used as a source of nutrients. Ancient processes have therefore been co-opted (the use of an existing biological structure or feature for a new function) for new functionalities. A summarizing model of the various steps that enabled phagosome antigen presentation is presented in Figure 6. This model highlights the fact that although antigen presentation is unique to evolutionary recent phagosomes (starting in jawed fishes about 450 million years ago), it uses and integrates molecular machines composed of proteins that emerged throughout evolution.
In summary, we present here the first large-scale comparative proteomics/phosphoproteomics study characterizing some of the key evolutionary steps that contributed to the remodeling of phagosomes during evolution. Functional properties of this organelle emerged by the remodeling of ancient molecules, the addition of novel components, the extensive adaption of protein phosphorylation sites and the duplication of existing proteins leading to the formation of molecular machines of mixed origin.
Amoeba use phagocytosis to internalize bacteria as a source of nutrients, whereas multicellular organisms utilize this process as a defense mechanism to kill microbes and, in vertebrates, initiate a sustained immune response. By using a large-scale approach to identify and compare the proteome and phosphoproteome of phagosomes isolated from distant organisms, and by comparative analysis over 39 taxa, we identified an ‘ancient' core of phagosomal proteins around which the immune functions of this organelle have likely organized. Our data indicate that a larger proportion of the phagosome proteome, compared with the whole cell proteome, has been acquired through gene duplication at a period coinciding with the emergence of innate and adaptive immunity. Our study also characterizes in detail the acquisition of novel proteins and the significant remodeling of the phagosome phosphoproteome that contributed to modify the core constituents of this organelle in evolution. Our work thus provides the first thorough analysis of the changes that enabled the transformation of the phagosome from a phagotrophic compartment into an organelle fully competent for antigen presentation.
PMCID: PMC2990642  PMID: 20959821
evolution; immunity; phosphoproteomics; phylogeny; proteomics
11.  Overexpression of Akt1 Enhances Adipogenesis and Leads to Lipoma Formation in Zebrafish 
PLoS ONE  2012;7(5):e36474.
Obesity is a complex, multifactorial disorder influenced by the interaction of genetic, epigenetic, and environmental factors. Obesity increases the risk of contracting many chronic diseases or metabolic syndrome. Researchers have established several mammalian models of obesity to study its underlying mechanism. However, a lower vertebrate model for conveniently performing drug screening against obesity remains elusive. The specific aim of this study was to create a zebrafish obesity model by over expressing the insulin signaling hub of the Akt1 gene.
Methodology/Principal Findings
Skin oncogenic transformation screening shows that a stable zebrafish transgenic of Tg(krt4Hsa.myrAkt1)cy18 displays severely obese phenotypes at the adult stage. In Tg(krt4:Hsa.myrAkt1)cy18, the expression of exogenous human constitutively active Akt1 (myrAkt1) can activate endogenous downstream targets of mTOR, GSK-3α/β, and 70S6K. During the embryonic to larval transitory phase, the specific over expression of myrAkt1 in skin can promote hypertrophic and hyperplastic growth. From 21 hour post-fertilization (hpf) onwards, myrAkt1 transgene was ectopically expressed in several mesenchymal derived tissues. This may be the result of the integration position effect. Tg(krt4:Hsa.myrAkt1)cy18 caused a rapid increase of body weight, hyperplastic growth of adipocytes, abnormal accumulation of fat tissues, and blood glucose intolerance at the adult stage. Real-time RT-PCR analysis showed the majority of key genes on regulating adipogenesis, adipocytokine, and inflammation are highly upregulated in Tg(krt4:Hsa.myrAkt1)cy18. In contrast, the myogenesis- and skeletogenesis-related gene transcripts are significantly downregulated in Tg(krt4:Hsa.myrAkt1)cy18, suggesting that excess adipocyte differentiation occurs at the expense of other mesenchymal derived tissues.
Collectively, the findings of this study provide direct evidence that Akt1 signaling plays an important role in balancing normal levels of fat tissue in vivo. The obese zebrafish examined in this study could be a new powerful model to screen novel drugs for the treatment of human obesity.
PMCID: PMC3356305  PMID: 22623957
12.  Double-stranded RNA-dependent Protein Kinase Links Pathogen Sensing with Stress and Metabolic Homeostasis 
Cell  2010;140(3):338.
As chronic inflammation is a hallmark of obesity, pathways that integrate nutrient and pathogen sensing pathways are of great interest in understanding the mechanisms of insulin resistance, type 2 diabetes, and other chronic metabolic pathologies. Here, we provide evidence that double-stranded RNA dependent protein kinase (PKR) can respond to nutrient signals as well as endoplasmic reticulum (ER) stress and coordinate the activity of other critical inflammatory kinases such as the c-Jun N-terminal kinase (JNK) to regulate insulin action and metabolism. PKR also directly targets and modifies insulin receptor substrate and hence integrates nutrients and insulin action with a defined pathogen response system. Dietary and genetic obesity features marked activation of PKR in adipose and liver tissues and absence of PKR alleviates metabolic deterioration due to nutrient or energy excess in mice. These findings demonstrate PKR as a critical component of an inflammatory complex that responds to nutrients and organelle dysfunction.
PMCID: PMC2820414  PMID: 20144759
13.  Implication of inflammatory signaling pathways in obesity-induced insulin resistance 
Obesity is characterized by the development of a low-grade chronic inflammatory state in different metabolic tissues including adipose tissue and liver. This inflammation develops in response to an excess of nutrient flux and is now recognized as an important link between obesity and insulin resistance. Several dietary factors like saturated fatty acids and glucose as well as changes in gut microbiota have been proposed as triggers of this metabolic inflammation through the activation of pattern-recognition receptors (PRRs), including Toll-like receptors (TLR), inflammasome, and nucleotide oligomerization domain (NOD). The consequences are the production of pro-inflammatory cytokines and the recruitment of immune cells such as macrophages and T lymphocytes in metabolic tissues. Inflammatory cytokines activate several kinases like IKKβ, mTOR/S6 kinase, and MAP kinases as well as SOCS proteins that interfere with insulin signaling and action in adipocytes and hepatocytes. In this review, we summarize recent studies demonstrating that PRRs and stress kinases are important integrators of metabolic and inflammatory stress signals in metabolic tissues leading to peripheral and central insulin resistance and metabolic dysfunction. We discuss recent data obtained with genetically modified mice and pharmacological approaches suggesting that these inflammatory pathways are potential novel pharmacological targets for the management of obesity-associated insulin resistance.
PMCID: PMC3539134  PMID: 23316186
obesity; insulin resistance; inflammation; adipose tissue; pattern-recognition receptors; stress kinases; macrophages
14.  Gene expression profiles in Atlantic salmon adipose-derived stromo-vascular fraction during differentiation into adipocytes 
BMC Genomics  2010;11:39.
Excessive fat deposition is one of the largest problems faced by salmon aquaculture industries, leading to production losses due to high volume of adipose tissue offal. In addition, increased lipid accumulation may impose considerable stress on adipocytes leading to adipocyte activation and production and secretion of inflammatory mediators, as observed in mammals.
Microarray and qPCR analyses were performed to follow transcriptome changes during adipogenesis in the primary culture of adipose stromo-vascular fraction (aSVF) of Atlantic salmon. Cellular heterogeneity decreased by confluence as evidenced by the down-regulation of markers of osteo/chondrogenic, myogenic, immune and vasculature lineages. Transgelin (TAGLN), a marker of the multipotent pericyte, was prominently expressed around confluence while adipogenic PPARγ was up-regulated already in subconfluent cells. Proliferative activity and subsequent cell cycle arrest were reflected in the fluctuations of pro- and anti-mitotic regulators. Marked regulation of genes involved in lipid and glucose metabolism and pathways producing NADPH and glycerol-3-phosphate (G3P) was seen during the terminal differentiation, also characterised by diverse stress responses. Activation of the glutathione and thioredoxin antioxidant systems and changes in the iron metabolism suggested the need for protection against oxidative stress. Signs of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) occured in parallel with the increased lipid droplet (LD) formation and production of secretory proteins (adipsin, visfatin). The UPR markers XBP1 and ATF6 were induced together with genes involved in ubiquitin-proteasome and lysosomal proteolysis. Concurrently, translation was suppressed as evidenced by the down-regulation of genes encoding elongation factors and components of the ribosomal machinery. Notably, expression changes of a panel of genes that belong to different immune pathways were seen throughout adipogenesis. The induction of AP1 (Jun, Fos), which is a master regulator of stress responses, culminated by the end of adipogenesis, concurrent with the maximal observed lipid deposition.
Our data point to an intimate relationship between metabolic regulation and immune responses in white adipocytes of a cold-blooded vertebrate. Stress imposed on adipocytes by LD formation and expansion is prominently reflected in the ER compartment and the activated UPR response could have an important role at visceral obesity in fish.
PMCID: PMC2824722  PMID: 20078893
15.  Interleukin-1 beta: a potential link between stress and the development of visceral obesity 
BMC Physiology  2012;12:8.
A disproportionate amount of body fat within the abdominal cavity, otherwise known as visceral obesity, best predicts the negative health outcomes associated with high levels body fat. Growing evidence suggests that repeated activation of the stress response can favor visceral fat deposition and that visceral obesity may induce low-grade, systemic inflammation which is etiologically linked to the pathogenesis of obesity related diseases such as cardiovascular disease and type 2 diabetes. While the obesity epidemic has fueled considerable interest in these obesity-related inflammatory diseases, surprisingly little research is currently focused on understanding the functions of inflammatory proteins in healthy, non-obese white adipose tissue (WAT) and their possible role in modulating stress-induced shifts in body fat distribution.
The current review presents evidence in support the novel hypothesis that stress-evoked interleukin-1 beta (IL-1β) signaling within subcutaneous adipose tissue, when repeatedly induced, contributes toward the development of visceral obesity. It is suggested that because acute stressor exposure differentially increases IL-1β levels within subcutaneous adipose relative to visceral adipose tissue in otherwise healthy, non-obese rats, repeated induction of this response may impair the ability of subcutaneous adipose tissue to uptake energy substrates, synthesize and retain triglycerides, and/or adapt to positive energy balance via hyperplasia. Consequently, circulating energy substrates may be disproportionately shunted to visceral adipose tissue for storage, thus driving the development of visceral obesity.
This review establishes the following key points: 1) body fat distribution outweighs the importance of total body fat when predicting obesity-related disease risk; 2) repeated exposure to stress can drive the development of visceral obesity independent of changes in body weight; 3) because of the heterogeneity of WAT composition and function, an accurate understanding of WAT responses requires sampling multiple WAT depots; 4) acute, non-pathogenic stressor exposure increases WAT IL-1β concentrations in a depot specific manner suggesting an adaptive, metabolic role for this cytokine; however, when repeated, stress-induced IL-1β in non-visceral WAT may result in functional impairments that drive the development of stress-induced visceral obesity.
PMCID: PMC3404929  PMID: 22738239
16.  PPARγ is a major driver of the accumulation and phenotype of adipose-tissue Treg cells 
Nature  2012;486(7404):549-553.
Obesity and type-2 diabetes (T2D) have increased dramatically over the past several decades, in parallel. One of the major links between these two disorders is chronic, low-grade inflammation 1. Prolonged nutrient excess promotes the accumulation and activation of leukocytes in visceral adipose tissue (VAT) and ultimately other tissues, which provokes metabolic abnormalities such as insulin resistance, T2D and fatty-liver disease. While invasion of VAT by pro-inflammatory macrophages is considered to be a key event driving adipose-tissue inflammation and insulin resistance, little is known about the roles of other immune-system cell-types in these processes. Recently, a unique population of VAT-resident regulatory T cells (Tregs) was implicated in control of the inflammatory state of adipose tissue and, thereby, insulin sensitivity 2. We have identified peroxisome proliferator-activated receptor gamma (PPARγ), the “master-regulator” of adipocyte differentiation, as a critical molecular orchestrator of VAT Treg accumulation, phenotype and function. Unexpectedly, PPARγ expression by VAT Tregs was necessary for complete restoration of insulin sensitivity in obese mice by the thiazolidinedione (TZD) drug, pioglitazone (Pio). These findings suggest a previously unknown cellular mechanism for this important class of T2D drugs, and provide proof-of-principle that discrete populations of Tregs with unique functions can be precisely targeted to therapeutic ends.
PMCID: PMC3387339  PMID: 22722857
regulatory T cell; adipose tissue; obesity; type-2 diabetes; nuclear receptor; TZD drug
17.  The inflammation highway: metabolism accelerates inflammatory traffic in obesity 
Immunological reviews  2012;249(1):218-238.
As humans evolved, perhaps the two strongest selection determinants of survival were a robust immune response able to clear bacterial, viral, and parasitic infection and an ability to efficiently store nutrients to survive times when food sources were scarce. These traits are not mutually exclusive. It is now apparent that critical proteins necessary for regulating energy metabolism such as peroxisome proliferator-activated receptors (PPARs), Toll-like receptors (TLRs), and fatty acid-binding proteins (FABPs) also act as links between nutrient metabolism and inflammatory pathway activation in immune cells. Obesity in humans is a symptom of energy imbalance: the scale has been tipped such that energy intake exceeds energy output and may be a result, in part, of evolutionary selection toward a phenotype characterized by efficient energy storage. As discussed in this review, obesity is a state of low-grade, chronic inflammation that promotes the development of insulin resistance and diabetes. Ironically, the formation of systemic and/or local, tissue-specific insulin resistance upon inflammatory cell activation may actually be a protective mechanism that co-evolved to repartition energy sources within the body during times of stress during infection. However, the point has been reached where a once beneficial adaptive trait has become detrimental to the health of the individual and an immense public health and economic burden. This article reviews the complex relationship between obesity, insulin resistance/diabetes, and inflammation, and while the liver, brain, pancreas, muscle, and other tissues are relevant, we focus specifically on how the obese adipose microenvironment can promote immune cell influx and sustain damaging inflammation that can lead to the onset of insulin resistance and diabetes. Finally, we address how substrate metabolism may regulate the immune response and discuss how fuel uptake and metabolism may be a targetable approach to limit or abrogate obesity-induced inflammation.
PMCID: PMC3422768  PMID: 22889225
obesity; diabetes; insulin resistance; macrophage; plasticity; metabolism
18.  Macrophage-Induced Adipose Tissue Dysfunction and the Preadipocyte: Should I Stay (and Differentiate) or Should I Go?123 
Advances in Nutrition  2013;4(1):67-75.
Adipose tissue can be regarded as a multidepot organ responsible for metabolic homeostasis by managing sophisticated energy transactions as well as by producing bioactive molecules that regulate insulin sensitivity and immune and vascular responses. Chronic nutrient excess expands adipose tissue, and concomitant variations in its cellular and matrix remodeling can affect the extent of the metabolic dysfunction that is associated with obesity. Preadipocytes, also termed adipose progenitor cells, play a pivotal role in determining whether a dysfunctional hypertrophic state arises as opposed to a hyperplastic process in which mature adipocytes remain relatively responsive. Obesity is associated with infiltration of macrophages, and these immune cells have been shown to communicate with preadipocytes to influence how they differentiate, survive, and proliferate. Understanding macrophage–preadipocyte interactions and their effect on adipose remodeling mechanisms may identify potential therapeutic molecular targets to improve adipose tissue function, even in the face of obesity.
PMCID: PMC3648741  PMID: 23319125
19.  Nutrient sensing hypothalamic TXNIP links nutrient excess to energy imbalance in mice 
Nutrient excess in obesity and diabetes is emerging as a common putative cause for multiple deleterious effects across diverse cell types, responsible for a variety of metabolic dysfunctions. The hypothalamus is acknowledged as an important regulator of whole body energy homeostasis, through both detection of nutrient availability and coordination of effectors that determine nutrient intake and utilization, thus preventing cellular and whole body nutrient excess. However, the mechanisms underlying hypothalamic nutrient detection and its impact on peripheral nutrient utilization remain poorly understood. Recent data suggest a role for thioredoxin-interacting protein (TXNIP) as a molecular nutrient sensor important in the regulation of energy metabolism, but the role of hypothalamic TXNIP in the regulation of energy balance has not been evaluated. Here we show in mice that thioredoxin interacting protein is expressed in nutrient sensing neurons of the mediobasal hypothalamus, responds to hormonal and nutrient signals, and regulates adipose tissue metabolism, fuel partitioning and glucose homeostasis. Hypothalamic expression of thioredoxin-interacting protein is induced by acute nutrient excess and in mouse models of obesity and diabetes, and downregulation of mediobasal hypothalamic thioredoxin-interacting protein expression prevents diet-induced obesity and insulin resistance. Thus, mediobasal hypothalamic thioredoxin-interacting protein plays a critical role in nutrient sensing and the regulation of fuel utilization.
PMCID: PMC3100164  PMID: 21508227
20.  SirT1 Regulates Adipose Tissue Inflammation 
Diabetes  2011;60(12):3235-3245.
Macrophage recruitment to adipose tissue is a reproducible feature of obesity. However, the events that result in chemokine production and macrophage recruitment to adipose tissue during states of energetic excess are not clear. Sirtuin 1 (SirT1) is an essential nutrient-sensing histone deacetylase, which is increased by caloric restriction and reduced by overfeeding. We discovered that SirT1 depletion causes anorexia by stimulating production of inflammatory factors in white adipose tissue and thus posit that decreases in SirT1 link overnutrition and adipose tissue inflammation.
We used antisense oligonucleotides to reduce SirT1 to levels similar to those seen during overnutrition and studied SirT1-overexpressing transgenic mice and fat-specific SirT1 knockout animals. Finally, we analyzed subcutaneous adipose tissue biopsies from two independent cohorts of human subjects.
We found that inducible or genetic reduction of SirT1 in vivo causes macrophage recruitment to adipose tissue, whereas overexpression of SirT1 prevents adipose tissue macrophage accumulation caused by chronic high-fat feeding. We also found that SirT1 expression in human subcutaneous fat is inversely related to adipose tissue macrophage infiltration.
Reduction of adipose tissue SirT1 expression, which leads to histone hyperacetylation and ectopic inflammatory gene expression, is identified as a key regulatory component of macrophage influx into adipose tissue during overnutrition in rodents and humans. Our results suggest that SirT1 regulates adipose tissue inflammation by controlling the gain of proinflammatory transcription in response to inducers such as fatty acids, hypoxia, and endoplasmic reticulum stress.
PMCID: PMC3219953  PMID: 22110092
21.  Inflammatory Concepts of Obesity 
Obesity, long considered a condition characterized by the deposition of inert fat, is now recognized as a chronic and systemic inflammatory disease, where adipose tissue plays a crucial endocrine role through the production of numerous bioactive molecules, collectively known as adipokines. These molecules regulate carbohydrate and lipid metabolism, immune function and blood coagulability, and may serve as blood markers of cardiometabolic risk. Local inflammatory loops operate in adipose tissue as a consequence of nutrient overload, and crosstalk among its cellular constituents-adipocytes, endothelial and immune cells-results in the elaboration of inflammatory mediators. These mediators promote important systemic effects that can result in insulin resistance, dysmetabolism and cardiovascular disease. The understanding that inflammation plays a critical role in the pathogenesis of obesity-derived disorders has led to therapeutic approaches that target different points of the inflammatory network induced by obesity.
PMCID: PMC3151511  PMID: 21837268
22.  HDAC9 Knockout Mice Are Protected From Adipose Tissue Dysfunction and Systemic Metabolic Disease During High-Fat Feeding 
Diabetes  2013;63(1):176-187.
During chronic caloric excess, adipose tissue expands primarily by enlargement of individual adipocytes, which become stressed with lipid overloading, thereby contributing to obesity-related disease. Although adipose tissue contains numerous preadipocytes, differentiation into functionally competent adipocytes is insufficient to accommodate the chronic caloric excess and prevent adipocyte overloading. We report for the first time that a chronic high-fat diet (HFD) impairs adipogenic differentiation, leading to accumulation of inefficiently differentiated adipocytes with blunted expression of adipogenic differentiation-specific genes. Preadipocytes from these mice likewise exhibit impaired adipogenic differentiation, and this phenotype persists during in vitro cell culture. HFD-induced impaired adipogenic differentiation is associated with elevated expression of histone deacetylase 9 (HDAC9), an endogenous negative regulator of adipogenic differentiation. Genetic ablation of HDAC9 improves adipogenic differentiation and systemic metabolic state during an HFD, resulting in diminished weight gain, improved glucose tolerance and insulin sensitivity, and reduced hepatosteatosis. Moreover, compared with wild-type mice, HDAC9 knockout mice exhibit upregulated expression of beige adipocyte marker genes, particularly during an HFD, in association with increased energy expenditure and adaptive thermogenesis. These results suggest that targeting HDAC9 may be an effective strategy for combating obesity-related metabolic disease.
PMCID: PMC3868044  PMID: 24101673
23.  Nutrient-Regulated Antisense and Intragenic RNAs Modulate a Signal Transduction Pathway in Yeast 
PLoS Biology  2008;6(12):e326.
The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to a change in nutrient availability. The PHO system is a well-studied case in the transcriptional regulation responding to nutritional changes in which a set of genes (PHO genes) is expressed to activate inorganic phosphate (Pi) metabolism for adaptation to Pi starvation. Pi starvation triggers an inhibition of Pho85 kinase, leading to migration of unphosphorylated Pho4 transcriptional activator into the nucleus and enabling expression of PHO genes. When Pi is sufficient, the Pho85 kinase phosphorylates Pho4, thereby excluding it from the nucleus and resulting in repression (i.e., lack of transcription) of PHO genes. The Pho85 kinase has a role in various cellular functions other than regulation of the PHO system in that Pho85 monitors whether environmental conditions are adequate for cell growth and represses inadequate (untimely) responses in these cellular processes. In contrast, Pho4 appears to activate some genes involved in stress response and is required for G1 arrest caused by DNA damage. These facts suggest the antagonistic function of these two players on a more general scale when yeast cells must cope with stress conditions. To explore general involvement of Pho4 in stress response, we tried to identify Pho4-dependent genes by a genome-wide mapping of Pho4 and Rpo21 binding (Rpo21 being the largest subunit of RNA polymerase II) using a yeast tiling array. In the course of this study, we found Pi- and Pho4-regulated intragenic and antisense RNAs that could modulate the Pi signal transduction pathway. Low-Pi signal is transmitted via certain inositol polyphosphate (IP) species (IP7) that are synthesized by Vip1 IP6 kinase. We have shown that Pho4 activates the transcription of antisense and intragenic RNAs in the KCS1 locus to down-regulate the Kcs1 activity, another IP6 kinase, by producing truncated Kcs1 protein via hybrid formation with the KCS1 mRNA and translation of the intragenic RNA, thereby enabling Vip1 to utilize more IP6 to synthesize IP7 functioning in low-Pi signaling. Because Kcs1 also can phosphorylate these IP7 species to synthesize IP8, reduction in Kcs1 activity can ensure accumulation of the IP7 species, leading to further stimulation of low-Pi signaling (i.e., forming a positive feedback loop). We also report that genes apparently not involved in the PHO system are regulated by Pho4 either dependent upon or independent of the Pi conditions, and many of the latter genes are involved in stress response. In S. cerevisiae, a large-scale cDNA analysis and mapping of RNA polymerase II binding using a high-resolution tiling array have identified a large number of antisense RNA species whose functions are yet to be clarified. Here we have shown that nutrient-regulated antisense and intragenic RNAs as well as direct regulation of structural gene transcription function in the response to nutrient availability. Our findings also imply that Pho4 is present in the nucleus even under high-Pi conditions to activate or repress transcription, which challenges our current understanding of Pho4 regulation.
Author Summary
How does a microorganism adapt to changes in its environment? Phosphate metabolism in the budding yeast Saccharomyces cerevisiae serves as a model for investigating mechanisms involved in physiological adaptation. The nutrient inorganic phosphate (Pi) is essential for building nucleic acids and phospholipids; when yeast cells are deprived of Pi, genes required for scavenging the nutrient are activated. This activation is mediated by the Pho4 transcription factor through its migration into or out of nucleus. The Pi-starvation (low-Pi) signal is transmitted by a class of inositol polyphosphate (IP) species, IP7, which is synthesized by one of two IP6 kinases, Vip1 or Kcs1. However, the IP7 made primarily by Vip1 is key in the signaling pathway. Here we report that under Pi starvation Pho4 binds within the coding sequence of KCS1 to activate transcription of both intragenic and antisense RNAs, resulting in the production of a truncated Kcs1 protein and the likely down-regulation of Kcs1 activity. Consequently Vip1 can produce more IP7 to enhance the low-Pi signaling and thus form a positive feedback loop. We have also demonstrated that Pho4 regulates, both positively and negatively, transcription of genes apparently uninvolved in cellular response to Pi starvation and that it sometimes does so independently of Pi conditions. These findings reveal mechanisms that go beyond the currently held model of Pho4 regulation.
During nutritional adaptation, transcriptional activation in yeast produces noncoding RNAs that allow the formation of a positive-feedback regulatory loop.
PMCID: PMC2605928  PMID: 19108609
24.  Transcriptional factors that promote formation of white adipose tissue 
Adipocytes are highly specialized cells that play a major role in energy homeostasis in vertebrate organisms. Excess adipocyte size or number is a hallmark of obesity, which is currently a global epidemic. Obesity is a major risk factor for the development of type II diabetes (T2DM), cardiovascular disease, and hypertension. Obesity and its related disorders result in dysregulation of the mechanisms that control the expression of metabolic and endocrine related genes in adipocytes. Therefore, understanding adipocyte differentiation is relevant not only for gaining insight into the pathogenesis of metabolic diseases, but also for identifying proteins or pathways which might be appropriate targets for pharmacological interventions. Significant advances towards an understanding of the regulatory processes involved in adipocyte differentiation have largely been made by the identification of transcription factors that contribute to the adipogenic process. It is important to note that the developmental origin of white and brown fat is distinct and different precursor cells are involved in the generation of these different types of adipose tissue (reviewed in Lefterova and Lazar, 2009; Seale et al., 2009). Several transcription factors, notably PPARγ, several members of the C/EBP and KLF families, STAT5, and SREBP-1c, have been shown to have significant roles in promoting adipogenesis. More comprehensive reviews on negative and positive regulators of adipogenesis have been published in the past year (reviewed in Christodoulides et al., 2009; Lefterova and Lazar, 2009). Though many proteins are known to negatively regulate adipogenesis, including Wnts, KLFs, the E2F family of transcription factors, CHOP, Delta-interacting protein A, ETO/MTG8, and members of the GATA and forkhead transcription factor families, this review will focus on transcription factors that positively impact the development of white adipose tissue.
PMCID: PMC3079373  PMID: 19733624
25.  Metabolically Protective Cytokines Adiponectin and Fibroblast Growth Factor-21 Are Increased by Acute Overfeeding in Healthy Humans 
PLoS ONE  2013;8(10):e78864.
Circulating levels of metabolically protective and adverse cytokines are altered in obese humans and rodent models. However, it is not clear whether these cytokines are altered rapidly in response to over-nutrition, or as a later consequence of the obese state.
Forty sedentary healthy individuals were examined prior to and at 3 and 28 days of high fat overfeeding (+1250 kCal/day, 45% fat). Insulin sensitivity (hyperinsulinaemic-euglycaemic clamp), adiposity, serum levels of adiponectin and fibroblast growth factor-21 (FGF21), fatty acid binding protein-4 (FABP4), lipocalin-2 and plasminogen activator factor-1 (PAI1) were assessed. Statistics were performed by repeated measures ANOVA.
Overfeeding increased weight, body fat and liver fat, fasting glucose, insulin and reduced insulin sensitivity by clamp (all P <0.05). Metabolically protective cytokines, adiponectin and FGF21 were increased at day 3 of overfeeding (P ≤0.001) and adiponectin was also elevated at day 28 (P=0.001). FABP4, lipocalin-2 and PAI-1 were not changed by overfeeding at either time point.
Metabolically protective cytokines, adiponectin and FGF-21, were increased by over nutrition and weight gain in healthy humans, despite increases in insulin resistance. We speculate that this was in attempt to maintain glucose homeostasis in a state of nutritional excess. PAI-I, FABP4 and lipocalin 2 were not altered by overfeeding suggesting that changes in these cytokines may be a later consequence of the obese state.
Clinical trial registration: (NCT00562393)
PMCID: PMC3799638  PMID: 24205333

Results 1-25 (965637)