Self-organized metabotyping of obese individuals identifies clusters responding differently to bariatric surgery.

Weight loss through bariatric surgery is efficient for treatment or prevention of obesity related diseases such as type 2 diabetes and cardiovascular disease. Long term weight loss response does, however, vary among patients undergoing surgery. Thus, it is difficult to identify predictive markers while most obese individuals have one or more comorbidities. To overcome such challenges, an in-depth multiple omics analyses including fasting peripheral plasma metabolome, fecal metagenome as well as liver, jejunum, and adipose tissue transcriptome were performed for 106 individuals undergoing bariatric surgery. Machine leaning was applied to explore the metabolic differences in individuals and evaluate if metabolism-based patients' stratification is related to their weight loss responses to bariatric surgery. Using Self-Organizing Maps (SOMs) to analyze the plasma metabolome, we identified five distinct metabotypes, which were differentially enriched for KEGG pathways related to immune functions, fatty acid metabolism, protein-signaling, and obesity pathogenesis. The gut metagenome of the most heavily medicated metabotypes, treated simultaneously for multiple cardiometabolic comorbidities, was significantly enriched in Prevotella and Lactobacillus species. This unbiased stratification into SOM-defined metabotypes identified signatures for each metabolic phenotype and we found that the different metabotypes respond differently to bariatric surgery in terms of weight loss after 12 months. An integrative framework that utilizes SOMs and omics integration was developed for stratifying a heterogeneous bariatric surgery cohort. The multiple omics datasets described in this study reveal that the metabotypes are characterized by a concrete metabolic status and different responses in weight loss and adipose tissue reduction over time. Our study thus opens a path to enable patient stratification and hereby allow for improved clinical treatments.

A systems biology approach to study non-alcoholic fatty liver (NAFL) in women with obesity.

Non-alcoholic fatty liver disease (NAFLD) is now the most frequent global chronic liver disease. Individuals with NAFLD exhibited an increased risk of all-cause mortality driven by extrahepatic cancers and liver and cardiovascular disease. Once the disease is established, women have a higher risk of disease progression and worse outcome. It is therefore critical to deepen the current knowledge on the pathophysiology of NAFLD in women. Here, we used a systems biology approach to investigate the contribution of different organs to this disease. We analyzed transcriptomics profiles of liver and adipose tissues, fecal metagenomes, and plasma metabolomes of 55 women with and without NAFLD. We observed differences in metabolites, expression of human genes, and gut microbial features between the groups and revealed that there is substantial crosstalk between these different omics sets. Multi-omics analysis of individuals with NAFLD may provide novel strategies to study the pathophysiology of NAFLD in humans.

Gut microbial characteristics in poor appetite and undernutrition: a cohort of older adults and microbiota transfer in germ-free mice.

BACKGROUND: Older adults are particularly prone to the development of poor appetite and undernutrition. Possibly, this is partly due to the aged gut microbiota. We aimed to evaluate the gut microbiota in relation to both poor appetite and undernutrition in community-dwelling older adults. Furthermore, we studied the causal effects of the microbiota on body weight and body composition by transferring faecal microbiota from cohort participants into germ-free mice. METHODS: First, we conducted a cross-sectional cohort study of 358 well-phenotyped Dutch community-dwelling older adults from the Longitudinal Aging Study Amsterdam. Data collection included body measurements, a faecal and blood sample, as well as extensive questionnaires on appetite, dietary intake, and nutritional status. Appetite was assessed by the Council of Nutrition Appetite Questionnaire (CNAQ) and undernutrition was defined by either a low body mass index (BMI) (BMI < 20 kg/m2 if <70 years or BMI < 22 kg/m2 if ≥70 years) or >5% body weight loss averaged over the last 2 years. Gut microbiota composition was determined with 16S rRNA sequencing. Next, we transferred faecal microbiota from 12 cohort participants with and without low BMI or recent weight loss into a total of 41 germ-free mice to study the potential causal effects of the gut microbiota on host BMI and body composition. RESULTS: The mean age (range) of our cohort was 73 (65-93); 58.4% was male. Seventy-seven participants were undernourished and 21 participants had poor appetite (CNAQ < 28). A lower abundance of the genus Blautia was associated with undernutrition (log2 fold change = -0.57, Benjamini-Hochberg-adjusted P = 0.008), whereas higher abundances of taxa from Lachnospiraceae, Ruminococcaceae UCG-002, Parabacteroides merdae, and Dorea formicigenerans were associated with poor appetite. Furthermore, participants with poor appetite or undernutrition had reduced levels of faecal acetate (P = 0.006 and 0.026, respectively). Finally, there was a trend for the mice that received faecal microbiota from older adults with low BMI to weigh 1.26 g less after 3 weeks (P = 0.086) and have 6.13% more lean mass (in % body weight, P = 0.067) than the mice that received faecal microbiota from older adults without low BMI or recent weight loss. CONCLUSIONS: This study demonstrates several associations of the gut microbiota with both poor appetite and undernutrition in older adults. Moreover, it is the first to explore a causal relation between the aged gut microbiota and body weight and body composition in the host. Possibly, microbiota-manipulating strategies will benefit older adults prone to undernutrition.

Hyperinsulinemia Is Highly Associated With Markers of Hepatocytic Senescence in Two Independent Cohorts.

Cellular senescence is an essentially irreversible growth arrest that occurs in response to various cellular stressors and may contribute to development of type 2 diabetes mellitus and nonalcoholic fatty liver disease (NAFLD). In this article, we investigated whether chronically elevated insulin levels are associated with cellular senescence in the human liver. In 107 individuals undergoing bariatric surgery, hepatic senescence markers were assessed by immunohistochemistry as well as transcriptomics. A subset of 180 participants from the ongoing Finnish Kuopio OBesity Surgery (KOBS) study was used as validation cohort. We found plasma insulin to be highly associated with various markers of cellular senescence in liver tissue. The liver transcriptome of individuals with high insulin revealed significant upregulation of several genes associated with senescence: p21, TGFß, PI3K, HLA-G, IL8, p38, Ras, and E2F. Insulin associated with hepatic senescence independently of NAFLD and plasma glucose. By using transcriptomic data from the KOBS study, we could validate the association of insulin with p21 in the liver. Our results support a potential role for hyperinsulinemia in induction of cellular senescence in the liver. These findings suggest possible benefits of lowering insulin levels in obese individuals with insulin resistance.

MyD88 Deficiency, but Not Gut Microbiota Depletion, Is Sufficient to Modulate the Blood-Brain Barrier Function in the Mediobasal Hypothalamus.

Circumventricular organs (CVOs), including the mediobasal hypothalamus (MBH), have an incomplete blood-brain barrier (BBB). In this study, we determined if the BBB function in the MBH is modulated by the gut microbiota or by the Toll-like receptor (TLR) adapter proteins TRIF or MyD88 signaling. By injecting mice with Evans blue, a marker for BBB permeability, we show that germ-free (GF) and conventionally raised (CONV-R) mice did not differ in the number of Evans blue-positive cells in MBH. Acute modulation of the gut microbiota did not change the number of Evans blue-positive cells. In contrast, CONV-R Myd88-/- and Trif-/- mice had a reduced number of cells in direct contact to the circulation compared to wildtype (WT) mice. This was accompanied by increased tight junction proteins in the blood vessels in Myd88-/- mice. To further characterize the BBB function, we injected WT and Myd88 -/- CONV-R mice as well as WT GF mice with monosodium glutamate (MSG), a neurotoxin that does not cross the BBB. While MSG caused vast cell death in the MBH in CONV-R and GF WT mice, Myd88 -/- mice were protected from such cell death suggesting that fewer cells are exposed to the neurotoxin in the Myd88 -/- mice. Taken together, our results suggest that MyD88 deficiency, but not gut microbiota depletion, is sufficient to modulate the BBB function in the MBH.

The Metabolic Role and Therapeutic Potential of the Microbiome.

We are host to an assembly of microorganisms that vary in structure and function along the length of the gut and from the lumen to the mucosa. This ecosystem is collectively known as the gut microbiota and significant efforts have been spent during the past 2 decades to catalog and functionally describe the normal gut microbiota and how it varies during a wide spectrum of disease states. The gut microbiota is altered in several cardiometabolic diseases and recent work has established microbial signatures that may advance disease. However, most research has focused on identifying associations between the gut microbiota and human diseases states and to investigate causality and potential mechanisms using cells and animals. Since the gut microbiota functions on the intersection between diet and host metabolism, and can contribute to inflammation, several microbially produced metabolites and molecules may modulate cardiometabolic diseases. Here we discuss how the gut bacterial composition is altered in, and can contribute to, cardiometabolic disease, as well as how the gut bacteria can be targeted to treat and prevent metabolic diseases.

The gut microbiota regulates hypothalamic inflammation and leptin sensitivity in Western diet-fed mice via a GLP-1R-dependent mechanism.

Mice lacking a microbiota are protected from diet-induced obesity. Previous studies have shown that feeding a Western diet causes hypothalamic inflammation, which in turn can lead to leptin resistance and weight gain. Here, we show that wild-type (WT) mice with depleted gut microbiota, i.e., germ-free (GF) and antibiotic-treated mice, have elevated levels of glucagon-like peptide-1 (GLP-1), are protected against diet-induced hypothalamic inflammation, and have enhanced leptin sensitivity when fed a Western diet. Using GLP-1 receptor (GLP-1R)-deficient mice and pharmacological inhibition of the GLP-1R in WT mice, we demonstrate that intact GLP-1R signaling is required for preventing hypothalamic inflammation and enhancing leptin sensitivity. Furthermore, we show that astrocytes express the GLP-1R, and deletion of the receptor in glial fibrillary acidic protein (GFAP)-expressing cells diminished the antibiotic-induced protection against diet-induced hypothalamic inflammation. Collectively, our results suggest that depletion of the gut microbiota attenuates diet-induced hypothalamic inflammation and enhances leptin sensitivity via GLP-1R-dependent mechanisms.

The role of the gut microbiota in development, function and disorders of the central nervous system and the enteric nervous system.

The gut microbiota has emerged as an environmental factor that modulates the development of the central nervous system (CNS) and the enteric nervous system (ENS). Before obtaining its own microbiota, eutherian foetuses are exposed to products and metabolites from the maternal microbiota. At birth, the infants are colonised by microorganisms. The microbial composition in early life is strongly influenced by the mode of delivery, the feeding method, the use of antibiotics and the maternal microbial composition. Microbial products and microbially produced metabolites act as signalling molecules that have direct or indirect effects on the CNS and the ENS. An increasing number of studies show that the gut microbiota can modulate important processes during development, including neurogenesis, myelination, glial cell function, synaptic pruning and blood-brain barrier permeability. Furthermore, numerous studies indicate that there is a developmental window early in life during which the gut microbial composition is crucial and perturbation of the gut microbiota during this period causes long-lasting effects on the development of the CNS and the ENS. However, other functions are readily modulated in adult animals, including microglia activation and neuroinflammation. Several neurobehavioural, neurodegenerative, mental and metabolic disorders, including Parkinson disease, autism spectrum disorder, schizophrenia, Alzheimer's disease, depression and obesity, have been linked to the gut microbiota. This review focuses on the role of the microorganisms in the development and function of the CNS and the ENS, as well as their potential role in pathogenesis.

Genes controlling the activation of natural killer lymphocytes are epigenetically remodeled in intestinal cells from germ-free mice.

Remodeling of the gut microbiota is implicated in various metabolic and inflammatory diseases of the gastrointestinal tract. We hypothesized that the gut microbiota affects the DNA methylation profile of intestinal epithelial cells (IECs) which could, in turn, alter intestinal function. In this study, we used mass spectrometry and methylated DNA capture to respectively investigate global and genome-wide DNA methylation of intestinal epithelial cells from germ-free (GF) and conventionally raised mice. In colonic IECs from GF mice, DNA was markedly hypermethylated. This was associated with a dramatic loss of ten-eleven-translocation activity, a lower DNA methyltransferase activity and lower circulating levels of the 1-carbon metabolite, folate. At the gene level, we found an enrichment for differentially methylated regions proximal to genes regulating the cytotoxicity of NK cells (false-discovery rate < 8.9E-6), notably genes regulating the cross-talk between NK cells and target cells, such as members of the NK group 2 member D ligand superfamily Raet. This distinct epigenetic signature was associated with a marked decrease in Raet1 expression and a loss of CD56+/CD45+ cells in the intestine of GF mice. Thus, our results indicate that altered activity of methylation-modifying enzymes in GF mice influences the IEC epigenome and modulates the crosstalk between IECs and NK cells. Epigenetic reprogramming of IECs may modulate intestinal function in diseases associated with altered gut microbiota.-Poupeau, A., Garde, C., Sulek, K., Citirikkaya, K., Treebak, J. T., Arumugam, M., Simar, D., Olofsson, L. E., Bäckhed, F., Barrès, R. Genes controlling the activation of natural killer lymphocytes are epigenetically remodeled in intestinal cells from germ-free mice.

Microbially Produced Imidazole Propionate Impairs Insulin Signaling through mTORC1.

Interactions between the gut microbiota, diet, and the host potentially contribute to the development of metabolic diseases. Here, we identify imidazole propionate as a microbially produced histidine-derived metabolite that is present at higher concentrations in subjects with versus without type 2 diabetes. We show that imidazole propionate is produced from histidine in a gut simulator at higher concentrations when using fecal microbiota from subjects with versus without type 2 diabetes and that it impairs glucose tolerance when administered to mice. We further show that imidazole propionate impairs insulin signaling at the level of insulin receptor substrate through the activation of p38γ MAPK, which promotes p62 phosphorylation and, subsequently, activation of mechanistic target of rapamycin complex 1 (mTORC1). We also demonstrate increased activation of p62 and mTORC1 in liver from subjects with type 2 diabetes. Our findings indicate that the microbial metabolite imidazole propionate may contribute to the pathogenesis of type 2 diabetes.

Gut microbiota regulates maturation of the adult enteric nervous system via enteric serotonin networks.

The enteric nervous system (ENS) is crucial for essential gastrointestinal physiologic functions such as motility, fluid secretion, and blood flow. The gut is colonized by trillions of bacteria that regulate host production of several signaling molecules including serotonin (5-HT) and other hormones and neurotransmitters. Approximately 90% of 5-HT originates from the intestine, and activation of the 5-HT4 receptor in the ENS has been linked to adult neurogenesis and neuroprotection. Here, we tested the hypothesis that the gut microbiota could induce maturation of the adult ENS through release of 5-HT and activation of 5-HT4 receptors. Colonization of germ-free mice with a microbiota from conventionally raised mice modified the neuroanatomy of the ENS and increased intestinal transit rates, which was associated with neuronal and mucosal 5-HT production and the proliferation of enteric neuronal progenitors in the adult intestine. Pharmacological modulation of the 5-HT4 receptor, as well as depletion of endogenous 5-HT, identified a mechanistic link between the gut microbiota and maturation of the adult ENS through the release of 5-HT and activation of the 5-HT4 receptor. Taken together, these findings show that the microbiota modulates the anatomy of the adult ENS in a 5-HT-dependent fashion with concomitant changes in intestinal transit.

Gut Microbiota-Dependent Modulation of Energy Metabolism.

The gut microbiota has emerged as an environmental factor that modulates the host's energy balance. It increases the host's ability to harvest energy from the digested food, and produces metabolites and microbial products such as short-chain fatty acids, secondary bile acids, and lipopolysaccharides. These metabolites and microbial products act as signaling molecules that modulate appetite, gut motility, energy uptake and storage, and energy expenditure. Several findings suggest that the gut microbiota can affect the development of obesity. Germ-free mice are leaner than conventionally raised mice and they are protected against diet-induced obesity. Furthermore, obese humans and rodents have an altered gut microbiota composition with less phylogeneic diversity compared to lean controls, and transplantation of the gut microbiota from obese subjects to germ-free mice can transfer the obese phenotype. Taken together, these findings indicate a role for the gut microbiota in obesity and suggest that the gut microbiota could be targeted to improve metabolic diseases like obesity. This review focuses on the role of the gut microbiota in energy balance regulation and its potential role in obesity.

Coordinated regulation of hepatic energy stores by leptin and hypothalamic agouti-related protein.

Like obesity, prolonged food deprivation induces severe hepatic steatosis; however, the functional significance of this phenomenon is not well understood. In this study, we show that the fall in plasma leptin concentration during fasting is required for the development of hepatic steatosis in mice. Removal of leptin receptors from AGRP neurons diminishes fasting-induced hepatic steatosis. Furthermore, the suppressive effects of leptin on fasting-induced hepatic steatosis are absent in mice lacking the gene encoding agouti-related protein (Agrp), suggesting that this function of leptin is mediated by AGRP. Prolonged fasting leads to suppression of hepatic sympathetic activity, increased expression of acyl CoA:diacylglycerol acyltransferase-2 in the liver, and elevation of hepatic triglyceride content and all of these effects are blunted in the absence of AGRP. AGRP deficiency, despite having no effects on feeding or body adiposity in the free-fed state, impairs triglyceride and ketone body release from the liver during prolonged fasting. Furthermore, reducing CNS Agrp expression in wild-type mice by RNAi protected against the development of hepatic steatosis not only during starvation, but also in response to consumption of a high-fat diet. These findings identify the leptin-AGRP circuit as a critical modulator of hepatic triglyceride stores in starvation and suggest a vital role for this circuit in sustaining the supply of energy from the liver to extrahepatic tissues during periods of prolonged food deprivation.

Modulation of AgRP-neuronal function by SOCS3 as an initiating event in diet-induced hypothalamic leptin resistance.

Chronic consumption of a fat-rich diet leads to attenuation of leptin signaling in hypothalamic neurons, a hallmark feature of cellular leptin resistance. To date, little is known about the temporal and spatial dysregulation of neuronal function under conditions of nutrient excess. We show that agouti-related protein (AgRP)-expressing neurons precede proopiomelanocortin neurons in developing diet-induced cellular leptin resistance. High-fat diet-induced up-regulation of suppressor of cytokine signaling-3 (SOCS3) occurs in AgRP neurons before proopiomelanocortin and other hypothalamic neurons. SOCS3 expression in AgRP neurons increases after 2 d of high-fat feeding, but reduces after switching to a low-fat diet for 1 d. Consistently, transgenic overexpression of SOCS3 in AgRP neurons produces metabolic phenotypes resembling those observed after short-term high-fat feeding. We further show that AgRP neurons are the predominant cell type situated outside the blood-brain barrier in the mediobasal hypothalamus. AgRP neurons are more responsive to low levels of circulating leptin, but they are also more prone to development of leptin resistance in response to a small increase in blood leptin concentrations. Collectively, these results suggest that AgRP neurons are able to sense slight changes in plasma metabolic signals, allowing them to serve as first-line responders to fluctuation of energy intake. Furthermore, modulation of SOCS3 expression in AgRP neurons may play a dynamic and physiological role in metabolic fine tuning in response to short-term changes of nutritional status.

Preliminary report: Zn-alpha2-glycoprotein genotype and serum levels are associated with serum lipids.

Zn-alpha2-glycoprotein (ZAG) is a serum protein implicated in cancer cachexia and lipolysis. Our aim was to investigate serum levels of ZAG and polymorphisms in the ZAG gene in relation to serum lipids in man. Serum levels of ZAG correlated with serum levels of cholesterol (P = .00088) in healthy subjects and during weight loss (P = .059). The ZAG genotype was associated with total cholesterol (P = .014) and low-density lipoprotein cholesterol (P = .026) in healthy subjects, and the associations were replicated in an additional cohort (P = .0017 and P = .060, respectively). Our data indicate that ZAG plays a role in lipid metabolism.

Functional role of suppressor of cytokine signaling 3 upregulation in hypothalamic leptin resistance and long-term energy homeostasis.

OBJECTIVE: Hypothalamic leptin resistance is found in most common forms of obesity, such as diet-induced obesity, and is associated with increased expression of suppressor of cytokine signaling 3 (Socs3) in the hypothalamus of diet-induced obese animals. This study aims to determine the functional consequence of Socs3 upregulation on leptin signaling and obesity, and to investigate whether Socs3 upregulation affects energy balance in a cell type-specific way. RESEARCH DESIGN AND METHODS: We generated transgenic mice overexpressing Socs3 in either proopiomelanocortin (POMC) or leptin receptor-expressing neurons, at levels similar to what is observed in diet-induced obesity. RESULTS: Upregulation of Socs3 in POMC neurons leads to impairment of STAT3 and mammalian target of rapamycin (mTOR)-S6K-S6 signaling, with subsequent leptin resistance, obesity, and glucose intolerance. Unexpectedly, Socs3 upregulation in leptin receptor neurons results in increased expression of STAT3 protein in mutant hypothalami, but does not lead to obesity. CONCLUSIONS: Our study establishes that Socs3 upregulation alone in POMC neurons is sufficient to cause leptin resistance and obesity. Socs3 upregulation impairs both STAT3 and mTOR signaling before the onset of obesity. The lack of obesity in mice with upregulated Socs3 in leptin receptor neurons suggests that Socs3's effect on energy balance could be cell type specific. Our study indicates that POMC neurons are important mediators of Socs3's effect on leptin resistance and obesity, but that other cell types or alteration of other signaling regulators could contribute to the development of obesity.

Functional role of c-Jun-N-terminal kinase in feeding regulation.

c-Jun-N-terminal kinase (JNK) is a signaling molecule that is activated by proinflammatory signals, endoplasmic reticulum (ER) stress, and other environmental stressors. Although JNK has diverse effects on immunological responses and insulin resistance in peripheral tissues, a functional role for JNK in feeding regulation has not been established. In this study, we show that central inhibition of JNK activity potentiates the stimulatory effects of glucocorticoids on food intake and that this effect is abolished in mice whose agouti-related peptide (AgRP) neurons are degenerated. JNK1-deficient mice feed more upon central administration of glucocorticoids, and glucocorticoid receptor nuclear immunoreactivity is enhanced in the AgRP neurons. JNK inhibition in hypothalamic explants stimulates Agrp expression, and JNK1-deficient mice exhibit increased Agrp expression, heightened hyperphagia, and weight gain during refeeding. Our study shows that JNK1 is a novel regulator of feeding by antagonizing glucocorticoid function in AgRP neurons. Paradoxically, JNK1 mutant mice feed less and lose more weight upon central administration of insulin, suggesting that JNK1 antagonizes insulin function in the brain. Thus, JNK may integrate diverse metabolic signals and differentially regulate feeding under distinct physiological conditions.

Functional requirement of AgRP and NPY neurons in ovarian cycle-dependent regulation of food intake.

In female mammals including rodents and humans, feeding decreases during the periovulatory period of the ovarian cycle, which coincides with a surge in circulating estrogen levels. Ovariectomy increases food intake, which can be normalized by estrogen treatment at a dose and frequency mimicking those during the estrous cycle. Furthermore, administration of estrogen to rodents potently inhibits food intake. Despite these well-known effects of estrogen, neuronal subtypes that mediate estrogen's anorexigenic effects have not been identified. In this study, we show that changes in hypothalamic expression of agouti-related protein (Agrp) and neuropeptide Y (Npy) coincide with the cyclic changes in feeding across the estrous cycle. These cyclic changes in feeding are abolished in mice with degenerated AgRP neurons even though these mice cycle normally. Central administration of 17beta-estradiol (E2) decreases food intake in controls but not in mice lacking the AgRP neurons. Furthermore, E2 treatment suppresses fasting-induced c-Fos activation in AgRP and NPY neurons and blunts the refeeding response. Surprisingly, although estrogen receptor alpha (ERalpha) is the key mediator of estrogen's anorexigenic effects, we find that expression of ERalpha is completely excluded from AgRP and NPY neurons in the mouse hypothalamus, suggesting that estrogen may regulate these neurons indirectly via presynaptic neurons that express ERalpha. This study indicates that neurons coexpressing AgRP and NPY are functionally required for the cyclic changes in feeding across estrous cycle and that AgRP and NPY neurons are essential mediators of estrogen's anorexigenic function.

CCAAT/enhancer binding protein alpha (C/EBPalpha) in adipose tissue regulates genes in lipid and glucose metabolism and a genetic variation in C/EBPalpha is associated with serum levels of triglycerides.

CONTEXT: CCAAT/enhancer binding protein alpha (C/EBPalpha) is a transcription factor involved in adipogenesis and hepatic glucose and lipid metabolism. OBJECTIVE: The aim of the study was to test the hypothesis that adipose tissue C/EBPalpha regulates genes in lipid and glucose metabolism and to test for an association between a polymorphism in C/EBPalpha and metabolic parameters. DESIGN AND METHODS: Adipose tissue C/EBPalpha mRNA expression was analyzed at four time points in obese subjects with (n = 12) and without (n = 12) the metabolic syndrome during caloric restriction (450 kcal/d for 16 wk) using DNA microarray and real-time PCR. Adenoviral overexpression of C/EBPalpha was used to identify genes regulated by C/EBPalpha in 3T3-L1 cells. Association between a genetic variation in C/EBPalpha (rs12691) and metabolic parameters was tested in the Swedish Obese Subjects (SOS) study (n = 528) and replicated in Finnish individuals from the Botnia type 2 diabetes study (n = 4,866). RESULTS: During caloric restriction, adipose tissue C/EBPalpha mRNA levels were reduced in subjects with the metabolic syndrome (P = 0.024) and correlated to metabolic parameters. In 3T3-L1 cells, C/EBPalpha regulated the expression of adiponectin; hexokinase 2; lipoprotein lipase; diacylglycerol O-acyltransferase 1 and 2; ATP-binding cassette, sub-family D, member 2; acyl-coenzyme A synthetase long-chain family member 1; CD36; and hydroxysteroid 11-beta dehydrogenase 1. Furthermore, the expression of the human homologs, except adiponectin, correlated to C/EBPalpha mRNA levels in human adipose tissue. The AA genotype of rs12691 was associated with higher serum triglyceride levels in the SOS study (P = 0.022), and this association was replicated in the Botnia study (P = 0.041). CONCLUSIONS: Adipose tissue C/EBPalpha regulates several genes in glucose and lipid metabolism, and a genetic variation in C/EBPalpha is associated with triglycerides in two independent populations.

Evaluation of reference genes for studies of gene expression in human adipose tissue.

OBJECTIVE: The aim of this study was to evaluate reference genes for expression studies of human adipose tissue. RESEARCH METHODS AND PROCEDURES: Using 52 human adipose tissue expression profiles (HU95), 10 putative reference genes with the lowest variation in expression levels were selected for further studies. Expression stability of these 10 novel and 5 previously established reference genes was evaluated by real-time reverse transcriptase-polymerase chain reaction analysis. For this purpose, 44 adipose tissue biopsies from 27 subjects were chosen to include a wide range of parameters such as sex, age, BMI, depot origin, biopsy procedure, and effects of nutrition. RESULTS: LRP10 was identified as the gene with the least variation in expression levels. The frequently used reference genes RPLP0, 18S rRNA, PPIA, ACTB, and GAPD were ranked as 4, 6, 7, 8, and 10, respectively. DISCUSSION: Our results suggest that LRP10 is a better choice as reference for expression studies of human adipose tissue compared with the most frequently used reference genes.