Audrey Leloire

Dysfunction of lipid sensor GPR120 leads to obesity in both mouse and human

Free fatty acids provide an important energy source as nutrients, and act as signalling molecules in various cellular processes. Several G-protein-coupled receptors have been identified as free-fatty-acid receptors important in physiology as well as in several diseases. GPR120 (also known as O3FAR1) functions as a receptor for unsaturated long-chain free fatty acids and has a critical role in various physiological homeostasis mechanisms such as adipogenesis, regulation of appetite and food preference. Here we show that GPR120-deficient mice fed a high-fat diet develop obesity, glucose intolerance and fatty liver with decreased adipocyte differentiation and lipogenesis and enhanced hepatic lipogenesis. Insulin resistance in such mice is associated with reduced insulin signalling and enhanced inflammation in adipose tissue. In human, we show that GPR120 expression in adipose tissue is significantly higher in obese individuals than in lean controls. GPR120 exon sequencing in obese subjects reveals a deleterious non-synonymous mutation (p.R270H) that inhibits GPR120 signalling activity. Furthermore, the p.R270H variant increases the risk of obesity in European populations. Overall, this study demonstrates that the lipid sensor GPR120 has a key role in sensing dietary fat and, therefore, in the control of energy balance in both humans and rodents.

Tryptophan metabolism activation by indoleamine 2,3-dioxygenase in adipose tissue of obese women: an attempt to maintain immune homeostasis and vascular tone.

Human obesity is characterized by chronic low-grade inflammation in white adipose tissue and is often associated with hypertension. The potential induction of indoleamine 2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme in tryptophan/kynurenine degradation pathway, by proinflammatory cytokines, could be associated with these disorders but has remained unexplored in obesity. Using immunohistochemistry, we detected IDO1 expression in white adipose tissue of obese patients, and we focused on its contribution in the regulation of vascular tone and on its immunoregulatory effects. Concentrations of tryptophan and kynurenine were measured in sera of 36 obese and 15 lean women. The expression of IDO1 in corresponding omental and subcutaneous adipose tissues and liver was evaluated. Proinflammatory markers and T-cell subsets were analyzed in adipose tissue via the expression of CD14, IL-18, CD68, TNFα, CD3ε, FOXP3 [a regulatory T-cell (Treg) marker] and RORC (a Th17 marker). In obese subjects, the ratio of kynurenine to tryptophan, which reflects IDO1 activation, is higher than in lean subjects. Furthermore, IDO1 expression in both adipose tissues and liver is increased and is inversely correlated with arterial blood pressure. Inflammation is associated with a T-cell infiltration in obese adipose tissue, with predominance of Th17 in the omental compartment and of Treg in the subcutaneous depot. The Th17/Treg balance is decreased in subcutaneous fat and correlates with IDO1 activation. In contrast, in the omental compartment, despite IDO1 activation, the Th17/Treg balance control is impaired. Taken together, our results suggest that IDO1 activation represents a local compensatory mechanism to limit obesity-induced inflammation and hypertension.

The Kynurenine pathway is activated in human obesity and shifted toward kynurenine monooxygenase activation

This study characterized the kynurenine pathway (KP) in human obesity by evaluating circulating levels of kynurenines and the expression of KP enzymes in adipose tissue.

Improved donor/acceptor BRET couples for monitoring β-arrestin recruitment to G protein-coupled receptors

We report highly sensitive bioluminescence resonance energy transfer (BRET) assays with optimized donor/acceptor couples. We combined the energy donors Renilla luciferase (Rluc) and the Rluc8 variant with the energy acceptors yellow fluorescent protein, the YPet variant and the Renilla green fluorescent protein (RGFP). Different donor/acceptor couples were tested in well‐established assays measuring ligand‐induced β‐arrestin (βARR) intramolecular rearrangements and recruitment to G protein‐coupled receptors. We show increased sensitivity with Rluc8/YPet and Rluc8/RGFP couples and measured previously undetectable BRET signals. These tools improve existing βARR assays and offer new options for the development of future BRET assays.

Alternative human liver transcripts of TCF7L2 bind to the gluconeogenesis regulator HNF4α at the protein level

Aims/hypothesisGene polymorphisms of TCF7L2 are associated with increased risk of type 2 diabetes and transcription factor 7-like 2 (TCF7L2) plays a role in hepatic glucose metabolism. We therefore addressed the impact of TCF7L2 isoforms on hepatocyte nuclear factor 4α (HNF4α) and the regulation of gluconeogenesis genes.MethodsLiver TCF7L2 transcripts were analysed by quantitative PCR in 33 non-diabetic and 31 type 2 diabetic obese individuals genotyped for TCF7L2 rs7903146. To analyse transcriptional regulation by TCF7L2, small interfering RNA transfection, luciferase reporter and co-immunoprecipitation assays were performed in human hepatoma HepG2 cells.ResultsIn livers of diabetic compared with normoglycaemic individuals, five C-terminal TCF7L2 transcripts showed increased expression. The type 2 diabetes risk allele of rs7903146 positively correlated with TCF7L2 expression in livers from normoglycaemic individuals only. In HepG2 cells, transcript and TCF7L2 protein levels were increased upon incubation in high glucose and insulin. Of the exon 13 transcripts, six were increased in a glucose dose-responsive manner. TCF7L2 transcriptionally regulated 29 genes related to glucose metabolism, including glucose-6-phosphatase. In cultured HepG2 cells, TCF7L2 did not regulate HNF4Α and FOXO1 transcription, but did affect HNF4α protein expression. The TCF7L2 isoforms T6 and T8 (without exon 13 and with exon 15/14, respectively) specifically interacted with HNF4α.Conclusions/interpretationThe different levels of expression of alternative C-terminal TCF7L2 transcripts in HepG2 cells, in livers of normoglycaemic individuals carrying the rs7901346 type 2 diabetes risk allele and in livers of diabetic individuals suggest that these transcripts play a role in the pathophysiology of type 2 diabetes. We also report for the first time a protein interaction in HepG2 cells between HNF4α and the T6 and T8 isoforms of TCF7L2, which suggests a distinct role for these specific alternative transcripts.

Identification of a Variable Number of Tandem Repeats Polymorphism and Characterization of LEF-1 Response Elements in the Promoter of the IDO1 Gene

Background Indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-limiting step of the kynurenine pathway that is an important component of immunomodulatory and neuromodulatory processes. The IDO1 gene is highly inducible by IFN-γ and TNF-α through interaction with cis-acting regulatory elements of the promoter region. Accordingly, functional polymorphisms in the IDO1 promoter could partly explain the interindividual variability in IDO expression that has been previously documented. Methodology/Principal Findings A PCR-sequencing strategy, applied to DNA samples from healthy Caucasians, allowed us to identify a VNTR polymorphism in the IDO1 promoter, which correlates significantly with serum tryptophan concentration, controlled partially by IDO activity, in female subjects, but not in males. Although this VNTR does not appear to affect basal or cytokine-induced promoter activity in gene reporter assays, it contains novel cis-acting elements. Three putative LEF-1 binding sites, one being located within the VNTR repeat motif, were predicted in silico and confirmed by chromatin immunoprecipitation. Overexpression of LEF-1 in luciferase assays confirmed an interaction between LEF-1 and the predicted transcription factor binding sites, and modification of the LEF-1 core sequence within the VNTR repeat motif, by site-directed mutagenesis, resulted in an increase in promoter activity. Conclusions/Significance The identification of a VNTR in the IDO1 promoter revealed a cis-acting element interacting with the most downstream factor of the Wnt signaling pathway, suggesting novel mechanisms of regulation of IDO1 expression. These data offer new insights, and suggest further studies, into the role of IDO in various pathological conditions, particularly in cancer where IDO and the Wnt pathway are strongly dysregulated.

Evidence for leptin receptor isoforms heteromerization at the cell surface.

MINT‐7714872: LEPRb (uniprotkb:P48357‐1) physically interacts (MI:0915) with LEPRc (uniprotkb:P48357‐2) by anti tag co‐immunoprecipitation (MI:0007)

Contribution of the low-frequency, loss-of-function p.R270H mutation in FFAR4 ( GPR120 ) to increased fasting plasma glucose levels

Background We previously reported that the low-frequency, loss-of-function variant p.R270H in FFAR4 encoding the lipid sensor GPR120 was associated with obesity. Gpr120-deficient mice develop obesity and both impaired fasting glucose and glucose intolerance under a high-fat diet. We aimed to assess the contribution of p.R270H to type 2 diabetes (T2D) risk and the variation of glucose-related traits. Methods We genotyped p.R270H in 8996 non-diabetic individuals (among whom 4523 had an oral glucose tolerance test (OGTT)) and in a T2D case–control study including 4725 cases and 4339 controls. The regression models were adjusted for age, sex and body mass index (BMI). Results We found a significant association between p.R270H and increased fasting glucose levels (β=0.092±0.05 mmol/L; p=4.13×10−4). Furthermore, p.R270H nominally contributed to decreased homeostasis model of pancreatic β-cell function (HOMA-B; β=−0.090±0.06; p=6.01×10−3). Despite a high statistical power, we did not find any significant association between p.R270H and T2D risk or the variation of fasting insulin levels, the homeostasis model of insulin resistance or OGTT-derived indices. Conclusions These results suggest that the low-frequency p.R270H variant which inhibits GPR120 activity might influence fasting glucose levels in a normal physiological range. This study does not exclude that other coding mutations in FFAR4 with stronger functional effect than p.R270H may be associated with T2D.

Low-dose exposure to bisphenols A, F and S of human primary adipocyte impacts coding and non-coding RNA profiles

Bisphenol A (BPA) exposure has been suspected to be associated with deleterious effects on health including obesity and metabolically-linked diseases. Although bisphenols F (BPF) and S (BPS) are BPA structural analogs commonly used in many marketed products as a replacement for BPA, only sparse toxicological data are available yet. Our objective was to comprehensively characterize bisphenols gene targets in a human primary adipocyte model, in order to determine whether they may induce cellular dysfunction, using chronic exposure at two concentrations: a “low-dose” similar to the dose usually encountered in human biological fluids and a higher dose. Therefore, BPA, BPF and BPS have been added at 10 nM or 10 μM during the differentiation of human primary adipocytes from subcutaneous fat of three non-diabetic Caucasian female patients. Gene expression (mRNA/lncRNA) arrays and microRNA arrays, have been used to assess coding and non-coding RNA changes. We detected significantly deregulated mRNA/lncRNA and miRNA at low and high doses. Enrichment in “cancer” and “organismal injury and abnormalities” related pathways was found in response to the three products. Some long intergenic non-coding RNAs and small nucleolar RNAs were differentially expressed suggesting that bisphenols may also activate multiple cellular processes and epigenetic modifications. The analysis of upstream regulators of deregulated genes highlighted hormones or hormone-like chemicals suggesting that BPS and BPF can be suspected to interfere, just like BPA, with hormonal regulation and have to be considered as endocrine disruptors. All these results suggest that as BPA, its substitutes BPS and BPF should be used with the same restrictions.

Post-Bariatric Surgery Changes in Quinolinic and Xanthurenic Acid Concentrations Are Associated with Glucose Homeostasis

Background An increase of plasma kynurenine concentrations, potentially bioactive metabolites of tryptophan, was found in subjects with obesity, resulting from low-grade inflammation of the white adipose tissue. Bariatric surgery decreases low-grade inflammation associated with obesity and improves glucose control. Objective Our goal was to determine the concentrations of all kynurenine metabolites after bariatric surgery and whether they were correlated with glucose control improvement. Design Kynurenine metabolite concentrations, analysed by liquid or gas chromatography coupled with tandem mass spectrometry, circulating inflammatory markers, metabolic traits, and BMI were measured before and one year after bariatric surgery in 44 normoglycemic and 47 diabetic women with obesity. Associations between changes in kynurenine metabolites concentrations and in glucose control and metabolic traits were analysed between baseline and twelve months after surgery. Results Tryptophan and kynurenine metabolite concentrations were significantly decreased one year after bariatric surgery and were correlated with the decrease of the usCRP in both groups. Among all the kynurenine metabolites evaluated, only quinolinic acid and xanthurenic acid were significantly associated with glucose control improvement. The one year delta of quinolinic acid concentrations was negatively associated with the delta of fasting glucose (p = 0.019) and HbA1c (p = 0.014), whereas the delta of xanthurenic acid was positively associated with the delta of insulin sensitivity index (p = 0.0018). Conclusion Bariatric surgery has induced a global down-regulation of kynurenine metabolites, associated with weight loss. Our results suggest that, since kynurenine monoxygenase diverts the kynurenine pathway toward the synthesis of xanthurenic acid, its inhibition may also contribute to glucose homeostasis.