Johanne Le Beyec

Hepatocyte Nuclear Factor 4α, a Key Factor for Homeostasis, Cell Architecture, and Barrier Function of the Adult Intestinal Epithelium

ABSTRACT Hepatocyte nuclear factor 4α (HNF-4α) is a transcription factor which is highly expressed in the intestinal epithelium from duodenum to colon and from crypt to villus. The homeostasis of this constantly renewing epithelium relies on an integrated control of proliferation, differentiation, and apoptosis, as well as on the functional architecture of the epithelial cells. In order to determine the consequences of HNF-4α loss in the adult intestinal epithelium, we used a tamoxifen-inducible Cre-loxP system to inactivate the Hnf-4a gene. In the intestines of adult mice, loss of HNF-4α led to an increased proliferation in crypts and to an increased expression of several genes controlled by the Wnt/β-catenin system. This control of the Wnt/β-catenin signaling pathway by HNF-4α was confirmed in vitro. Cell lineage was affected, as indicated by an increased number of goblet cells and an impairment of enterocyte and enteroendocrine cell maturation. In the absence of HNF-4α, cell-cell junctions were destabilized and paracellular intestinal permeability increased. Our results showed that HNF-4α modulates Wnt/β-catenin signaling and controls intestinal epithelium homeostasis, cell function, and cell architecture. This study indicates that HNF-4α regulates the intestinal balance between proliferation and differentiation, and we hypothesize that it might act as a tumor suppressor.

Melanocortin-4 receptor mutations and polymorphisms do not affect weight loss after bariatric surgery.

Bariatric surgery is the most effective long term weight-loss therapy for severe and morbidly obese patients. Melanocortin-4 Receptor (MC4R) mutations, the most frequent known cause of monogenic obesity, affect the regulation of energy homeostasis. The impact of such mutations on weight loss after bariatric surgery is still debated. The objective is to determine the impact of MC4R status on weight loss in obese subjects over one year after bariatric surgery. A total of 648 patients, who were referred to bariatric surgery in a single clinical nutrition department, were genotyped for their MC4R status. The following four groups were categorized: functional MC4R mutations, MC4R single nucleotide polymorphisms (SNPs): Val103Ile (V103L) and Ile251Leu (I251L), MC4R variant rs17782313 (downstream of MC4R) and MC4R SNP A-178C on the promoter. Each patient was matched with two randomly paired controls without mutation. Matching factors were age, sex, baseline weight and type of surgery procedure (Roux-en-Y gastric bypass and adjustable gastric banding). We compared weight loss between cases and controls at 3, 6 and 12 months after surgery. Among 648 patients, we identified 9 carriers of functional MC4R mutations, 10 carriers of MC4R V103L and I251L SNPs, 7 carriers of the rs17792313 variant and 22 carriers of the A-178C SNP. Weight loss at 3, 6 and 12 months did not differ between cases and controls, whatever the MC4R mutations. This is the first case-control study to show that MC4R mutations and polymorphisms do not affect weight loss and body composition over one year after bariatric surgery.

Epidermal growth factor receptor is involved in enterocyte anoikis through the dismantling of E-cadherin-mediated junctions

Enterocytes of the intestinal epithelium are continually regenerated. They arise from precursor cells in crypts, migrate along villi, and finally die, 3-4 days later, when they reach the villus apex. Their death is thought to occur by anoikis, a form of apoptosis induced by cell detachment, but the mechanism of this process remains poorly understood. We have previously shown that a key event in the onset of anoikis in normal enterocytes detached from the basal lamina is the disruption of adherens junctions mediated by E-cadherin (Fouquet S, Lugo-Martinez VH, Faussat AM, Renaud F, Cardot P, Chambaz J, Pincon-Raymond M, Thenet S. J Biol Chem 279: 43061-43069, 2004). Here we have further investigated the mechanisms underlying this disassembly of the adherens junctions. We show that disruption of the junctions occurs through endocytosis of E-cadherin and that this process depends on the tyrosine-kinase activity of the epidermal growth factor receptor (EGFR). Activation of EGFR was detected in detached enterocytes before E-cadherin disappearance. Specific inhibition of EGFR by tyrphostin AG-1478 maintained E-cadherin and its cytoplasmic partners beta- and alpha-catenin at cell-cell contacts and decreased anoikis. Finally, EGFR activation was evidenced in the intestinal epithelium in vivo, in rare individual cells, which were shown to lose their interactions with the basal lamina. We conclude that EGFR is activated as enterocytes become detached from the basal lamina, and that this mechanism contributes to the disruption of E-cadherin-dependent junctions leading to anoikis. This suggests that EGFR participates in the physiological elimination of the enterocytes.

Evaluation of a melanocortin-4 receptor (MC4R) agonist (Setmelanotide) in MC4R deficiency.

Objective Pro-opiomelanocortin (POMC)-derived peptides act on neurons expressing the Melanocortin 4 receptor (MC4R) to reduce body weight. Setmelanotide is a highly potent MC4R agonist that leads to weight loss in diet-induced obese animals and in obese individuals with complete POMC deficiency. While POMC deficiency is very rare, 1–5% of severely obese individuals harbor heterozygous mutations in MC4R. We sought to assess the efficacy of Setmelanotide in human MC4R deficiency. Methods We studied the effects of Setmelanotide on mutant MC4Rs in cells and the weight loss response to Setmelanotide administration in rodent studies and a human clinical trial. We annotated the functional status of 369 published MC4R variants. Results In cells, we showed that Setmelanotide is significantly more potent at MC4R than the endogenous ligand alpha-melanocyte stimulating hormone and can disproportionally rescue signaling by a subset of severely impaired MC4R mutants. Wild-type rodents appear more sensitive to Setmelanotide when compared to MC4R heterozygous deficient mice, while MC4R knockout mice fail to respond. In a 28-day Phase 1b clinical trial, Setmelanotide led to weight loss in obese MC4R variant carriers. Patients with POMC defects upstream of MC4R show significantly more weight loss with Setmelanotide than MC4R deficient patients or obese controls. Conclusions Setmelanotide led to weight loss in obese people with MC4R deficiency; however, further studies are justified to establish whether Setmelanotide can elicit clinically meaningful weight loss in a subset of the MC4R deficient obese population.

Enhanced Ghrelin Levels and Hypothalamic Orexigenic AgRP and NPY Neuropeptide Expression in Models of Jejuno-Colonic Short Bowel Syndrome

Short bowel syndrome (SBS) patients developing hyperphagia have a better outcome. Gastrointestinal endocrine adaptations help to improve intestinal functions and food behaviour. We investigated neuroendocrine adaptations in SBS patients and rat models with jejuno-ileal (IR-JI) or jejuno-colonic (IR-JC) anastomosis with and without parenteral nutrition. Circulating levels of ghrelin, PYY, GLP-1, and GLP-2 were determined in SBS rat models and patients. Levels of mRNA for proglucagon, PYY and for hypothalamic neuropeptides were quantified by qRT-PCR in SBS rat models. Histology and immunostaining for Ki67, GLP-1 and PYY were performed in SBS rats. IR-JC rats, but not IR-JI, exhibited significantly higher crypt depths and number of Ki67-positive cells than sham. Fasting and/or postprandial plasma ghrelin and PYY concentrations were higher, or tend to be higher, in IR-JC rats and SBS-JC patients than in controls. Proglucagon and Pyy mRNA levels were significantly enhanced in IR-JC rats. Levels of mRNA coding hypothalamic orexigenic NPY and AgRP peptides were significantly higher in IR-JC than in sham rats. We demonstrate an increase of plasma ghrelin concentrations, major changes in hypothalamic neuropeptides levels and greater induction of PYY in SBS-JC rats and patients suggesting that jejuno-colonic continuity creates a peculiar environment promoting further gut-brain adaptations.

Hepatic Nuclear Factor-4, a Key Transcription Factor at the Crossroads Between Architecture and Function of Epithelia

Hepatic nuclear factor-4 (HNF-4) is a transcription factor and a member of the large family of nuclear receptors. It was first cloned from liver but is expressed also in kidney, pancreas and intestine. Three genes encoding three isoforms have been identified, HNF- 4α and γ, in mammals, drosophila and xenopus and HNF-4β, exclusively in xenopus. HNF-4α is the best studied isoform, especially in liver. Such studies put HNF-4α at the crossroads between architecture and function of epithelia, as it induces expression of cell/cell junction proteins while it also controls glucido-lipidic metabolism and drug metabolizing enzyme genes. Furthermore, mutations in the HNF-4α gene lead to a metabolic disease in humans, Maturity Onset Diabetes of the Young-1 (MODY-1). The existence of a “true ligand” is not clearly established but a “structural” fatty acid is present in the ligand binding pocket of HNF-4α and γ. Consequently, activity of HNF-4 can be modulated by the interaction with co-regulators or by post-translational modifications. Then, HNF-4 is a potential direct or indirect target for pharmacologic drugs, with a special interest for the intestinal epithelium which is the primary site of metabolic control, due to its roles in nutrient absorption and in sensing energy. The patents related to the HNF-4α gene are also discussed in this article.

Fasting levels of glicentin are higher in Roux-en-Y gastric bypass patients exhibiting postprandial hypoglycemia during a meal test

BACKGROUND Post-bypass postprandial hypoglycemia (PPH) is a frequent complication of Roux-en-Y Gastric Bypass (RYGB) but predictors remain poorly identified and are needed to assess individual risk. After RYGB, exaggerated secretion of glucagon-like peptide-1 (GLP-1) and insulin could lead to PPH, but other proglucagon-derived peptides, including glicentin and glucagon, could also contribute to this phenomenon. OBJECTIVES To identify biological hypoglycemia in relation to the secretion of proglucagon-derived peptides during a mixed-meal test (MMT) in RYGB patients. SETTING University hospital. METHODS Twenty RYGB patients reporting symptoms consistent with PPH were examined 36.9 ± 5.1 months after surgery. Plasma levels of glucose, c-peptide, glucagon, GLP-1 and glicentin were assessed before and during MMT. Patients with postprandial hypoglycemia ≤3 mM (54 mg/dL) during MMT were assigned to HYPO group and compared with patients not exhibiting hypoglycemia (NONHYPO group). RESULTS Seven patients displayed hypoglycemia ≤3 mM (HYPO) during the MMT. Lower fasting glycemia (4.5 mM versus 5.3 mM, P<.05) and higher fasting glicentin (22.6 pM versus 14.0 pM, P<.05) were observed in HYPO versus NONHYPO patients. Fasting glicentin was inversely correlated with postprandial nadir glucose. Examining the receiver-operating characteristics curve analysis, a cutoff of 17.2 pM for fasting glicentin identified PPH with 85.7% sensitivity and 53.8% specificity. All patients exhibited a similar increase of postprandial GLP-1, glucagon, and glicentin secretions that correlated with each other. CONCLUSIONS These results suggest that fasting glicentin is a potential biomarker to examine in operated-obese patients at risk of developing PPH. Further studies are needed before proposing fasting glicentin as a predictive factor of PPH.