Overexpression of gastric leptin precedes adipocyte leptin during high-fat diet and is linked to 5HT-containing enterochromaffin cells.

OBJECTIVES: In obesity, while hyperleptinemia highly correlates with excess fat mass, the status of gastric leptin remains unknown. Here, we investigated the expression of leptin in stomach biopsies of obese humans and analyzed the temporal changes of gastric leptin expression in response to diet-induced obesity and its impact on 5-hydroxytryptamine (5HT)-producing cells. METHODS: Enterochromaffin (EC) cells and expression of leptin, PAX4 (critical factor for EC specification), tryptophane hydroxylase-1 (TPH1, the peripheral rate-limiting enzyme for 5HT) and 5HT were examined by immunofluorescence, quantitative real-time PCR, radioimmunoassay, respectively, in stomach and duodenum biopsies from 19 obese and 14 normo-weighed individuals, and in mucosa scrapings from C57Bl6/J diet-induced obese mice, leptin-deficient ob/ob mice and intestine-specific leptin receptor isoform B-deficient mice. RESULTS: Gastric mucosa of obese subjects displays an increased expression of leptin (LEP mRNA by fivefold and protein by twofold, P<0.01), TPH1 ((1.75-2.73, 95% confidence interval (CI)) vs (0.38-0.67, 95% CI); P<0.01) and PAX4 ((1.33-2.11, 95%CI) vs (0.62-0.81, 95% CI); P<0.01) as compared with normo-weighed individuals. In diet-induced obese mice, the overexpressions of gastric leptin, antral Pax4, Tph1 and increased EC cell number occurred before the onset of obesity and hyperleptinemia (reflect of adipocyte leptin production). In addition, leptin deficiency was associated with reduced Pax4 mRNA, whereas oral leptin treatment enhanced both Tph1 and Pax4 mRNA. Finally, mice with an intestine-specific deletion of leptin signaling exhibit significant decrease in duodenal mucosa 5HT content. CONCLUSIONS: These data demonstrate that gastric leptin is upregulated in obese individuals. RESULTS from high-fat diet mice showed that overexpression of gastric leptin that is linked to gut '5HT pathway' occurred before the onset of obesity and expansion of fat mass. This may be relevant in the pathophysiology of obesity.

Association between melanocortin-4 receptor mutations and eating behaviors in obese patients: a case--control study.

Melanocortin-4 receptor (MC4R) gene mutations are involved in the leptin-melanocortin pathways that control food intake. The effect of these mutations on eating behavior phenotypes is still debated. To determine the association between functional MC4R mutations and eating behaviors, dietary intake and physical activity, we sequenced the MC4R gene in 4653 obese adults. Among them, 19 adults carriers of functional MC4R mutation were matched on age, sex and body mass index with two randomly-paired controls without MC4R mutation (n=57). We found that eating behaviors and physical activity did not differ between groups. In particular, cases were not at increased risk of binge eating disorders. Subjects carriers of MC4R mutation reported a higher proportion of dietary carbohydrates intakes (43.2±7.1 and 39.2±8.1% of total energy intake, respectively, P=0.048) and a lower proportion of dietary lipids (34.3±6.7 and 38.5±6.7% of total energy intake, respectively, P=0.018). In conclusion, mutation carriers differ from controls by a higher consumption of carbohydrates counterbalanced by a lower consumption of lipids expressed as percentage of total energy intake. However, functional MC4R mutations do not have a higher risk of compulsive eating contrary to what was previously suggested.

Illegitimate expression of apolipoprotein A-II in Caco-2 cells is due to chromatin organization.

Transcriptional activity of the human apolipoprotein (apo) A-II promoter has been reported in transiently transfected Caco-2 cells, but not in the intestine in vivo. In the present study we established that the transcription of a stably transfected reporter gene under the control of the -911/+29 human apo A-II, decreases with the onset of the differentiation process. This decrease paralleled that of the expression of the endogenous apo A-II gene. The decrease in apo A-II expression is also followed by a marked increase in the expression of the intestine-specific apo A-IV gene, analyzed here as a marker of enterocytic differentiation. Using clonal glucose metabolic variants of Caco-2 cells we have also observed that the lowest levels of apo A-II mRNA are associated with the lowest rates of glucose consumption. The illegitimate apo A-II transcriptional activity observed in Caco-2 cells is linked to the presence of DNase-I hypersensitive sites within the enhancer. This reflects a chromatin organization which allows, in Caco-2 cells as in the liver, the communication between the apo A-II enhancer and the proximal promoter, unlike what is observed in intestinal epithelial cells.

The -700/-310 fragment of the apolipoprotein A-IV gene combined with the -890/-500 apolipoprotein C-III enhancer is sufficient to direct a pattern of gene expression similar to that for the endogenous apolipoprotein A-IV gene.

Spatial gene expression in the intestine is mediated by specific regulatory sequences. The three genes of the apoA-I/C-III/A-IV cluster are expressed in the intestine following cephalocaudal and crypt-to-villus axes. Previous studies have shown that the -780/-520 enhancer region of the apoC-III gene directs the expression of the apoA-I gene in both small intestinal villi and crypts, implying that other unidentified elements are necessary for a normal intestinal pattern of apoA-I gene expression. In this study, we have characterized transgenic mice expressing the chloramphenicol acetyltransferase gene under the control of different regions of the apoC-III and apoA-IV promoters. We found that the -890/+24 apoC-III promoter directed the expression of the reporter gene in crypts and villi and did not follow a cephalocaudal gradient of expression. In contrast, the -700/+10 apoA-IV promoter linked to the -500/-890 apoC-III enhancer directed the expression of the reporter gene in enterocytes with a pattern of expression similar to that of the endogenous apoA-IV gene. Furthermore, linkage of the -700/-310 apoA-IV distal promoter region to the -890/+24 apoC-III promoter was sufficient to restore the appropriate pattern of intestinal expression of the reporter gene. These findings demonstrate that the -700/-310 distal region of the apoA-IV promoter contains regulatory elements that, in combination with proximal promoter elements and the -500/-890 enhancer, are necessary and sufficient to restrict apoC-III and apoA-IV gene expression to villus enterocytes of the small intestine along the cephalocaudal axis.

A complete epithelial organization of Caco-2 cells induces I-FABP and potentializes apolipoprotein gene expression.

The culture of Caco-2 cells on plastic support impairs the expression of several genes involved in lipid metabolism. We describe culture conditions that permit the expression of the I-FABP gene and better expression of the apolipoprotein A-I, C-III, and A-IV genes. Basal lamina deposited on filters as well as the nature of nutrients on the apical side differentially modulated mRNA expression of I-FABP, APOBEC-1, and apolipoprotein genes. Growing cells on a filter led to functional polarization, illustrated by a secretion of apo B at the basal side, which induced the expression of the I-FABP, APOBEC-1, and apo A-IV genes and highly increased the expression of the apo C-III gene. Moreover, basal lamina deposited on the filter enhances the mRNA expression of apo A-I. Apo C-III and A-IV mRNA levels were decreased when cells were grown on a filter covered with basal lamina in the presence of a medium deprived of protein and lipid on the apical side, whereas these conditions had no effect on I-FABP, apo A-I, and APOBEC-1 mRNA levels. The addition of lipid micelles on the apical side had various effects, according to the genes. Caco-2 cells cultured under the conditions described here closely resembled enterocytes and represent a useful tool for studying the regulation of genes involved in lipid metabolism.