Mu-opioid receptors and dietary protein stimulate a gut-brain neural circuitry limiting food intake.

Intestinal gluconeogenesis is involved in the control of food intake. We show that mu-opioid receptors (MORs) present in nerves in the portal vein walls respond to peptides to regulate a gut-brain neural circuit that controls intestinal gluconeogenesis and satiety. In vitro, peptides and protein digests behave as MOR antagonists in competition experiments. In vivo, they stimulate MOR-dependent induction of intestinal gluconeogenesis via activation of brain areas receiving inputs from gastrointestinal ascending nerves. MOR-knockout mice do not carry out intestinal gluconeogenesis in response to peptides and are insensitive to the satiety effect induced by protein-enriched diets. Portal infusions of MOR modulators have no effect on food intake in mice deficient for intestinal gluconeogenesis. Thus, the regulation of portal MORs by peptides triggering signals to and from the brain to induce intestinal gluconeogenesis are links in the satiety phenomenon associated with alimentary protein assimilation.

Intestinal absorption of sphingosine: new insights on generated ceramide species using stable isotope tracing in vitro.

Dietary sphingomyelin (SM) has been reported to favorably modulate postprandial lipemia. Mechanisms underlying these beneficial effects on cardiovascular risk markers are not fully elucidated. Rodent studies showed that tritiated SM was hydrolyzed in the intestinal lumen into ceramides (Cer) and further to sphingosine (SPH) and fatty acids (FA) that were absorbed by the intestine. Our objective was to investigate the uptake and metabolism of SPH and/or tricosanoic acid (C23:0), the main FA of milk SM, as well as lipid secretion in Caco-2/TC7 cells cultured on semipermeable inserts. Mixed micelles (MM) consisting of different digested lipids and taurocholate were prepared without or with SPH, SPH and C23:0 (SPH+C23:0), or C23:0. Triglycerides (TG) were quantified in the basolateral medium, and sphingolipids were analyzed by tandem mass spectrometry. TG secretion increased 11-fold in all MM-incubated cells compared with lipid-free medium. Apical supply of SPH-enriched MM led to increased concentrations of total Cer in cells, and coaddition of C23:0 in SPH-enriched MM led to a preferential increase of C23:0 Cer and C23:0 SM. Complementary experiments using deuterated SPH demonstrated that SPH-d9 was partly converted to sphingosine-1-phosphate-d9, Cer-d9, and SM-d9 within cells incubated with SPH-enriched MM. A few Cer-d9 (2% of added SPH-d9) was recovered in the basolateral medium of (MM+SPH)-incubated cells, especially C23:0 Cer-d9 in (MM+SPH+C23:0)-enriched cells. In conclusion, present results indicate that MM enriched with (SPH+C23:0), such as found in postprandial micelles formed after milk SM ingestion, directly impacts sphingolipid endogenous metabolism in enterocytes, resulting in the secretion of TG-rich particles enriched with C23:0 Cer.

Probiotic from human breast milk, Lactobacillus fermentum, promotes growth in animal model of chronic malnutrition.

BACKGROUND: Chronic undernutrition leads to growth hormone resistance and poor growth in children, which has been shown to be modulated by microbiota. We studied whether Lactobacillus fermentum CECT5716 (Lf CECT5716), isolated from mother's breast milk, could promote juvenile growth through the modulation of lipid absorption in a model of starvation. METHODS: Germ-free (GF) Drosophila melanogaster larvae were inoculated with Lf CECT5716 in conditions of undernutrition with and without infant formula. The impact of Lf CECT5716 on larval growth was assessed 7 days after egg laying (AED) by measuring the larval size and on maturation by measuring the emergence of pupae during 21 days AED. For lipid absorption test, Caco2/TC7 intestinal cells were incubated with Lf CECT5716 and challenged with mixed lipid micelles. RESULTS: The mono-associated larvae with Lf CECT5716 were significantly longer than GF larvae (3.7 vs 2.5 mm; p < 0.0001). The effect was maintained when Lf CECT5716 was added to the infant formula. The maturation time of larvae was accelerated by Lf CECT5716 (12 vs 13.2 days; p = 0.01). Lf CECT5716 did not have significant impact on lipid absorption in Caco2/TC7 cells. CONCLUSIONS: Lf CECT5716 is a growth-promoting strain upon undernutrition in Drosophila, with a maintained effect when added to an infant formula but without effect on lipid absorption in vitro.

Milk Polar Lipids Affect In Vitro Digestive Lipolysis and Postprandial Lipid Metabolism in Mice.

BACKGROUND: Polar lipid (PL) emulsifiers such as milk PLs (MPLs) may affect digestion and subsequent lipid metabolism, but focused studies on postprandial lipemia are lacking. OBJECTIVE: We evaluated the impact of MPLs on postprandial lipemia in mice and on lipid digestion in vitro. METHODS: Female Swiss mice were gavaged with 150 µL of an oil-in-water emulsion stabilized with 5.7 mg of either MPLs or soybean PLs (SPLs) and killed after 1, 2, or 4 h. Plasma lipids were quantified and in the small intestine, gene expression was analyzed by reverse transcriptase-quantitative polymerase chain reaction. Emulsions were lipolyzed in vitro using a static human digestion model; triglyceride (TG) disappearance was followed by thin-layer chromatography. RESULTS: In mice, after 1 h, plasma TGs tended to be higher in the MPL group than in the SPL group (141 µg/mL vs. 90 µg/mL; P = 0.07) and nonesterified fatty acids (NEFAs) were significantly higher (64 µg/mL vs. 44 µg/mL; P < 0.05). The opposite was observed after 4 h with lower TGs (21 µg/mL vs. 35 µg/mL; P < 0.01) and NEFAs (20 µg/mL vs. 32 µg/mL; P < 0.01) in the MPL group compared with the SPL group. This was associated at 4 h with a lower gene expression of apolipoprotein B (Apob) and Secretion Associated, Ras related GTPase 1 gene homolog B (Sar1b), in the duodenum of MPL mice compared with SPL mice (P < 0.05). In vitro, during the intestinal phase, TGs were hydrolyzed more in the MPL emulsion than in the SPL emulsion (decremental AUCs were 1750%/min vs. 180%/min; P < 0.01). MPLs enhance lipid intestinal hydrolysis and promote more rapid intestinal lipid absorption and sharper kinetics of lipemia. CONCLUSIONS: Postprandial lipemia in mice can be modulated by emulsifying with MPLs compared with SPLs, partly through differences in chylomicron assembly, and TG hydrolysis rate as observed in vitro. MPLs may thereby contribute to the long-term regulation of lipid metabolism.

Postprandial lipid and vascular responses following consumption of a commercially-relevant interesterified palmitic acid-rich spread in comparison to functionally-equivalent non-interesterified spread and spreadable butter: a randomised controlled trial in healthy adults.

Background: Interesterification is an industrial processing technique used widely where hard fats are essential for functionality and consumer acceptability, e.g. margarines and lower fat spreads. Objective: The aim of this study was to compare acute cardiovascular effects of functionally equivalent spreads (similar solid fat content) made with interesterified (IE) or non-IE palm-based fats, or spreadable butter. Methods: A randomised, controlled, 4-armed crossover, double-blind study (25 men, 25 women; 35-75 years; healthy; mean BMI 24.5, SD 3.8), compared effects of mixed nutrient meals containing 50 g fat from functionally equivalent products [IE spread, non-IE spread and spreadable butter (SB), with rapeseed oil (RO) as a reference treatment: with 16.7%, 27.9%, 19.3% and 4% palmitic acid, respectively] on 8 h postprandial changes in plasma triacylglycerol (TAG) and endothelial dysfunction (flow-mediated dilatation; FMD). Circulating reactive oxygen species (estimated using a neutrophil oxidative burst assay), glucose, insulin, NEFA, lipoprotein particle profiles, inflammatory markers (glycoprotein acetylation (Glyc-A) and IL-6), and biomarkers of endotoxemia were measured. Results: Postprandial plasma TAG concentrations after test meals were similar. However following RO versus the 3 spreads, there were significantly higher postprandial apolipoprotein B concentrations, and small HDL and LDL particle concentrations, and lower postprandial extra-large, large, and medium HDL particle concentrations, as well as smaller average HDL and LDL particle sizes. There were no differences following IE compared to the other spreads. Postprandial FMD% did not decrease after high-fat test meals, and there were no differences between treatments. Postprandial serum IL-6 increased similarly after test meals, but RO provoked a greater increase in postprandial concentrations of glycoprotein acetyls (GlycA), as well as 8 h sCD14, an endotoxemia marker. All other postprandial outcomes were not different between treatments. Conclusions: In healthy adults, a commercially-available IE-based spread did not evoke a different postprandial triacylglycerol, lipoprotein subclass, oxidative stress, inflammatory or endotoxemic response to functionally-equivalent, but compositionally-distinct alternative spreads. Clinical trial registry number: NCT03438084 (https://ClinicalTrials.gov).

Validation of Knock-Out Caco-2 TC7 Cells as Models of Enterocytes of Patients with Familial Genetic Hypobetalipoproteinemias.

Abetalipoproteinemia (FHBL-SD1) and chylomicron retention disease (FHBL-SD3) are rare recessive disorders of lipoprotein metabolism due to mutations in MTTP and SAR1B genes, respectively, which lead to defective chylomicron formation and secretion. This results in lipid and fat-soluble vitamin malabsorption, which induces severe neuro-ophthalmic complications. Currently, treatment combines a low-fat diet with high-dose vitamin A and E supplementation but still fails in normalizing serum vitamin E levels and providing complete ophthalmic protection. To explore these persistent complications, we developed two knock-out cell models of FHBL-SD1 and FHBL-SD3 using the CRISPR/Cas9 technique in Caco-2/TC7 cells. DNA sequencing, RNA quantification and Western blotting confirmed the introduction of mutations with protein knock-out in four clones associated with i) impaired lipid droplet formation and ii) defective triglyceride (-57.0 ± 2.6% to -83.9 ± 1.6%) and cholesterol (-35.3 ± 4.4% to -60.6 ± 3.5%) secretion. A significant decrease in α-tocopherol secretion was also observed in these clones (-41.5 ± 3.7% to -97.2 ± 2.8%), even with the pharmaceutical forms of vitamin E: tocopherol-acetate and tocofersolan (α-tocopheryl polyethylene glycol succinate 1000). MTTP silencing led to a more severe phenotype than SAR1B silencing, which is consistent with clinical observations. Our cellular models thus provide an efficient tool to experiment with therapeutic strategies and will allow progress in understanding the mechanisms involved in lipid metabolism.

Inhibition of intestinal FXR activity as a possible mechanism for the beneficial effects of a probiotic mix supplementation on lipid metabolism alterations and weight gain in mice fed a high fat diet.

Supplementation with probiotics has emerged as a promising therapeutic tool to manage metabolic diseases. We investigated the effects of a mix of Bifidobacterium animalis subsp. lactis LA804 and Lactobacillus gasseri LA806 on high-fat (HF) diet -induced metabolic disease in mice. Supplementation with the probiotic mix in HF diet-fed mice (HF-Pr2) reduced weight and fat mass gains, decreased hepatic lipid accumulation, and lowered plasma triglyceride peak during an oral lipid tolerance test. At the molecular level, the probiotic mix protected against HF-induced rise in mRNA levels of genes related to lipid uptake, metabolism, and storage in the liver and white adipose tissues, and strongly decreased mRNA levels of genes related to inflammation in the white adipose tissue and to oxidative stress in the liver. Regarding intestinal homeostasis, the probiotic mix did not prevent HF-induced gut permeability but slightly modified microbiota composition without correcting the dysbiosis induced by the HF diet. Probiotic supplementation also modified the cecal bile acid (BA) profile, leading to an increase in the Farnesoid-X-Receptor (FXR) antagonist/agonist ratio between BA species. In agreement, HF-Pr2 mice exhibited a strong inhibition of FXR signaling pathway in the ileum, which was associated with lipid metabolism protection. This is consistent with recent reports proposing that inhibition of intestinal FXR activity could be a potent mechanism to overcome metabolic disorders. Altogether, our results demonstrate that the probiotic mix evaluated, when administered preventively to HF diet-fed mice could limit obesity and associated lipid metabolism disorders, likely through the inhibition of FXR signaling in the intestinal tract.

Role of circulating sphingolipids in lipid metabolism: Why dietary lipids matter.

Sphingolipids are structural components of cell membranes and lipoproteins but also act as signaling molecules in many pathophysiological processes. Although sphingolipids comprise a small part of the plasma lipidome, some plasma sphingolipids are recognized as implicated in the development of metabolic diseases and cardiovascular diseases. Plasma sphingolipids are mostly carried out into lipoproteins and may modulate their functional properties. Lipids ingested from the diet contribute to the plasma lipid pool besides lipids produced by the liver and released from the adipose tissue. Depending on their source, quality and quantity, dietary lipids may modulate sphingolipids both in plasma and lipoproteins. A few human dietary intervention studies investigated the impact of dietary lipids on circulating sphingolipids and lipid-related cardiovascular risk markers. On the one hand, dietary saturated fatty acids, mainly palmitic acid, may increase ceramide concentrations in plasma, triglyceride-rich lipoproteins and HDL. On the other hand, milk polar lipids may decrease some molecular species of sphingomyelins and ceramides in plasma and intestine-derived chylomicrons. Altogether, different dietary fatty acids and lipid species can modulate circulating sphingolipids vehicled by postprandial lipoproteins, which should be part of future nutritional strategies for prevention of cardiovascular diseases.

Targeted deletion of liver glucose-6 phosphatase mimics glycogen storage disease type 1a including development of multiple adenomas.

BACKGROUND AND AIMS: Glycogen storage disease type 1a (GSD1a) is an inherited disease caused by a deficiency in the catalytic subunit of the glucose-6 phosphatase enzyme (G6Pase). GSD1a is characterized by hypoglycaemia, hyperlipidemia, and lactic acidosis with associated hepatic (including hepatocellular adenomas), renal, and intestinal disorders. A total G6pc (catalytic subunit of G6Pase) knock-out mouse model has been generated that mimics the human pathology. However, these mice rarely live longer than 3 months and long-term liver pathogenesis cannot be evaluated. Herein, we report the long-term characterization of a liver-specific G6pc knock-out mouse model (L-G6pc(-/-)). METHODS: We generated L-G6pc(-/-) mice using an inducible CRE-lox strategy and followed up the development of hepatic tumours using magnetic resonance imaging. RESULTS: L-G6pc(-/-) mice are viable and exhibit normoglycemia in the fed state. They develop hyperlipidemia, lactic acidosis, and uricemia during the first month after gene deletion. However, these plasmatic parameters improved after 6 months. L-G6pc(-/-) mice develop hepatomegaly with glycogen accumulation and hepatic steatosis. Using an MRI approach, we could detect hepatic nodules with diameters of less than 1 mm, 9 months after induction of deficiency. Hepatic nodules (1 mm) were detected in 30-40% of L-G6pc(-/-) mice at 12 months. After 18 months, all L-G6pc(-/-) mice developed multiple hepatocellular adenomas of 1-10 mm diameter. CONCLUSIONS: This is the first report of a viable animal model of the hepatic pathology of GSD1a, including the late development of hepatocellular adenomas.

Human milk pasteurisation reduces pre-lipolysis but not digestive lipolysis and moderately decreases intestinal lipid uptake in a combination of preterm infant in vitro models.

Donor human milk, pasteurised for safety reasons, is the first alternative for feeding preterm infants when mothers' own milk is unavailable. Breastmilk pasteurisation impact on lipid digestion and absorption was evaluated by a static in vitro digestion model for preterm infants coupled with intestinal absorption using Caco-2/TC7 cells. Lipid absorption was quantified by digital image analysis of lipid droplets, by measurement of basolateral triglyceride concentration and by analysing the expression of major genes involved. After in vitro digestion, lipolysis extent was 13% lower in pasteurised human milk (PHM) than in raw human milk (RHM). In Caco-2/TC7 cells, the number of lipid droplets was identical for both milk types, while the mean droplet area was 17% smaller with PHM. Altogether, pasteurisation decreased the pre-lipolysis of human milk. This initial difference in free fatty acid amount was only partially buffered by the subsequent processes of in vitro digestion and cellular lipid absorption.

Acute effects of milk polar lipids on intestinal tight junction expression: towards an impact of sphingomyelin through the regulation of IL-8 secretion?

Milk polar lipids (MPL) are specifically rich in milk sphingomyelin (MSM) which represents 24% of MPL. Beneficial effects of MPL or MSM have been reported on lipid metabolism, but information on gut physiology is scarce. Here we assessed whether MPL and MSM can impact tight junction expression. Human epithelial intestinal Caco-2/TC7 cells were incubated with mixed lipid micelles devoid of MSM (Control) or with 0.2 or 0.4 mM of MSM via pure MSM or via total MPL. C57Bl/6 mice received 5 or 10 mg of MSM via MSM or via MPL (oral gavage); small intestinal segments were collected after 4 h. Impacts on tight junction and cytokine expressions were assessed by qPCR; IL-8 and IL-8 murine homologs (Cxcl1, Cxcl2) were analyzed. In vitro, MSM increased tight junction expression (Occludin, ZO-1) vs Control, unlike MPL. However, no differences were observed in permeability assays (FITC-dextran, Lucifer yellow). MSM increased the secretion and gene expression of IL-8 but not of other inflammatory cytokines. Moreover, cell incubation with IL-8 induced an overexpression of tight junction proteins. In mice, mRNA level of Cxcl1 and Cxcl2 in the ileum were increased after gavage with MSM vs NaCl but not with MPL. Altogether, these results suggest a specific action of MSM on intestinal tight junction expression, possibly mediated by IL-8. Our study provides clues to shed light on the beneficial effects of MPL on intestinal functions and supports the need for further mechanistic exploration of the direct vs indirect effects of MSM and IL-8 on the gut barrier.

Dietary emulsifiers from milk and soybean differently impact adiposity and inflammation in association with modulation of colonic goblet cells in high-fat fed mice.

SCOPE: Enhanced adiposity and metabolic inflammation are major features of obesity that could be impacted by dietary emulsifiers. We investigated in high-fat fed mice the effects of using a new polar lipid (PL) emulsifier from milk (MPL) instead of soybean lecithin (soybean PL [SPL]) on adipose tissue and intestinal mucosa function. METHODS AND RESULTS: Four groups of C57BL6 mice received for 8 wks a low-fat (LF) diet or a high-fat diet devoid of PLs or an high-fat diet including MPL (high-fat-MPL) or SPL (high-fat-SPL). Compared with high-fat diet, high-fat-SPL diet increased white adipose tissue (WAT) mass (p < 0.05), with larger adipocytes (p < 0.05) and increased expression of tumor necrosis factor alpha, monochemoattractant protein-1, LPS-binding protein, and leptin (p < 0.05). This was not observed with high-fat-MPL diet despite similar dietary intakes and increased expression of fatty acid transport protein 4 and microsomal TG transfer protein, involved in lipid absorption, in upper intestine (p < 0.05). High-fat-MPL mice had a lower expression in WAT of cluster of differentiation 68, marker of macrophage infiltration, versus high-fat and high-fat-SPL mice (p < 0.05), and more goblet cells in the colon (p < 0.05). CONCLUSIONS: Unlike SPL, MPL in the high-fat diet did not induce WAT hypertrophy and inflammation but increased colonic goblet cells. This supports further clinical exploration of different sources of dietary emulsifiers in the frame of obesity outbreak.

Inactivation and intracellular retention of the human I183N mutated melanocortin 3 receptor associated with obesity.

Melanocortins are known to be involved in the regulation of feeding behavior. These hormones mediate their effects through G protein-coupled receptors (GPCRs) by stimulating adenylate cyclase. The melanocortin 3 receptor (MC3R) in the melanocortin receptor (MCR) family has been identified as a neural receptor subtype mainly expressed in the brain in mammals. Until now, only one heterozygous mutation (I183N) has been identified in the coding region of this receptor in two obese patients of the same family. In this study, we reported the functional characterization of the I183N mutated MC3R compared with that of the wild-type MC3R after transfection in HEK293 cells. Our results showed that the I183N mutation totally abolished the activity of the mutated receptor to generate intracellular cAMP. Furthermore, confocal microscopy observation revealed that the mutation induced an intracellular retention of the mutated receptor. Moreover, we demonstrated for the first time by co-transfection studies that the mutated receptor could reduce the wild-type receptor activity through a dominant negative effect.

Expression of the human melanocortin-2 receptor in different eukaryotic cells.

The human melanocortin-2 receptor (hMC2R) is mainly present in the adrenal cortex and has been difficult to express in heterologous cells. The hMC2R fused to the EGFP at its C-terminus has been stably transfected in the murine M3 melanoma and HEK293 cells. In the M3 cells, the hMC2R-EGFP was well-addressed to the cell membrane and functional whereas in the HEK293 cells, the hMC2R-EGFP was retained intracellularly. These results suggest that some specific factors, missing in cells, which do not express any melanocortin receptor, are involved in the correct addressing of the hMC2R to the cell membrane.

A gut-brain neural circuit controlled by intestinal gluconeogenesis is crucial in metabolic health.

OBJECTIVES: Certain nutrients positively regulate energy homeostasis via intestinal gluconeogenesis (IGN). The objective of this study was to evaluate the impact of a deficient IGN in glucose control independently of nutritional environment. METHODS: We used mice deficient in the intestine glucose-6 phosphatase catalytic unit, the key enzyme of IGN (I-G6pc (-/-) mice). We evaluated a number of parameters involved in energy homeostasis, including insulin sensitivity (hyperinsulinemic euglycaemic clamp), the pancreatic function (insulin secretion in vivo and in isolated islets) and the hypothalamic homeostatic function (leptin sensitivity). RESULTS: Intestinal-G6pc (-/-) mice exhibit slight fasting hyperglycaemia and hyperinsulinemia, glucose intolerance, insulin resistance and a deteriorated pancreatic function, despite normal diet with no change in body weight. These defects evoking type 2 diabetes (T2D) derive from the basal activation of the sympathetic nervous system (SNS). They are corrected by treatment with an inhibitor of α-2 adrenergic receptors. Deregulation in a key target of IGN, the homeostatic hypothalamic function (highlighted here through leptin resistance) is a mechanistic link. Hence the leptin resistance and metabolic disorders in I-G6pc (-/-) mice are corrected by rescuing IGN by portal glucose infusion. Finally, I-G6pc (-/-) mice develop the hyperglycaemia characteristic of T2D more rapidly under high fat/high sucrose diet. CONCLUSIONS: Intestinal gluconeogenesis is a mandatory function for the healthy neural control of glucose homeostasis.

Familial glucocorticoid deficiency type 2 in two neonates.

Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder characterized by glucocorticoid deficiency, elevated plasma adrenocorticotropic hormone (ACTH) levels and preserved aldosterone/renin secretion. Adrenocorticotropic receptor mutations occur in about 40% of patients (FGD type 1), whereas the rest of the cases are associated with a normal receptor (FGD type 2). More than 50 cases have been reported in the literature so far. We report two cases of type 2 FGD from two related families who presented in the newborn period with varying clinical features. Direct sequencing of the genomic DNA of the patients failed to reveal mutations or other defects in the coding sequence of the ACTH receptor. Linkage analysis excluded mutations on the MC2-R gene outside the coding region. To our knowledge, these are the first two cases of FGD reported from the Middle East.

Intestinal gluconeogenesis is crucial to maintain a physiological fasting glycemia in the absence of hepatic glucose production in mice.

OBJECTIVE: Similar to the liver and kidneys, the intestine has been strongly suggested to be a gluconeogenic organ. However, the precise contribution of the intestine to endogenous glucose production (EGP) remains to be determined. To define the quantitative role of intestinal gluconeogenesis during long-term fasting, we compared changes in blood glucose during prolonged fasting in mice with a liver-deletion of the glucose-6 phosphatase catalytic (G6PC) subunit (LKO) and in mice with a combined deletion of G6PC in both the liver and the intestine (ILKO). MATERIALS/METHODS: The LKO and ILKO mice were studied after 6h and 40 h of fasting by measuring metabolic and hormonal plasmatic parameters, as well as the expression of gluconeogenic enzymes in the liver, kidneys and intestine. RESULTS: After a transient hypoglycemic episode (approximately 60 mg/dL) because of their incapacity to mobilize liver glycogen, the LKO mice progressively re-increased their plasma glucose to reach a glycemia comparable to that of wild-type mice (90 mg/dL) from 30 h of fasting. This increase was associated with a rapid induction of renal and intestinal gluconeogenic gene expression, driven by glucagon, glucocorticoids and acidosis. The ILKO mice exhibited a similar induction of renal gluconeogenesis. However, these mice failed to re-increase their glycemia and maintained a plasma glucose level of only 60 mg/dL throughout the 48 h-fasting period. CONCLUSIONS: These data indicate that intestinal glucose production is essential to maintain glucose homeostasis in the absence of hepatic glucose production during fasting. These data provide a definitive quantitative estimate of the capacity of intestinal gluconeogenesis to sustain EGP during long-term fasting.

Protein-induced satiety is abolished in the absence of intestinal gluconeogenesis.

Protein-enriched diets are well known to initiate satiety effects in animals and humans. It has been recently suggested that this might be dependent on the induction of gluconeogenesis in the intestine. The resulting intestinal glucose release, detected by a "so-called" glucose sensor located within the walls of the portal vein and connected to peripheral afferents, activates hypothalamic nuclei involved in the regulation of food intake, in turn initiating a decrease in hunger. To definitively demonstrate the role of intestinal gluconeogenesis in this mechanism, we tested the food intake response to a protein-enriched diet in mice with an intestine-specific deletion (using an inducible Cre/loxP strategy) of the glucose-6 phosphatase gene (I-G6pc(-/-) mice) encoding the mandatory enzyme for glucose production. There was no effect on food intake in I-G6pc(-/-) mice fed on a standard rodent diet compared to their wild-type counterparts. After switching to a protein-enriched diet, the food intake of wild-type mice decreased significantly (by about 20% of daily calorie intake), subsequently leading to a decrease of 12 ± 2% of initial body weight after 8 days. On the contrary, I-G6pc(-/-) mice were insensitive to the satiety effect induced by a protein-enriched diet and preserved their body weight. These results provide molecular evidence of the causal role of intestinal gluconeogenesis in the satiety phenomenon initiated by protein-enriched diets.

ACTH-induced Cl(-) current in bovine adrenocortical cells: correlation with cortisol secretion.

ACTH has been shown to depolarize bovine adrenal zona fasciculata cells by inhibiting a K(+) current. The effects of this hormone on such cells have been reexamined using perforated and standard patch recording methods. In current clamp experiments, ACTH (10 nM) induced a membrane depolarization to -36 +/- 1 mV (n = 56), which was mimicked by forskolin (10 microM) or by 8-(4-chlorophenylthio)-cAMP (8 mM). ACTH-induced membrane depolarizations were associated in the majority of cells with an increase in membrane conductance. In the other cells, these membrane responses could occur without change or could be correlated with a transient or with a continuous Cs(+)-sensitive decrease in membrane conductance. The depolarizations associated with an increase in membrane conductance were depressed by Cl(-) current inhibitors diphenylamine-2-carboxylic acid (DPC; 1 mM), anthracene-9-carboxylic acid (9-AC; 1 mM), DIDS (400 microM), verapamil (100 microM), and glibenclamide (20 microM). In voltage-clamped Cs(+)-loaded cells, ACTH activated a time-independent current that displayed an outward rectification and reversed at -21.5 mV +/- 2 (n = 6). This current, observed in the presence of internal EGTA (5 mM), was depressed in low Cl(-) external solution and was inhibited by DPC, 9-AC, DIDS, 5-nitro-2-(3-phenylpropylamino)benzoic acid, verapamil, and glibenclamide. ACTH-stimulated cortisol secretion was blocked by Cl(-) channel inhibitors DIDS (400 microM) and DPC (1 mM). The present results reveal that, in addition to inhibiting a K(+) current, ACTH activates in bovine zona fasciculata cells a Ca(2+)-insensitive, cAMP-dependent Cl(-) current. This Cl(-) current is involved in the ACTH-induced membrane depolarization, which seems to be a crucial step in stimulating steroidogenesis.

Stable expression of human melanocortin 3 receptor fused to EGFP in the HEK293 cells.

Among the melanocortins alpha-MSH is known to be involved in feeding behavior. These hormones mediate their effects through G protein-coupled receptors by stimulating adenylate cyclase. In this study, we have developed an in vitro expression model for human melanocortin 3 receptor (hMC3R) tagged at its C terminus with EGFP. The corresponding chimeric cDNA was stably expressed in HEK293 cells. The selected clones expressing the hMC3R-EGFP exhibited cell surface fluorescence and responded to NDP-MSH stimulation by producing cAMP in a dose-dependent manner (EC(50): 0.3 nM). Binding studies revealed a single class of binding sites with a K(D) of 2.24 nM. Moreover, Agouti-related protein was also demonstrated to be an antagonist of the hMC3R-EGFP. Thus, the hMC3R tagged with EGFP stably expressed in HEK293 cells, exhibiting the same characteristics than the wild-type hMC3R, is the only model of expression of this receptor allowing its direct localization inside living cells.