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).

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.

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.

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.

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.

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.

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.