Metabolic effects in mice of cream processing: Direct ultra-high-temperature process lowers high-fat-induced adipose tissue inflammation.

Although UHT heat treatment is being optimized to improve the stability and functional properties of dairy products, its metabolic effects remain scarcely known. As such, we studied the effect of the type of UHT process on lipid metabolism, intestinal barrier, and inflammation in mice. Nine-week-old male C57Bl/6J mice were fed a diet composed of nonlipidic powder mixed with different UHT dairy creams (final: 13% milkfat) for 1 or 4 wk. All creams contained 0.02% of thickener (carrageenan) and were treated via either (1) classical indirect heating process (Th), (2) indirect process at higher temperature (Th+), or (3) direct process by steam injection (ThD). Plasma, epididymal adipose tissue (EAT), and intestine were analyzed. Multivariate principal component analyses were used to identify differential effects of processes. Th+ differed by a globally higher liver damage score compared with that of the other creams. After 4 wk, the duodenal expression of lipid absorption genes fatty acid binding protein 4 (Fatp4) and microsomal triglycerides transfer protein (Mttp) was lower in the Th+ versus Th group. Expression in the colon of tight junction protein zonula occludens 1 (Zo1) and of some endoplasmic reticulum stress markers was lower in both Th+ and ThD versus the Th group. In EAT, ThD had lower gene expression of several inflammatory markers after 4 wk. Some differential effects may be related to heat-induced physicochemical changes of creams. The type of cream UHT process differentially affected metabolic parameters in mice after a 4-wk fat-rich diet, partly due to cream structure. Altogether, direct steam injection process induced the lowest early markers of high-fat-induced metabolic inflammation in EAT.

Metabolic effects in mice of cream formulation: Addition of both thickener and emulsifier does not alter lipid metabolism but modulates mucus cells and intestinal endoplasmic reticulum stress.

Additives stabilize or improve the organoleptic or functional properties (or both) of many dairy products including whipping cream. Their influence on the metabolic effect of dairy cream is scarcely known. We tested the hypothesis that added emulsifier (lactic acid esters of mono- and diglycerides; MAG/DAG), thickener (carrageenan, CGN), or both, could modify the metabolic effect, notably in the intestine and liver. Nine-week-old male C57Bl/6J mice were fed UHT cream (indirect treatment) mixed with nonlipidic powder (final: 13% milkfat) for 1 or 4 wk. We compared creams (1) without additive (Ctl), (2) with thickener (Th), 0.02% of κ-CGN, and (3) with both thickener and emulsifier, 0.1% of MAG/DAG esters (Th/Em). We analyzed plasma parameters, intestine, and liver. Fasting glycemia, insulinemia, triglyceridemia, nonesterified fatty acids, body weight gain, and liver weight did not differ among groups. After 1 wk, Th/Em had higher expression in the duodenum of some of the genes involved in (1) intestinal lipid absorption and (2) tight junction proteins versus Ctl and Th. After 4 wk, mucus cell number in the small intestine was higher in Th/Em versus Ctl and Th. Genes involved in endoplasmic reticulum (ER) stress in the duodenum were more expressed in Th/Em after 1 wk. After 4 wk, in the colon, a higher expression of ER stress genes was observed for Th versus Th/Em and Ctl. Liver damage score was not altered by additives. Adding both CGN (0.02%) and MAG/DAG esters (0.1%) in dairy cream did not result in deleterious outcomes in mice after 4 wk regarding lipid metabolism, intestinal permeability, and liver disorders. The longer term effect of intestinal ER stress modulation deserves further investigation.

Gene expression profiling in peripheral blood cells of patients with rheumatoid arthritis in response to anti-TNF-alpha treatments.

The efficacy of anti-TNF-α therapies highlights the role of TNF-α in the pathogenesis of rheumatoid arthritis (RA). However, the mechanism of action of these agents is poorly understood at the molecular level. The aim of this study was to characterize the effects of anti-TNF-α treatment on the global gene expression profile in peripheral blood mononuclear cells (PBMCs) of responder RA patients. Changes in gene expression were determined using oligonucleotide microarrays (25,341 genes) in PBMCs obtained before and after 12 wk of treatment with either etanercept or adalimumab from responder RA patients. Two hundred fifty-one genes displayed significant changes (false discovery rate < 0.1%) in expression level (178 upregulations with mean fold change = 1.5 and 73 downregulations with mean fold change = -1.50) after 12 wk of treatment. Importantly, the expression of several genes, including those coding for the calcium binding proteins S100A12 and A8, CD14 antigen, Selectin P, or ribosomal protein L39, reported to be upregulated in RA patients, were found to be decreased after anti-TNF-α treatment. Globally, inflammation, immune response, apoptosis, protein synthesis, and mitochondrial oxido-reduction were the most affected pathways in response to anti-TNF-α treatment. The obtained gene expression signature in PBMCs provides new information to better understand the mechanisms of action of anti-TNF-α treatment in RA patients.

Claudin 11 deficiency in mice results in loss of the Sertoli cell epithelial phenotype in the testis.

Tissue integrity relies on barriers formed between epithelial cells. In the testis, the barrier is formed at the initiation of puberty by a tight junction complex between adjacent Sertoli cells, thereby defining an adluminal compartment where meiosis and spermiogenesis occur. Claudin 11 is an obligatory protein for tight junction formation and barrier integrity in the testis. It is expressed by Sertoli cells, and spermatogenesis does not proceed beyond meiosis in its absence, resulting in male sterility. Sertoli cell maturation--arrest of proliferation and expression of proteins to support germ cell development--parallels tight junction assembly; however, the pathophysiology underlying the loss of tight junctions in the mature testis remains largely undefined. Here, using immunohistochemistry and microarrays we demonstrate that adult Cldn11(-/-) mouse Sertoli cells can proliferate while maintaining expression of mature markers. Sertoli cells detach from the basement membrane, acquire a fibroblast cell shape, are eliminated through the lumen together with apoptotic germ cells, and are found in epididymis. These changes are associated with tight junction regulation as well as actin-related and cell cycle gene expression. Thus, Cldn11(-/-) Sertoli cells exhibit a unique phenotype whereby loss of tight junction integrity results in loss of the epithelial phenotype.

The effects of an in utero exposure to 2,3,7,8-tetrachloro-dibenzo-p-dioxin on male reproductive function: identification of Ccl5 as a potential marker.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like compounds are widely encountered toxic substances suspected of interfering with the endocrine systems of humans and wildlife, and of contributing to the loss of fertility. In this study, we determined the changes in testicular gene expression caused by in utero exposure to TCDD along with the intra-testicular testosterone levels, epididymal sperm reserves, daily sperm production (DSP) and testis histology. To this purpose, female pregnant Sprague-Dawley rats orally received TCDD (10, 100 or 200 ng/kg body weight) or vehicle at embryonic day 15, and the offspring was killed throughout development. Hepatic Cyp1a1 gene expression was measured in the offspring to confirm the exposure to TCDD. The gross histology of the testes and intra-testicular testosterone levels were normal among the studied groups. Sperm reserves were altered in 67-day-old rats of the TCDD-200 group, but not in 145-day-old animals or in the other TCDD-exposed groups. Nonetheless, fertility was not altered in males of the TCDD-200 group, and the F2 males generated had normal sperm reserves and DSP. Microarray analysis permitted the identification of eight differentially expressed genes in the 4-week-old testes of the TCDD-200 compared with that of the control group (cut-off value +/- 1.40), including the down-regulated chemokine Ccl5/Rantes. Inhibition of Ccl5/Rantes gene expression was observed throughout development in the TCDD-200 group, and at 67 and 145 days in the TCDD-100 group (animals of younger ages were not examined). Ccl5/Rantes gene expression was mostly confined in Leydig cells. F2 males generated from males of the TCDD-200 group had normal levels of Ccl5/Rantes in testis and Cyp1a1 in liver, which might indicate that Ccl5/Rantes is a marker of TCDD exposure in testis such as Cyp1a1 in liver. In conclusion, we demonstrated a decrease in Ccl5/Rantes RNA levels and a transitory decline in sperm reserves in the testes of rats of TCDD-dosed dams.

Microarray analysis of genes with impaired insulin regulation in the skeletal muscle of type 2 diabetic patients indicates the involvement of basic helix-loop-helix domain-containing, class B, 2 protein (BHLHB2).

AIMS/HYPOTHESIS: One of the major processes by which insulin exerts its multiple biological actions is through gene expression regulation. Thus, the identification of transcription factors affected by insulin in target tissues represents an important challenge. The aim of the present study was to gain a greater insight into this issue through the identification of transcription factor genes with insulin-regulated expression in human skeletal muscle. METHODS: Using microarray analysis, we defined the sets of genes modulated during a 3 h hyperinsulinaemic-euglycaemic clamp (2 mU min(-1) kg(-1)) in the skeletal muscle of insulin-sensitive control volunteers and in moderately obese insulin-resistant type 2 diabetic patients. RESULTS: Of the 1,529 and 1,499 genes regulated during the clamp in control and diabetic volunteers, respectively, we identified 30 transcription factors with impaired insulin-regulation in type 2 diabetic patients. Analysis of the promoters of the genes encoding these factors revealed a possible contribution of the transcriptional repressor basic helix-loop-helix domain-containing, class B, 2 protein (BHLHB2), insulin regulation of which is strongly altered in the muscle of diabetic patients. Gene ontology analysis of BHLHB2 target genes, identified after BHLHB2 overexpression in human primary myotubes, demonstrated that about 10% of the genes regulated in vivo during hyperinsulinaemia are potentially under the control of this repressor. The data also suggested that BHLHB2 is situated at the crossroads of a complex transcriptional network that is able to modulate major metabolic and biological pathways in skeletal muscle, including the regulation of a cluster of genes involved in muscle development and contraction. CONCLUSIONS/INTERPRETATION: We have identified BHLHB2 as a potential novel mediator of insulin transcriptional action in human skeletal muscle.

MicroRNAs from urinary extracellular vesicles are non-invasive early biomarkers of diabetic nephropathy in type 2 diabetes patients with the 'Asian Indian phenotype'.

AIMS: MicroRNAs (miRNAs) from extracellular vesicles (EVs) have been proposed as promising biomarkers for a number of diseases. In this study, their potential as urine-based biomarkers of diabetic nephropathy (DN) was assessed. METHODS: MiRNAs were profiled in urinary EVs from 160 fasting subjects with normal glucose tolerance (NGT) and in T2DM patients with either microalbumininuria (MIC) or macroalbuminuria (MAC). RESULTS: A total of 73 miRNAs detected in urinary EVs (NGT) were predicted to target important functions for kidney homoeostasis, thereby validating their use as indicators of kidney dysfunction. Indeed, a urinary EV miRNA signature was found to comprise increased levels of let-7i-3p, miR-24-3p and miR-27b-3p, and decreased levels of miR-15b-5p, to identify patients with MIC. ROC curve analysis confirmed this ability to identify MIC in normo-albuminuria T2DM (T2DM-NA) patients and to differentiate between MAC and T2DM patients. These miRNAs were also predicted to target protein networks involved in the Wnt/ß-catenin signalling cascade, activin receptor signalling and cell differentiation/proliferation, and correlated with eGRF, HbA1c, serum creatinine, urea, albumin and blood pressure. Concentrations of miR-30a-5p were specifically modified in urinary EVs from patients with MAC, but not MIC, suggesting that miR-30a-5p could be related to severe kidney damage. CONCLUSION: Urinary EV miRNAs correlate with the degree of MIC. As they are also thought to regulate pathways that are targets of pharmacological agents to prevent DN (reticulum stress, activin receptors), they may also serve as non-invasive 'liquid biopsies' to stratify patients at risk of developing MAC and to monitor treatment efficacy.

High expression of CPT1b in skeletal muscle in metabolically healthy older subjects.

AIM: Ageing is often associated with metabolic abnormalities such as insulin resistance, although some people remain metabolically healthy throughout their lives. The aim of this study was to gain more insight into metabolic health with increasing age. METHODS: Two groups of robust and of frail subjects, respectively, were identified based on a composite ageing indicator and recruited from the French SU.VI.MAX 2 cohort of older disease-free subjects. In all, 14 men and 12 women, aged 67±4 years, with similar anthropometric and metabolic characteristics at baseline (BMI: 24.5±2.9kg.m-2) were included in the Compaliclamp study. Skeletal muscle biopsy was performed to assess expression of a set of metabolic and sirtuin (SIRT) genes. Also, whole-body substrate oxidation and insulin sensitivity were determined using the euglycaemic-hyperinsulinaemic clamp and indirect calorimetry techniques. RESULTS: Robust subjects were more insulin-sensitive, oxidized more lipid in a fasting state and stored more glucose during the euglycaemic - hyperinsulinaemic clamp than did frail subjects. At the gene-expression level in skeletal muscle, carnitine palmitoyltransferase 1b (CPT1b) messenger RNA (mRNA) levels were around four times higher in the robust compared with frail counterparts. Moreover, both SIRT2 and SIRT6 expression was lower in robust subjects and correlated with CPT1b expression. CONCLUSION: CPT1b overexpression could be helping to maintain metabolic health with increasing age. Thus, it is suggested that targeting CPT1b expression might be an interesting strategy to counteract frailty at an early stage. In addition, future studies should examine the role of sirtuin in CPT1b expression regulation.

Reduced fibre size, capillary supply and mitochondrial activity in constitutional thinness' skeletal muscle.

AIM: Constitutional thinness (CT) is a rare condition of natural low body weight, with no psychological issues, no marker of undernutrition and a resistance to weight gain. This study evaluated the skeletal muscle phenotype of CT women by comparison with a normal BMI control group. METHODS: Ten CT women (BMI < 17.5 kg/m2 ) and 10 female controls (BMI: 18.5-25 kg/m2 ) underwent metabolic and hormonal assessment along with muscle biopsies to analyse the skeletal muscular fibres pattern, capillarity, enzymes activities and transcriptomics. RESULTS: Constitutional thinness displayed similar energy balance metabolic and hormonal profile to controls. Constitutional thinness presented with lower mean area of all the skeletal muscular fibres (-24%, P = .01) and percentage of slow-twitch type I fibres (-25%, P = .02, respectively). Significant downregulation of the mRNA expression of several mitochondrial-related genes and triglycerides metabolism was found along with low cytochrome c oxidase (COX) activity and capillary network in type I fibres. Pre- and post-mitochondrial respiratory chain enzymes levels were found similar to controls. Transcriptomics also revealed downregulation of cytoskeletal-related genes. CONCLUSION: Diminished type I fibres, decreased mitochondrial and metabolic activity suggested by these results are discordant with normal resting metabolic rate of CT subjects. Downregulated genes related to cytoskeletal proteins and myocyte differentiation could account for CT's resistance to weight gain.

Atrial fibrillation is associated with hypermethylation in human left atrium, and treatment with decitabine reduces atrial tachyarrhythmias in spontaneously hypertensive rats.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. As the molecular mechanisms underlying the pathology are largely unknown, this cardiac arrhythmia remains difficult to treat. To identify specific molecular actors involved in AF, we have performed a transcriptomic analysis on left atrium (LA) from patients with valvular heart disease with or without AF. We showed that 1627 genes had altered basal expression level in LA tissue of AF patients compared with the control group. The significantly enriched gene ontology biological process "anatomical structure morphogenesis" contained the highest number of genes in line with changes in structure that occur when the human heart remodels following AF development (ie, LA dilatation and interstitial fibrosis). We then focused the study on Pitx2 (paired-like homeodomain 2), being the most altered transcription factor in LA from AF patients and from which compelling evidence have indicated that its reduced expression can be considered as a marker for the disease. In addition, its expression was inversely correlated with LA size. We demonstrated that AF is associated with Pitx2 promoter hypermethylation both in humans and arrhythmic aging spontaneously hypertensive rats. Chronic administration of a DNA methylation inhibitor (ie, 5-Aza-2'-deoxycitidine) improved ECG arrhythmic profiles and superoxide dismutase activities and reduced fibrosis in the left ventricle of spontaneously hypertensive rats. Taken together, these data support the notion that AF is associated with epigenetic changes in LA and provide a proof-of-concept that hypomethylating agents have to be considered in the treatment of atrial arrhythmias.

Androgen-regulated microRNA-135a decreases prostate cancer cell migration and invasion through downregulating ROCK1 and ROCK2.

Androgen signaling, via the androgen receptor (AR), is crucial in mediating prostate cancer (PCa) initiation and progression. Identifying new downstream effectors of the androgens/AR pathway will allow a better understanding of these mechanisms and could reveal novel biomarkers and/or therapeutic agents to improve the rate of patient survival. We compared the microRNA expression profiles in androgen-sensitive LNCaP cells stimulated or not with 1 nM R1881 by performing a high-throughput reverse transcriptase-quantitative PCR and found that miR-135a was upregulated. After androgen stimulation, we showed that AR directly activates the transcription of miR-135a2 gene by binding to an androgen response element in the promoter region. Our findings identify miR-135a as a novel effector in androgens/AR signaling. Using xenograft experiments in chick embryos and adult male mice, we showed that miR-135a overexpression decreases in vivo invasion abilities of prostate PC-3 cells. Through in vitro wound-healing migration and invasion assays, we demonstrated that this effect is mediated through downregulating ROCK1 and ROCK2 expression, two genes that we characterized as miR-135a direct target genes. In human surgical samples from prostatectomy, we observed that miR-135a expression was lower in tumoral compared with paired adjacent normal tissues, mainly in tumors classified with a high Gleason score (⩾8). Moreover, miR-135a expression is lower in invasive tumors, showing extraprostatic extension, as compared with intraprostatic localized tumors. In tumor relative to normal glands, we also showed a more frequently higher ROCK1 protein expression determined using a semi-quantitative immunohistochemistry analysis. Therefore, in tumor cells, the lower miR-135a expression could lead to a higher ROCK1 protein expression, which could explain their invasion abilities. The highlighted relationship between miR-135a expression level and the degree of disease aggressiveness suggests that miR-135a may be considered as a prognostic marker in human PCa.

Impact of various emulsifiers on ALA bioavailability and chylomicron synthesis through changes in gastrointestinal lipolysis.

Formulating healthy food rich in omega 3 fatty acids requires prior knowledge of the parameters influencing their bioavailability and their metabolic fate. In this context, we studied the effects of various emulsifiers widely used in the food industry, on the gastrointestinal lipolysis of flaxseed oil emulsions in an in vitro model and on the intestinal absorption and lymphatic secretion of alpha-linolenic acid (ALA) in rats. In vitro data showed that the emulsification of flaxseed oil with soya lecithin improved the gastric lipolysis of the oil (+30%), while the presence of Tween 80 or of sodium caseinate decreased it (-80% and -40%, respectively). The in vivo data demonstrated that the intestinal absorption and the lymphatic secretion of ALA were improved with soya lecithin (Cmax = 24 mg mL(-1)) and reduced in the presence of sodium caseinate (Cmax = 7 mg mL(-1)) compared to unemulsified flaxseed oil (Cmax = 16 mg mL(-1)); Tween 80 had no effect. In addition, the synthesized chylomicrons were notably larger and more numerous with soya lecithin whereas they were smaller in the presence of sodium caseinate (p < 0.05). This study shows that the intestinal bioavailability of ALA was increased by the emulsification of flaxseed oil with soya lecithin via an improved lipolysis, favouring the intestinal absorption of ALA and the secretion of many large chylomicrons in lymph.

Regulation of energy metabolism and mitochondrial function in skeletal muscle during lipid overfeeding in healthy men.

CONTEXT/OBJECTIVE: The aim of this study was to evaluate the regulation of the fuel partitioning and energy metabolism in skeletal muscle during lipid overfeeding in healthy men. Design/Participants/Intervention: Thirty-nine healthy volunteers were overfed for 56 days with a high-fat diet (3180 kJ/d). Energy metabolism (indirect calorimetry) was characterized in the fasting state and during a test meal before and at the end of the diet. Skeletal muscle biopsies were taken at day 0 and day 56. MAIN OUTCOME MEASURES: Change in gene expression, mitochondrial respiration, nicotinamide adenine dinucleotide (NAD(+)) content, and acetylation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in skeletal muscle was measured. RESULTS: Overfeeding increased body weight (+2.6 kg) and fat mass concomitantly with a shift in the use of substrates as energy fuel toward preferential oxidation of carbohydrates instead of lipids. Changes in lipid metabolic gene expression supported this observation, with a reduction in pyruvate dehydrogenase kinase 4 expression that could be the consequences of decreased NAD(+) concentration and reduced deacetylase activity of the sirtuins, as supported by hyperacetylation of PGC-1α after overfeeding. Interestingly, this reduction of the sirtuin PGC-1α pathway was associated with increased mitochondrial gene expression and higher respiration rate under these conditions. CONCLUSION: Adaptation to lipid overfeeding and regulation of fuel partitioning in human muscle appear to rely on a dissociation between the regulatory functions of the sirtuin-PGC-1α pathway on fatty acid oxidation and on mitochondrial regulation. This may facilitate lipid storage during a period of positive energy balance while maintaining mitochondrial functions and oxidative capacities.

Subcutaneous adipose tissue remodeling during the initial phase of weight gain induced by overfeeding in humans.

CONTEXT: Deciphering the early processes occurring in adipose tissue during weight gain is a major issue for understanding the development of fat mass and obesity. Experimental overfeeding in humans is a unique situation to tackle these events. OBJECTIVE: Our aim was to identify the pathways involved in sc adipose tissue remodeling during the initial phase of weight gain. RESEARCH DESIGN AND METHODS: Forty-four healthy men were involved in an overfeeding protocol with a lipid-enriched diet (+760 kcal/d) for 2 months. Subcutaneous abdominal adipose tissue biopsies were taken for histology, transcriptomics, and Western blotting in the basal state, after 14 d, and at the end of the protocol. RESULTS: Overfeeding significantly increased body weight (+2.5 kg) and fat mass. Reorganization of gene expression patterns occurred in adipose tissue with an up-regulation of numerous genes involved in lipid metabolism and storage, followed by clusters of genes related to angiogenesis and extracellular matrix remodeling. Histological examination showed increased microvascular density and connective tissue deposition after 56 d of overfeeding, with no changes in the number of macrophages or inflammatory cells. Inhibition of the canonical Wnt/ß-catenin signaling pathway and induction of the renin-angiotensin system might be implicated in the remodeling of sc adipose tissue. CONCLUSIONS: We characterize the coordinated and time-dependent processes that occur in human adipose tissue during the early phase of weight gain in healthy subjects and identify pathways representing potential targets in pathologies of adipose development, including obesity.