Frédéric Raymond

Germ-free C57BL/6J mice are resistant to high-fat-diet-induced insulin resistance and have altered cholesterol metabolism

Recent studies showed that germ-free (GF) mice are resistant to obesity when consuming a high-fat, high-carbohydrate Western diet. However, it remains unclear what mechanisms are involved in the antiobesity phenotype and whether GF mice develop insulin resistance and dyslipidemia with high-fat (HF) feeding. In the present study, we compared the metabolic consequences of HF feeding on GF and conventional (conv) C57BL/6J mice. GF mice consumed fewer calories, excreted more fecal lipids, and weighed significantly less than conv mice. GF/HF animals also showed enhanced insulin sensitivity with improved glucose tolerance, reduced fasting and nonfasting insulinemia, and increased phospho-Akt((Ser-473)) in adipose tissue. In association with enhanced insulin sensitivity, GF/HF mice had reduced plasma TNF-α and total serum amyloid A concentrations. Reduced hypercholesterolemia, a moderate accretion of hepatic cholesterol, and an increase in fecal cholesterol excretion suggest an altered cholesterol metabolism in GF/HF mice. Pronounced nucleus SREBP2 proteins and up-regulation of cholesterol biosynthesis genes indicate that enhanced cholesterol biosynthesis contributed to the cholesterol homeostasis in GF/HF mice. Our results demonstrate that fewer calorie consumption and increased lipid excretion contributed to the obesity-resistant phenotype of GF/HF mice and reveal that insulin sensitivity and cholesterol metabolism are metabolic targets influenced by the gut microbiota.

Loss of fibronectin from the aged stem cell niche affects the regenerative capacity of skeletal muscle in mice

Age-related changes in the niche have long been postulated to impair the function of somatic stem cells. Here we demonstrate that the aged stem cell niche in skeletal muscle contains substantially reduced levels of fibronectin (FN), leading to detrimental consequences for the function and maintenance of muscle stem cells (MuSCs). Deletion of the gene encoding FN from young regenerating muscles replicates the aging phenotype and leads to a loss of MuSC numbers. By using an extracellular matrix (ECM) library screen and pathway profiling, we characterize FN as a preferred adhesion substrate for MuSCs and demonstrate that integrin-mediated signaling through focal adhesion kinase and the p38 mitogen-activated protein kinase pathway is strongly de-regulated in MuSCs from aged mice because of insufficient attachment to the niche. Reconstitution of FN levels in the aged niche remobilizes stem cells and restores youth-like muscle regeneration. Taken together, we identify the loss of stem cell adhesion to FN in the niche ECM as a previously unknown aging mechanism.

Transcriptome and translational signaling following endurance exercise in trained skeletal muscle: impact of dietary protein

Postexercise protein feeding regulates the skeletal muscle adaptive response to endurance exercise, but the transcriptome guiding these adaptations in well-trained human skeletal muscle is uncharacterized. In a crossover design, eight cyclists ingested beverages containing protein, carbohydrate and fat (PTN: 0.4, 1.2, 0.2 g/kg, respectively) or isocaloric carbohydrate and fat (CON: 1.6, 0.2 g/kg) at 0 and 1 h following 100 min of cycling. Biopsies of the vastus lateralis were collected at 3 and 48 h following to determine the early and late transcriptome and regulatory signaling responses via microarray and immunoblot. The top gene ontology enriched by PTN were: muscle contraction, extracellular matrix--signaling and structure, and nucleoside, nucleotide, and nucleic acid metabolism (3 and 48 h); developmental processes, immunity, and defense (3 h); glycolysis, lipid and fatty acid metabolism (48 h). The transcriptome was also enriched within axonal guidance, actin cytoskeletal, Ca2+, cAMP, MAPK, and PPAR canonical pathways linking protein nutrition to exercise-stimulated signaling regulating extracellular matrix, slow-myofibril, and metabolic gene expression. At 3 h, PTN attenuated AMPKα1Thr172 phosphorylation but increased mTORC1Ser2448, rps6Ser240/244, and 4E-BP1-γ phosphorylation, suggesting increased translation initiation, while at 48 h AMPKα1Thr172 phosphorylation and PPARG and PPARGC1A expression increased, supporting the late metabolic transcriptome, relative to CON. To conclude, protein feeding following endurance exercise affects signaling associated with cell energy status and translation initiation and the transcriptome involved in skeletal muscle development, slow-myofibril remodeling, immunity and defense, and energy metabolism. Further research should determine the time course and posttranscriptional regulation of this transcriptome and the phenotype responding to chronic postexercise protein feeding.

Influence of gut microbiota on mouse B2 B cell ontogeny and function

A complex interplay between the microbiota and the host immune system is evidenced to shape the immune system throughout life, but little is known about the microbial effect on key players of the adaptive immune system, the B2 B cells. In the presented study, we have evaluated the effect of commensal bacteria on B cell ontogeny and function, with the focus on B2 B cells of spleen and Peyers patches. We have compared germ-free mice to mice that are exposed to a normal complex bacterial community from the day of birth and combined classical immunological assessment with advanced genome-wide expression profiling. Despite a preservation of all B cell subsets and phenotype, our results show that microbiota strongly impact mucosal B cell physiology and lead to higher serum Ig concentrations. We show that this microbial influence comprises downregulation of transcription factors involved in early B cell activation steps and upregulation of genes and proteins involved in later stages of B cell response. In summary, we show an influence of the gut microbiota on function of mucosal B2 B cells, involving mechanisms downstream of B cell activation and proliferation.

Comparative gene expression profiling between human cultured myotubes and skeletal muscle tissue.

BackgroundA high-sensitivity DNA microarray platform requiring nanograms of RNA input facilitates the application of transcriptome analysis to individual skeletal muscle (SM) tissue samples. Culturing myotubes from SM-biopsies enables investigating transcriptional defects and assaying therapeutic strategies. This study compares the transcriptome of aneurally cultured human SM cells versus that of tissue biopsies.ResultsWe used the Illumina expression BeadChips to determine the transcriptomic differences between tissue and cultured SM samples from five individuals. Changes in the expression of several genes were confirmed by QuantiGene Plex assay or reverse transcription real-time PCR. In cultured myotubes compared to the tissue, 1216 genes were regulated: 583 down and 633 up. Gene ontology analysis showed that downregulated genes were mainly associated with cytoplasm, particularly mitochondria, and involved in metabolism and the muscle-system/contraction process. Upregulated genes were predominantly related to cytoplasm, endoplasmic reticulum, and extracellular matrix. The most significantly regulated pathway was mitochondrial dysfunction. Apoptosis genes were also modulated. Among the most downregulated genes detected in this study were genes encoding metabolic proteins AMPD1, PYGM, CPT1B and UCP3, muscle-system proteins TMOD4, MYBPC1, MYOZ1 and XIRP2, the proteolytic CAPN3 and the myogenic regulator MYF6. Coordinated reduced expression of five members of the GIMAP gene family, which form a cluster on chromosome 7, was shown, and the GIMAP4-reduction was validated. Within the most upregulated group were genes encoding senescence/apoptosis-related proteins CDKN1A and KIAA1199 and potential regulatory factors HIF1A, TOP2A and CCDC80.ConclusionsCultured muscle cells display reductive metabolic and muscle-system transcriptome adaptations as observed in muscle atrophy and they activate tissue-remodeling and senescence/apoptosis processes.

A robust neuromuscular system protects rat and human skeletal muscle from sarcopenia

Declining muscle mass and function is one of the main drivers of loss of independence in the elderly. Sarcopenia is associated with numerous cellular and endocrine perturbations, and it remains challenging to identify those changes that play a causal role and could serve as targets for therapeutic intervention. In this study, we uncovered a remarkable differential susceptibility of certain muscles to age-related decline. Aging rats specifically lose muscle mass and function in the hindlimbs, but not in the forelimbs. By performing a comprehensive comparative analysis of these muscles, we demonstrate that regional susceptibility to sarcopenia is dependent on neuromuscular junction fragmentation, loss of motoneuron innervation, and reduced excitability. Remarkably, muscle loss in elderly humans also differs in vastus lateralis and tibialis anterior muscles in direct relation to neuromuscular dysfunction. By comparing gene expression in susceptible and non-susceptible muscles, we identified a specific transcriptomic signature of neuromuscular impairment. Importantly, differential molecular profiling of the associated peripheral nerves revealed fundamental changes in cholesterol biosynthetic pathways. Altogether our results provide compelling evidence that susceptibility to sarcopenia is tightly linked to neuromuscular decline in rats and humans, and identify dysregulation of sterol metabolism in the peripheral nervous system as an early event in this process.

Sialic Acid Utilisation and Synthesis in the Neonatal Rat Revisited

Background Milk is the sole source of nutrients for neonatal mammals and is generally considered to have co-evolved with the developmental needs of the suckling newborn. One evolutionary conserved constituent of milk and present on many glycoconjugates is sialic acid. The brain and colon are major sites of sialic acid display and together with the liver also of synthesis. Methodology/Principal Findings In this study we examined in rats the relationship between the sialic acid content of milk and the uptake, utilization and synthesis of sialic acid in suckling pups. In rat milk sialic acid was found primarily as 3′sialyllactose and at highest levels between 3 and 10 days postpartum and that decreased towards weaning. In the liver of suckling pups sialic acid synthesis paralleled the increase in milk sialic acid reaching and keeping maximum activity from postnatal day 5 onwards. In the colon, gene expression profiles suggested that a switch from sialic acid uptake and catabolism towards sialic acid synthesis and utilization occurred that mirrored the change of sialic acid in milk from high to low expression. In brain sialic acid related gene expression profiles did not change to any great extent during the suckling period. Conclusions/Significance Our results support the views that (i) when milk sialic acid levels are high, in the colon this sialic acid is catabolized to GlcNAc that in turn may be used as such or used as substrate for sialic acid synthesis and (ii) when milk sialic acid levels are low the endogenous sialic acid synthetic machinery in colon is activated.

Mfn1 Deficiency in the Liver Protects Against Diet-Induced Insulin Resistance and Enhances the Hypoglycemic Effect of Metformin

Mitochondrial function can be influenced by mitochondrial shape and connectivity with other cellular organelles through fusion and fission processes. Disturbances in mitochondrial architecture and mitochondrial fusion-related genes are observed in situations of type 2 diabetes and obesity, leading to a highly fissioned mitochondrial network. To directly test the effect of reduced mitochondrial fusion on hepatic metabolism, we generated mice with a liver-specific deletion of the Mfn1 gene (Mfn1LKO) and monitored their energy homeostasis, mitochondrial function, and susceptibility to diet-induced insulin resistance. Livers from Mfn1LKO mice displayed a highly fragmented mitochondrial network. This was coupled to an enhanced mitochondrial respiration capacity and a preference for the use of lipids as the main energy source. Although Mfn1LKO mice are similar to control mice fed a low-fat diet, they are protected against insulin resistance induced by a high-fat diet. Importantly, Mfn1 deficiency increased complex I abundance and sensitized animals to the hypoglycemic effect of metformin. Our results suggest that targeting Mfn1 could provide novel avenues to ameliorate glucose homeostasis in obese patients and improve the effectiveness of metformin.

Biomarkers of browning of white adipose tissue and their regulation during exercise- and diet-induced weight loss

Background: A hypothesis exists whereby an exercise- or dietary-induced negative energy balance reduces human subcutaneous white adipose tissue (scWAT) mass through the formation of brown-like adipocyte (brite) cells. However, the validity of biomarkers of brite formation has not been robustly evaluated in humans, and clinical data that link brite formation and weight loss are sparse. Objectives: We used rosiglitazone and primary adipocytes to stringently evaluate a set of biomarkers for brite formation and determined whether the expression of biomarker genes in scWAT could explain the change in body composition in response to exercise training combined with calorie restriction in obese and overweight women (n = 79). Design: Gene expression was derived from exon DNA microarrays and preadipocytes from obesity-resistant and -sensitive mice treated with rosiglitazone to generate candidate brite biomarkers from a microarray. These biomarkers were evaluated against data derived from scWAT RNA from obese and overweight women before and after supervised exercise 5 d/wk for 16 wk combined with modest calorie restriction (∼0.84 MJ/d). Results: Forty percent of commonly used brite gene biomarkers exhibited an exon or strain-specific regulation. No biomarkers were positively related to weight loss in human scWAT. Greater weight loss was significantly associated with less uncoupling protein 1 expression (P = 0.006, R2 = 0.09). In a follow-up global analysis, there were 161 genes that covaried with weight loss that were linked to greater CCAAT/enhancer binding protein α activity (z = 2.0, P = 6.6 × 10−7), liver X receptor α/β agonism (z = 2.1, P = 2.8 × 10−7), and inhibition of leptin-like signaling (z = −2.6, P = 3.9 × 10−5). Conclusion: We identify a subset of robust RNA biomarkers for brite formation and show that calorie-restriction–mediated weight loss in women dynamically remodels scWAT to take on a more-white rather than a more-brown adipocyte phenotype.

Protein-leucine ingestion activates a regenerative inflammo-myogenic transcriptome in skeletal muscle following intense endurance exercise

Protein-leucine supplement ingestion following strenuous endurance exercise accentuates skeletal-muscle protein synthesis and adaptive molecular responses, but the underlying transcriptome is uncharacterized. In a randomized single-blind triple-crossover design, 12 trained men completed 100 min of high-intensity cycling then ingested 70/15/180/30 g protein-leucine-carbohydrate-fat (15LEU), 23/5/180/30 g (5LEU), or 0/0/274/30 g (CON) beverages during the first 90 min of a 240 min recovery period. Vastus lateralis muscle samples (30 and 240 min postexercise) underwent transcriptome analysis by microarray followed by bioinformatic analysis. Gene expression was regulated by protein-leucine in a dose-dependent manner affecting the inflammatory response and muscle growth and development. At 30 min, 15LEU and 5LEU vs. CON activated transcriptome networks with gene-set functions involving cell-cycle arrest (Z-score 2.0-2.7, P < 0.01), leukocyte maturation (1.7, P = 0.007), cell viability (2.4, P = 0.005), promyogenic networks encompassing myocyte differentiation and myogenin (MYOD1, MYOG), and a proteinaceous extracellular matrix, adhesion, and development program correlated with plasma lysine, arginine, tyrosine, taurine, glutamic acid, and asparagine concentrations. High protein-leucine dose (15LEU-5LEU) activated an IL-1I-centered proinflammatory network and leukocyte migration, differentiation, and survival functions (2.0-2.6, <0.001). By 240 min, the protein-leucine transcriptome was anti-inflammatory and promyogenic (IL-6, NF- β, SMAD, STAT3 network inhibition), with overrepresented functions including decreased leukocyte migration and connective tissue development (-1.8-2.4, P < 0.01), increased apoptosis of myeloid and muscle cells (2.2-3.0, P < 0.002), and cell metabolism (2.0-2.4, P < 0.01). The analysis suggests protein-leucine ingestion modulates inflammatory-myogenic regenerative processes during skeletal muscle recovery from endurance exercise. Further cellular and translational research is warranted to validate amino acid-mediated myeloid and myocellular mechanisms within skeletal-muscle functional plasticity.