Louise Deldicque

Inulin-type fructans with prebiotic properties counteract GPR43 overexpression and PPARγ-related adipogenesis in the white adipose tissue of high-fat diet-fed mice

Inulin-type fructans (ITF) are nondigestible/fermentable carbohydrates which are able - through the modification of the gut microbiota - to counteract high-fat (HF) diet-induced obesity, endotoxemia and related-metabolic alterations. However, their influence on adipose tissue metabolism has been poorly studied until now. The aim of this study was to assess the influence of ITF supplementation on adipose tissue metabolism, by focusing on a G protein-coupled receptor (GPR), GPR43, as a potential link between gut fermentation processes and white adipose tissue development. Male C57bl6/J mice were fed a standard diet or an HF diet without or with ITF (0.2 g/day per mouse) during 4 weeks. The HF diet induced an accumulation of large adipocytes, promoted peroxisome proliferator activated receptor gamma (PPARγ)-activated differentiation factors and led to a huge increase in GPR43 expression in the subcutaneous adipose tissue. All those effects were blunted by ITF treatment, which modulated the gut microbiota in favor of bifidobacteria at the expense of Roseburia spp. and of Clostridium cluster XIVa. The dietary modulation of GPR43 expression seems independent of endotoxemia, in view of data obtained in vivo (acute and chronic lipopolysaccharides treatment). In conclusion, ITF, which promote gut fermentation, paradoxically counteract GPR43 overexpression induced in the adipose tissue by an HF diet, a phenomenon that correlates with a beneficial effect on adiposity and with potential decrease in PPARγ-activated processes.

Modulation of autophagy and ubiquitin-proteasome pathways during ultra-endurance running

In this study, the coordinated activation of ubiquitin-proteasome pathway (UPP), autophagy-lysosomal pathway (ALP), and mitochondrial remodeling including mitophagy was assessed by measuring protein markers during ultra-endurance running exercise in human skeletal muscle. Eleven male, experienced ultra-endurance athletes ran for 24 h on a treadmill. Muscle biopsy samples were taken from the vastus lateralis muscle 2 h before starting and immediately after finishing exercise. Athletes ran 149.8 ± 16.3 km with an effective running time of 18 h 42 min ( ± 41 min). The phosphorylation state of Akt (-74 ± 5%; P < 0.001), FOXO3a (-49 ± 9%; P < 0.001), mTOR Ser2448 (-32 ± 14%; P = 0.028), and 4E-BP1 (-34 ± 7%; P < 0.001) was decreased, whereas AMPK phosphorylation state increased by 247 ± 170% (P = 0.042). Proteasome β2 subunit activity increased by 95 ± 44% (P = 0.028), whereas the activities associated with the β1 and β5 subunits remained unchanged. MuRF1 protein level increased by 55 ± 26% (P = 0.034), whereas MAFbx protein and ubiquitin-conjugated protein levels did not change. LC3bII increased by 554 ± 256% (P = 0.005), and the form of ATG12 conjugated to ATG5 increased by 36 ± 17% (P = 0.042). The mitochondrial fission marker phospho-DRP1 increased by 110 ± 47% (P = 0.003), whereas the fusion marker Mfn1 and the mitophagy markers Parkin and PINK1 remained unchanged. These results fit well with a coordinated regulation of ALP and UPP triggered by FOXO3 and AMPK during ultra-endurance exercise.

Regulation of mTOR by amino acids and resistance exercise in skeletal muscle

Resistance exercise disturbs skeletal muscle homeostasis leading to activation of catabolic and anabolic processes within the muscle cell. A current challenge of exercise biology is to describe the molecular mechanisms of regulation by which contractile activity stimulates net protein breakdown during exercise and net protein synthesis during recovery. Muscle growth is optimized by combining exercise and appropriate nutritional strategies, such as amino acid (AA) and carbohydrate ingestion. The effects are integrated at the level of one central regulatory protein, mTOR (mammalian target of rapamycin). mTOR is a complex protein integrating signals of the energetic status of the cell and environmental stimuli to control protein synthesis, protein breakdown and therefore cell growth. mTOR is known to be activated by insulin, and the mechanisms involved are well documented. The ways by which exercise and AA lead to mTOR activation remain partially unclear. Exercise and AA use different signalling pathways upstream of mTOR. Exercise seems to recruit partially the same pathway as insulin, whereas AA could act more directly on mTOR. During resistance exercise, the activity of mTOR could be acutely blunted by AMP-activated protein kinase (AMPK), thus inhibiting protein synthesis and enhancing AA availability for energy metabolism. During recovery, the inhibition of mTOR by AMPK is suppressed, and its activation is maximized by the presence of AA. There appears to be a requirement for a minimal concentration of plasma insulin to stimulate muscle protein synthesis in response to resistance exercise and AA ingestion.

The unfolded protein response is activated in skeletal muscle by high-fat feeding: potential role in the downregulation of protein synthesis

High-fat diets are known to decrease muscle protein synthesis, the adaptation to overload, and insulin sensitivity. Conditions that disrupt endoplasmic reticulum (ER) homeostasis lead to the activation of the unfolded protein response (UPR) that is associated with decreases in protein synthesis, chronic inflammation, and insulin resistance. The purpose of the present study was to establish whether ER stress is induced by a high-fat diet in skeletal muscle and whether ER stress can decrease mTORC1 activity and protein synthesis in muscle cells. Two independent protocols of high-fat feeding activated the UPR in mice. In the first study, mice consuming a high-fat diet containing 70% fat and <1% carbohydrates for 6 wk showed higher markers of the UPR (BiP, IRE1α, and MBTPS2) in the soleus and in the tibialis anterior muscles and ATF4 in the tibialis anterior (P < 0.05). In the second study, a 20-wk high-fat diet containing 46% fat and 36% carbohydrates also increased BiP, IRE1α, and phospho-PERK protein and the expression of ATF4, CHOP, and both the spliced and unspliced forms of XBP1 in the plantar flexors (P < 0.05). In C(2)C(12) muscle cells, tunicamycin, thapsigargin, and palmitic acid all increased UPR markers and decreased phosphorylation of S6K1 (P < 0.05). Collectively, these data show that a high-fat diet activates the UPR in mouse skeletal muscle in vivo. In addition, in vitro studies indicate that palmitic acid, and other well-known ER stress inducers, triggered the UPR in myogenic cells and led to a decrease in protein synthesis and mTORC1 activity.

Hepatic n-3 polyunsaturated fatty acid depletion promotes steatosis and insulin resistance in mice : genomic analysis of cellular targets.

Patients with non-alcoholic fatty liver disease are characterised by a decreased n-3/n-6 polyunsaturated fatty acid (PUFA) ratio in hepatic phospholipids. The metabolic consequences of n-3 PUFA depletion in the liver are poorly understood. We have reproduced a drastic drop in n-3 PUFA among hepatic phospholipids by feeding C57Bl/6J mice for 3 months with an n-3 PUFA depleted diet (DEF) versus a control diet (CT), which only differed in the PUFA content. DEF mice exhibited hepatic insulin resistance (assessed by euglycemic-hyperinsulinemic clamp) and steatosis that was associated with a decrease in fatty acid oxidation and occurred despite a higher capacity for triglyceride secretion. Microarray and qPCR analysis of the liver tissue revealed higher expression of all the enzymes involved in lipogenesis in DEF mice compared to CT mice, as well as increased expression and activation of sterol regulatory element binding protein-1c (SREBP-1c). Our data suggest that the activation of the liver X receptor pathway is involved in the overexpression of SREBP-1c, and this phenomenon cannot be attributed to insulin or to endoplasmic reticulum stress responses. In conclusion, n-3 PUFA depletion in liver phospholipids leads to activation of SREBP-1c and lipogenesis, which contributes to hepatic steatosis.

Endoplasmic reticulum stress markers and ubiquitin–proteasome pathway activity in response to a 200-km run.

PURPOSE This study investigated whether a 200-km run modulates signaling pathways implicated in cellular stress in skeletal muscle, with special attention paid to the endoplasmic reticulum (ER) stress and to the activation of the ubiquitin-proteasome pathway. METHODS Eight men ran 200 km (28 h 03 min ± 2 h 01 min). Two muscle biopsies were obtained from the vastus lateralis muscle 2 wk before and 3 h after the race. Mitogen-activated protein kinase, ubiquitin-proteasome pathway, ER stress, inflammation, and oxidative stress markers were assayed by Western blot analysis or by quantitative real-time polymerase chain reaction. Chymotrypsin-like activity of the proteasome was measured by a fluorimetric assay. RESULTS Phosphorylation states of extracellular signal-related kinase 1/2 (+401% ± 173.8%, P = 0.027) and c-Jun N-terminal (+149% ± 61.9%, P = 0.023) increased after the race, whereas p38 phosphorylation remained unchanged. Increases in BiP (+235% ± 94.7%, P = 0.021) and in the messenger RNA level of total (+138% ± 31.2%, P = 0.002) and spliced X-box binding protein 1 (+241% ± 53.3%, P = 0.001) indicated the presence of ER stress. Transcripts of inflammatory markers interleukin-6 (+403% ± 96.1%, P = 0.002) and tumor necrosis factor-α (+233% ± 58.4%, P = 0.003) as well as oxidative stress markers metallothionein 1F (+519% ± 258.3%, P = 0.042), metallothionein 1H (+666% ± 157.5%, P = 0.002), and nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) (+162% ± 60.5%, P = 0.016) were increased. The messenger RNA level of the ubiquitin ligases muscle-specific RING finger 1 (+583% ± 244.3%, P = 0.024) and muscle atrophy F-box (+249% ± 83.8%, P = 0.011) and the C2 proteasome subunit (+116% ± 40.6%, P = 0.012) also increased. Surprisingly, the amount of ubiquitin-conjugated proteins and the chymotrypsin-like activity of the proteasome were decreased by 20% ± 8.3% (P = 0.025) and 21% ± 4.4% (P = 0.001), respectively. The expression of ubiquitin-specific protease 28 deubiquitinase was increased (+81% ± 37.9%, P = 0.034). CONCLUSIONS In the skeletal muscle, a 200-km run activates the expression of ubiquitin ligases muscle-specific RING finger 1 and muscle atrophy F-box as well as various cellular stresses, among which are ER stress, oxidative stress, and inflammation. Meanwhile, compensatory mechanisms seem also triggered: the unfolded protein response is up-regulated, and the chymotrypsin-like activity of the proteasome is repressed.

Toll-Like Receptor 4 Knockout Mice Are Protected against Endoplasmic Reticulum Stress Induced by a High-Fat Diet

The purpose of this study was to investigate whether toll-like receptor 4 (TLR4) is implicated in the development of endoplasmic reticulum stress (ER stress) observed after a high-fat diet (HFD) in liver, skeletal muscle and adipose tissue. TLR4−/− and C57BL/6J wild-type mice (WT) were fed with chow or HFD (45% calories from fat) during 18 weeks. An oral glucose tolerance-test was performed. The animals were sacrificed in a fasted state and the tissues were removed. TLR4 deletion protected from body weight gain and glucose intolerance induced by HFD whereas energy intake was higher in transgenic mice suggesting larger energy expenditure. HFD induced an ER stress in skeletal muscle, liver and adipose tissue of WT mice as assessed by BiP, CHOP, spliced and unspliced XBP1 and phospho-eIF2α. TLR4−/− mice were protected against HFD-induced ER stress. Then, we investigated the main signaling downstream of TLR4 namely the NF-κB pathway, expecting to identify the mechanism by which TLR4 is able to activate ER stress. The mRNA levels of cytokines regulated by NF-κB namely TNFα, IL-1β and IL-6, were not changed after HFD and phospho-IκB-α (ser 32) was not changed. Our results indicate that TLR4 is essential for the development of ER stress related to HFD. Nevertheless, the NFκ-B pathway does not seem to be directly implicated. The reduced fat storage in TLR4−/− mice could explain the absence of an ER stress after HFD.

Activation of autophagy in human skeletal muscle is dependent on exercise intensity and AMPK activation

In humans, nutrient deprivation and extreme endurance exercise both activate autophagy. We hypothesized that cumulating fasting and cycling exercise would potentiate activation of autophagy in skeletal muscle. Well‐trained athletes were divided into control (n = 8), low‐intensity (LI, n = 8), and high‐intensity (HI, n = 7) exercise groups and submitted to fed and fasting sessions. Muscle biopsy samples were obtained from the vastus lateralis before, at the end, and 1 h after a 2 h LI or HI bout of exercise. Phosphorylation of ULK1Ser317 was higher after exercise (P< 0.001). In both the fed and the fasted states, LC3bII protein level and LC3bII/I were decreased after LI and HI (P < 0.05), while p62/ SQSTM1 was decreased only 1 h after HI (P < 0.05), indicating an increased autophagic flux after HI. The autophagic transcriptional program was also activated, as evidenced by the increased level of LC3b, p62/ SQSTM1, GabarapL1, and Cathepsin L mRNAs observed after HI but not after LI. The increased autophagic flux after HI exercise could be due to increased AMP‐activated protein kinase α (AMPKα) activity, as both AMPKαThr72 and ACCSer79 had a higher phosphorylation state after HI (P < 0.001). In summary, the most effective strategy to activate autophagy in human skeletal muscle seems to rely on exercise intensity more than diet.— Schwalm, C., Jamart, C., Benoit, N., Naslain, D., Prémont, C., Prévet, J., Van Thienen, R., Deldicque, L., Francaux, M. Activation of autophagy in human skeletal muscle is dependent on exercise intensity and AMPK activation. FASEB J. 29, 3515‐3526 (2015). www.fasebj.org

Antagonistic effects of leucine and glutamine on the mTOR pathway in myogenic C2C12 cells

Summary.This study compared the effects of leucine and glutamine on the mTOR pathway, on protein synthesis and on muscle-specific gene expression in myogenic C2C12 cells. Leucine increased the phosphorylation state of mTOR, on both Ser2448 and Ser2481, and its downstream effectors, p70S6k, S6 and 4E-BP1. By contrast, glutamine decreased the phosphorylation state of mTOR on Ser2448, p70S6k and 4E-BP1, but did not modify the phosphorylation state of mTOR on Ser2481 and S6. Whilst the phosphorylation state of the mTOR pathway is usually related to protein synthesis, the incorporation of labelled methionine/cysteine was only transiently modified by leucine and was unaltered by glutamine. However, these two amino acids affected the mRNA levels of desmin, myogenin and myosin heavy chain in a time-dependant manner. In conclusion, leucine and glutamine have opposite effects on the mTOR pathway. Moreover, they induce modification of muscle-specific gene expression, unrelated to their effects on the mTOR/p70S6k pathway.

Training in the fasted state improves glucose tolerance during fat-rich diet

A fat‐rich energy‐dense diet is an important cause of insulin resistance. Stimulation of fat turnover in muscle cells during dietary fat challenge may contribute to maintenance of insulin sensitivity. Exercise in the fasted state markedly stimulates energy provision via fat oxidation. Therefore, we investigated whether exercise training in the fasted state is more potent than exercise in the fed state to rescue whole‐body glucose tolerance and insulin sensitivity during a period of hyper‐caloric fat‐rich diet. Healthy male volunteers (18–25 y) received a hyper‐caloric (∼+30% kcal day−1) fat‐rich (50% of kcal) diet for 6 weeks. Some of the subjects performed endurance exercise training (4 days per week) in the fasted state (F; n= 10), whilst the others ingested carbohydrates before and during the training sessions (CHO; n= 10). The control group did not train (CON; n= 7). Body weight increased in CON (+3.0 ± 0.8 kg) and CHO (+1.4 ± 0.4 kg) (P < 0.01), but not in F (+0.7 ± 0.4 kg, P= 0.13). Compared with CON, F but not CHO enhanced whole‐body glucose tolerance and the Matsuda insulin sensitivity index (P < 0.05). Muscle GLUT4 protein content was increased in F (+28%) compared with both CHO (P= 0.05) and CON (P < 0.05). Furthermore, only training in F elevated AMP‐activated protein kinase α phosphorylation (+25%) as well as up‐regulated fatty acid translocase/CD36 and carnitine palmitoyltransferase 1 mRNA levels compared with CON (∼+30%). High‐fat diet increased intramyocellular lipid but not diacylglycerol and ceramide contents, either in the absence or presence of training. This study for the first time shows that fasted training is more potent than fed training to facilitate adaptations in muscle and to improve whole‐body glucose tolerance and insulin sensitivity during hyper‐caloric fat‐rich diet.