Identifying regulatory mechanisms that influence inflammation in metabolic tissues is critical for developing novel metabolic disease treatments. Here, we investigated the role of microRNA-146a ...(miR-146a) during diet-induced obesity in mice. miR-146a is reduced in obese and type 2 diabetic patients and our results reveal that miR-146a-/- mice fed a high-fat diet (HFD) have exaggerated weight gain, increased adiposity, hepatosteatosis, and dysregulated blood glucose levels compared to wild-type controls. Pro-inflammatory genes and NF-κB activation increase in miR-146a-/- mice, indicating a role for this miRNA in regulating inflammatory pathways. RNA-sequencing of adipose tissue macrophages demonstrated a role for miR-146a in regulating both inflammation and cellular metabolism, including the mTOR pathway, during obesity. Further, we demonstrate that miR-146a regulates inflammation, cellular respiration and glycolysis in macrophages through a mechanism involving its direct target Traf6. Finally, we found that administration of rapamycin, an inhibitor of mTOR, was able to rescue the obesity phenotype in miR-146a-/- mice. Altogether, our study provides evidence that miR-146a represses inflammation and diet-induced obesity and regulates metabolic processes at the cellular and organismal levels, demonstrating how the combination of diet and miRNA genetics influences obesity and diabetic phenotypes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Key points
Five days of bed rest resulted in a reduction in leg lean mass and strength in older adults.
After bed rest, older (but not younger) adults had reduced amino acid‐induced anabolic ...sensitivity (blunted muscle protein synthesis; MPS) and enhanced markers associated with the ubiquitin proteasome and autophagy–lysosomal systems (increase in molecular markers related to muscle proteolysis).
Younger adults did not lose leg lean mass (via DXA) after 5 days of bed rest despite blunted amino acid‐induced mTORC1 signalling and increased skeletal muscle REDD1, REDD2 and MURF1 mRNA expression.
Exercise rehabilitation restored bed rest‐induced deficits in lean mass, strength, nutrient‐induced protein anabolism (protein synthesis and mTORC1 signalling) and select muscle proteolytic markers in older adults.
Bed rest‐induced muscle loss and impaired muscle recovery may contribute to age‐related sarcopenia. It is unknown if there are age‐related differences in muscle mass and muscle anabolic and catabolic responses to bed rest. A secondary objective was to determine if rehabilitation could reverse bed rest responses. Nine older and fourteen young adults participated in a 5‐day bed rest challenge (BED REST). This was followed by 8 weeks of high intensity resistance exercise (REHAB). Leg lean mass (via dual‐energy X‐ray absorptiometry; DXA) and strength were determined. Muscle biopsies were collected during a constant stable isotope infusion in the postabsorptive state and after essential amino acid (EAA) ingestion on three occasions: before (PRE), after bed rest and after rehabilitation. Samples were assessed for protein synthesis, mTORC1 signalling, REDD1/2 expression and molecular markers related to muscle proteolysis (MURF1, MAFBX, AMPKα, LC3II/I, Beclin1). We found that leg lean mass and strength decreased in older but not younger adults after bedrest (P < 0.05) and was restored after rehabilitation. EAA‐induced mTORC1 signalling and protein synthesis increased before bed rest in both age groups (P < 0.05). Although both groups had blunted mTORC1 signalling, increased REDD2 and MURF1 mRNA after bedrest, only older adults had reduced EAA‐induced protein synthesis rates and increased MAFBX mRNA, p‐AMPKα and the LC3II/I ratio (P < 0.05). We conclude that older adults are more susceptible than young persons to muscle loss after short‐term bed rest. This may be partially explained by a combined suppression of protein synthesis and a marginal increase in proteolytic markers. Finally, rehabilitation restored bed rest‐induced deficits in lean mass and strength in older adults.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Short-term muscle disuse induces significant muscle loss in older adults and in some reports may be more accelerated with aging. Identifying muscle transcriptional events in response to bed rest may ...help identify therapeutic targets to offset muscle loss. Therefore, we compared the muscle transcriptome between young and older adults after bed rest and identified candidate targets related to changes in muscle loss. RNA was sequenced (HiSeq, Illumina; DESeq, R) from muscle biopsies obtained from young n = 9; 23 yr (SD 3) and older n = 18; 68 yr (SD 6) adults before and after 5-day bed rest. Significantly altered pathways in both young and old subjects relating to mechanosensing and cell adhesion (Actin Cytoskeleton Signaling, ILK Signaling, RhoA Signaling, and Integrin Signaling) were altered (activation z score) to a greater extent in old subjects. Hepatic Fibrosis/Hepatic Stellate Cell Activation was the top regulated pathway significantly altered only in the old. Fifty-one differentially regulated genes were only altered in the young after bed rest and resembled a gene expression profile like that in the old at baseline. Inflammation and muscle wasting genes (CXCL2, GADD45A) were uniquely increased in the old after bed rest, and the macrophage gene MAFB decreased in the old and correlated with the change in leg lean mass. In summary, skeletal muscle dysregulation during bed rest in the old may be driven by alterations in molecules related to fibrosis, inflammation, and cell adhesion. This information may aid in the development of mechanistic-based therapies to combat muscle atrophy during short-term disuse. NEW & NOTEWORTHY Using RNA sequencing and bioinformatics approaches, we identified that older adult skeletal muscle was characterized by dysregulated pathways associated with fibrosis, inflammation (upregulated), and cell adhesion and mechanosensing (downregulated) pathways, with a subset of genes differentially regulated in old and young muscle after bed rest that may describe predisposition to muscle loss. Unique upregulated genes only expressed in old muscle after bed rest indicated increased inflammation and muscle wasting (CXCL2, GADD45A) and decreased MAFB correlated with the change in leg lean mass.
Loss of muscle mass and strength after disuse followed by impaired muscle recovery commonly occurs with aging. Metformin (MET) and leucine (LEU) individually have shown positive effects in skeletal ...muscle during atrophy conditions but have not been evaluated in combination nor tested as a remedy to enhance muscle recovery following disuse atrophy in aging. The purpose of this study was to determine if a dual treatment of metformin and leucine (MET + LEU) would prevent disuse‐induced atrophy and/or promote muscle recovery in aged mice and if these muscle responses correspond to changes in satellite cells and collagen remodeling. Aged mice (22–24 months) underwent 14 days of hindlimb unloading (HU) followed by 7 or 14 days of reloading (7 or 14 days RL). MET, LEU, or MET + LEU was administered via drinking water and were compared to Vehicle (standard drinking water) and ambulatory baseline. We observed that during HU, MET + LEU resolved whole body grip strength and soleus muscle specific force decrements caused by HU. Gastrocnemius satellite cell abundance was increased with MET + LEU treatment but did not alter muscle size during disuse or recovery conditions. Moreover, MET + LEU treatment alleviated gastrocnemius collagen accumulation caused by HU and increased collagen turnover during 7 and 14 days RL driven by a decrease in collagen IV content. Transcriptional pathway analysis revealed that MET + LEU altered muscle hallmark pathways related to inflammation and myogenesis during HU. Together, the dual treatment of MET and LEU was able to increase muscle function, satellite cell content, and reduce collagen accumulation, thus improving muscle quality during disuse and recovery in aging.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The loss of skeletal muscle mass during aging, sarcopenia, increases the risk for falls and dependence. Resistance exercise (RE) is an effective rehabilitation technique that can improve muscle mass ...and strength; however, older individuals are resistant to the stimulation of muscle protein synthesis (MPS) with traditional high-intensity RE. Recently, a novel rehabilitation exercise method, low-intensity RE, combined with blood flow restriction (BFR), has been shown to stimulate mammalian target of rapamycin complex 1 (mTORC1) signaling and MPS in young men. We hypothesized that low-intensity RE with BFR would be able to activate mTORC1 signaling and stimulate MPS in older men. We measured MPS and mTORC1-associated signaling proteins in seven older men (age 70+/-2 yr) before and after exercise. Subjects were studied identically on two occasions: during BFR exercise bilateral leg extension exercise at 20% of 1-repetition maximum (1-RM) with pressure cuff placed proximally on both thighs and inflated at 200 mmHg and during exercise without the pressure cuff (Ctrl). MPS and phosphorylation of signaling proteins were determined on successive muscle biopsies by stable isotopic techniques and immunoblotting, respectively. MPS increased 56% from baseline after BFR exercise (P<0.05), while no change was observed in the Ctrl group (P>0.05). Downstream of mTORC1, ribosomal S6 kinase 1 (S6K1) phosphorylation and ribosomal protein S6 (rpS6) phosphorylation increased only in the BFR group after exercise (P<0.05). We conclude that low-intensity RE in combination with BFR enhances mTORC1 signaling and MPS in older men. BFR exercise is a novel intervention that may enhance muscle rehabilitation to counteract sarcopenia.
Impaired muscle recovery (size and strength) following a disuse period commonly occurs in older adults. Many of these individuals are not able to adequately exercise due to pain and logistic ...barriers. Thus, nutritional and pharmacological therapeutics, that are translatable, are needed to promote muscle recovery following disuse in older individuals. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) may be a suitable therapeutic target due to pleiotropic regulation of skeletal muscle. This review focuses on nutritional and pharmacological interventions that target PGC-1α and related Sirtuin 1 (SIRT1) and 5' AMP-activated protein kinase (AMPKα) signaling in muscle and thus may be rapidly translated to prevent muscle disuse atrophy and promote recovery. In this review, we present several therapeutics that target PGC-1α in skeletal muscle such as leucine, β-hydroxy-β-methylbuyrate (HMB), arginine, resveratrol, metformin and combination therapies that may have future application to conditions of disuse and recovery in humans.
Departments of 1 Physical Therapy, 2 Internal Medicine, and 3 Surgery, 4 Division of Rehabilitation Sciences, University of Texas Medical Branch, Galveston, Texas
Submitted 7 September 2007
; ...accepted in final form 3 December 2007
We recently showed that resistance exercise and ingestion of essential amino acids with carbohydrate (EAA+CHO) can independently stimulate mammalian target of rapamycin (mTOR) signaling and muscle protein synthesis in humans. Providing an EAA+CHO solution postexercise can further increase muscle protein synthesis. Therefore, we hypothesized that enhanced mTOR signaling might be responsible for the greater muscle protein synthesis when leucine-enriched EAA+CHOs are ingested during postexercise recovery. Sixteen male subjects were randomized to one of two groups (control or EAA+CHO). The EAA+CHO group ingested the nutrient solution 1 h after resistance exercise. mTOR signaling was assessed by immunoblotting from repeated muscle biopsy samples. Mixed muscle fractional synthetic rate (FSR) was measured using stable isotope techniques. Muscle protein synthesis and 4E-BP1 phosphorylation during exercise were significantly reduced ( P < 0.05). Postexercise FSR was elevated above baseline in both groups at 1 h but was even further elevated in the EAA+CHO group at 2 h postexercise ( P < 0.05). Increased FSR was associated with enhanced phosphorylation of mTOR and S6K1 ( P < 0.05). Akt phosphorylation was elevated at 1 h and returned to baseline by 2 h in the control group, but it remained elevated in the EAA+CHO group ( P < 0.05). 4E-BP1 phosphorylation returned to baseline during recovery in control but became elevated when EAA+CHO was ingested ( P < 0.05). eEF2 phosphorylation decreased at 1 and 2 h postexercise to a similar extent in both groups ( P < 0.05). Our data suggest that enhanced activation of the mTOR signaling pathway is playing a role in the greater synthesis of muscle proteins when resistance exercise is followed by EAA+CHO ingestion.
muscle protein synthesis; mammalian target of rapamycin; essential amino acids
Address for reprint requests and other correspondence: B. B. Rasmussen, Dept. of Physical Therapy, Division of Rehabilitation Sciences, Univ. of Texas Medical Branch, 301 Univ. Blvd., Galveston, TX 77555-1144 (e-mail: blrasmus{at}utmb.edu )
Muscle protein synthesis and mTORC1 signalling are concurrently stimulated following muscle contraction in humans. In an effort
to determine whether mTORC1 signalling is essential for regulating ...muscle protein synthesis in humans, we treated subjects
with a potent mTORC1 inhibitor (rapamycin) prior to performing a series of high-intensity muscle contractions. Here we show
that rapamycin treatment blocks the early (1â2 h) acute contraction-induced increase (â¼40%) in human muscle protein synthesis.
In addition, several downstream components of the mTORC1 signalling pathway were also blunted or blocked by rapamycin. For
instance, S6K1 phosphorylation (Thr421/Ser424) was increased post-exercise 6-fold in the control group while being unchanged
with rapamycin treatment. Furthermore, eEF2 phosphorylation (Thr56) was reduced by â¼25% post-exercise in the control group
but phosphorylation following rapamycin treatment was unaltered, indicating that translation elongation was inhibited. Rapamycin
administration prior to exercise also reduced the ability of raptor to associate with mTORC1 during post-exercise recovery.
Surprisingly, rapamycin treatment prior to resistance exercise completely blocked the contraction-induced increase in the
phosphorylation of ERK1/2 (Thr202/Tyr204) and blunted the increase in MNK1 (Thr197/202) phosphorylation. However, the phosphorylation
of a known target of MNK1, eIF4E (Ser208), was similar in both groups ( P > 0.05) which is consistent with the notion that rapamycin does not directly inhibit MAPK signalling. We conclude that mTORC1
signalling is, in part, playing a key role in regulating the contraction-induced stimulation of muscle protein synthesis in
humans, while dual activation of mTORC1 and ERK1/2 stimulation may be required for full stimulation of human skeletal muscle
protein synthesis.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Sarcopenia, the loss of skeletal muscle mass during aging, increases the risk for falls and dependency. Resistance exercise (RE) training is an effective treatment to improve muscle mass and strength ...in older adults, but aging is associated with a smaller amount of training-induced hypertrophy. This may be due in part to an inability to stimulate muscle-protein synthesis (MPS) after an acute bout of RE. We hypothesized that older adults would have impaired mammalian target of rapamycin complex (mTORC)1 signaling and MPS response compared with young adults after acute RE.
We measured intracellular signaling and MPS in 16 older (mean 70 ± 2 years) and 16 younger (27 ± 2 years) subjects. Muscle biopsies were sampled at baseline and at 3, 6 and 24 hr after exercise. Phosphorylation of regulatory signaling proteins and MPS were determined on successive muscle biopsies by immunoblotting and stable isotopic tracer techniques, respectively.
Increased phosphorylation was seen only in the younger group (P< 0.05) for several key signaling proteins after exercise, including mammalian target of rapamycin (mTOR), ribosomal S6 kinase (S6K)1, eukaryotic initiation factor 4E-binding protein (4E-BP)1 and extracellular signal-regulated kinase (ERK)1/2, with no changes seen in the older group (P >0.05). After exercise, MPS increased from baseline only in the younger group (P< 0.05), with MPS being significantly greater than that in the older group (P <0.05).
We conclude that aging impairs contraction-induced human skeletal muscle mTORC1 signaling and protein synthesis. These age-related differences may contribute to the blunted hypertrophic response seen after resistance-exercise training in older adults, and highlight the mTORC1 pathway as a key therapeutic target to prevent sarcopenia.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Impaired recovery of aged muscle following a disuse event is an unresolved issue facing the older adult population. Although investigations in young animals have suggested that rapid regrowth of ...skeletal muscle following a disuse event entails a coordinated involvement of skeletal muscle macrophages, this phenomenon has not yet been thoroughly tested as an explanation for impaired muscle recovery in aging. To examine this hypothesis, young (4-5 mo) and old (24-26 mo) male mice were examined as controls following 2 wk of hindlimb unloading (HU) and following 4 (RL4) and 7 (RL7) days of reloading after HU. Muscles were harvested to assess muscle weight, myofiber-specifc cross-sectional area, and skeletal muscle macrophages via immunofluorescence. Flow cytometry was used on gastrocnemius and soleus muscle (at RL4) single-cell suspensions to immunophenotype skeletal muscle macrophages. Our data demonstrated impaired muscle regrowth in aged compared with young mice following disuse, which was characterized by divergent muscle macrophage polarization patterns and muscle-specifc macrophage abundance. During reloading, young mice exhibited the classical increase in M1-like (MHC II
CD206
) macrophages that preceeded the increase in percentage of M2-like macrophages (MHC II
CD206
); however, old mice did not demonstrate this pattern. Also, at RL4, the soleus demonstrated reduced macrophage abundance with aging. Together, these data suggest that dysregulated macrophage phenotype patterns in aged muscle during recovery from disuse may be related to impaired muscle growth. Further investigation is needed to determine whether the dysregulated macrophage response in the old during regrowth from disuse is related to a reduced ability to recruit or activate specific immune cells.
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