Lpaatδ/Agpat4 deficiency impairs maximal force contractility in soleus and alters fibre type in extensor digitorum longus muscle.

Lysophosphatidic acid acyltransferase (LPAAT) δ/acylglycerophosphate acyltransferase 4 is a mitochondrial enzyme and one of five homologues that catalyze the acyl-CoA-dependent synthesis of phosphatidic acid (PA) from lysophosphatidic acid. We studied skeletal muscle LPAATδ and found highest levels in soleus, a red oxidative fibre-type that is rich in mitochondria, and lower levels in extensor digitorum longus (EDL) (white glycolytic) and gastrocnemius (mixed fibre-type). Using Lpaatδ-deficient mice, we found no change in soleus or EDL mass, or in treadmill time-to-exhaustion compared to wildtype littermates. There was, however, a significant reduction in the proportion of type I and type IIA fibres in EDL but, surprisingly, not soleus, where these fibre-types predominate. Also unexpectedly, there was no impairment in force generation by EDL, but a significant reduction by soleus. Oxidative phosphorylation and activity of complexes I, I + II, III, and IV in soleus mitochondria was unchanged and therefore could not explain this effect. However, pyruvate dehydrogenase activity was significantly reduced in Lpaatδ-/- soleus and EDL. Analysis of cellular lipids indicated no difference in soleus triacylglycerol, but specific elevations in soleus PA and phosphatidylethanolamine levels, likely due to a compensatory upregulation of Lpaatß and Lpaatε in Lpaatδ-/- mice. An anabolic effect for PA as an activator of skeletal muscle mTOR has been reported, but we found no change in serine 2448 phosphorylation, indicating reduced soleus force generation is unlikely due to the loss of mTOR activation by a specific pool of LPAATδ-derived PA. Our results identify an important role for LPAATδ in soleus and EDL.

Glucose metabolism during the acute prostate cancer treatment trajectory: The influence of age and obesity.

BACKGROUND & AIMS: Obesity and age, key risk factors for aggressive prostate cancer, are associated with insulin resistance. Glucose-related parameters in patients with aggressive prostate cancer were compared with 2 reference groups: men of similar age and body mass index (BMI) without cancer, and healthy young men. Acute changes in these parameters following radiation treatment were also evaluated. METHODS: Nine patients with aggressive prostate cancer underwent metabolic assessments prior to treatment (baseline), 7 and 33 weeks post-baseline (post-treatment initiation). Baseline measures were compared with the 2 reference groups. Evaluations included: 1) fasting and oral glucose tolerance test (OGTT) blood samples for glucose, C-peptide, and insulin, 2) fasting blood samples for triglycerides, cholesterols, leptin, adiponectin, IL-6, and TNF-α, 3) body composition, 4) nutrition, and 5) physical activity. RESULTS: At baseline, patients had normal fasting glucose concentrations (<5.6 mM; 4.9 ± 1.2 mM) but impaired 2-h OGTT glucose concentrations (>7.8 mM; 8.7 ± 2.9 mM). Both reference groups had normal fasting (matched males: 4.2 ± 0.5 mM; young males: 3.7 ± 0.4 mM) and 2-h OGTT glucose concentrations (matched males: 5.6 ± 1.8 mM; young males: 3.1 ± 0.1 mM) that were significantly lower than patient values. During the OGTT, patients had higher insulin (120 min) and C-peptide (45, 60, 90, 120 min) concentrations compared to the matched males. At 7 weeks, 2-h OGTT glucose concentrations in patients improved to healthy ranges without changes in insulin, C-peptide, IGF-1, IGFBP-3 or other metabolic parameters. CONCLUSIONS: At baseline patients with aggressive prostate cancer demonstrated impaired glucose tolerance compared with men of similar age and body size. Following treatment, glucose tolerance improved in the absence of changes in expected modifiers of glucose metabolism. These improvements may be related to treatment.

The effect of ARC ablation on skeletal muscle morphology, function, and apoptotic signaling during aging.

Augmented apoptotic signaling can result in degradation of skeletal muscle proteins and loss of myonuclei, ultimately contributing to muscle atrophy and contractile dysfunction. Apoptosis repressor with caspase recruitment domain (ARC) is an anti-apoptotic protein highly expressed in skeletal muscle. Here we examined the role of ARC on age-related skeletal muscle apoptosis and wasting by utilizing an ARC-deficient mouse model. Aged mice displayed a number of morphological, phenotypic, and contractile alterations in both soleus and plantaris muscle with aging. Although no differences were found in proteolytic enzyme activity, ARC protein decreased while several anti-apoptotic proteins (e.g., BCL2, BCLXL, HSP70, and XIAP) and the release of mitochondrial housed protein (i.e., SMAC, AIF) increased in aged muscle. Importantly, ARC KO mice had low muscle weights and fewer fibers in soleus, with 2-year-old ARC KO mice displaying lower mitochondrial BCL2 protein along with augmented release of CYTC and SMAC in red/oxidative muscle. Overall, these results indicate that aged skeletal muscle undergoes atrophy as well as contractile and fiber type composition alterations despite an increase in anti-apoptotic protein expression. Although some mitochondrial-specific apoptotic alterations occurred in skeletal muscle due to ARC ablation over the lifespan, our data suggest that ARC may not have a large influence during skeletal muscle aging.

Mice Deficient in lysophosphatidic acid acyltransferase delta (Lpaatδ)/acylglycerophosphate acyltransferase 4 (Agpat4) Have Impaired Learning and Memory.

We previously characterized LPAATδ/AGPAT4 as a mitochondrial lysophosphatidic acid acyltransferase that regulates brain levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Here, we report that Lpaatδ-/- mice display impaired spatial learning and memory compared to wild-type littermates in the Morris water maze and our investigation of potential mechanisms associated with brain phospholipid changes. Marker protein immunoblotting suggested that the relative brain content of neurons, glia, and oligodendrocytes was unchanged. Relative abundance of the important brain fatty acid docosahexaenoic acid was also unchanged in phosphatidylserine, phosphatidylglycerol, and cardiolipin, in agreement with prior data on PC, PE and PI. In phosphatidic acid, it was increased. Specific decreases in ethanolamine-containing phospholipids were detected in mitochondrial lipids, but the function of brain mitochondria in Lpaatδ-/- mice was unchanged. Importantly, we found that Lpaatδ-/- mice have a significantly and drastically lower brain content of the N-methyl-d-asparate (NMDA) receptor subunits NR1, NR2A, and NR2B, as well as the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1, compared to wild-type mice. However, general dysregulation of PI-mediated signaling is not likely responsible, since phospho-AKT and phospho-mTOR pathway regulation was unaffected. Our findings indicate that Lpaatδ deficiency causes deficits in learning and memory associated with reduced NMDA and AMPA receptors.

EARLY CRT MONITORING USING TIME-DOMAIN OPTICAL COHERENCE TOMOGRAPHY DOES NOT ADD TO VISUAL ACUITY FOR PREDICTING VISUAL LOSS IN PATIENTS WITH CENTRAL RETINAL VEIN OCCLUSION TREATED WITH INTRAVITREAL RANIBIZUMAB: A Secondary Analysis of Trial Data.

PURPOSE: Our primary purpose was to assess the clinical (predictive) validity of central retinal thickness (CRT) and best corrected visual acuity (BCVA) at 1 week and 1 month after starting treatment with ranibizumab for central retinal vein occlusion. The authors also assessed detectability of response to treatment. METHODS: The authors used data from 325 participants in the CRUISE study, which included measurement of time-domain CRT and BCVA at baseline, 1 week, 1 month, and 6 months postrandomization. Analysis of covariance models were fitted to assess clinical validity, and distributions of change were constructed to assess detectability of response. RESULTS: There was no evidence that 1-week CRT, and very strong evidence that 1-week BCVA were associated with baseline-adjusted BCVA at 6 months (P = 0.17 and P < 0.001, respectively). There was strong evidence that both 1-month CRT and 1-month BCVA were associated with baseline-adjusted 6-month BCVA (P = 0.005 and P < 0.001, respectively), but simultaneous adjustment found evidence of independent association only for BCVA (P = 0.71 and P < 0.001 for CRT and BCVA, respectively). Detectability of response tended to be higher for CRT than BCVA at 1 week and 1 month but by 6 months these were equivalent for CRT and BCVA. CONCLUSION: In this study, BCVA monitoring of treated central retinal vein occlusion patients seemed more informative than time-domain optical coherence tomography monitoring.

Shell neurons of the master circadian clock coordinate the phase of tissue clocks throughout the brain and body.

BACKGROUND: Daily rhythms in mammals are programmed by a master clock in the suprachiasmatic nucleus (SCN). The SCN contains two main compartments (shell and core), but the role of each region in system-level coordination remains ill defined. Herein, we use a functional assay to investigate how downstream tissues interpret region-specific outputs by using in vivo exposure to long day photoperiods to temporally dissociate the SCN. We then analyze resulting changes in the rhythms of clocks located throughout the brain and body to examine whether they maintain phase synchrony with the SCN shell or core. RESULTS: Nearly all of the 17 tissues examined in the brain and body maintain phase synchrony with the SCN shell, but not the SCN core, which indicates that downstream oscillators are set by cues controlled specifically by the SCN shell. Interestingly, we also found that SCN dissociation diminished the amplitude of rhythms in core clock gene and protein expression in brain tissues by 50-75 %, which suggests that light-driven changes in the functional organization of the SCN markedly influence the strength of rhythms in downstream tissues. CONCLUSIONS: Overall, our results reveal that body clocks receive time-of-day cues specifically from the SCN shell, which may be an adaptive design principle that serves to maintain system-level phase relationships in a changing environment. Further, we demonstrate that lighting conditions alter the amplitude of the molecular clock in downstream tissues, which uncovers a new form of plasticity that may contribute to seasonal changes in physiology and behavior.

Functional, morphological, and apoptotic alterations in skeletal muscle of ARC deficient mice.

Apoptotic signaling plays an important role in the development and maintenance of healthy skeletal muscle. However, dysregulation of apoptotic signals in skeletal muscle is associated with atrophy and loss of function. Apoptosis repressor with caspase recruitment domain (ARC) is a potent anti-apoptotic protein that is highly expressed in skeletal muscle; however, its role in this tissue has yet to be elucidated. To investigate whether ARC deficiency has morphological, functional, and apoptotic consequences, skeletal muscle from 18 week-old wild-type and ARC knockout (KO) mice was studied. In red muscle (soleus), we found lower maximum tetanic force, as well as a shift towards a greater proportion of type II fibers in ARC KO mice. Furthermore, the soleus of ARC KO mice exhibited lower total, as well as fiber type-specific cross sectional area in type I and IIA fibers. Interestingly, these changes in ARC KO mice corresponded with increased DNA fragmentation, albeit independent of caspase or calpain activation. However, cytosolic fractions of red muscle from ARC KO mice had higher apoptosis inducing factor content, suggesting increased mitochondrial-mediated, caspase-independent apoptotic signaling. This was confirmed in isolated mitochondrial preparations, as mitochondria from skeletal muscle of ARC KO mice were more susceptible to calcium stress. Interestingly, white muscle from ARC KO mice showed no signs of altered apoptotic signaling or detrimental morphological differences. Results from this study suggest that even under basal conditions ARC influences muscle apoptotic signaling, phenotype, and function, particularly in slow and/or oxidative muscle.

High-fat feeding does not induce an autophagic or apoptotic phenotype in female rat skeletal muscle.

Apoptosis and autophagy are critical in normal skeletal muscle homeostasis; however, dysregulation can lead to muscle atrophy and dysfunction. Lipotoxicity and/or lipid accumulation may promote apoptosis, as well as directly or indirectly influence autophagic signaling. Therefore, the purpose of this study was to examine the effect of a 16-week high-fat diet on morphological, apoptotic, and autophagic indices in oxidative and glycolytic skeletal muscle of female rats. High-fat feeding resulted in increased fat pad mass, altered glucose tolerance, and lower muscle pAKT levels, as well as lipid accumulation and reactive oxygen species generation in soleus muscle; however, muscle weights, fiber type-specific cross-sectional area, and fiber type distribution were not affected. Moreover, DNA fragmentation and LC3 lipidation as well as several apoptotic (ARC, Bax, Bid, tBid, Hsp70, pBcl-2) and autophagic (ATG7, ATG4B, Beclin 1, BNIP3, p70 s6k, cathepsin activity) indices were not altered in soleus or plantaris following high-fat diet. Interestingly, soleus muscle displayed small increases in caspase-3, caspase-8, and caspase-9 activity, as well as higher ATG12-5 and p62 protein, while both soleus and plantaris muscle showed dramatically reduced Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP) levels. In conclusion, this work demonstrates that 16 weeks of high-fat feeding does not affect tissue morphology or induce a global autophagic or apoptotic phenotype in skeletal muscle of female rats. However, high-fat feeding selectively influenced a number of apoptotic and autophagic indices which could have implications during periods of enhanced muscle stress.

Resveratrol supplementation does not augment performance adaptations or fibre-type-specific responses to high-intensity interval training in humans.

The present study examined the effect of concurrent exercise training and daily resveratrol (RSV) supplementation (150 mg) on training-induced adaptations following low-dose high-intensity interval training (HIIT). Sixteen recreationally active (∼22 years, ∼51 mL·kg(-1)·min(-1)) men were randomly assigned in a double-blind fashion to either the RSV or placebo group with both groups performing 4 weeks of HIIT 3 days per week. Before and after training, participants had a resting muscle biopsy taken, completed a peak oxygen uptake test, a Wingate test, and a submaximal exercise test. A main effect of training (p < 0.05) and interaction effect (p < 0.05) on peak aerobic power was observed; post hoc pairwise comparisons revealed that a significant (p < 0.05) increase occurred in the placebo group only. Main effects of training (p < 0.05) were observed for both peak oxygen uptake (placebo - pretraining: 51.3 ± 1.8, post-training: 54.5 ± 1.5 mL·kg(-1)·min(-1), effect size (ES) = 0.93; RSV - pretraining: 49.6 ± 2.2, post-training: 52.3 ± 2.5 mL·kg(-1)·min(-1), ES = 0.50) and Wingate peak power (placebo: pretraining: 747 ± 39, post-training: 809 ± 31 W, ES = 0.84; RSV - pretraining: 679 ± 39, post-training: 691 ± 43 W, ES = 0.12). Fibre-type distribution was unchanged, while a main effect of training (p < 0.05) was observed for succinate dehydrogenase activity and glycogen content, but not α-glycerophosphate dehydrogenase activity or intramuscular lipids in type I and IIA fibres. The fold change in PGC-1α, SIRT1, and SOD2 gene expression following training was significantly (p < 0.05) lower in the RSV group than placebo. These results suggest that concurrent exercise training and RSV supplementation may alter the normal training response induced by low-volume HIIT.

Fibre-specific responses to endurance and low volume high intensity interval training: striking similarities in acute and chronic adaptation.

The current study involved the completion of two distinct experiments. Experiment 1 compared fibre specific and whole muscle responses to acute bouts of either low-volume high-intensity interval training (LV-HIT) or moderate-intensity continuous endurance exercise (END) in a randomized crossover design. Experiment 2 examined the impact of a six-week training intervention (END or LV-HIT; 4 days/week), on whole body and skeletal muscle fibre specific markers of aerobic and anaerobic capacity. Six recreationally active men (Age: 20.7 ± 3.8 yrs; VO2peak: 51.9 ± 5.1 mL/kg/min) reported to the lab on two separate occasions for experiment 1. Following a muscle biopsy taken in a fasted state, participants completed an acute bout of each exercise protocol (LV-HIT: 8, 20-second intervals at ∼ 170% of VO2peak separated by 10 seconds of rest; END: 30 minutes at ∼ 65% of VO2peak), immediately followed by a muscle biopsy. Glycogen content of type I and IIA fibres was significantly (p<0.05) reduced, while p-ACC was significantly increased (p<0.05) following both protocols. Nineteen recreationally active males (n = 16) and females (n = 3) were VO2peak-matched and assigned to either the LV-HIT (n = 10; 21 ± 2 yrs) or END (n = 9; 20.7 ± 3.8 yrs) group for experiment 2. After 6 weeks, both training protocols induced comparable increases in aerobic capacity (END: Pre: 48.3 ± 6.0, Mid: 51.8 ± 6.0, Post: 55.0 ± 6.3 mL/kg/min LV-HIT: Pre: 47.9 ± 8.1, Mid: 50.4 ± 7.4, Post: 54.7 ± 7.6 mL/kg/min), fibre-type specific oxidative and glycolytic capacity, glycogen and IMTG stores, and whole-muscle capillary density. Interestingly, only LV-HIT induced greater improvements in anaerobic performance and estimated whole-muscle glycolytic capacity. These results suggest that 30 minutes of END exercise at ∼ 65% VO2peak or 4 minutes of LV-HIT at ∼ 170% VO2peak induce comparable changes in the intra-myocellular environment (glycogen content and signaling activation); correspondingly, training-induced adaptations resulting for these protocols, and other HIT and END protocols are strikingly similar.

Microglial cell activation increases saturated and decreases monounsaturated fatty acid content, but both lipid species are proinflammatory.

Neuroinflammation is a component of age-related neurodegenerative diseases and cognitive decline. Saturated (SFA) and monounsaturated (MUFA) fatty acids are bioactive molecules that may play different extrinsic and intrinsic roles in neuroinflammation, serving as exogenous ligands for cellular receptors, or endogenous components of cell structural, energetic and signaling pathways. We determined the fatty acyl profile of BV2 microglial cells before and after acute activation with lipopolysaccharide (LPS). We also investigated the effect of SFA and MUFA pretreatment on the production of an invasive, neurotoxic phenotype in BV2 cells. Acute activation of BV2 microglia resulted in an increase in the relative content of SFA (12:0, 16:0, 18:0, 20:0, 22:0, and 24:0 increased significantly), and a relative decrease in the content of MUFA (16:1n7, 18:1n7, 18:1n9, 20:1n9, 24:1n9 decreased significantly). In agreement, the major stearoyl-CoA desaturase (SCD) isoform in BV2 cells, SCD2, was significantly down-regulated by LPS. We next treated cells with SFA (16:0 or 18:0) or MUFA (16:1n7 or 18:1n9), and found that levels of secreted IL6 were increased, as was secreted MMP9-mediated proteolytic activity. To test the functional significance, we treated SH-SY5Y neuronal cells with conditioned medium from BV2 cells pretreated with fatty acids, and found a small but significant induction of cell death. Our findings suggest differential intrinsic roles for SFA and MUFA in activated microglial cells, but similar extrinsic roles for these fatty acid species in inducing activation. Expansion of SFA is important during microglial cell activation, but either supplemental SFA or MUFA may contribute to chronic low-grade neuroinflammation.

Induction of mitochondrial biogenesis protects against caspase-dependent and caspase-independent apoptosis in L6 myoblasts.

Apoptotic signaling plays an important role in skeletal muscle degradation, atrophy, and dysfunction. Mitochondria are central executers of apoptosis by directly participating in caspase-dependent and caspase-independent cell death signaling. Given the important apoptotic role of mitochondria, altering mitochondrial content could influence apoptosis. Therefore, we examined the direct effect of modest, but physiological increases in mitochondrial biogenesis and content on skeletal muscle apoptosis using a cell culture approach. Treatment of L6 myoblasts with SNAP or AICAR (5h/day for 5days) significantly increased PGC-1, AIF, cytochrome c, and MnSOD protein content as well as MitoTracker staining. Following induction of mitochondrial biogenesis, L6 myoblasts displayed decreased sensitivity to apoptotic cell death as well as reduced caspase-3 and caspase-9 activation following exposure to staurosporine (STS) and C2-ceramide. L6 myoblasts with higher mitochondrial content also exhibited reduced apoptosis and AIF release following exposure to hydrogen peroxide (H2O2). Analysis of several key apoptosis regulatory proteins (ARC, Bax, Bcl-2, XIAP), antioxidant proteins (catalase, MnSOD, CuZnSOD), and reactive oxygen species (ROS) measures (DCF and MitoSOX fluorescence) revealed that these mechanisms were not responsible for the observed cellular protection. However, myoblasts with higher mitochondrial content were less sensitive to Ca(2+)-induced mitochondrial permeability transition pore formation (mPTP) and mitochondrial membrane depolarization. Collectively, these data demonstrate that increased mitochondrial content at physiological levels provides protection against apoptotic cell death by decreasing caspase-dependent and caspase-independent signaling through influencing mitochondrial Ca(2+)-mediated apoptotic events. Therefore, increasing mitochondrial biogenesis/content may represent a potential therapeutic approach in skeletal muscle disorders displaying increased apoptosis.

Elevated skeletal muscle apoptotic signaling following glutathione depletion.

Oxidative stress has a well-established role in numerous intracellular signaling pathways, including apoptosis. Glutathione is an important cellular antioxidant and is the most abundant low molecular weight thiol in the cell. Although previous work has shown a link between glutathione and apoptosis, this relationship has not been defined in skeletal muscle. The present investigation examined the effect of glutathione depletion on skeletal muscle apoptotic signaling, and mitochondrial apoptotic-susceptibility. Administration of L: -buthionine-[S,R]-sulfoximine (BSO; 30 mM in drinking water for 10 days) caused glutathione depletion in whole muscle and isolated mitochondria, as well as elevated muscle catalase protein content and reactive oxygen species (ROS) generation. Glutathione depletion was associated with elevated DNA fragmentation, mitochondrial Bax levels, Poly(ADP-ribose) polymerase (PARP) cleavage, and calpain activity; however, caspase-3, -8, and -9 activity were not altered. BSO administration was also associated with higher cytosolic and nuclear protein levels of apoptosis-inducing factor (AIF), but not cytochrome c, second mitochondria-derived activator of caspase (Smac), or endonuclease G (EndoG). In addition, isolated mitochondria from BSO animals demonstrated significantly lower membrane potential, increased Ca(2+)-induced permeability transition pore opening, and greater basal and ROS-induced AIF and cytochrome c release. These results demonstrate that glutathione depletion in skeletal muscle increases caspase-independent signaling, as well as augments mitochondrial-associated apoptotic events to subsequent cell death stimuli.