Citrate Synthase Protein Expression Research Articles

Physical activity of mice on dietary sulfur amino acid restriction is influenced by age of diet initiation and biological sex

Please use the attached pdf file for corrections.Sulfur amino acid restriction (SAAR)—the reduction of methionine and cysteine concentrations either in the diet or by genetic manipulation—promotes health span and extends lifespan, but its effects on physical activity remain unclear. We investigated whether age of diet initiation and biological sex could influence physical activity in mice fed either a control diet (CF, 0.86% methionine w/w) or SAAR (0.12% methionine w/w). Quadriceps femoris muscle mass is smaller in SAAR than in CF mice. Young mice fed a chronic SAAR diet at 8 weeks of age exhibited improved wire hang and running wheel activities compared to young CF mice, while aged mice showed comparable results. The effects of chronic SAAR on physical activity was mildly influenced by sex as observed in middle-aged male SAAR mice who showed minor improvements than CF males while middle-aged females displayed no discernible effects. Muscle mass is minimally affected by changes in markers of protein synthesis, autophagy and atrophy. Improvements to physical activity in young SAAR mice could be partially attributed to increased skeletal muscle mitochondrial activity. Furthermore, SAAR in C2C12 myotubes increased citrate synthase protein expression and enhanced succinyl dehydrogenase enzyme activity compared to CF myotubes. Overall, our data reveal that SAAR can improve mouse physical activity without compromising muscle proteostasis. This is partially due to enhanced mitochondrial activity, but the effects are influenced by age of diet initiation and sex.

Skeletal muscle mitochondrial fragmentation and impaired bioenergetics from nutrient overload are prevented by carbon monoxide.

Nutrient excess increases skeletal muscle oxidant production and mitochondrial fragmentation that may result in impaired mitochondrial function, a hallmark of skeletal muscle insulin resistance. This led us to explore whether an endogenous gas molecule, carbon monoxide (CO), which is thought to prevent weight gain and metabolic dysfunction in mice consuming high-fat diets, alters mitochondrial morphology and respiration in C2C12 myoblasts exposed to high glucose (15.6 mM) and high fat (250 µM BSA-palmitate) (HGHF). Also, skeletal muscle mitochondrial morphology, distribution, respiration, and energy expenditure were examined in obese resistant (OR) and obese prone (OP) rats that consumed a high-fat and high-sucrose diet for 10 wk with or without intermittent low-dose inhaled CO and/or exercise training. In cells exposed to HGHF, superoxide production, mitochondrial membrane potential (ΔΨm), mitochondrial fission regulatory protein dynamin-related protein 1 (Drp1) and mitochondrial fragmentation increased, while mitochondrial respiratory capacity was reduced. CO decreased HGHF-induced superoxide production, Drp1 protein levels and mitochondrial fragmentation, maintained ΔΨm, and increased mitochondrial respiratory capacity. In comparison with lean OR rats, OP rats had smaller skeletal muscle mitochondria that contained disorganized cristae, a normal mitochondrial distribution, but reduced citrate synthase protein expression, normal respiratory responses, and a lower energy expenditure. The combination of inhaled CO and exercise produced the greatest effect on mitochondrial morphology, increasing ADP-stimulated respiration in the presence of pyruvate, and preventing a decline in resting energy expenditure. These data support a therapeutic role for CO and exercise in preserving mitochondrial morphology and respiration during metabolic overload.

Effects of photoperiod on morphology and function in testis and epididymis of Cricetulus barabensis.

Photoperiod regulates the seasonal reproductive rhythms of mammals by influencing the development and function of sexual organs; however, the underlying mechanism remains unclear. We examined the morphology and functioning of the main sex organs of striped dwarf hamsters (Cricetulus barabensis) under different photoperiods (short daylight [SD], moderate daylight [MD], and long daylight [LD]) and further investigated the underlying molecular mechanisms. There was an inverse correlation between blood melatonin levels and photoperiod in the order SD > MD > LD. Decreases in body and tissue weights were observed under SD, whereas testis and epididymis weights between MD and LD were comparable. The diameters of the spermatogenic tubules, thickness of the spermatogenic epithelium, and the number of spermatogonia and Sertoli cells decreased under SD, whereas the serum-luteinizing hormone, follicle-stimulating hormone, and fecal testosterone concentrations decreased under LD. In SD, bax/bcl2 protein expression increased in the testes and decreased in the epididymides, whereas LC3II/LC3I remained unchanged in the testes and increased in the epididymides compared with the MD group. In LD, bax/bcl2 and LC3II/LC3I protein expression levels were unchanged in the testes but were decreased in the epididymides. In SD and LD, adenosine triphosphate synthase and citrate synthase protein expression levels were unchanged in the testes but were decreased in the epididymides. Drp1 and Mff protein expression increased in the testes and decreased in the epididymides. Overall, different regulatory mechanisms in the testis and epididymis led to degeneration under SD and maintenance under LD, preferentially protecting mitochondrial function in the testis by regulating mitochondrial fission.

Comparative pharmacoproteomics reveals potential targets for berberine, a promising therapy for colorectal cancer

Berberine (BBR), a natural isoquinoline alkaloid, has been shown to be a promising therapeutic agent for colorectal cancer (CRC), but the molecular mechanism remains unclear. Here, we used mass spectrometry-based label-free proteomics to explore the potential targets of BBR in CRC cells. Comprehensive proteomic profiles demonstrated that of 8051 identified proteins, 503 and 277 differentially expressed proteins (DEPs) were screened out of CACO2 and LOVO cells, respectively. 83 DEPs were overlapped and most of these were down-regulated. A pathway enrichment analysis pinpointed mitochondrial translation, respiratory electron transport and the citric acid (TCA) cycle as biological effectors. The data of proteomics was subsequently confirmed by citrate synthase (CS), Tu translation elongation factor (TUFM), pentatricopeptide repeat domain 3 (PTCD3) and mitochondrial ribosomal protein L48 (MRPL 48) protein measurement. CS protein expression in CRC cells and tissues was higher than it was in normal specimens. Additionally, forcible downregulation of CS led to remarkable cell proliferation inhibition. Taken together, we concluded that the anticancer effects of BBR are attributable to mitochondrial protein synthesis, TCA and respiratory electron transport inhibition and that CS might be a useful therapeutic target in CRC treatment.

In vitro comparison of hydroxocobalamin (B12a) and the mitochondrial directed therapy by a succinate prodrug in a cellular model of cyanide poisoning

The objective of this study was to compare the use of hydroxocobalamin (B12a) and a succinate prodrug to evaluate for improvement in mitochondrial function in an in vitro model of cyanide poisoning. Peripheral blood mononuclear cells (PBMC) and human aortic smooth muscle cells (HASMC) incubated with 50 mM of sodium cyanide (CN) for five minutes serving as the CN group compared to controls. We investigated the following: (1) Mitochondrial respiration; (2) Superoxide and mitochondrial membrane potential with microscopy; (3) Citrate synthase protein expression. All experiments were performed with a cell concentration of 2−3 × 106 cells/ml for both PBMC and HASMC. There were four conditions: (1) Control (no exposure); (2) Cyanide (exposure only); (3) B12a (cyanide exposure followed by B12a treatment); (4) NV118 (cyanide followed by NV118 treatment). In this study the key findings include: (1) Improvement in key mitochondrial respiratory states with the succinate prodrug (NV118) but not B12a; (2) Attenuation of superoxide production with treatment of NV118 but not with B12a treatment; (3) The changes in respiration were not secondary to increased mitochondrial content as measured by citrate synthase; (4) The use of easily accessible human blood cells showed similar mitochondrial response to both cyanide and treatment to HASMC. The use of a succinate prodrug to circumvent partial CIV inhibition by cyanide with clear reversal of cellular respiration and superoxide production that was not attributed to changes in mitochondrial content not seen by the use of B12a.

Gastric Bypass Surgery Improves the Skeletal Muscle Ceramide/S1P Ratio and Upregulates the AMPK/ SIRT1/ PGC-1α Pathway in Zucker Diabetic Fatty Rats.

Roux-en-Y gastric bypass (RYGB) is associated with remission of type 2 diabetes. However, the cellular and molecular mechanisms remain unknown. We hypothesized that RYGB would increase peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), sirtuin-1 (SIRT1), AMPK/pAMPK, and citrate synthase (CS) protein expression and decrease insulin resistance and these changes would bemediated by sphingolipids, including ceramides and the sphingolipid metabolite sphingosine-1 phosphate (S1P). Male ZDF rats were randomized to RYGB (n = 7) or sham surgery (n = 7) and harvested after 28days. Total tissue ceramide, ceramide subspecies (C14:0, C16:0, C18:0, C18:1, C20:0, C24:0, and C24:1), and S1P were quantified in the white gastrocnemius muscle using LC-ESI-MS/MS after separation with HPLC. Total SIRT1, AMPK, PGC-1α, and CS protein expression were measured by Western blot. Body weight, fasting glucose, insulin, and HOMA-IR decreased significantly after RYGB compared with sham control. These changes were paralleled by lower total ceramide (483.7 ± 32.3 vs. 280.1 ± 38.8nmol/g wwt), C18:0 ceramide subspecies (P < 0.05), higher S1P (0.83 ± 0.05 vs. 1.54 ± 0.21nmol/g wwt, P < 0.05), and a lower ceramide/S1P ratio (P < 0.05) in the RYGB versus sham group. AMPK, pAMPK, SIRT1, PGC-1α, and CS protein expression was also higher after RYGB (P < 0.05). The ceramide/S1P ratio correlated with weight loss (r = 0.48, P = 0.08), insulin resistance (r = 0.61, P = 0.02), PGC-1α (r = - 0.51, P < 0.06), CS (r = - 0.63, P = 0.01), and SIRT1 (r = - 0.54, P < 0.04). Our data demonstrate that sphingolipid balance, and increased AMPK, SIRT1, PGC-1α, and CS protein expression are part of the mechanism that contributes to the remission of diabetes after RYGB surgery.

Estrogen‐related Receptor α Overexpression Drives a Metabolic and Gene Expression Program Mimicking Aerobic Exercise in Skeletal Muscle

Aerobic exercise is considered one of the most effective interventions to prevent obesity and type 2 diabetes (T2D). Exercise increases skeletal muscle insulin sensitivity by stimulating mitochondrial respiration and fatty acid oxidation through coordinated regulation of metabolic genes. Transcriptional pathways that regulate exercise‐induced metabolic gene programs present an attractive class of therapeutic targets to combat obesity and related metabolic diseases. We and others have shown that the Estrogen‐related Receptor α (ERRα) nuclear receptor regulates mitochondrial biogenesis, respiration and fatty acid oxidation and is required for metabolic reprogramming in response to exercise training. The objective of these studies is to determine whether ERRα over‐expression is sufficient to mediate exercise‐induced adaptations in muscle. We generated a conditional ERRα transgenic mouse line that expresses the Flag‐tagged human ERRα in skeletal muscle‐specific manner when crossed with Mck‐Cre recombinase mice (ERRαMOE). We examined gene expression (trancript and protein) and characterized the metabolic phentoype of the ERRαMOE mice. ERRαMOE mice exhibited a reddening of the superficial gastrocnemius and quadriceps, characteristic of oxidative muscles, accompanied by a reciprocal change in myosin heavy chain (MHC) IIb (−35%) and IIa (+80%) expression. Mitochondrial content was higher in skeletal muscle and heart, but not in non‐muscle tissues, based on mtDNA quantitation and citrate synthase protein expression. Consistently, basal (+32%) and maximal (+70%) O2 consumption rates were increased in FDB fibers isolated from ERRα‐MOE compared to WT. Signaling through the AMP‐activated kinase (AMPK) pathway, which mediates exercise‐dependent metabolic reprogramming, was unchanged in the basal state; however, maximal activation by AICAR was enhanced in ERRα‐overexpressing myotubes. We assessed systemic glucose homeostasis in adult chow fed mice. Although fasting glucose (6 hr) was slightly lower in ERRαMOE, no significant difference in glucose tolerance was observed. However, the results of insulin tolerance tests showed that ERRαMOE mice were more insulin sensitive than WT. In future studies we will investigate whether ERRα activation in skeletal muscle protects against high fat diet‐induced insulin resistance. Based on the results from these studies, ERRα over‐expression mimics the effects of exercise on skeletal muscle mitochondrial respiratory capacity and improves whole body insulin sensitivity.Support or Funding InformationSupported by Diabetes and Metabolic Research Institute (DMRI) and Shared Resource Pilot Awards from the City of Hope and NIH U24DK097748This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oxidative capacity and glycogen content increase more in arm than leg muscle in sedentary women after intense training

The hypothesis that the adaptive capacity is higher in human upper- than lower-body skeletal muscle was tested. Furthermore, the hypothesis that more pronounced adaptations in upper-body musculature can be achieved by "low-volume high-intensity" compared with "high-volume low-intensity" exercise training was evaluated. A group of sedentary premenopausal women aged 45 ± 6 yr (± SD) with expected high adaptive potential in both upper- and lower-extremity muscle groups participated. After random allocation to high-intensity swimming (HIS, n = 21), moderate-intensity swimming (MOS, n = 21), soccer (SOC, n = 21) or a nontraining control group (CON, n = 20), the training groups completed three workouts per week for 15 wk. Resting muscle biopsies were obtained from the vastus lateralis muscle and deltoideus muscle before and after the intervention. After the training intervention, a larger (P < 0.05) increase existed in deltoideus muscle of the HIS group compared with vastus lateralis muscle of the SOC group for citrate synthase maximal activity (95 ± 89 vs. 27 ± 34%), citrate synthase protein expression (100 ± 29 vs. 31 ± 44%), 3-hydroxyacyl-CoA dehydrogenase maximal activity (35 ± 43 vs. 3 ± 25%), muscle glycogen content (63 ± 76 vs. 20 ± 51%), and expression of mitochondrial complex II, III, and IV. Additionally, HIS caused higher (P < 0.05) increases than MOS in deltoideus muscle citrate synthase maximal activity, citrate synthase protein expression, and muscle glycogen content. In conclusion, the deltoideus muscle has a higher adaptive potential than the vastus lateralis muscle in sedentary women, and "high-intensity low-volume" training is a more efficient regime than "low-intensity high-volume" training for increasing the aerobic capacity of the deltoideus muscle.

Effect Of Exercise Training Mode On Oxidative Capacity In Human Arm And Leg Skeletal Muscle

The exercise-induced adaptation pattern in oxidative capacity in human skeletal is highly dependent on the type of training performed. However, information on the impact of different exercise training protocols on muscle responses in arm versus leg is sparse. PURPOSE: To examined effects of different whole-body exercise training modes on oxidative capacity in arm and leg human skeletal muscle. METHODS: Sedentary premenopausal women (n=83; age; 45±2 (±SEM) yrs, height; 165±1 cm, weight; 79.2±1.5 kg, body fat; 42.9±0.6 %) were randomized into high-intensity swimming (HIS, n=21), moderate-intensity swimming (MOS, n=21), football training intervention-groups (FTG, n=21), and control (CON, n=20). Intervention-groups completed 3 sessions/wk for 15 wks. Pre and post-intervention muscle biopsies were obtained at rest from arm (m. deltoideus) and leg (m. vastus lateralis). RESULTS: Maximal citrate synthase (CS) activity in intervention-groups increased by 44±59% overall (P<0.001; n=82), with only HIS displaying a higher (P<0.05) increase in arm than leg muscle (95±89% vs. 52±80%, respectively). In leg, CS protein expression increased in intervention-groups (31-55%, P<0.05), while arm, CS protein expression was increased in FTG and HIS (49-100%, P<0.01) only. Maximal arm 3-Hydroxyacyl-CoA Dehydrogenase (HAD) activity post-training was elevated in FTG, MOS and HIS (20-35%), while only HIS had an increase (18±30%, P<0.05) in leg. HIS and MOS increased (P<0.05) arm mitochondrial respiratory chain complex 1-5, and FTG only had an increase (P<0.05) in complex 5, while groups-specific adaptations occurred in the leg muscle. In HIS the increase in arm complex 2 and 4 was higher (P<0.05) than in leg. Resting muscle glycogen was elevated post-HIS in arm only (63±76%, P<0.001). CONCLUSION: In conclusion, markers of oxidative capacity in arm and leg human skeletal muscle respond differently to exercise training. In general, Krebs cycle capacity, marked by CS, increased in both arm and leg muscle, while the increase in β-oxidation capacity primarily occurred in arm. The greatest improvement in mitochondrial electron-transport capacity was in arm muscles after swim-training, with the overall greatest effect after high-intensity swim-training. Supported by a grant (ST-05710) from The Faroese Research Council.

SIRT3, a mitochondrial NAD⁺-dependent deacetylase, is involved in the regulation of myoblast differentiation.

Sirtuin 3 (SIRT3), one of the seven mammalian sirtuins, is a mitochondrial NAD+-dependent deacetylase known to control key metabolic pathways. SIRT3 deacetylases and activates a large number of mitochondrial enzymes involved in the respiratory chain, in ATP production, and in both the citric acid and urea cycles. We have previously shown that the regulation of myoblast differentiation is tightly linked to mitochondrial activity. Since SIRT3 modulates mitochondrial activity, we decide to address its role during myoblast differentiation. For this purpose, we first investigated the expression of endogenous SIRT3 during C2C12 myoblast differentiation. We further studied the impact of SIRT3 silencing on both the myogenic potential and the mitochondrial activity of C2C12 cells. We showed that SIRT3 protein expression peaked at the onset of myoblast differentiation. The inhibition of SIRT3 expression mediated by the stable integration of SIRT3 short inhibitory RNA (SIRT3shRNA) in C2C12 myoblasts, resulted in: 1) abrogation of terminal differentiation - as evidenced by a marked decrease in the myoblast fusion index and a significant reduction of Myogenin, MyoD, Sirtuin 1 and Troponin T protein expression - restored upon MyoD overexpression; 2) a decrease in peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and citrate synthase protein expression reflecting an alteration of mitochondrial density; and 3) an increased production of reactive oxygen species (ROS) mirrored by the decreased activity of manganese superoxide dismutase (MnSOD). Altogether our data demonstrate that SIRT3 mainly regulates myoblast differentiation via its influence on mitochondrial activity.