Citrate Synthase Enzyme Activity Research Articles

Calcification in sea urchin larvae is associated with low metabolic costs.

The energetic costs to generate calcium carbonate skeletons and shells in marine organisms remain largely speculative due to the scarcity of empirical data. However, this information is critical to estimate energetic limitations of marine calcifiers that can explain their sensitivities to changes in sea water carbonate chemistry in past, present and future marine systems. The cost of calcification was evaluated using larval stages of the purple sea urchin, Strongylocentrotus purpuratus. We developed a skeleton re-mineralization assay, in which the skeleton was dissolved in live larvae followed by a re-mineralization over a few days. During skeleton re-mineralization, energetic costs were estimated through the measurement of key metabolic parameters including whole animal metabolic rates, citrate synthase (CS) enzyme activities and mRNA expression as well as mitochondrial densities in the calcifying primary mesenchyme cells (PMCs). Minor increases in a CS activity and a 10-15% increase in mitochondrial densities in PMCs were observed in re-mineralizing larvae as compared to control larvae. Re-mineralization under three different pH conditions (pH 8.1, pH 7.6 and pH 7.1) decreased with decreasing pH accompanied by pronounced increases in CS expression levels and increased mitochondrial densities in PMCs at pH 7.6. Despite a prominent increase in mitochondrial density of primary mesenchyme cells, particularly in the calcifying cohort of this cell type, this work demonstrated a low overall metabolic response to increased mineralization rates on the whole animal level under both, high and low pH conditions. We conclude that calcification in sea urchin larvae is compromised under low pH conditions, associated with low energetic efforts to fuel compensatory processes.

The impact of resistance training on memory, gait and oxidative stress during periestropause in rats

Aging, especially in female, is complex, involving various factors such as reproductive sensitivity, cognitive and functional decline, and an imbalance in the redox system. This study aims to assess the effectiveness of long-term resistance training as a non-pharmacological strategy to mitigate the impairment of recognition memory, hippocampal redox state, and ambulation in aging female Wistar rats during the periestropause period. Thirty Wistar rats aged 17 months, in periestropause, were distributed into non-trained (NT) and resistance training (RT; stair climbing 3 times per week for 4 months) groups. Before (17 months) and after (21 months) of the RT period, the rats underwent tests for ambulation, elevated plus maze (EPM), open field, and object recognition. Biochemical and histological analyses were conducted on the hippocampus of these animals. Analysis of the results revealed that at 21 months, females in the NT group (21Mo/NT) exhibited a decreased in length (p=0.0458) and an increased in past width (p<0.0479) compared to their measurements at 17 months. However, after 4 months of RT, the female rats aged 21 months (21Mo/RT group) experienced changes in gait components, showing an increase in length (p<0.0008) and a decrease in stride width. Regarding memory, the object recognition test indicated potential cognitive improvement in 21Mo/RT animals, with significant interaction between intervention and age across all three stages of the test (total exploration time, p=0.0001; Test 1, p=0.0003; Test 2, p=0.0014). This response was notable compared to animals in the 21Mo/NT group, which showed a decline in memory capacity (p<0.01). The data showed a significant difference in relation to the age of the animals (p<0.01). The hippocampal redox state markers showed reduced lipid oxidative (p=0.028), catalase (p=0.022), and superoxide dismutase (p=0.0067) in the RT group compared to the NT group. Hippocampal cells from the 21Mo/RT group showed increased citrate synthase enzyme activity (p<0.05) and Nissl body staining (p<0.05). The results of this study demonstrate that RT performed during the periestropause phase leads to significant improvements in functional abilities, cognitive performance, and neuroplasticity in aging female rats.

Characterization of Sex-Dependent Differences in Brain Microvascular Bioenergetics

Background: Women experience increased incidence of stroke-related deaths and lifetime risk of Alzheimer's disease (AD). Microvascular dysfunction has been implicated in the pathogenesis of ischemic stroke and AD. Our objective is to investigate the sex-dependent differences in bioenergetics of young mouse brain microvessels (BMVs). Methods: BMVs were isolated from young male and female mice (3-4 months), using a combination of gradient centrifugation and filtration (300μm and 40μm filters) methods. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BMVs were measured utilizing the Agilent Seahorse XFe 24 analyzer. The levels of proteins and mRNAs of mitochondrial respiratory complexes were measured by western blot and RT-PCR, respectively. Citrate synthase (CS) enzyme activity and ATP levels were determined in lysates of BMVs. Results: Real-Time ATP Rate assay demonstrated a decreased total ATP production rate and ATP contribution from both glycolysis and oxidative phosphorylation (OxPhos) in BMVs from females compared to males. Mitochondrial stress test assay revealed significantly lower values of several mitochondrial respiratory parameters, including basal respiration, ATP production, maximal respiration, spare respiratory capacity, and proton leak in BMVs from females compared to males. Glycolytic Rate assay uncovered reduced basal glycolysis, proton efflux rate (PER) originating from basal glycolysis, and the mitoOCR/glycoPER ratio in BMVs from females compared to males. Interestingly, no sex-dependent differences were observed in basal PER and post-2-DG acidification rates in BMVs. Mito Fuel Flex Test assay showed no sex-dependent difference in the utilization of glucose, glutamine, and long-chain fatty acids as fuel substrates in BMVs. ATP measurements confirmed the reduced ATP levels in BMVs from females compared to males. Measurements of CS activity revealed no sex-dependent difference in the BMVs. Lastly, Western blot and RT-PCR experiments indicated no differences in either protein or mRNA levels of various mitochondrial complex protein levels.Conclusions: Female BMVs showed reduced mitochondrial respiratory and glycolytic parameters compared to male BMVs although relative contribution of various substrates for energy production were not different. These sex-dependent differences involving glycolysis and OxPhos make the microvasculature vulnerable to injury in females. Funding sources: NIH grants AG074489 and NS114286 (PVGK and RM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Restricting bioenergetic efficiency enhances longevity and mitochondrial redox capacity in Drosophila melanogaster.

Mitochondria are essential for survival and as such, impairments in organelle homeostasis significantly accelerate age-related morbidity and mortality. Here, we determined the contribution of bioenergetic efficiency to life span and health span in Drosophila melanogaster utilizing the mitochondrial uncoupler BAM15. Life span was determined in flies fed a normal diet (ND) or high fat diet (HFD) supplemented with vehicle or BAM15. Locomotor function was determined by negative geotaxis assay in middle-aged flies fed vehicle or BAM15 under ND or HFD conditions. Redox capacity (high-resolution respirometry/fluorometry), citrate synthase (enzyme activity), mtDNA content (qPCR), gene expression (qPCR), and protein expression (western blot) were assessed in flight muscle homogenates of middle-aged flies fed vehicle or BAM15 ND. The molar ratio of H2O2 and O2 (H2O2:O2) in a defined respiratory state was calculated as a measure of redox balance. BAM15 extended life span by 9% on ND and 25% on HFD and improved locomotor activity by 125% on ND and 53% on HFD. Additionally, BAM15 enhanced oxidative phosphorylation capacity supported by pyruvate + malate, proline, and glycerol 3-phosphate. Concurrently, BAM15 enhanced the mitochondrial H2O2 production rate, reverse electron flow from mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) to Complex I, mGPDH, and Complex I without altering the H2O2:O2 ratio. BAM15 upregulated transcriptional signatures associated with mitochondrial function and fitness as well as antioxidant defense. BAM15-mediated restriction of bioenergetic efficiency prolongs life span and health span in Drosophila fed a ND or HFD. Improvements in life span and health span in ND were supported by synergistic enhancement of muscular redox capacity.

Does chronic exposure to moderate hypoxia affect growth, physiological and oxidative stress biomarkers in adult spotted wolffish (Anarhichas minor) and the hybrid A. minor × A. lupus?

AbstractBoth in their natural habitat (estuaries, marine coastal environment, ocean depths and freshwater lakes) or in captivity conditions (land‐based, open‐sea aquaculture and aquariums), exposure to hypoxia can have direct detrimental effects on fish growth, reproduction, behaviour and health. The objective of this study was to investigate the effects of chronic exposure of spotted wolffish (Anarhichas minor) (∼1.4 kg) and the interspecific hybrid (A. minor × A. lupus) (∼600 g) to moderate hypoxia levels (dissolved oxygen [DO]: 40%, 50% and 60% saturation) in comparison to a normoxia group (control 100%) over a period of 14 weeks at ∼7.5°C. The trials were conducted as a common‐garden experiment (both fish groups reared together) in quadruplicate (4 tanks per DO level = 16 tanks). Fish performance (specific growth rate, feed intake [FI]) and physiological status (condition factor [K] and hepatosomatic index, haematocrit) were monitored at different intervals. The metabolic enzyme activity of citrate synthase (CS), lactate dehydrogenase (LDH) and pyruvate kinase (PK) was measured as well as antioxidant enzymes glutathione reductase and catalase. Oxidative stress indices were assessed by the quantification of thiobarbituric acid reactive substances (TBARS) and the measurement of the enzymatic activity level of aconitase. Significant reduction in growth rate occurred at DO 40%. DO was identified as a major driver of FI. Our results indicate a very strong adaptation to hypoxia as (1) exposures to DO 40% were not severe enough to affect aerobic or anaerobic metabolism capacity or to significantly induce oxidative stress and (2) metabolic organization of the muscle tissue, as expressed by LDH, CS and PK is not affected by DO levels down to 40%. However, at the lowest DO levels (40% and 50%), hybrid fish displayed significantly better growth than the spotted wolffish.

Differential effects of Western diet and traumatic muscle injury on skeletal muscle metabolic regulation in male and female mice.

This study was designed to develop an understanding of the pathophysiology of traumatic muscle injury in the context of Western diet (WD; high fat and high sugar) and obesity. The objective was to interrogate the combination of WD and injury on skeletal muscle mass and contractile and metabolic function. Male and female C57BL/6J mice were randomized into four groups based on a two-factor study design: (1) injury (uninjured vs. volumetric muscle loss [VML]) and (2) diet (WD vs. normal chow [NC]). Electrophysiology was used to test muscle strength and metabolic function in cohorts of uninjured+NC, uninjured+WD, VML+NC and VML+WD at 8weeks of intervention. VML-injured male and female mice both exhibited decrements in muscle mass (-17%, P<0.001) and muscle strength (-28%, P<0.001); however, VML+WD females had a 28% greater muscle mass compared to VML+NC females (P=0.034), a compensatory response not detected in males. VML-injured male and female mice both had lower carbohydrate- and fat-supported muscle mitochondrial respiration (JO2 ) and less electron conductance through the electron transport system (ETS); however, male VML-WD had 48% lower carbohydrate-supported JO2 (P=0.014) and 47% less carbohydrate-supported electron conductance (P=0.026) compared to male VML+NC, and this diet-injury phenotype was not present in females. ETS electron conductance starts with complex I and complex II dehydrogenase enzymes at the inner mitochondrial membrane, and male VML+WD had 31% less complex I activity (P=0.004) and 43% less complex II activity (P=0.005) compared to male VML+NC. This was a diet-injury phenotype not present in females. Pyruvate dehydrogenase (PDH), β-hydroxyacyl-CoA dehydrogenase, citrate synthase, α-ketoglutarate dehydrogenase and malate dehydrogenase metabolic enzyme activities were evaluated as potential drivers of impaired JO2 in the context of diet and injury. There were notable male and female differential effects in the enzyme activity and post-translational regulation of PDH. PDH enzyme activity was 24% less in VML-injured males, independent of diet (P<0.001), but PDH enzyme activity was not influenced by injury in females. PDH enzyme activity is inhibited by phosphorylation at serine-293 by PDH kinase 4 (PDK4). In males, there was greater total PDH, phospho-PDHser293 and phospho-PDH-to-total PDH ratio in WD mice compared to NC, independent of injury (P≤0.041). In females, PDK4 was 51% greater in WD compared to NC, independent of injury (P=0.025), and was complemented by greater phospho-PDHser293 (P=0.001). Males are more susceptible to muscle metabolic dysfunction in the context of combined WD and traumatic injury compared to females, and this may be due to impaired metabolic enzyme functions.

#4668 CITRATE SYNTHASE LACTYLATION PROMOTES THE TRANSITION FROM ACUTE KIDNEY INJURY TO CHRONIC KIDNEY DISEASE BY ACTIVATING NLRP3 INFLAMMASOME

Abstract Background and Aims Renal interstitial inflammation has been postulated to play a critical role in the acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Recent studies have shown that protein lactylation caused by lactate accumulation can regulate chronic organ injury. The purpose of this study was to investigated the role and mechanism of protein lactlylation in AKI-CKD transition. Method Severe renal ischemia-reperfusion(I/R) injury (severe AKI) was constructed by bilateral renal ischemia for 35min. The lactylation enhancer rotenone and the lactylation inhibitor oxamate were used to verify the effect of protein lactylation. Lactylated proteomics was used to detect the changes of lactylated proteins in renal cortex at different time points (day 0, 3, and 7), and the lactylated proteins related to kidney injury were screened for verification. Results The pathological staining results showed that the severe AKI mice were most severely injuried at day 3, partially recovered at day 7, and developed renal interstitial fibrosis at day 28. The Serum creatinine (SCr) levels did not return to baseline at day 28 after I/R injury. The levels of lactate and protein lactylation modification detected by Elisa and western blot analysis showed the same tendency as the above pathological injury, indicating that protein lactylation might contribute to the chronic transition of severe AKI. The renal injury score was significantly increased at day 3 and the percentage of renal interstitial fibrosis was significantly increased at day 28 after treatment with protein lactylation enhancer. IL-1b and IL-18 were found to be the most significantly enhanced inflammatory cytokines in the blood of mice, and the NLRP3 inflammasome activation was significantly enhanced in renal tissue at 3 days. The in vitro results further confirmed these findings. We found that protein lactylation might activate the NLRP3 inflammasome, therefore leading to AKI-CKD transition. Meanwhile, lactylation inhibitor had a protective effect on AKI-CKD transition. Lactylated proteins were detected by lactylated proteomics analysis at day 0, 3 and 7 after I/R, and the enrichment of differential lactylated proteins was analyzed. The results showed that the differential proteins with significant difference mainly existed in the tricarboxylic acid cycle. One of them is citrate synthase (CS), a key rate-limiting enzyme in the tricarboxylic acid cycle. Citrate synthase lactylation level increased significantly on day 3 after AKI. Among the three lactylation sites of CS, the further screening revealed that CS-K370 is the most significant. We constructed mutations of CS-K370 lactylation sites to verify its effects on the enzyme activity and function. Results showed in hypoxia/reoxygenation(H/R) model for mice renal tubular epithelial cells (mRTECs), compared with control group, the enzyme activity of CS in K370T group (modified status) decreased significantly, while in K370R group (unmodified status) did not decrease significantly. Moreover, the lactylation modification of CS-K370 can lead to the activation of NLRP3 inflammasome. The above results indicated that the lactylation of CS-K370 site inhibited the enzyme activity of CS and activated NLRP3 inflammasome. Conclusion Renal protein lactylation promotes activation of NLRP3 inflammasome, leading to AKI-CKD transition. Citrate synthase may be the key lactylated protein.

Hypoxia acclimation improves mitochondrial efficiency in the aerobic swimming muscle of red drum (Sciaenops ocellatus)

Environmental hypoxia (low dissolved oxygen) is a significant threat facing fishes. As fishes require oxygen to efficiently produce ATP, hypoxia can significantly limit aerobic capacity. However, some fishes show respiratory flexibility that rescues aerobic performance, including plasticity in mitochondrial performance. This plasticity may result in increased mitochondrial efficiency (e.g., less proton leak), increased oxygen storage capacity (increased myoglobin), and oxidative capacity (e.g., higher citrate synthase activity) under hypoxia. We acclimated a hypoxia-tolerant fish, red drum (Sciaenops ocellatus), to 8-days of constant hypoxia to induce a hypoxic phenotype. Fish were terminally sampled for cardiac and red muscle tissue to quantify oxidative phosphorylation, proton leak, and maximum respiration in tissue from both hypoxia-acclimated and control fish. Tissue was also collected to assess the plasticity of citrate synthase enzyme activity and mRNA expression for select oxygen storage and antioxidant pathway transcripts. We found that mitochondrial respiration rates were not affected by hypoxia exposure in cardiac tissue, though citrate synthase activity and myoglobin expression were higher following hypoxia acclimation. Interestingly, measures of mitochondrial efficiency in red muscle significantly improved in hypoxia-acclimated individuals. Hypoxia-acclimated fish had significantly higher OXPHOS Control Efficiency, OXPHOS Capacity and Coupling Control Ratios (i.e., LEAK/OXPHOS). There was no significant change to citrate synthase activity or myoglobin expression in red muscle. Overall, these results suggest that red muscle mitochondria of hypoxia-acclimated fish more efficiently utilize oxygen, which may explain previous reports in red drum of improved aerobic swimming performance in the absence of improved maximum metabolic rate following hypoxia acclimation.

The cardioprotective effect of human glucagon-like peptide-1 receptor agonist (semaglutide) on cisplatin-induced cardiotoxicity in rats: Targeting mitochondrial functions, dynamics, biogenesis, and redox status pathways.

The cardiotoxic effect of chemotherapeutic agents as cisplatin has become a major issue recently. Interference with mitochondrial dynamics, biogenesis, redox status, and apoptosis are the most possible underlying mechanisms. Semaglutide is a human glucagon-like peptide-1 receptor agonist (GLP-1R), which is used primarily for the treatment of DM. Various recent studies have investigated (GLP-1R) role in cardiovascular diseases due to antiapoptotic and antioxidant effects. The current study aimed to investigate the curative role of semaglutide's against cisplatin- induced cardiotoxicity and its relation to mitochondrial functions, dynamics, biogenesis, apoptosis, and redox status pathways. The study included 30 male rats divided into three groups: control, cisplatin-induced cardiotoxicity, and cisplatin-induced cardiotoxicity treated with semaglutide. At the end of the experiment heart index, serum cardiotoxicity markers, SOD, GPX activities and H2 O2 level were estimated. Mitochondrial transmembrane potential, complex I and citrate synthase enzyme activities, ATP level, Mfn2 in addition to PGC-1 α levels were assessed as biogenesis markers. Mitophagy markers PINK1 and Parkin mRNA gene expression were estimated. Histopathological examination of cardiac muscles of all studied groups and immunoassay of P53 and caspase 3 in cardiac tissue were examined to assess apoptosis. Cisplatin has disturbed mitochondrial function and dynamics, dysregulate redox status and induced mitophagy and apoptosis, in the other hand semaglutide treatment has normalized dysregulated mitochondrial function and dynamics, redox status and suppressed mitophagy and apoptosis. Semaglutide has ameliorative effect against cisplatin- induced cardiotoxicity via modulation of mitochondrial functions, dynamics, biogenesis, apoptosis, and redox status pathways.

Acute resistance exercise training does not augment mitochondrial remodelling in master athletes or untrained older adults.

Background: Ageing is associated with alterations to skeletal muscle oxidative metabolism that may be influenced by physical activity status, although the mechanisms underlying these changes have not been unraveled. Similarly, the effect of resistance exercise training (RET) on skeletal muscle mitochondrial regulation is unclear. Methods: Seven endurance-trained masters athletes ([MA], 74 ± 3years) and seven untrained older adults ([OC]. 69 ± 6years) completed a single session of knee extension RET (6 x 12 repetitions, 75% 1-RM, 120-s intra-set recovery). Vastus lateralis muscle biopsies were collected pre-RET, 1h post-RET, and 48h post-RET. Skeletal muscle biopsies were analyzed for citrate synthase (CS) enzyme activity, mitochondrial content, and markers of mitochondrial quality control via immunoblotting. Results: Pre-RET CS activity and protein content were ∼45% (p < .001) and ∼74% greater in MA compared with OC (p = .006). There was a significant reduction (∼18%) in CS activity 48h post-RET (p < .05) in OC, but not MA. Pre-RET abundance of individual and combined mitochondrial electron transport chain (ETC) complexes I-V were significantly greater in MA compared with OC, as were markers of mitochondrial fission and fusion dynamics (p-DRP-1Ser616, p-MFFSer146, OPA-1 & FIS-1, p < .05 for all). Moreover, MA displayed greater expression of p-AMPKThr172, PGC1α, TFAM, and SIRT-3 (p < .05 for all). Notably, RET did not alter the expression of any marker of mitochondrial content, biogenesis, or quality control in both OC and MA. Conclusion: The present data suggest that long-term aerobic exercise training supports superior skeletal muscle mitochondrial density and protein content into later life, which may be regulated by greater mitochondrial quality control mechanisms and supported via superior fission-fusion dynamics. However, a single session of RET is unable to induce mitochondrial remodelling in the acute (1h post-RET) and delayed (48h post-RET) recovery period in OC and MA.

Short communication: Acute hypoxia does not alter mitochondrial abundance in naked mole-rats

Hypoxia poses a significant energetic challenge and most species exhibit metabolic remodelling when exposed to prolonged hypoxia. One component of this remodelling is mitochondrial biogenesis/mitophagy, which alter mitochondrial abundance and helps to adjust metabolic throughput to match changes in energy demands in hypoxia. However, how acute hypoxia impacts mitochondrial abundance in hypoxia-tolerant species is poorly understood. To help address this gap, we exposed hypoxia-tolerant naked mole-rats to 3 h of normoxia or acute hypoxia (5% O2) and measured changes in mitochondrial abundance using two well-established markers: citrate synthase (CS) enzyme activity and mitochondrial DNA (mtDNA) abundance. We found that neither marker changed with hypoxia in brain, liver, or kidney, suggesting that mitochondrial biogenesis is not initiated during acute hypoxia in these tissues. Conversely in skeletal muscle, the ratio of CS activity to total protein decreased 50% with hypoxia. However, this change was likely driven by an increase in soluble protein density in hypoxia because CS activity was unchanged relative to wet tissue weight and the mtDNA copy number was unchanged. To confirm this, we examined skeletal muscle mitochondria using transmission electron microscopy and found no change in mitochondrial volume density. Taken together with previous studies of mitochondrial respiratory function, our present findings suggest that naked mole-rats primarily rely on tissue-specific functional remodelling of metabolic pathways and mitochondrial respiratory throughput, and not physical changes in mitochondrial number or volume, to adjust to short-term hypoxic exposure.

Effects of Broad-Spectrum Antibiotic Treatment or Germ-Free Status on Endurance Performance and Exercise Adaptations in Mice.

Endurance exercise alters the gut microbiome independently of diet. The extent to which gut microbes are responsible for physiologic adaptations to exercise training is unknown. The purpose of these experiments was to determine the role of gut microbes in performance and muscle adaptation to 6 wk of voluntary wheel running (VWR) in mice. We depleted microbes with broad-spectrum antibiotic (ABX) treatment and used germ-free (GF) mice to determine effects on adaptations to VWR. Male and female C57Bl/6 mice ( n = 56) were assigned to daily VWR or sedentary conditions. After the intervention, treadmill endurance and glucose tolerance were assessed, and gastrocnemius and soleus tissues were harvested and analyzed for citrate synthase (CS) enzyme activity and expression of exercise training-sensitive genes. ABX treatment and GF status resulted in VWR volumes ~22% and 26% lower than controls, respectively. Analysis of variance revealed that, although VWR increased treadmill endurance, ABX had no effect. GF status significantly reduced treadmill performance in trained GF mice after training. VWR increased gastrocnemius CS enzyme activity in all groups, and ABX and GF status did not reduce the VWR effect. VWR also increased muscle expression of PGC1a, but this was not affected by ABX treatment. ABX treatment and GF status reduced VWR behavior but did not affect VWR-induced adaptations in endurance capacity, CS activity, or expression of muscle metabolic genes. However, GF status reduced endurance capacity. These data indicated that reducing microbes in adulthood does not inhibit endurance training adaptations in C57Bl/6 mice, but that GF mice possess a reduced responsiveness to endurance exercise training, perhaps because of a developmental defect associated with lack of microbes from birth.

Kombucha tea improves glucose tolerance and reduces hepatic steatosis in obese mice

Nonalcoholic fatty liver disease (NAFLD), often associated with obesity, is becoming one of the most common liver diseases worldwide. It is estimated to affect one billion individuals and may be present in approximately 25% of the population globally. NAFLD is viewed as a hepatic manifestation of metabolic syndrome, with humans and animal models presenting dyslipidemia, hypertension, and diabetes. The gut-liver axis has been considered the main pathogenesis branch for NAFLD development. Considering that foods or beverages could modulate the gastrointestinal tract, immune system, energy homeostasis regulation, and even the gut-liver axis, we conducted an exploratory study to analyze the effects of kombucha probiotic on hepatic steatosis, glucose tolerance, and hepatic enzymes involved in carbohydrate and fat metabolism using a pre-clinical model. The diet-induced obese mice presented glucose intolerance, hyperinsulinemia, hepatic steatosis, increased collagen fiber deposition in liver vascular spaces, and upregulated TNF-alpha and SREBP-1 gene expression. Mice receiving the kombucha supplement displayed improved glucose tolerance, reduced hyperinsulinemia, decreased citrate synthase and phosphofructokinase-1 enzyme activities, downregulated G-protein-coupled bile acid receptor, also known as TGR5, and farnesol X receptor gene expression, and attenuated steatosis and hepatic collagen fiber deposition. The improvement in glucose tolerance was accompanied by the recovery of acute insulin-induced liver AKT serine phosphorylation. Thus, it is possible to conclude that this probiotic drink has a beneficial effect in reducing the metabolic alterations associated with diet-induced obesity. This probiotic beverage deserves an extension of studies to confirm or refute its potentially beneficial effects.

Effects Of Broad-spectrum Antibiotic Administration And Germ-free Status On Biochemical And Endurance Adaptations To Regular Exercise In Mouse Muscle Tissue

PURPOSE: The gut microbiota modulates a variety of physiologic processes. Recent studies from our group have shown that regular endurance exercise alters the composition of the gut microbiome and metabolome in mice and humans, and we hypothesized that the presence of microbes would affect endurance training adaptations in muscular endurance capacity, mitochondrial activity and gene expression in mice. METHODS: To discern differences in adaptations from 6 wks of voluntary wheel running (VWR), we depleted microbes with antibiotics (ABX) and used germ free (GF) mice to compare to control (CON). Male and female C57Bl/6 mice of all groups underwent daily VWR or sedentary (SED) conditions in a 2 x 3 design (VWR/SED, CON/ABX/GF, n = 56). After the intervention, treadmill endurance was assessed and gastrocnemius and soleus tissue were harvested and analyzed for activity of key Krebs cycle enzyme Citrate Synthase (CS) and expression of genes indicating mitochondrial adaptations respectively. RESULTS: Two way ANOVA revealed that VWR increased treadmill endurance, ABX had no effect, and that GF status significantly reduced performance. Additionally, VWR increased Citrate Synthase enzyme activity in gastrocnemius muscle tissue in all groups, and ABX and GF status did not reduce VWR’s effect or have an effect of their own. VWR also tended to increase expression of genes associated with increased mitochondrial activity (PGC-1α, CS, Succinate Dehydrogenase) in soleus tissue, but ABX had no effect on these observations. CONCLUSIONS: We conclude that ABX treatment and GF status do not affect VWR induced adaptations in endurance capacity, gastrocnemius mitochondrial activity or expression of metabolic genes in soleus tissue, but that GF status significantly hinders endurance capacity. This indicates that removing gut microbes does not inhibit muscular endurance training adaptations, but that germ free mice possess hindered endurance exercise capacity.

Peripheral blood mononuclear cell mitochondrial enzyme activity is associated with parity and lactation performance in early lactation Holstein dairy cows

Mitochondria are central to metabolism and are the primary energy producers for all biosynthesis, including lactation. The objectives of this study were to determine if high- and low-producing dairy cows exhibit differences in peripheral blood mononuclear cell mitochondrial enzyme activities of citrate synthase, complex I, complex IV, and complex V during early lactation and, thus, to determine whether those differences were related to differences in lactation performance in the dairy cow. Fifty-six Holstein cows were assigned to 1 of 4 groups: (1) primiparous high, (2) primiparous low, (3) multiparous high, or (4) multiparous low. Primiparous and multiparous cows were analyzed separately. Then, cows were divided into high or low production groups for each production parameter [peak milk, average milk, energy-corrected milk (ECM), fat-corrected milk (FCM), milk lactose, milk fat, milk protein, total solids (TS), solids-not-fat, feed efficiency, and somatic cell count (SCC)]. For all data analysis, production parameters are expressed as yields (kg/d) and SCC (103 cells/mL). High and low production groups were defined by their respective mean production parameters for the 56 cows, with below average cows defined as low and above average cows defined as high. Whole blood samples were collected at one time point, approximately 70 d in milk at 0800 h, and processed for crude mitochondrial extracts from peripheral blood mononuclear cells to determine the activity rates of mitochondrial enzymes. Milk samples were collected 9 times (3 d, 3 times per d) during the week of blood collection and analyzed for major components (fat, protein, lactose, TS, and SCC). Multiparous cows had lower citrate synthase activity than primiparous cows across all production parameters. High-producing cows had greater complex I activity for peak milk, milk yield, ECM, FCM, milk fat, TS, and feed efficiency, and greater complex V activity for ECM, FCM, milk lactose, milk fat, and TS across parities. These findings imply that the most influential respiratory chain enzymes on the level of milk production are those responsible for electron transport chain initialization and ATP production.

No effect of five days of bed rest or short-term resistance exercise prehabilitation on markers of skeletal muscle mitochondrial content and dynamics in older adults.

Bed rest (BR) results in significant impairments in skeletal muscle metabolism. Mitochondrial metabolism is reportedly highly sensitive to disuse, with dysregulated fission‐fusion events and impaired oxidative function previously reported. The effects of clinically relevant short‐term BR (≤5 days) on mitochondrial protein expression are presently unclear, as are the effects of exercise prehabilitation as a potential counteractive intervention. The present study examined the effects of a 5‐day period of BR and short‐term resistance exercise prehabilitation (ST‐REP) on mitochondrial‐protein content. Ten older men (71 ± 4 years) underwent 5 days of BR, completing four sessions of high‐volume unilateral resistance exercise prehabilitation over 7 days beforehand. Muscle biopsies were obtained from the vastus lateralis in the non‐exercised control and exercised legs, both pre‐ and post‐prehabilitation and pre‐ and post‐BR, to determine changes in citrate synthase enzyme activity and the expression of key proteins in the mitochondrial electron transport chain and molecular regulators of fission‐fusion dynamics, biosynthesis, and mitophagy. We observed no significant effect of either BR or ST‐REP on citrate synthase protein content, enzyme activity, or ETC complex I‐V protein content. Moreover, we observed no significant changes in markers of mitochondrial fission and fusion (p‐DRP1S616, p‐DRP1S637, p‐DRP1S616/S637 ratio, p‐MFFS146, Mitofillin, OPA1, or MFN2 (p > 0.05 for all). Finally, we observed no differences in markers of biosynthesis (p‐AMPKT172, p‐ACCS79, PGC1a, TFAM) or mitophagy‐related signaling (ULK‐1, BNIP3/NIX, LC3B I/II) (p > 0.05 for all). In contrast to previous longer‐term periods of musculoskeletal disuse (i.e., 7–14 days), a clinically relevant, 5‐day period of BR resulted in no significant perturbation in muscle mitochondrial protein signaling in healthy older adults, with no effect of ST‐REP in the week prior to BR. Accordingly, disuse‐induced muscle atrophy may precede alterations in mitochondrial content.

Multi-Omics Analysis of Low-Temperature Fruiting Highlights the Promising Cultivation Application of the Nutrients Accumulation in Hypsizygus marmoreus.

Hypsizygus marmoreus is a representative edible mushroom with low-temperature fruiting after a long postripening (LFLP). Clarifying the mechanism of LFLP and applying a rigorous low-temperature-limited process will optimize the mushroom cultivation process. This study performed an integrative multi-omics analysis of the molecular mechanism of LFLP in combination with genetic, physiological, and cultivation confirmation. The results showed that the amino acid content was increased during LFLP, mainly arginine. pH analysis showed acidification in the postripening stage and alkalization in the substrates of the reproductive growth stage. An enzyme activity test confirmed the increased enzyme activity of arginase and citrate synthase in the postripening stage. Weighted gene coexpression network analysis of the transcriptome and metabolomics indicated that pH variation is correlated mainly with changes in citrate and arginine. Multi-omics reveals a straightforward way of providing enriched materials for amino acid biosynthesis, namely, synergistically elevating citric acid and arginine through enhanced activity of the arginine synthesis branch pathway in the citrate cycle. Our study confirmed that GCN2 mediated metabolic adaptation by enhancing protein translation, highlighting its regulatory role during LFLP. Exogenously added citric acid and arginine shortened the postripening period by 10 days and increased the fruiting body yield by 10.2~15.5%. This research sheds light on the molecular mechanism of LFLP in H. marmoreus and highlights the promising application of nutrient accumulation in high-efficiency cultivation.

Abstract 3779: Patient-derived iPSCs reveal pharmacologic targeting mitochondrial respiratory complex I for treating Rothmund-Thomson syndrome associated osteosarcoma

Abstract Background: Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by a rash that progresses to poikiloderma; small stature; skeletal anomalies; sparse hair, eyelashes, and/or eyebrows, juvenile cataracts, and an increased risk to cancer. Type 2 RTS patients with biallelic RECQL4 pathogenic variants have a significantly increased incidence of osteosarcoma. However, there is currently no available RTS model that recapitulates the bone malignancy phenotype in this disease, severely limiting the ability of researchers to explore new treatment avenues. Here, we generated RTS patient-derived induced pluripotent stem cells (iPSCs) to dissect the pathological signaling leading to RTS patient-associated osteosarcoma. Methods: iPSCs were reprogrammed from fibroblasts of four individuals from two RTS families, each with one affected RTS proband with RECQL4 mutations and one unaffected parent. These patient iPSCs were differentiated to mesenchymal stem cells (MSCs) and then to osteoblasts, the cell-of-origin of osteosarcoma. All of these MSCs and osteoblasts were subjected to transcriptome analysis by RNA-seq. Based on enriched mitochondrial respiratory gene signatures, we also systematically analyzed the enzymatic activities of the major mitochondrial respiratory protein complexes including complex I/II/III and citrate synthase enzyme activity in RTS osteoblasts by Seahorse assay. Finally, we tested the potential therapeutic effect of clinical oxidative phosphorylation complex I inhibitor IACS-010759 to treat RTS cells and dissect the pharmacological mechanisms involved. Results: RTS iPSC-derived osteoblasts showed defective osteogenic differentiation and a gain of in vitro tumorigenic ability. Transcriptome analysis of RTS osteoblasts revealed decreased bone morphogenesis gene expression but aberrantly upregulated mitochondrial complex I gene expression. Metabolic assays of RTS osteoblasts demonstrated elevated mitochondrial respiratory complex I function, increased oxidative phosphorylation, but decreased ATP production. Inhibition of mitochondrial respiratory complex I activity by IACS-010759 selectively suppressed cellular respiration and cell viability of RTS osteoblasts. Furthermore, systems analysis of IACS-010759-induced changes to RTS osteoblasts suggested that inhibition of mitochondrial complex I in RTS osteoblasts leads to enhanced cell senescence, decreased MAPK signaling and cell cycle associated genes. Conclusion: In summary, we established an RTS iPSC disease platform to dissect the pathological mechanisms involved in RTS-associated osteosarcoma. Our data suggested that mitochondrial complex I is a potential therapeutic target for RTS-associated osteosarcoma and provides future insights for clinical treatment strategies. Citation Format: An Xu, Brittany E. Jewell, Dandan Zhu, Mo-Fan Huang, Yi-Hung Chen, Linchao Lu, Ruiying Zhao, Lisa L. Wang, Dung-Fang Lee. Patient-derived iPSCs reveal pharmacologic targeting mitochondrial respiratory complex I for treating Rothmund-Thomson syndrome associated osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3779.

No Differences Between 12 Weeks of Block- vs. Traditional-Periodized Training in Performance Adaptations in Trained Cyclists

The purpose of this study was to compare the effects of 12 weeks load-matched block periodization (BP, n = 14), using weekly concentration of high- (HIT), moderate- (MIT), and low- (LIT) intensity training, with traditional periodization (TP, n = 16) using a weekly, cyclic progressive increase in training load of HIT-, MIT-, and LIT-sessions in trained cyclists (peak oxygen uptake: 58 ± 8 ml·kg−1·min−1). Red blood cell volume increased 10 ± 16% (p = 0.029) more in BP compared to TP, while capillaries around type I fibers increased 20 ± 12% (p = 0.002) more in TP compared to BP from Pre to Post12. No other group differences were found in time-trial (TT) performances or muscular-, or hematological adaptations. However, both groups improved 5 and 40-min TT power by 9 ± 9% (p < 0.001) and 8 ± 9% (p < 0.001), maximal aerobic power (Wmax) and power output (PO) at 4 mmol·L−1 blood lactate (W4mmol), by 6 ± 7 (p = 0.001) and 10 ± 12% (p = 0.001), and gross efficiency (GE) in a semi-fatigued state by 0.5 ± 1.1%-points (p = 0.026). In contrast, GE in fresh state and VO2peak were unaltered in both groups. The muscle protein content of β-hydroxyacyl (HAD) increased by 55 ± 58% in TP only, while both TP and BP increased the content of cytochrome c oxidase subunit IV (COXIV) by 72 ± 34%. Muscle enzyme activities of citrate synthase (CS) and phosphofructokinase (PFK) were unaltered. TP increased capillary-to-fiber ratio and capillary around fiber (CAF) type I by 36 ± 15% (p < 0.001) and 17 ± 8% (p = 0.025), respectively, while BP increased capillary density (CD) by 28 ± 24% (p = 0.048) from Pre to Post12. The present study shows no difference in performance between BP and “best practice”-TP of endurance training intensities using a cyclic, progressively increasing training load in trained cyclists. However, hematological and muscle capillary adaptations may differ.

Cow–calf efficiency of beef cows grazing different herbage allowances of rangelands: hepatic mechanisms related to energy efficiency

Context Improvement in efficiency of energy utilisation of cow–calf systems could benefit beef production profitability and sustainability. Energy efficiency has been associated with mitochondrial function; therefore, hepatic mitochondrial function and oxidative stress could play a key role in energy efficiency of beef cows. Aim We evaluated the effect of two herbage allowances from rangelands (Campos biome) on cow–calf energy efficiency, hepatic mitochondrial density and function, and oxidative stress of purebred (Hereford and Aberdeen Angus) and reciprocal F1 crossbred beef cows. Methods Mature cows (n = 32) were used in a complete randomised block design with a factorial arrangement of herbage allowance (2.5 vs 4 kg dry matter/day; low vs high) and cow genotype (pure vs crossbred) over 3 years. At the end of the third year, cows were slaughtered at 190 ± 10 days postpartum. Liver was dissected and weighed, and samples were collected and snap-frozen pending analysis of mitochondrial density and oxidative stress markers. Estimated cow–calf energy efficiency was calculated by using total cow estimated metabolisable energy intake as input and calf energy retained at weaning as output. Key results Cow–calf energy efficiency was greater (P ≤ 0.07) for high than low herbage allowance and for crossbred than purebred cows. Mitochondrial density biomarkers (hepatic citrate synthase enzyme activity, citrate synthase mRNA, and mitochondrial:nuclear DNA ratio) were greater (P ≤ 0.03) for high than low herbage allowance. Plasma pro-oxidants and plasma antioxidant capacity were greater (P ≤ 0.07) for crossbred than purebred cows. Plasma oxidative stress index and expression of hepatic 4-hydroxynonenal protein adducts were affected (P ≤ 0.06) by herbage allowance × cow genotype interaction. Conclusion Greater cow–calf energy efficiency was associated with greater hepatic mitochondrial density without differences in mitochondrial function. Contrary to expectation, greater efficiency of crossbred than purebred cows was associated with increased hepatic oxidative damage, which probably reflects greater liver metabolic activity in crossbreds. Implications Herbage allowance and cow genotype affect cow–calf efficiency, hepatic mitochondrial function and oxidative stress markers. Greater efficiency of crossbred cows seems associated with increased hepatic oxidative damage.