Increased ATP Production Research Articles

Ca2+ pushes and pulls energetics to maintain ATP balance in atrial cells: computational insights.

To maintain atrial function, ATP supply-to-demand matching must be tightly controlled. Ca2+ can modulate both energy consumption and production. In light of evidence suggesting that Ca2+ affects energetics through "push" (activating metabolite flux and enzymes in the Krebs cycle to push the redox flux) and "pull" (acting directly on ATP synthase and driving the redox flux through the electron transport chain and increasing ATP production) pathways, we investigated whether both pathways are necessary to maintain atrial ATP supply-to-demand matching. Rabbit right atrial cells were electrically stimulated at different rates, and oxygen consumption and flavoprotein fluorescence were measured. To gain mechanistic insight into the regulators of ATP supply-to-demand matching in atrial cells, models of atrial electrophysiology, Ca2+ cycling and force were integrated with a model of mitochondrial Ca2+ and a modified model of mitochondrial energy metabolism. The experimental results showed that oxygen consumption increased in response to increases in the electrical stimulation rate. The model reproduced these findings and predicted that the increase in oxygen consumption is associated with metabolic homeostasis. The model predicted that Ca2+ must act both in "push" and "pull" pathways to increase oxygen consumption. In contrast to ventricular trabeculae, no rapid time-dependent changes in mitochondrial flavoprotein fluorescence were measured upon an abrupt change in workload. The model reproduced these findings and predicted that the maintenance of metabolic homeostasis is due to the effects of Ca2+ on ATP production. Taken together, this work provides evidence of Ca2+ "push" and "pull" activity to maintain metabolic homeostasis in atrial cells.

Inversion of a paradigm: The positive roles of plant phenolics in dairy goat nutrition

The paradigm prevailing in the nineties, based on studies with goats fed tannin-rich browse as sole feed, was that dietary tannins are associated with impaired feed intake, increased fecal loss of N, and impaired NDF digestibility, hence, impaired productivity, with much research aiming at alleviating these negative effects. However, dairy goats fed plant phenolic-rich diets, and supplemented with concentrates will voluntarily ingest diets comprising more than 4% of tannins, without impairment of total feed intake and milk yield. Also, supplementing them with Poly (ethylene-glycol) MW 4000 is not needed. All plant phenolics, including condensed tannins, are partly digested, inferring that phenolic derivatives are absorbed and may reach tissues. In some studies, diets rich in plant phenolics are associated with the production of milk richer in protein and fat, reduced urea content, and improved curdling performance. Feeding goats with plant phenolics-rich diets is generally associated with inhibited bio-hydrogenation (BH) of fatty acids (FA) in the rumen, resulting in decreased saturated FA, increased polyunsaturated FA (PUFA) and, in particular C18:3–n3 but also a modified composition of de novo synthesized FA in goat milk that suggest direct action of phenolics on the mammary gland. Knowledge of the absorption of plant phenolics is still scarce for goats but even modest daily intakes seem to be associated with increased phenolic derivatives and anti-oxidative activity in plasma and milk. Dietary phenolic compounds may induce a local effect in the mammary gland and its production units (the mammary epithelium cells) encompassing increased cellular triglyceride and casein contents, and increased ATP production and non-mitochondrial oxygen consumption. Plant phenolics and their derivatives divert energy to production of milk fat, protein and lactose, with less energy directed to cellular damage control. Dietary plant phenolics are associated with improved goat health and production of healthier milk. In addition, dietary plant phenolics have anti-parasitic properties and are environment-friendly as they decrease gut methane emission and they increase fecal N at the expense of rapidly decomposable urinary N. Therefore, the attitude of scientists towards feeding plant phenolics has evoluted from mostly negative to mostly positive.

5-Aminolevulinic acid bypasses mitochondrial complex I deficiency and corrects physiological dysfunctions in Drosophila.

Complex I (CI) deficiency in mitochondrial oxidative phosphorylation (OXPHOS) is the most common cause of mitochondrial diseases, and limited evidence-based treatment options exist. Although CI provides the most electrons to OXPHOS, complex II (CII) is another entry point of electrons. Enhancement of this pathway may compensate for a loss of CI; however, the effects of boosting CII activity on CI deficiency are unclear at the animal level. 5-Aminolevulinic acid (5-ALA) is a crucial precursor of heme, which is essential for CII, complex III, complex IV (CIV) and cytochrome c activities. Here, we show that feeding a combination of 5-ALA hydrochloride and sodium ferrous citrate (5-ALA-HCl + SFC) increases ATP production and suppresses defective phenotypes in Drosophila with CI deficiency. Knockdown of sicily, a Drosophila homolog of the critical CI assembly protein NDUFAF6, caused CI deficiency, accumulation of lactate and pyruvate and detrimental phenotypes such as abnormal neuromuscular junction development, locomotor dysfunctions and premature death. 5-ALA-HCl + SFC feeding increased ATP levels without recovery of CI activity. The activities of CII and CIV were upregulated, and accumulation of lactate and pyruvate was suppressed. 5-ALA-HCl + SFC feeding improved neuromuscular junction development and locomotor functions in sicily-knockdown flies. These results suggest that 5-ALA-HCl + SFC shifts metabolic programs to cope with CI deficiency. Bullet outline 5-Aminolevulinic acid (5-ALA-HCl + SFC) increases ATP production in flies with complex I deficiency.5-ALA-HCl + SFC increases the activities of complexes II and IV.5-ALA-HCl + SFC corrects metabolic abnormalities and suppresses the detrimental phenotypes caused by complex I deficiency.

1689-P: Macrophage SUCLA2-Coupled Glutamine Metabolism Drives Obesity

Activation and infiltration of macrophages in adipose tissue are closely linked to obesity and are implicated in the pathogenesis of metabolic diseases. Glutamine is an essential energy resource that supports the inflammatory activation of macrophages, however, the underlying mechanism remains unknown. We found that AMP-activated protein kinase (AMPK) was the key regulator for mitochondrial glutamine metabolism. Once inflammatory activation of macrophages, increased ATP production from glutaminolysis inhibited the activity of AMPK, then removed its inhibitory effect of glutamine metabolism, resulting in the succinate accumulation derived IL-1β production and secretion. Mechanically, macrophage AMPK phosphorylated at Ser60 of succinyl-CoA synthetase β subunit (SUCLA2) that was responsible for catalyzing the succinate production, decreases its activity resulting in the reduction of the succinate accumulation derived IL-1β production and secretion correspondingly. Moreover, myeloid cell-specific knockout of the α subunit of AMPK exacerbated obesity and insulin resistance through IL-1β production and secretion in macrophages. Both the obstruction of the IL-1β signaling pathway by the neutralizing antibody and the decreasing of IL-1β production and secretion by myeloid cell-specific knockout of IL-1β could repair insulin resistance and obesity derived by AMPK deficiency in macrophages. Our study revealed that AMPK-mediated SUCLA2 phosphorylation drove the mitochondrial glutamine metabolism and the IL-1β production and secretion in macrophages, and targeting macrophage AMPK-SUCLA2 pathway might be a potential strategy for obesity and insulin resistance therapy. Disclosure H.Jiang: None.

Trimetazidine Affects Mitochondrial Calcium Uniporter Expression to Restore Ischemic Heart Function via Reactive Oxygen Species/NFκB Pathway Inhibition.

Studies have demonstrated the roles of trimetazidine beyond being an antianginal agent in ischemic heart disease (IHD) treatment associated with mechanisms of calcium regulation. Our recent studies revealed that mitochondrial calcium uniporter (MCU, the pore-forming unit responsible for mitochondrial calcium entrance) inhibition provided cardioprotective effects for failing hearts. Because trimetazidine and MCU are associated with calcium homeostasis, we hypothesized that trimetazidine may affect MCU to restore the failing heart function. In the present study, we tested this hypothesis in the context of cardiac ischemia in vivo and in vitro. The IHD model was established in male C57BL/6 mice followed by trimetazidine administration intraperitoneally at 20 mg/kg q.o.d for 8 weeks. In vitro studies were performed in a hypoxia model using primary rat neonate cardiomyocytes. The mice survival outcomes and heart function, pathohistologic, and biological changes were analyzed. The results demonstrated that trimetazidine treatment resulted in longer life spans and heart function improvement accompanied by restoration of mitochondrial calcium levels and increase in ATP production via MCU down-regulation. Studies in vitro further showed that trimetazidine treatment and MCU inhibition decreased reactive oxygen species (ROS) production, inhibited the NFκB pathway, and protected the cardiomyocytes from hypoxic injury, and vice versa. Thus, the present study unveils a unique mechanism in which trimetazidine is involved in ameliorating the ischemic failing heart via MCU down-regulation and the following mitochondrial calcium homeostasis restoration, ROS reduction, and cardiomyocyte protection through NFκB pathway inhibition. This mechanism provides a novel explanation for the treatment effects of trimetazidine on IHD.

Noncanonical role of singleminded-2s in mitochondrial respiratory chain formation in breast cancer

Dysregulation of cellular metabolism is a hallmark of breast cancer progression and is associated with metastasis and therapeutic resistance. Here, we show that the breast tumor suppressor gene SIM2 promotes mitochondrial oxidative phosphorylation (OXPHOS) using breast cancer cell line models. Mechanistically, we found that SIM2s functions not as a transcription factor but localizes to mitochondria and directly interacts with the mitochondrial respiratory chain (MRC) to facilitate functional supercomplex (SC) formation. Loss of SIM2s expression disrupts SC formation through destabilization of MRC Complex III, leading to inhibition of electron transport, although Complex I (CI) activity is retained. A metabolomic analysis showed that knockout of SIM2s leads to a compensatory increase in ATP production through glycolysis and accelerated glutamine-driven TCA cycle production of NADH, creating a favorable environment for high cell proliferation. Our findings indicate that SIM2s is a novel stabilizing factor required for SC assembly, providing insight into the impact of the MRC on metabolic adaptation and breast cancer progression.

Resilient CD8 +T cells maintain a high cytotoxic capacity by balancing ROS and energy needs via ME1 upregulation

Abstract Cytotoxic T lymphocytes (CTL) are indispensable in anti-tumor immunity. Although CTLs are prone to exhaustion in patients with advanced cancer, T cell resiliency explains the presence of tumor-reactive CTLs that are less exhausted, capable of cytolytic function, expansion, and rebound in response to immunotherapy to reject metastatic malignancies. However, the features of resilient T cells have not been clearly defined. In this report, we demonstrate that peripheral CX3CR1 +CD8 +T cells with low mitochondrial membrane potential rebounded CTL function quickly after radiation therapy in patients with large tumor burden portraying their functional resiliency. Furthermore, CX3CR1 +CD8 +T cell with low, but not high, mitochondrial membrane potential are highly cytotoxic, accumulate less reactive oxygen species (ROS), and express more Malic enzyme 1 (ME1). ME1 overexpression increases ATP production in a glycolysis-independent manner while concurrently curtailing excessive ROS in activated CD8 +T cells; and expands CX3CR1 +NKG7 +effector CD8 +T cells with enhanced cytotoxicity. Importantly, transfection of ME1 mRNA promotes tumoricidal activity in CD8 +T cells from patients with advanced cancers. Our study reveals a mechanism used by CTLs to balance excessive ROS via ME1 to maintain a metabolic and functional resiliency. Modification of ME1 expression in CTLs may be a novel method to improve the efficacy of cancer immunotherapy by preventing T cell exhaustion. Supported by grants from Mayo Clinic Foundation and NIH (R01CA 256927-2)

Combining supervised machine learning with statistics reveals differential gene expression patterns related to energy metabolism in the jejuna of chickens divergently selected for antibody response to sheep red blood cells

Since the 1970s, 2 lines of White Leghorn chickens, HAS and LAS, have been continuously divergently selected for 5-day postinjection antibody titer to injection with sheep red blood cells (SRBC). Antibody response is a complex genetic trait and characterizing differences in gene expression could facilitate better understanding of physiological changes due to selection and antigen exposure. At 41 d of age, randomly selected HAS and LAS chickens, which had been coraised from hatch, were either injected with SRBC (HASI and LASI) or kept as the noninjected cohort (HASN and LASN). Five days later, all were euthanized, and samples collected from the jejunum for RNA isolation and sequencing. Resulting gene expression data were analyzed combining traditional statistics with machine learning to obtain signature gene lists for functional analysis. Differences in ATP production and cellular processes were observed in the jejunum between lines and following SRBC injection. HASN vs. LASN exhibited upregulation of ATP production, immune cell motility, and inflammation. LASI exhibits upregulation of ATP production and protein synthesis vs. LASN, reflective of what was observed in HASN vs. LASN. In contrast, no corresponding upregulation of ATP production was observed in HASI vs. HASN, and most other cellular processes appear inhibited. Without exposure to SRBC, gene expression in the jejunum indicates HAS generates more ATP than LAS, suggesting HAS maintains a "primed" system; and gene expression of HASI vs. HASN further suggests this basal ATP production is sufficient for robust antibody responses. Conversely, LASI vs. LASN jejunal gene expression implies a physiological need for increased ATP production with only minimal correlating antibody production. The results of this experiment provide insight into energetic resource needs and allocations in the jejunum in response to genetic selection and antigen exposure in HAS and LAS which may help explain phenotypic differences observed in antibody response.

Effect of L-carnitine in Ameliorating Lipopolysaccharide-Induced Cardiomyocyte Injury via MAPK Signaling.

The present study aimed to elucidate whether L-carnitine (LC) protected H9c2 cells and its underlying mechanisms. Cell counting kit-8 (CCK-8) assay was used to evaluate cell viability. Apoptosis, cell morphology, and lactate dehydrogenase (LDH) assessment were used to prove effects of lipopolysaccharide (LPS) and LC on H9c2 cells. RT-qPCR and western blot assays were hired to evaluate the mRNA and protein expression levels, respectively. ELISA assay was performed to determine the released protein levels. Reactive oxygen species (ROS) level was evaluated by immunofluorescence and flow cytometry. LC was revealed to protect H9c2 cells against LPS-induced injury as indicated by increased cell viability, reduced apoptosis ratio and LDH level. LC treatment also reduced BAX expression as well as up-regulated Bcl-2 expression under LPS treatment. Mechanically, LC reduced oxidative stress and ameliorated the mitochondrial injury through modulating extracellular signal-regulated kinase 1/2 and c-Jun N-terminal protein kinase c-Jun N-terminal protein kinase phosphorylation levels as indicated by decreased membrane potential, increased ATP production and mtDNA expression. We found that LC ameliorates LPS-induced cardiomyocyte injury by abrogating cell apoptosis ratio, ROS levels, as well as mitochondrial dysfunction via mitogen-activated protein kinase signaling. Our findings revealed a potential drug for sepsis or LPS-induced cardiomyocyte injury.

Sodium Glucose Cotransporter 2 (SGLT2) Inhibitor Ameliorate Metabolic Disorder and Obesity Induced Cardiomyocyte Injury and Mitochondrial Remodeling.

Sodium-glucose transporter 2 inhibitors (SGLT2is) exert significant cardiovascular and heart failure benefits in type 2 diabetes mellitus (DM) patients and can help reduce cardiac arrhythmia incidence in clinical practice. However, its effect on regulating cardiomyocyte mitochondria remain unclear. To evaluate its effect on myocardial mitochondria, C57BL/6J mice were divided into four groups, including: (1) control, (2) high fat diet (HFD)-induced metabolic disorder and obesity (MDO), (3) MDO with empagliflozin (EMPA) treatment, and (4) MDO with glibenclamide (GLI) treatment. All mice were sacrificed after 16 weeks of feeding and the epicardial fat secretome was collected. H9c2 cells were treated with the different secretomes for 18 h. ROS production, Ca2+ distribution, and associated proteins expression in mitochondria were investigated to reveal the underlying mechanisms of SGLT2is on cardiomyocytes. We found that lipotoxicity, mitochondrial ROS production, mitochondrial Ca2+ overload, and the levels of the associated protein, SOD1, were significantly lower in the EMPA group than in the MDO group, accompanied with increased ATP production in the EMPA-treated group. The expression of mfn2, SIRT1, and SERCA were also found to be lower after EMPA-secretome treatment. EMPA-induced epicardial fat secretome in mice preserved a better cardiomyocyte mitochondrial biogenesis function than the MDO group. In addition to reducing ROS production in mitochondria, it also ameliorated mitochondrial Ca2+ overload caused by MDO-secretome. These findings provide evidence and potential mechanisms for the benefit of SGLT2i in heart failure and arrhythmias.

Echinochrome A Prevents Diabetic Nephropathy by Inhibiting the PKC-Iota Pathway and Enhancing Renal Mitochondrial Function in db/db Mice.

Echinochrome A (EchA) is a natural bioproduct extracted from sea urchins, and is an active component of the clinical drug, Histochrome®. EchA has antioxidant, anti-inflammatory, and antimicrobial effects. However, its effects on diabetic nephropathy (DN) remain poorly understood. In the present study, seven-week-old diabetic and obese db/db mice were injected with Histochrome (0.3 mL/kg/day; EchA equivalent of 3 mg/kg/day) intraperitoneally for 12 weeks, while db/db control mice and wild-type (WT) mice received an equal amount of sterile 0.9% saline. EchA improved glucose tolerance and reduced blood urea nitrogen (BUN) and serum creatinine levels but did not affect body weight. In addition, EchA decreased renal malondialdehyde (MDA) and lipid hydroperoxide levels, and increased ATP production. Histologically, EchA treatment ameliorated renal fibrosis. Mechanistically, EchA suppressed oxidative stress and fibrosis by inhibiting protein kinase C-iota (PKCι)/p38 mitogen-activated protein kinase (MAPK), downregulating p53 and c-Jun phosphorylation, attenuating NADPH oxidase 4 (NOX4), and transforming growth factor-beta 1 (TGFβ1) signaling. Moreover, EchA enhanced AMPK phosphorylation and nuclear factor erythroid-2-related factor 2 (NRF2)/heme oxygenase 1 (HO-1) signaling, improving mitochondrial function and antioxidant activity. Collectively, these findings demonstrate that EchA prevents DN by inhibiting PKCι/p38 MAPK and upregulating the AMPKα/NRF2/HO-1 signaling pathways in db/db mice, and may provide a therapeutic option for DN.

Use of a gene expression signature to identify trimetazidine for repurposing to treat bipolar depression.

The aim of this study was to repurpose a drug for the treatment of bipolar depression. A gene expression signature representing the overall transcriptomic effects of a cocktail of drugs widely prescribed to treat bipolar disorder was generated using human neuronal-like (NT2-N) cells. A compound library of 960 approved, off-patent drugs were then screened to identify those drugs that affect transcription most similar to the effects of the bipolar depression drug cocktail. For mechanistic studies, peripheral blood mononuclear cells were obtained from a healthy subject and reprogrammed into induced pluripotent stem cells, which were then differentiated into co-cultured neurons and astrocytes. Efficacy studies were conducted in two animal models of depressive-like behaviours (Flinders Sensitive Line rats and social isolation with chronic restraint stress rats). The screen identified trimetazidine as a potential drug for repurposing. Trimetazidine alters metabolic processes to increase ATP production, which is thought to be deficient in bipolar depression. We showed that trimetazidine increased mitochondrial respiration in cultured human neuronal-like cells. Transcriptomic analysis in induced pluripotent stem cell-derived neuron/astrocyte co-cultures suggested additional mechanisms of action via the focal adhesion and MAPK signalling pathways. In two different rodent models of depressive-like behaviours, trimetazidine exhibited antidepressant-like activity with reduced anhedonia and reduced immobility in the forced swim test. Collectively our data support the repurposing of trimetazidine for the treatment of bipolar depression.

Spike Protein Impairs Mitochondrial Function in Human Cardiomyocytes: Mechanisms Underlying Cardiac Injury in COVID-19.

COVID-19 has a major impact on cardiovascular diseases and may lead to myocarditis or cardiac failure. The clove-like spike (S) protein of SARS-CoV-2 facilitates its transmission and pathogenesis. Cardiac mitochondria produce energy for key heart functions. We hypothesized that S1 would directly impair the functions of cardiomyocyte mitochondria, thus causing cardiac dysfunction. Through the Seahorse Mito Stress Test and real-time ATP rate assays, we explored the mitochondrial bioenergetics in human cardiomyocytes (AC16). The cells were treated without (control) or with S1 (1 nM) for 24, 48, and 72 h and we observed the mitochondrial morphology using transmission electron microscopy and confocal fluorescence microscopy. Western blotting, XRhod-1, and MitoSOX Red staining were performed to evaluate the expression of proteins related to energetic metabolism and relevant signaling cascades, mitochondrial Ca2+ levels, and ROS production. The 24 h S1 treatment increased ATP production and mitochondrial respiration by increasing the expression of fatty-acid-transporting regulators and inducing more negative mitochondrial membrane potential (Δψm). The 72 h S1 treatment decreased mitochondrial respiration rates and Δψm, but increased levels of reactive oxygen species (ROS), mCa2+, and intracellular Ca2+. Electron microscopy revealed increased mitochondrial fragmentation/fission in AC16 cells treated for 72 h. The effects of S1 on ATP production were completely blocked by neutralizing ACE2 but not CD147 antibodies, and were partly attenuated by Mitotempo (1 µM). S1 might impair mitochondrial function in human cardiomyocytes by altering Δψm, mCa2+ overload, ROS accumulation, and mitochondrial dynamics via ACE2.

BDNF mimetic 7,8-dihydroxyflavone rescues rotenone-induced cytotoxicity in cardiomyocytes by ameliorating mitochondrial dysfunction.

The relationship between mitochondrial dysfunction and cardiovascular disease pathogenesis is well recognized. 7,8-Dihydroxyflavone (7,8-DHF), a mimetic of brain-derived neurotrophic factor, inhibits mitochondrial impairments and improves cardiac function. However, the regulatory role of 7,8-DHF in the mitochondrial function of cardiomyocytes is not fully understood. To investigate the potential mito-protective effects of 7,8-DHF in cardiomyocytes, we treated H9c2 or HL-1cells with the mitochondrial respiratory complex I inhibitor rotenone (Rot) as an in vitro model of mitochondrial dysfunction. We found that 7,8-DHF effectively eliminated various concentrations of Rot-induced cell death and reduced lactate dehydrogenase release. 7,8-DHF significantly improved mitochondrial membrane potential and inhibited mitochondrial reactive oxygen species. Moreover, 7,8-DHF decreased routine and leak respiration, restored protein levels of mitochondrial complex I-IV, and increased ATP production in Rot-treated H9c2 cells. The protective role of 7,8-DHF in Rot-induced damage was validated in HL-1cells. Nuclear phosphorylation protein expression of signal transducer and activator of transcription 3 (STAT3) was significantly increased by 7,8-DHF. The present study suggests that 7,8-DHF rescues Rot-induced cytotoxicity by inhibiting mitochondrial dysfunction and promoting nuclear translocation of p-STAT3 in cardiomyocytes, thus nominating 7,8-DHF as a new pharmacological candidate agent against mitochondrial dysfunction in cardiac diseases.

Effect of Dl-3-n-butylphthalide on mitochondrial Cox7c in models of cerebral ischemia/reperfusion injury.

Several studies have demonstrated the protective effect of dl-3-n-Butylphthalide (NBP) against cerebral ischemia, which may be related to the attenuation of mitochondrial dysfunction. However, the specific mechanism and targets of NBP in cerebral ischemia/reperfusion remains unclear. In this study, we used a chemical proteomics approach to search for targets of NBP and identified cytochrome C oxidase 7c (Cox7c) as a key interacting target of NBP. Our findings indicated that NBP inhibits mitochondrial apoptosis and reactive oxygen species (ROS) release and increases ATP production through upregulation of Cox7c. Subsequently, mitochondrial respiratory capacity was improved and the HIF-1α/VEGF pathway was upregulated, which contributed to the maintenance of mitochondrial membrane potential and blood brain barrier integrity and promoting angiogenesis. Therefore, our findings provided a novel insight into the mechanisms underlying the neuroprotective effects of NBP, and also proposed for the first time that Cox7c exerts a critical role by protecting mitochondrial function.

Investigating the changes of the components of the Krebs cycle in patients with type 2 diabetes treated with squalene

Background: Type 2 diabetes mellitus is a chronic disease that impairs the body’s ability to regulate glucose. Recent studies have shown that squalene, a bioactive compound, has shown promising potential in increasing ATP levels for diabetic patients and aged individuals.Objective: Our main goal was to evaluate the cellular effects of different doses of squalene on the intermediates and enzymes of Krebs cycle, in order to determine if squalene increases ATP production among groups of people with type 2 diabetes. The intermediates and enzymes that are being studied are acetyl coenzyme (A-CoA), alpha ketoglutarate dehydrogenase (AKGDH), calcium ion (Ca2+), citrate synthase (CS), isocitrate dehydrogenase, oxaloacetate, and pyruvate dehydrogenase complex component (PDH).Methods: In this study, 30 healthy volunteers were selected as the healthy control group (group 1) and 120 volunteers with type 2 diabetes mellitus were selected. Subjects with diabetes were randomly divided into 4 groups. Group 2 was untreated with squalene and groups 3, 4, and 5 were treated with 200, 400 and 600 mg of squalene, respectively for 84 days. Intermediates and enzymes of the Krebs cycle as well as calcium ion were assayed on days 1, 14, 28, 56, and 84 according to the relevant protocols in all groups.Results: The squalene-treated diabetic groups were compared to group 2 that was not treated any squalene to determine the differences of the parameters. Throughout these 84 days, it was observed that only calcium levels increased in the diabetic patients with high statistical difference (P < 0.05). The other parameters: acetyl coenzyme, alpha ketoglutarate dehydrogenase, citrate synthase, isocitrate dehydrogenase, oxaloacetate, and pyruvate dehydrogenase did not have a significant difference (P > 0.05). Conclusion: Based on the findings of this study, the addition of various doses of squalene to a diabetic patient's diet increases the amount of calcium found in their metabolic process in relation to the Krebs cycle. As calcium is responsible for stimulating the Krebs Cycle, it is evident that squalene plays an important part in ATP production.Keywords: squalene, type 2 diabetes, Krebs cycle, calcium, ATP

The mitochondrial chaperone TRAP-1 regulates the glutamine metabolism in tumor cells

Understanding cancer cell metabolism always provides information on hidden dimensions of tumor adaptations. Warburg's theory that cancer cells opt for aerobic glycolysis over the mitochondrial oxidative phosphorylation (OXPHOS) system is widely accepted. However, the hypothesis does not explain the mitochondrion's role in these cells. Here, we demonstrate that intact mitochondria are used for anaplerotic functions and ATP production by utilizing glutamine with the help of mitochondrial chaperone TRAP-1 (Tumor Necrosis Factor Receptor-associated Protein 1). TRAP-1 otherwise promotes aerobic glycolysis by lowering the mitochondrial OXPHOS in the presence of glucose. Here, we show that TRAP-1 maintains mitochondrial integrity and augments glutamine metabolism upon glucose deprivation to meet the cellular energy demand. The enhanced PER and ECAR correlating with increased ATP production suggest that glutamine fuels mitochondria in the presence of TRAP-1. We also found that TRAP1-dependent glutamine utilization involves the HIF2α-SLC1A5-GLS axis and is independent of hypoxia. Subsequently, we show that the metastatic potential of tumor cells is linked with glucose utilization, whereas the proliferative potential is linked with both glucose and glutamine utilization. Our findings establish that TRAP-1 contributes to enhanced glutamine utilization through the HIF2α-SLC1A5-GLS axis. Our results endow that TRAP-1 inhibitors can be potential drug candidates to combat tumor metabolism. Therefore, their use, either alone or in combination with existing chemotherapeutic agents, may target tumor metabolism and improve anticancer treatment response.

A novel therapeutic strategy against mitochondrial respiratory chain dysfunction-linked diseases.

Mitochondrial diseases result from a decreased oxidative phosphorylation (OXPHOS) that leads to a broad spectrum of incurable pathologies. Our goal was to understand whether membrane permeant small molecule(s) can be exploited to treat OXPHOS-related diseases as an alternative to gene therapy. Therefore, we selected some molecules for their ability to replace the redox functions of complex III and among them identified pyocyanin as a promising agent. Pyocyanin is a bacterial redox cycler that can shuttle electrons from reduced coenzyme Q to cytochrome c, acting as an electron shunt. Sub-μM dose of pyocyanin is harmless, restores respiration and increases ATP production in Ttc19-/- mouse embryonic fibroblasts as well as in fibroblasts from patients harboring pathogenic mutations in three different assembly/stabilization factors of complex III (namely, TTC19, BCS1L and LYRM7). The drug normalized the mitochondrial membrane potential, mildly increased ROS production, and triggered mitochondrial biogenesis. These in vitro effects were confirmed in both Drosophila melanogasterTTC19KO, in Danio rerioTTC19KD. Here we show that pyocyanin and its newly synthesized derivative with enhanced life-time and tissue distribution exhibited a benefit in Ttc19 KO mouse model as well. Indeed, in all these models, administration of low, non-toxic concentration of pyocyanin significantly ameliorated movement proficiency, without inducing toxicity. Likewise, pyocyanin, able to receive electrons from NADH, showed a beneficial effect also in the case of cells and mice with complex I disease. Our results point to exploitation of redox cyclers for therapy against diseases due to OXPHOS dysfunction.

Differing modes of growth performance induced by hypoxia and effects on oxidative defense and glycolysis metabolism in two marine Brachionus rotifer species

Hypoxia is often a concern in closed, recirculating aquaculture facilities. Here, we examined the effects of hypoxia (0.3–1.0 mg O2/L; two days of continuous exposure) on growth and metabolism in two marine rotifers, Brachionus plicatilis and Brachionus rotundiformis, having differing sizes and environmental acclimation abilities. The obtained results revealed that the super-small (SS)-type rotifer B. rotundiformis possesses higher tolerance to hypoxia during reproduction than the large (L)-type B. plicatilis. Exposure to hypoxia significantly inhibited the growth of B. plicatilis, whereas it had no influence on B. rotundiformis. While adaptive strategies, such as smaller body size and enhanced swimming speed, were detected in B. rotundiformis. To identify the underlying mechanisms of the phenomena, the activity of key biomarkers and enzymes involved in oxidative defense and glycolysis (energy) was examined. In B. plicatilis, although the level of antioxidants increased, hypoxia still induced the production of high levels of reactive oxygen species (ROS) and hypoxia-inducible factors (single-minded homolog 1 and aryl hydrocarbon receptor nuclear translocator). However, in B. rotundiformis, enhanced defensive responses (increment of manganese superoxide dismutase and glutathione S-transferase levels) successfully maintained the balance of ROS during hypoxia. Regarding energy metabolism, the expression of six key biomarkers involved in the glycolytic pathway was all significantly upregulated in B. plicatilis. Both B. plicatilis and B. rotundiformis increased ATP production, by 1.9- and 1.2-fold, respectively. Unlike B. plicatilis, B. rotundiformis may have used the produced ATP to swim faster and maintain reproductive homeostasis. The difference in resistance and growth/molecular responses to hypoxic exposure between B. plicatilis and B. rotundiformis may be attributed to their disparate mass-specific metabolic rates and habitat-evolutionary adaptations. The greater tolerance for hypoxia is beneficial to the rapid and high-density cultivation of B. rotundiformis in aquaculture facilities.

Development of a Mitochondrial Targeting Lipid Nanoparticle Encapsulating Berberine.

Delivering drugs to mitochondria, the main source of energy in neurons, can be a useful therapeutic strategy for the treatment of neurodegenerative diseases. Berberine (BBR), an isoquinoline alkaloid, acts on mitochondria and is involved in mechanisms associated with the normalization and regulation of intracellular metabolism. Therefore, BBR has attracted considerable interest as a possible therapeutic drug for neurodegenerative diseases. While BBR has been reported to act on mitochondria, there are few reports on the efficient delivery of BBR into mitochondria. This paper reports on the mitochondrial delivery of BBR using a lipid nanoparticle (LNP), a "MITO-Porter" that targets mitochondria, and its pharmacological action in Neuro2a cells, a model neuroblastoma. A MITO-Porter containing encapsulated BBR (MITO-Porter (BBR)) was prepared. Treatment with MITO-Porter (BBR) increased the amount of BBR that accumulated in mitochondria compared with a treatment with naked BBR. Treatment with MITO-Porter (BBR) resulted in increased ATP production in Neuro2a cells, which are important for maintaining life phenomena, compared with treatment with naked BBR. Treatment with MITO-Porter (BBR) also increased the level of expression of mitochondrial ubiquitin ligase (MITOL), which is involved in mitochondrial quality control. Our findings indicate that increasing the accumulation of BBR into mitochondria is important for inducing enhanced pharmacological actions. The use of this system has the potential for being important in terms of the regulation of the metabolic mechanism of mitochondria in nerve cells.