Differential In Vitro Effects of SGLT2 Inhibitors on Mitochondrial Oxidative Phosphorylation, Glucose Uptake and Cell Metabolism.
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SUBMITTER: Zugner E
PROVIDER: S-EPMC9319636 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Abstract Background: Friedreich’s Ataxia (FA) is a neurodegenerative disease caused by disruptions in the gene encoding frataxin, a protein involved in formation of the iron-sulfur clusters needed for mitochondrial oxidative phosphorylation (OXPHOS). FA confers an increased risk for diabetes mellitus, but the mechanisms underlying this propensity are not well understood. Design: Cross-sectional study (clinicaltrials.gov: NCT02920671) Methods: Participants were non-diabetic individuals with FA (ages 18y-<65y) and healthy controls with a similar distribution of age, sex, BMI, and ancestry. OXPHOS capacity of calf muscle was assessed using a non-invasive, exercise-based MRI technique, creatine chemical exchange saturation transfer (CrCEST); results for lateral gastrocnemius (LG), the most consistently exercised muscle, are shown. After an overnight fast, a stable isotope tracer-enhanced oral glucose tolerance test was done. Body composition was assessed using dual energy x-ray absorptiometry. Linear regression analyses of the entire sample tested the association between outcomes and covariates (results are shown as standardized coefficient β). Results: This interim analysis has n=34 participants (11 FA, 23 control). Participants with FA were 61% male, median age 27y (IQI, 23-39), median BMI 26.9 kg/m2 (IQI, 24.1-29.4), and median visceral fat mass 0.50 kg (IQI, 0.35-0.70). Controls were 61% male, median age 29y (IQI, 26-38), median BMI 24.6 kg/m2 (IQI, 21.7-28.5), and median visceral fat mass 0.45 kg (IQI, 0.27-0.64). Fasting glucose was higher in FA (91 vs. 82 mg/dL, p=0.006). Lactate was similar at baseline and increased after oral glucose in both, but remained higher after 180min in FA (at 210min, 1.13 vs. 0.88 mmol/L, p=0.01). Post-exercise CrCEST recovery time constant (τCr) was increased 2.2-fold in FA (p=0.02), consistent with decreased OXPHOS capacity. In univariate analyses, whole body insulin sensitivity (WBISI) was decreased in FA (β -1.14, p=0.003), decreased with longer τCr, (β -0.48, p=0.01), and decreased with higher visceral fat (β -0.55, p=0.004). In multivariate analyses, these associations were attenuated when FA diagnosis (β -0.85, p=0.06) and τCr (β -0.25, p=0.25) were included in the same model, but were persistent when FA diagnosis (β -0.93, p=0.008) and visceral fat (β -0.43, p=0.01) were included in the same model. In univariate analyses, lactate area under the curve (AUC) was nominally increased in FA (β 0.67, p=0.11), and significantly positively associated with longer τCr (β 0.51, p=0.008). Conclusions: Individuals with a genetic mitochondrial disorder conferring increased diabetes risk have decreased whole body insulin sensitivity that may be mediated by decreased skeletal muscle OXPHOS capacity. Studies in rare disorders may provide insights into the role of skeletal muscle metabolism in the pathogenesis of Type 2 diabetes. Funding: NIH/NIDDK Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.
| S-EPMC6550843 | biostudies-literature
Project description:Aminoglycoside antibiotics, like gentamicin, continue to be clinically essential worldwide to treat life-threatening bacterial infections. Yet, the ototoxic and nephrotoxic side-effects of these drugs remain serious complications. A major site of gentamicin uptake and toxicity resides within kidney proximal tubules that also heavily express electrogenic sodium-glucose transporter-2 (SGLT2; SLC5A2) in vivo. We hypothesized that SGLT2 traffics gentamicin, and promotes cellular toxicity. We confirmed in vitro expression of SGLT2 in proximal tubule-derived KPT2 cells, and absence in distal tubule-derived KDT3 cells. D-glucose competitively decreased the uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescent analog of glucose, and fluorescently-tagged gentamicin (GTTR) by KPT2 cells. Phlorizin, an SGLT2 antagonist, strongly inhibited uptake of 2-NBDG and GTTR by KPT2 cells in a dose- and time-dependent manner. GTTR uptake was elevated in KDT3 cells transfected with SGLT2 (compared to controls); and this enhanced uptake was attenuated by phlorizin. Knock-down of SGLT2 expression by siRNA reduced gentamicin-induced cytotoxicity. In vivo, SGLT2 was robustly expressed in kidney proximal tubule cells of heterozygous, but not null, mice. Phlorizin decreased GTTR uptake by kidney proximal tubule cells in Sglt2+/- mice, but not in Sglt2-/- mice. However, serum GTTR levels were elevated in Sglt2-/- mice compared to Sglt2+/- mice, and in phlorizin-treated Sglt2+/- mice compared to vehicle-treated Sglt2+/- mice. Loss of SGLT2 function by antagonism or by gene deletion did not affect gentamicin cochlear loading or auditory function. Phlorizin did not protect wild-type mice from kanamycin-induced ototoxicity. We conclude that SGLT2 can traffic gentamicin and contribute to gentamicin-induced cytotoxicity.
| S-EPMC4182564 | biostudies-literature
Project description:Serine hydroxymethyltransferase 2 (SHMT2), which catalyzes the conversion of serine to glycine and one-carbon transfer reactions in mitochondria, is significantly upregulated in glioblastoma (GBM). However, the mechanism by which the stability of SHMT2 gene expression is maintained to drive GBM tumorigenesis has not been clarified. Herein, through microarray screening, we identified that HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) modulates the SHMT2 level in various GBM cell lines. Serine catabolism and mitochondrial oxidative phosphorylation activities were decreased by HOTAIRM1 inhibition. Mechanistically, according to our mass spectrometry and eCLIP-seq results, HOTAIRM1 can bind to PTBP1 and IGF2BP2. Furthermore, HOTAIRM1 maintains the stability of SHMT2 by promoting the recognition of an m6A site and the interaction of PTBP1/IGF2BP2 with SHMT2 mRNA. The stability of HOTAIRM1 can also be enhanced and results in positive feedback regulation to support the progression of GBM. Thus, targeting HOTAIRM1 could be a promising metabolic therapy for GBM.
| S-EPMC9389257 | biostudies-literature
Project description:Tumor mitochondria have heightened protein folding quality control, but the regulators of this process and how they impact cancer traits are not completely understood. Here we show that the ATP-directed mitochondrial protease, LonP1 is upregulated by stress conditions, including hypoxia, in tumor, but not normal cells. In mitochondria, LonP1 is phosphorylated by Akt on Ser173 and Ser181, enhancing its protease activity. Interference with this pathway induces accumulation of misfolded subunits of electron transport chain complex II and complex V, resulting in impaired oxidative bioenergetics and heightened ROS production. Functionally, this suppresses mitochondrial trafficking to the cortical cytoskeleton, shuts off tumor cell migration and invasion, and inhibits primary and metastatic tumor growth, in vivo. These data identify LonP1 as a key effector of mitochondrial reprogramming in cancer and potential therapeutic target.
| S-EPMC6814529 | biostudies-literature
Project description:Maternal inheritance of mitochondria creates a sex-specific selective sieve through which mitochondrial mutations harmful to males but not females accumulate and contribute to sexual differences in longevity and disease susceptibility. Because eggs and sperm are under disruptive selection, sperm are predicted to be particularly vulnerable to the genetic load generated by maternal inheritance, yet evidence for mitochondrial involvement in male fertility is limited and controversial. Here, we exploit the coexistence of two divergent mitochondrial haplogroups (A and B2) in a Neotropical arachnid to investigate the role of mitochondria in sperm competition. DNA profiling demonstrated that B2-carrying males sired more than three times as many offspring in sperm competition experiments than A males, and this B2 competitive advantage cannot be explained by female mitochondrial haplogroup or male nuclear genetic background. RNA-Seq of testicular tissues implicates differential expression of mitochondrial oxidative phosphorylation (OXPHOS) genes in the B2 competitive advantage, including a 22-fold upregulation of atp8 in B2 males. Previous comparative genomic analyses have revealed functionally significant amino acid substitutions in differentially expressed genes, indicating that the mitochondrial haplogroups differ not only in expression but also in DNA sequence and protein functioning. However, mitochondrial haplogroup had no effect on sperm number or sperm viability, and, when females were mated to a single male, neither male haplogroup, female haplogroup nor the interaction between male/female haplogroup significantly affected female reproductive success. Our findings therefore suggest that mitochondrial effects on male reproduction may often go undetected in noncompetitive contexts and may prove more important in nature than is currently appreciated.
| S-EPMC6904504 | biostudies-literature
Project description:The sodium-dependent glucose transporters 2 (SGLT2) plays important role in renal reabsorption of urinal glucose back to plasma for maintaining glucose homeostasis. The approval of SGLT2 inhibitors for treatment of type 2 diabetes highlights the SGLT2 as a feasible and promising drug target in recent years. Current methods for screening SGLT2 inhibitors are complex, expensive and labor intensive. Particularly, these methods cannot directly measure nonradioactive glucose uptake in endogenous SGLT2-expressing kidney cells. In present work, human kidney cells, HK-2, was incubated with a fluorescent D-glucose derivant 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) and the fluorescent intensity of 2-NBDG was employed to measure the amount of glucose uptake into the cells. By optimizing the passages of HK-2 cells, 2-NBDG concentration and incubation time, and by measuring glucose uptake treated by Dapagliflozin, a clinical drug of SGLT2 inhibitors, we successfully developed a new assay for measuring glucose uptake through SGLT2. The nonradioactive microplate and microscope-based high-throughput screening assay for measuring glucose can be a new method for screening of SGLT2 inhibitors and implied for other cell assays for glucose measurement extensively.
| S-EPMC6328422 | biostudies-literature
Project description:BackgroundSodium-glucose co-transporters (SGLT) inhibitors (SGLT2i) showed many beneficial effects at the cardiovascular level. Several mechanisms of action have been identified. However, no data on their capability to act via epigenetic mechanisms were reported. Therefore, this study aimed to investigate the ability of SGLT2 inhibitors (SGLT2i) to induce protective effects at the cardiovascular level by acting on DNA methylation.MethodsTo better clarify this issue, the effects of empagliflozin (EMPA) on hyperglycemia-induced epigenetic modifications were evaluated in human ventricular cardiac myoblasts AC16 exposed to hyperglycemia for 7 days. Therefore, the effects of EMPA on DNA methylation of NF-κB, SOD2, and IL-6 genes in AC16 exposed to high glucose were analyzed by pyrosequencing-based methylation analysis. Modifications of gene expression and DNA methylation of NF-κB and SOD2 were confirmed in response to a transient SGLT2 gene silencing in the same cellular model. Moreover, chromatin immunoprecipitation followed by quantitative PCR was performed to evaluate the occupancy of TET2 across the investigated regions of NF-κB and SOD2 promoters.ResultsSeven days of high glucose treatment induced significant demethylation in the promoter regions of NF-kB and SOD2 with a consequent high level in mRNA expression of both genes. The observed DNA demethylation was mediated by increased TET2 expression and binding to the CpGs island in the promoter regions of analyzed genes. Indeed, EMPA prevented the HG-induced demethylation changes by reducing TET2 binding to the investigated promoter region and counteracted the altered gene expression. The transient SGLT2 gene silencing prevented the DNA demethylation observed in promoter regions, thus suggesting a role of SGLT2 as a potential target of the anti-inflammatory and antioxidant effect of EMPA in cardiomyocytes.ConclusionsIn conclusion, our results demonstrated that EMPA, mainly acting on SGLT2, prevented DNA methylation changes induced by high glucose and provided evidence of a new mechanism by which SGLT2i can exert cardio-beneficial effects.
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Project description:Metabolic cycles are a fundamental element of cellular and organismal function. Among the most critical in higher organisms is the Cori Cycle, the systemic cycling between lactate and glucose. Here, skeletal muscle-specific Mitochondrial Pyruvate Carrier (MPC) deletion in mice diverted pyruvate into circulating lactate. This switch disinhibited muscle fatty acid oxidation and drove Cori Cycling that contributed to increased energy expenditure. Loss of muscle MPC activity led to strikingly decreased adiposity with complete muscle mass and strength retention. Notably, despite decreasing muscle glucose oxidation, muscle MPC disruption increased muscle glucose uptake and whole-body insulin sensitivity. Furthermore, chronic and acute muscle MPC deletion accelerated fat mass loss on a normal diet after high fat diet-induced obesity. Our results illuminate the role of the skeletal muscle MPC as a whole-body carbon flux control point. They highlight the potential utility of modulating muscle pyruvate utilization to ameliorate obesity and type 2 diabetes.
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Project description:Cancer cells generally rely mostly on glycolysis rather than oxidative phosphorylation (OXPHOS) for ATP production. In fact, they are particularly sensitive to glycolysis inhibition and glucose depletion. On the other hand mitochondrial dysfunctions, involved in the onset of the Warburg effect, are sometimes also associated with the resistance to apoptosis that characterizes cancer cells. Therefore, combined treatments targeting both glycolysis and mitochondria function, exploiting peculiar tumor features, might be lethal for cancer cells. In this study, we show that glucose deprivation and mitochondrial Complex I inhibitors synergize in inducing cancer cell death. In particular, our results reveal that low doses of Complex I inhibitors, ineffective on immortalized cells and in high glucose growth, become specifically cytotoxic on cancer cells deprived of glucose. Importantly, the cytotoxic effect of the inhibitors on cancer cells is strongly enhanced by forskolin, a PKA pathway activator, that we have previously shown to stimulate OXPHOS. Taken together, we demonstrate that induction in cancer cells of a switch from a glycolytic to a more respirative metabolism, obtained by glucose depletion or mitochondrial activity stimulation, strongly increases their sensitivity to low doses of mitochondrial Complex I inhibitors. Our findings might be a valuable approach to eradicate cancer cells.
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Project description:Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor BNIP3L/Nix is responsive to lipotoxicity and accumulates in response to a high-fat (HF) feeding. To provide a better understanding of this observation, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the reported roles of these phospholipids and BNIP3L in mitochondrial dynamics, we investigated aberrant mitochondrial turnover as a mechanism of impaired myocyte insulin signaling. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that BNIP3L accumulation triggers mitochondrial depolarization, calcium-dependent activation of DNM1L/DRP1, and mitophagy. In addition, BNIP3L can inhibit insulin signaling through activation of MTOR-RPS6KB/p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and RHEB. Finally, we demonstrate that BNIP3L-induced mitophagy and impaired glucose uptake can be reversed by direct phosphorylation of BNIP3L by PRKA/PKA, leading to the translocation of BNIP3L from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of BNIP3L, mitochondrial turnover, and impaired myocyte insulin signaling during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggest a mechanism by which exercise or pharmacological activation of PRKA may overcome myocyte insulin resistance.Abbreviations: BCL2: B cell leukemia/lymphoma 2; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; DNM1L/DRP1: dynamin 1-like; FUNDC1: FUN14 domain containing 1; IRS1: insulin receptor substrate 1; MAP1LC3A/LC3: microtubule-associated protein 1 light chain 3 alpha; MFN1: mitofusin 1; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; OPA1: OPA1 mitochondrial dynamin like GTPase; PDE4i: phosphodiesterase 4 inhibitor; PLD1: phospholipase D1; PLD6: phospholipase D family member 6; PRKA/PKA: protein kinase, AMP-activated; PRKCD/PKCδ: protein kinase C, delta; PRKCQ/PKCθ: protein kinase C, theta; RHEB: Ras homolog enriched in brain; RPS6KB/p70S6K: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; YWHAB/14-3-3β: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta.
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