Increase In Oxygen Consumption Rate Research Articles

Effects of 5-aminolevulinic acid and sodium ferrous citrate on fibroblasts from individuals with mitochondrial diseases

Mitochondrial respiratory chain complexes II, III, and IV and cytochrome c contain haem, which is generated by the insertion of Fe2+ into protoporphyrin IX. 5-Aminolevulinic acid (ALA) combined with sodium ferrous citrate (SFC) was reported to enhance haem production, leading to respiratory complex and haem oxygenase-1 (HO-1) upregulation. Here, we investigated the effects of different concentrations of ALA and SFC alone or in combination (ALA/SFC) on fibroblasts from 8 individuals with mitochondrial diseases and healthy controls. In normal fibroblasts, expression levels of oxidative phosphorylation (OXPHOS) complex subunits and corresponding genes were upregulated only by ALA/SFC. Additionally, the increased oxygen consumption rate (OCR) and ATP levels in normal fibroblasts were more obvious after treatment with ALA/SFC than after treatment with ALA or SFC. OXPHOS complex proteins were enhanced by ALA/SFC, whereas OCR and ATP levels were increased in 6 of the 8 patient-derived fibroblasts. Further, HO-1 protein and mRNA levels were enhanced by ALA/SFC in all fibroblasts. The relative mtDNA copy number was increased by ALA/SFC. Thus, our findings indicate that ALA/SFC is effective in elevating OXPHOS, HO-1 protein, and mtDNA copy number, resulting in an increase in OCR and ATP levels, which represents a promising therapeutic option for mitochondrial diseases.

Auraptene Mitigates Parkinson's Disease-Like Behavior by Protecting Inhibition of Mitochondrial Respiration and Scavenging Reactive Oxygen Species.

Current therapeutics for Parkinson’s disease (PD) are only effective in providing relief of symptoms such as rigidity, tremors and bradykinesia, and do not exert disease-modifying effects by directly modulating mitochondrial function. Here, we investigated auraptene (AUR) as a potent therapeutic reagent that specifically protects neurotoxin-induced reduction of mitochondrial respiration and inhibits reactive oxygen species (ROS) generation. Further, we explored the mechanism and potency of AUR in protecting dopaminergic neurons. Treatment with AUR significantly increased the viability of substantia nigra (SN)-derived SN4741 embryonic dopaminergic neuronal cells and reduced rotenone-induced mitochondrial ROS production. By inducing antioxidant enzymes AUR treatment also increased oxygen consumption rate. These results indicate that AUR exerts a protective effect against rotenone-induced mitochondrial oxidative damage. We further assessed AUR effects in vivo, investigating tyrosine hydroxylase (TH) expression in the striatum and substantia nigra of MPTP-induced PD model mice and behavioral changes after injection of AUR. AUR treatment improved movement, consistent with the observed increase in the number of dopaminergic neurons in the substantia nigra. These results demonstrate that AUR targets dual pathogenic mechanisms, enhancing mitochondrial respiration and attenuating ROS production, suggesting that the preventative potential of this natural compound could lead to improvement in PD-related neurobiological changes.

Effects of vitamin D on primary human skeletal muscle cell proliferation, differentiation, protein synthesis and bioenergetics

The active form of Vitamin D (1,25(OH)2D), has been suggested to have a regulatory role in skeletal muscle function and metabolism, however, the effects and mechanisms of vitamin D (VitD) action in this tissue remain to be fully established. In this study, we have used primary human skeletal muscle myoblast (HSMM) cells that display typical characteristics of human skeletal muscle function and protein levels, to investigate the effects of the active form of VitD on proliferation, differentiation, protein synthesis and bioenergetics. Myoblast cells were treated with 100 nM of VitD for 24 h, 48 h, 72 h and five days (cells were differentiated into myotubes) and then analyses were performed. We report that VitD inhibits myoblast proliferation and enhances differentiation by altering the expression of myogenic regulatory factors. In addition, we found that protein synthesis signaling improved in myotubes after VitD treatment in the presence of insulin. We also report an increase in oxygen consumption rate after 24 h of treatment in myoblasts and after 5 days of treatment in myotubes after VitD exposure. VitD significantly impacted HSMM myogenesis, as well as protein synthesis in the presence of insulin.

Abstract 3483: The African-specific S47 variant of the p53 tumor suppressor gene alters cell metabolism

Abstract The TP53 gene, often referred to as the guardian of the genome, is the most frequently mutated gene in human cancer. This gene encodes the tumor suppressor p53, a master regulator of various processes such as programmed cell death, growth arrest and senescence. However, there is increasing evidence that highlights the role of p53 in tumor suppression with regards to maintaining metabolic homeostasis. Our lab has identified a polymorphic variant of p53 that encodes a serine residue instead of a proline at amino acid 47 (hereafter S47). This variant is most prevalent in African and African-American individuals, and mice and humans carrying this variant have increased cancer risk and impaired response to therapy. Using both mouse models and human cells, we have found that S47 cells have significantly altered metabolism compared to the WT counterpart. Specifically, S47 mice have increased weight and increased lean content compared to WT mice. These mice also show increased fitness in treadmill studies. Analyses of cell metabolism in S47 murine and human cells indicate that S47 cells show increased glycolysis and glycolytic flux, along with increased oxygen consumption rate on a Seahorse analyzer. The combined data support the premise that the S47 variant confers increased cancer risk but also increased metabolic fitness; the latter may explain the high rate of this variant in certain regions of Africa. By clearly elucidating the roles of WT p53 and the S47 variant in the context of metabolism, we hope to uncover new therapeutic avenues and ultimately enable more personalized medicine approaches for individuals who carry this variant. Citation Format: Keerthana Gnanapradeepan, Subhasree Basu, Che-Pei Kung, Thibaut Barnoud, Madeline Good, William Quinn, Joyce Lee, Kathryn Wellen, Zachary Schug, Joseph Baur, Donna George, Maureen Murphy. The African-specific S47 variant of the p53 tumor suppressor gene alters cell metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3483.

Nutritional preconditioning induced by astragaloside Ⅳ on isolated hearts and cardiomyocytes against myocardial ischemia injury via improving Bcl-2-mediated mitochondrial function

Ischemic preconditioning and pharmacological preconditioning are common strategies to prevent lethal myocardial injury, especially nutritional preconditioning (NPC). In this study, we investigated the effects of astragaloside IV (Ast), as an NPC agent, on myocardium suffered anoxia/reoxygenation (A/R) injury. Rats received 5 mg/kg Ast daily for 3 weeks by intragastric administration. Then, hearts were harvested and underwent A/R treatment using a Langendorff apparatus. Ast- pretreatment significantly promoted functional recovery of the myocardium, reduced infarct size, and oxidative stress, and decreased the apoptotic index. Similar findings were demonstrated in H9c2 cardiomyocytes that were pretreated with Ast for 24 h. Moreover, Ast-pretreatment significantly upregulated Bcl-2 expression, especially in mitochondria. The effects of Ast treatment against A/R injury were also reflected by increased antioxidant potential, inhibited reactive oxygen species (ROS) burst, increased oxygen consumption rate, maintained mitochondrial membrane potential (MMP), inhibited mitochondrial permeability transition pore (mPTP) opening, and prevented apoptosis. Selective inhibition of Bcl-2 by ABT-737 decreased myocardial injury protection of Ast. Ast-pretreatment resulted in NPC- related effects against A/R, and mitochondria may be the target of a cascade of events elicited by upregulating Bcl-2 expression, promoting translocation of Bcl-2 into mitochondria, maintaining MMP, inhibiting ROS bursts, thereby leading to recovery of mitochondrial respiration, preventing mPTP opening, decreasing cytochrome C release, preventing apoptosis, and ultimately alleviating myocardial injury.

1990-P: STAT1 Knockdown in CD11c+ Cells Prevents Adipose Tissue Inflammation and Metabolic Dysfunction in Mice on High-Fat Diet

Background: Classically activated M1-like CD11c+ macrophages/dendritic cells (MDCs) are increased in obese adipose tissue (AT) and may contribute to AT inflammation and the development of insulin resistance in obesity. STAT1 is a key transcription factor for MDC polarization into M1-like phenotypes. Here, we examined the role of STAT1 in obesity-induced AT MDC polarization and inflammation and insulin resistance in mice. Methods: Mice with specific knockout (KO) of STAT1 in CD11c+ MDCs were generated by crossbreeding STAT1fl/fl and CD11c-Cre mice. CD11c/STAT1 KO and littermate controls were fed high-fat diet (HFD, 16 weeks) to induce obesity and evaluated for immune cells and browning/beige adipogenesis in perigonadal (pAT) and inguinal (iAT) AT and metabolic functions. Results: Compared to control mice, KO mice on HFD had similar body weight. Analyses of AT immune cells revealed that compared to controls, KO mice had a decrease in M1-like proinflammatory polarization but an increase in M2-like polarization of macrophages and reduced CD8+ T cell number in pAT, and significant increases in the proportion of IL-5+ Th2 cells and eosinophils (CD170+) in pAT and iAT (p<0.05). Furthermore, compared to control mice, KO mice showed significantly increased iAT expression of browning markers (Ucp-1, Cidea, and prdm16) and had increased oxygen consumption rate but lower respiratory exchange ratio, indicating higher energy expenditure and increased fat utilization as energy source. Moreover, KO mice, as compared to controls, had significant reductions in triglyceride content in the liver and skeletal muscle and exhibited improved insulin sensitivity and glucose tolerance examined by insulin and glucose tolerance test (p<0.05). Conclusion: STAT1 plays an important role in obesity-induced MDC M1-like polarization and AT inflammation and contributes to insulin resistance and metabolic dysfunctions in obese mice. Disclosure A. Kalathookunnel Antony: None. X. Perrard: None. Z. Lian: None. J. Perrard: None. C.M. Ballantyne: Consultant; Self; Abbott, Akcea Therapeutics, Amarin Corporation, Amgen Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Esperion, Gilead Sciences, Inc., Matinas BioPharma, Merck & Co., Inc., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Regeneron Pharmaceuticals, Roche Diagnostic USA, Sanofi. Research Support; Self; Abbott, Akcea Therapeutics, Amarin Corporation, Amgen Inc., Esperion, Novartis Pharmaceuticals Corporation, Regeneron Pharmaceuticals, Roche Diagnostic USA, Sanofi. Other Relationship; Self; Roche Diagnostic USA. H. Wu: None. Funding American Diabetes Association (1-17-IBS-082 to H.W.)

288-LB: Effects of the Hepatic Mitochondrial Calcium Uniporter on Hepatic Gluconeogenesis and Mitochondrial Metabolism in Awake Mice

Increased hepatic glucose production, due to increased rates of gluconeogenesis, is a contributor to both fasting and postprandial hyperglycemia in patients with T2D. Recent evidence implicates a role for hepatic calcium signaling in mediating glucagon's effect to stimulate glucose production and mitochondrial oxidation; however, the cellular and molecular mechanisms by which glucagon promotes these alterations remain poorly understood. We hypothesize that an acute physiological increase in plasma glucagon concentrations will increase cytosolic and mitochondrial calcium concentrations, promoting increases in rates of hepatic mitochondrial oxidation and gluconeogenesis in a transcription-independent manner. To test this hypothesis, we used mice with a liver-specific loss of the mitochondrial calcium uniporter (MCU), which is the main transporter that allows rapid transport of calcium into the mitochondrial matrix. We found that glucagon increased oxygen consumption rate by 80% (p<0.0001) in WT hepatocytes, while this effect was abrogated in MCU KO hepatocytes. We also measured hepatic mitochondrial oxidation (VCS) in vivo using Positional Isotopomer NMR Tracer Analysis (PINTA) and found a 74% decrease in VCS (p=0.05) in MCU KO mice compared to WT mice. This suggests that tightly regulated mitochondrial calcium signaling is necessary for regulating hepatic mitochondrial oxidation. Next, we measured intrahepatic lipolysis and hepatic gluconeogenesis in vitro and in vivo. We found that glucagon increased lipolysis (p=0.05) and glucose production (p=0.024) by ∼1.5 fold in MCU KO compared to WT hepatocytes. Furthermore, in vivo PINTA results showed that glucagon increased pyruvate carboxylase flux (VPC) by 68% (p=0.08) in MCU KO mice. Taken together, these results suggest that mitochondrial calcium signaling through MCU is necessary to regulate the effect of glucagon to modulate hepatic gluconeogenesis and mitochondrial oxidation. Disclosure J. Lee: None. L. Goedeke: None. Y. Zhang: None. R.J. Perry: Research Support; Self; AstraZeneca. R. Goodman: None. V. Mootha: Advisory Panel; Self; Janssen Pharmaceuticals, Inc. Consultant; Self; 5AM Ventures. Other Relationship; Self; Raze Therapeutics. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding National Institutes of Health

MiR-150-5p regulates melanoma proliferation, invasion and metastasis via SIX1-mediated Warburg Effect

Aerobic glycolysis is a hallmark of cancer. Sine oculis homeobox 1 (SIX1), a key transcription factor in terms of regulating aerobic glycolysis (the Warburg Effect), plays a critical role in tumorigenesis of various cancer types, including breast cancer, liver cancer, and lung cancer. However, the upstream regulating mechanisms of SIX1 in melanoma remain to be determined. MicroRNAs (miRNAs) have emerged as key regulators in tumorigenesis and progression. Here, we initially showed that microRNA-150-5p (miR-150-5p) inhibits SIX1 expression by directly targeting its 3′-UTR in melanoma cells. miR-150-5p suppressed melanoma cell proliferation, migration, and invasion through inhibition of SIX1. Mechanistically, miR-150-5p dampens glycolysis by decreasing the glucose uptake, lactate production, ATP generation, and extracellular acidification rate (ECAR), and increasing oxygen consumption rate (OCR) by targeting SIX1. Importantly, glycolysis regulated by miR-150-5p/SIX1 axis is critical for its regulation of melanoma growth and metastasis both in vitro and in vivo. Collectively, our study demonstrates the importance of miR-150-5p/SIX1 axis in melanoma, which could be a promising therapeutic target in melanoma.

Development of novel human lactate dehydrogenase A inhibitors: High-throughput screening, synthesis, and biological evaluations

Human lactate dehydrogenase A (LDHA) plays a critical role in the glycolytic process, making the enzyme an ideal of anti-cancer drug target. Herein, we report the discovery of novel potent LDHA inhibitors by screening an in-house library. The hit-to-lead modification enabled us to identify compound 24c, which inhibited LDHA activity with an EC50 value of 90 nM, and reduced MiaPaCa-2 cancer cell proliferation with an IC50 value of 2.1 μM. In line with the in vitro anticancer activity, 24c suppressed the tumor growth at a dose of 10 mg/kg in a MiaPaCa-2 cells xenograft model, but with little effect to the mice weight. Moreover, 24c strongly inhibited MiaPaCa-2 cell colonies formation, induced MiaPaCa-2 cell apoptosis, and arrested MiaPaCa-2 cell cycle at G2 phase. In addition, the mitochondrial bioenergetics analysis suggested that 24c could reprogram cancer cell metabolic pathways from glycolysis to oxidation phosphorylation, which verified by decreasing the extracellular acidification rates and lactate formation, and increasing oxygen consumption rate in cancer cell. All these results indicate 24c is a promising metabolic modulator for the anticancer drug development.

Heterozygous knockdown insulin‐like growth factor‐1 receptor (IGF‐1R) regulates mitochondrial functions and prevents colitis and colorectal cancer

Although insulin‐like growth factor‐1 receptor (IGF‐1R) has been accepted as a major determinant of tumorigenesis, the mechanisms of its roles in the pathogenesis of cancers have remained elusive. Herein, we demonstrate that IGF‐1R plays pivotal roles in regulation of mitochondrial functions and resistance to oxidative stress in colitis and colorectal tumorigenesis. Heterozygous knockdown IGF‐1R attenuated azoxymethane (AOM)/dextran sulfate sodium (DSS)‐induced colitis and colitis associated cancer (CAC) in Igf1r+/− mice. Knockdown of IGF‐1R confers the resistance to oxidative stress‐induced damage on colorectal epithelium through protection the mitochondrial dynamics and structures. Knockdown of IGF‐1R develops the function of mitochondrial fusion under oxidative stress. Mechanically, an increase of mitochondrial coupling index RCI and oxidative phosphorylation index ADP/O was seen in the epithelial cells of Igf1r+/− mice. Seahorse XFe‐24 analyzer analysis of mitochondrial bioenergetics showed an increase of oxygen consumption rate (OCR) and a decrease of extracellular acidification rate (ECAR) in Igf1r+/− cells. Further analysis suggests the protection mechanisms of colorectal epithelial cells from oxidative stress through activating mitochondrial respiratory chain and the LKB1/AMPK pathways. These results support the roles of IGF‐1R at the nexus between oxidative damage and mitochondrial function and connect colitis and colorectal tumorigenesis.Support or Funding InformationThis work was supported by Natural Science Foundation of China (81673449, 91629303).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic and oncogenic adaptations to pyruvate dehydrogenase inactivation in fibroblasts

Eukaryotic cell metabolism consists of processes that generate available energy, such as glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (Oxphos), and those that consume it, including macromolecular synthesis, the maintenance of ionic gradients, and cellular detoxification. By converting pyruvate to acetyl-CoA (AcCoA), the pyruvate dehydrogenase (PDH) complex (PDC) links glycolysis and the TCA cycle. Surprisingly, disrupting the connection between glycolysis and the TCA cycle by inactivation of PDC has only minor effects on cell replication. However, the molecular basis for this metabolic re-equilibration is unclear. We report here that CRISPR/Cas9-generated PDH-knockout (PDH-KO) rat fibroblasts reprogrammed their metabolism and their response to short-term c-Myc (Myc) oncoprotein overexpression. PDH-KO cells replicated normally but produced surprisingly little lactate. They also exhibited higher rates of glycolysis and Oxphos. In addition, PDH-KO cells showed altered cytoplasmic and mitochondrial pH, redox states, and mitochondrial membrane potential (ΔΨM). Conditionally activated Myc expression affected some of these parameters in a PDH-dependent manner. PDH-KO cells had increased oxygen consumption rates in response to glutamate, but not to malate, and were depleted in all TCA cycle substrates between α-ketoglutarate and malate despite high rates of glutaminolysis, as determined by flux studies with isotopically labeled glutamine. Malate and pyruvate were diverted to produce aspartate, thereby potentially explaining the failure to accumulate lactate. We conclude that PDH-KO cells maintain proliferative capacity by utilizing glutamine to supply high rates of AcCoA-independent flux through the bottom portion of the TCA cycle while accumulating pyruvate and aspartate that rescue their redox defects.

Hypoxic Conditions in Crown Galls Induce Plant Anaerobic Responses That Support Tumor Proliferation.

Agrobacterium tumefaciens infection of wounded plant tissues causes the formation of crown gall tumors. Upon infection, genes encoded on the A. tumefaciens tumor inducing plasmid are integrated in the plant genome to induce the biosynthesis of auxin and cytokinin, leading to uncontrolled cell division. Additional sequences present on the bacterial T-DNA encode for opine biosynthesis genes, which induce the production of opines that act as a unique carbon and nitrogen source for Agrobacterium. Crown galls therefore become a very strong sink for photosynthate. Here we found that the increased metabolic demand in crown galls causes an increase in oxygen consumption rate, which leads to a steep drop in the internal oxygen concentration. Consistent with this, plant hypoxia-responsive genes were found to be significantly upregulated in crown galls compared to uninfected stem tissue. Following this observation, we aimed at understanding whether the low-oxygen response pathway, mediated by group VII ethylene response factor (ERF-VII) transcription factors, plays a role in the development of crown galls. We found that quintuple knock-out mutants of all ERF-VII members, which are incapable of inducing the hypoxic response, show reduced crown gall symptoms. Conversely, mutant genotypes characterized by constitutively high levels of hypoxia-associated transcripts, displayed more severe crown gall symptoms. Based on these results, we concluded that uncontrolled cell proliferation of crown galls established hypoxic conditions, thereby requiring adequate anaerobic responses of the plant tissue to support tumor growth.

Telmisartan induces browning of fully differentiated white adipocytes via M2 macrophage polarization

Telmisartan is a well-known anti-hypertensive drug acting as an angiotensin 2 receptor blocker (ARB), but it also possesses partial PPARγ agonistic activity and induces insulin sensitivity. In the present study, we investigated the effects of telmisartan on macrophage polarization in association with its browning capacity, because PPARγ plays a key role in M2 polarization and in the browning of white adipocytes. Telmisartan induced M2 marker expression in murine macrophages concentration dependently, which was confirmed by flow cytometry. Both PPARγ and PPARδ activations appear to be responsible for telmisartan-induced M2 polarization. Telmisartan-treated conditioned medium (Tel-CM) of RAW264.7 cells and of bone marrow derived macrophages (BMDM) induced the expressions of browning markers in fully differentiated white adipocytes with reduced lipid droplets, and increased oxygen consumption rate and mitochondrial biogenesis. Levels of catecholamines (CA) released into the conditioned medium as well as intracellular tyrosine hydroxylase (TH) mRNAs were found to be increased by telmisartan, and browning effects of Tel-CM were lessened by β3 receptor antagonist (L-748,337), suggesting CA secreted into CM play a role in Tel-CM-induced adipocyte browning. Acute administration of telmisartan (2 weeks, p.o.) to C57BL/6J mice increased the expressions of browning markers and M2 markers in white adipose tissues, whereas macrophage depletion by clodronate liposome pretreatment attenuated the telmisartan-induced expressions of browning markers. Together, telmisartan was observed to induce the browning of fully differentiated white adipocytes, at least in part, via PPAR activation-mediated M2 polarization.

Study of diffusive- and convective-transport mediated microtumor growth in a controlled microchamber.

In this paper, we report on using mass transport to control nutrition supply of colorectal cancer cells for developing a microtumor in a confined microchamber. To mimic the spatial heterogeneity of a tumor, two microfluidic configurations based on resistive circuits are designed. One has a convection-dominated microchamber to simulate the tumor region proximal to leaky blood vessels. The other has a diffusion-dominated microchamber to mimic the tumor core that lacks blood vessels and nutrient supply. Thus, the time for nutrition to fill the microchamber can vary from tens of minutes to several hours. Results show that cells cultured under a diffusive supply of nutrition have a high glycolytic rate and a nearly constant oxygen consumption rate. In contrast, cells cultured under convective supply of nutrition have a gradual increase of oxygen consumption rate with a low glycolytic rate. This suggests that cancer cells have distinct reactions under different mass transport and nutrition supply. Using these two microfluidic platforms to create different rate of nutrition supply, it is found that a continuous microtumor that almost fills the mm-size microchamber can be developed under a low-nutrient supply environment, but not for the convective condition. It also is demonstrated that microchannels can simulate the delivery of anti-cancer drugs to the microtumor under controlled mass-transport. This method provides a means to develop a larger scale microtumor in a lab-on-a-Chip system for post development and stimulations, and microchannels can be applied to control the physical and chemical environment for anti-cancer drug screening.

Heterozygous knockout insulin-like growth factor-1 receptor (IGF-1R) regulates mitochondrial functions and prevents colitis and colorectal cancer

Although insulin-like growth factor-1 receptor (IGF-1R) has been accepted as a major determinant of cancers, its biological roles and corresponding mechanisms in tumorigenesis have remained elusive. Herein, we demonstrate that IGF-1R plays pivotal roles in the regulation of mitochondrial respiratory chain and functions during colitis and tumorigenesis. Heterozygous knockout IGF-1R attenuated azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis and colitis associated cancer (CAC) in Igf1r+/- mice. Heterozygous knockout IGF-1R confers resistance to oxidative stress-induced damage on colorectal epithelial cells by protecting mitochondrial dynamics and structures. IGF-1R low expression improves the biological function of mitochondrial fusion under oxidative stress. Mechanically, an increase in respiratory coupling index (RCI) and oxidative phosphorylation index (ADP/O) was seen in colorectal epithelial cells of Igf1r+/- mice. Seahorse XFe-24 analyzer analysis of mitochondrial bioenergetics demonstrated an increase in oxygen consumption rate (OCR) and a decrease of extracellular acidification rate (ECAR) in Igf1r+/- cells. Further analysis suggests the protection mechanisms of Igf1r+/- cells from oxidative stress through the activation of the mitochondrial respiratory chain and LKB1/AMPK pathways. These results highlight the biological roles of IGF-1R at the nexus between oxidative damage and mitochondrial function and a connection between colitis and colorectal cancer.

Detection of respiration changes inside biofilms with microelectrodes during exposure to antibiotics

In order to evaluate the influence of antibiotics on the respiration of subpopulations in biofilms, dissolved oxygen microelectrodes were used to determine the oxygen concentration inside E. Coli biofilms. Based on the results, the oxygen consumption rate varying with depth in the biofilms was calculated before and after the treatment with antibiotics. For both ceftriaxone sodium and chloramphenicol, significant attenuation of oxygen consumption was identified when the system reached steady state. However, by monitoring the oxygen concentration change induced by ceftriaxone sodium, an increased oxygen consumption rate and a shortened oxygen penetration depth were identified in the first few hours, which indicated the accelerated respiration of the subpopulation. Followed by cellular death, the induced respiration acceleration seems to be part of the action mechanism of ceftriaxone sodium. Compared to the planktonic cells, the biofilms showed much higher tolerance to ceftriaxone sodium, which may be attributed to the extended hypoxic zone induced by the accelerated respiration.

Vitamin D produces a perilipin 2-dependent increase in mitochondrial function in C2C12 myotubes

Vitamin D has been connected with increased intramyocellular lipid (IMCL) and has also been shown to increase mitochondrial function and insulin sensitivity. Evidence suggests that perilipin 2 (PLIN2), a perilipin protein upregulated with calcitriol treatment, may be integral to managing increased IMCL capacity and lipid oxidation in skeletal muscle. Therefore, we hypothesized that PLIN2 is required for vitamin D induced IMCL accumulation and increased mitochondrial oxidative function. To address this hypothesis, we treated C2C12 myotubes with 100 nM calcitriol (the active form of vitamin D) and/or PLIN2 siRNA in a four group design and analyzed markers of IMCL accumulation and metabolism using qRT-PCR, cytochemistry, and oxygen consumption assay. Expression of PLIN2, but not PLIN3 or PLIN5 mRNA was increased with calcitriol, and PLIN2 induction was prevented with siRNA knockdown without compensation by other perilipins. PLIN2 knockdown did not appear to prevent lipid accumulation. Calcitriol treatment increased mRNA expression of triglyceride synthesizing genes DGAT1 and DGAT2 and also lipolytic genes ATGL and CGI-58. PLIN2 knockdown decreased the expression of CGI-58 and CPT1, and was required for calcitriol-induced upregulation of DGAT2. Calcitriol increased oxygen consumption rate while PLIN2 knockdown decreased oxygen consumption rate. PLIN2 was required for a calcitriol-induced increase in oxygen consumption driven by mitochondrial complex II. We conclude that calcitriol increases mitochondrial function in myotubes and that this increase is at least in part mediated by PLIN2.

Benzoylaconine induces mitochondrial biogenesis in mice via activating AMPK signaling cascade.

The traditional Chinese medicine "Fuzi" (Aconiti Lateralis Radix Praeparata) and its three representative alkaloids, aconitine (AC), benzoylaconine (BAC), and aconine, have been shown to increase mitochondrial mass. Whether Fuzi has effect on mitochondrial biogenesis and the underlying mechanisms remain unclear. In the present study, we focused on the effect of BAC on mitochondrial biogenesis and the underlying mechanisms. We demonstrated that Fuzi extract and its three components AC, BAC, and aconine at a concentration of 50 μM significantly increased mitochondrial mass in HepG2 cells. BAC (25, 50, 75 μM) dose-dependently promoted mitochondrial mass, mtDNA copy number, cellular ATP production, and the expression of proteins related to the oxidative phosphorylation (OXPHOS) complexes in HepG2 cells. Moreover, BAC dose-dependently increased the expression of proteins involved in AMPK signaling cascade; blocking AMPK signaling abolished BAC-induced mitochondrial biogenesis. We further revealed that BAC treatment increased the cell viability but not the cell proliferation in HepG2 cells. These in vitro results were verified in mice treated with BAC (10 mg/kg per day, ip) for 7 days. We showed that BAC administration increased oxygen consumption rate in mice, but had no significant effect on intrascapular temperature. Meanwhile, BAC administration increased mtDNA copy number and OXPHOS-related protein expression and activated AMPK signaling in the heart, liver, and muscle. These results suggest that BAC induces mitochondrial biogenesis in mice through activating AMPK signaling cascade. BAC may have the potential to be developed as a novel remedy for some diseases associated with mitochondrial dysfunction.

VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function.

Dysregulated mitochondrial function is associated with the pathology of a wide range of diseases including renal disease and cancer. Thus, investigating regulators of mitochondrial function is of particular interest. Previous work has shown that the von Hippel-Lindau tumor suppressor protein (pVHL) regulates mitochondrial biogenesis and respiratory chain function. pVHL is best known as an E3-ubiquitin ligase for the α-subunit of the hypoxia inducible factor (HIF) family of dimeric transcription factors. In normoxia, pVHL recognizes and binds hydroxylated HIF-α (HIF-1α and HIF-2α), targeting it for ubiquitination and proteasomal degradation. In this way, HIF transcriptional activity is tightly controlled at the level of HIF-α protein stability. At least 80% of clear cell renal carcinomas exhibit inactivation of the VHL gene, which leads to HIF-α protein stabilization and constitutive HIF activation. Constitutive HIF activation in renal carcinoma drives tumor progression and metastasis. Reconstitution of wild-type VHL protein (pVHL) in pVHL-defective renal carcinoma cells not only suppresses HIF activation and tumor growth, but also enhances mitochondrial respiratory chain function via mechanisms that are not fully elucidated. Here, we show that pVHL regulates mitochondrial function when re-expressed in pVHL-defective 786O and RCC10 renal carcinoma cells distinct from its regulation of HIF-α. Expression of CHCHD4, a key component of the disulphide relay system (DRS) involved in mitochondrial protein import within the intermembrane space (IMS) was elevated by pVHL re-expression alongside enhanced expression of respiratory chain subunits of complex I (NDUFB10) and complex IV (mtCO-2 and COX IV). These changes correlated with increased oxygen consumption rate (OCR) and dynamic changes in glucose and glutamine metabolism. Knockdown of HIF-2α also led to increased OCR, and elevated expression of CHCHD4, NDUFB10, and COXIV in 786O cells. Expression of pVHL mutant proteins (R200W, N78S, D126N, and S183L) that constitutively stabilize HIF-α but differentially promote glycolytic metabolism, were also found to differentially promote the pVHL-mediated mitochondrial phenotype. Parallel changes in mitochondrial morphology and the mitochondrial network were observed. Our study reveals a new role for pVHL in regulating CHCHD4 and mitochondrial function in renal carcinoma cells.

The dipeptidyl peptidase-4 (DPP-4) inhibitor teneligliptin enhances brown adipose tissue function, thereby preventing obesity in mice.

To clarify the effects of a dipeptidyl peptidase‐4 (DPP‐4) inhibitor on whole‐body energy metabolism, we treated mice fed a high‐fat diet (HFD) with teneligliptin, a clinically available DPP‐4 inhibitor. Teneligliptin significantly prevented HFD‐induced obesity and obesity‐associated metabolic disorders. It also increased oxygen consumption rate and upregulated uncoupling protein 1 (UCP1) expression in both brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT), suggesting that it enhances BAT function. Soluble DPP‐4 inhibited β‐adrenoreceptor‐stimulated UCP1 expression in primary adipocytes, and this inhibition was prevented in the presence of teneligliptin, or an extracellular signal‐related kinase inhibitor. These results indicate that soluble DPP‐4 inhibits β‐adrenoreceptor‐stimulated UCP1 induction and that chronic DPP‐4 inhibitor treatment may prevent obesity through the activation of BAT function.