The Niemann-Pick C1 gene interacts with a high-fat diet to promote weight gain through differential regulation of central energy metabolism pathways.

A genome-wide association study (GWAS) reported that common variation in the human Niemann-Pick C1 gene (NPC1) is associated with morbid adult obesity. This study was confirmed using our BALB/cJ Npc1 mouse model, whereby heterozygous mice (Npc1+/- ) with decreased gene dosage were susceptible to weight gain when fed a high-fat diet (HFD) compared with homozygous normal mice (Npc1+/+ ) fed the same diet. The objective for our current study was to validate this Npc1 gene-diet interaction using statistical modeling with fitted growth trajectories, conduct body weight analyses for different measures, and define the physiological basis responsible for weight gain. Metabolic phenotype analysis indicated no significant difference between Npc1+/+ and Npc1+/- mice fed a HFD for food and water intake, oxygen consumption, carbon dioxide production, locomotor activity, adaptive thermogenesis, and intestinal lipid absorption. However, the livers from Npc1+/- mice had significantly increased amounts of mature sterol regulatory element-binding protein-1 (SREBP-1) and increased expression of SREBP-1 target genes that regulate glycolysis and lipogenesis with an accumulation of triacylglycerol and cholesterol. Moreover, white adipose tissue from Npc1+/- mice had significantly decreased amounts of phosphorylated hormone-sensitive lipase with decreased triacylglycerol lipolysis. Consistent with these results, cellular energy metabolism studies indicated that Npc1+/- fibroblasts had significantly increased glycolysis and lipogenesis, in addition to significantly decreased substrate (glucose and endogenous fatty acid) oxidative metabolism with an accumulation of triacylglycerol and cholesterol. In conclusion, these studies demonstrate that the Npc1 gene interacts with a HFD to promote weight gain through differential regulation of central energy metabolism pathways.

Effect of novel dietary supplement on metabolism in vitro and in vivo.

Obesity is an increasingly prevalent and preventable morbidity with multiple behavioral, surgical and pharmacological interventions currently available. Commercial dietary supplements are often advertised to stimulate metabolism and cause rapid weight and/or fat loss, although few well-controlled studies have demonstrated such effects. We describe a commercially available dietary supplement (purportedly containing caffeine, catechins, and other metabolic stimulators) on resting metabolic rate in humans, and on metabolism, mitochondrial content, and related gene expression in vitro. Human males ingested either a placebo or commercially available supplement (RF) in a randomized double-blind placebo-controlled cross-over fashion. Metabolic rate, respiratory exchange ratio, and blood pressure were measured hourly for 3 h post-ingestion. To investigate molecular effects, human rhabdomyosarcoma cells (RD) and mouse myocytes (C2C12) were treated with various doses of RF for various durations. RF enhanced energy expenditure and systolic blood pressure in human males without altering substrate utilization. In myocytes, RF enhanced metabolism, metabolic gene expression, and mitochondrial content suggesting RF may target common energetic pathways which control mitochondrial biogenesis. RF appears to increase metabolism immediately following ingestion, although it is unclear if RF provides benefits beyond those provided by caffeine alone. Additional research is needed to examine safety and efficacy for human weight loss.

Towards a personalized surgical margin for breast conserving surgery-Implications of field cancerization in local recurrence.

The amount of normal tissue that should be excised during breast conserving surgery is widely debated. Tissue adjacent to breast tumors, although histologically normal, possesses many of the molecular abnormalities found in tumor tissues. Here, we propose that the ideal physical distance for a surgical margin may not be universal. Rather, an adequate surgical margin likely varies from patient to patient, depending on the biology of the tissue that remains after surgery. J. Surg. Oncol. 2017;115:109-115. © 2017 Wiley Periodicals, Inc.

Effects of the exercise-inducible myokine irisin on malignant and non-malignant breast epithelial cell behavior in vitro.

Exercise has been shown to reduce risk and improve prognosis of several types of cancers. Irisin is a myokine linked to exercise and lean body mass, which is thought to favorably alter metabolism systemically, potentially providing benefit for metabolic disease (including cancer). We evaluated the effects of various concentrations of irisin (with and without post-translational modifications) on malignant and non-malignant breast epithelial cell number, migration and viability. Irisin significantly decreased cell number, migration and viability in malignant MDA-MB-231 cells, without affecting non-malignant MCF-10a cells. Moreover, irisin enhanced the cytotoxic effect of doxorubicin (Dox) when added to a wide spectrum of irisin concentrations in the malignant cell type (with simultaneous reduction in Dox uptake), which was not observed in non-malignant MCF-10a cells. Additionally, we found that irisin decreases malignant cell viability in part through stimulation of caspase activity leading to apoptotic death. Interestingly, we found that irisin suppresses NFκB activation, an opposite effect of other myokines such as tumor necrosis factor alpha (TNF-α). Our observations suggest that irisin may offer therapeutic benefits for breast cancer prevention and treatment possibly through an anti-inflammatory response, induction of apoptotic cell death, or through enhanced tumor sensitivity to common antineoplastic agents such as Dox.

ß-alanine suppresses malignant breast epithelial cell aggressiveness through alterations in metabolism and cellular acidity in vitro.

BACKGROUND: Deregulated energetics is a property of most cancer cells. This phenomenon, known as the Warburg Effect or aerobic glycolysis, is characterized by increased glucose uptake, lactate export and extracellular acidification, even in the presence of oxygen. ß-alanine is a non-essential amino acid that has previously been shown to be metabolized into carnosine, which functions as an intracellular buffer. Because of this buffering capacity, we investigated the effects of ß-alanine on the metabolic cancerous phenotype. METHODS: Non-malignant MCF-10a and malignant MCF-7 breast epithelial cells were treated with ß-alanine at 100 mM for 24 hours. Aerobic glycolysis was quantified by measuring extracellular acidification rate (ECAR) and oxidative metabolism was quantified by measuring oxygen consumption rate (OCR). mRNA of metabolism-related genes was quantified by qRT-PCR with corresponding protein expression quantified by immunoblotting, or by flow cytometry which was verified by confocal microscopy. Mitochondrial content was quantified using a mitochondria-specific dye and measured by flow cytometry. RESULTS: Cells treated with ß-alanine displayed significantly suppressed basal and peak ECAR (aerobic glycolysis), with simultaneous increase in glucose transporter 1 (GLUT1). Additionally, cells treated with ß-alanine exhibited significantly reduced basal and peak OCR (oxidative metabolism), which was accompanied by reduction in mitochondrial content with subsequent suppression of genes which promote mitochondrial biosynthesis. Suppression of glycolytic and oxidative metabolism by ß-alanine resulted in the reduction of total metabolic rate, although cell viability was not affected. Because ß-alanine treatment reduces extracellular acidity, a constituent of the invasive microenvironment that promotes progression, we investigated the effect of ß-alanine on breast cell viability and migration. ß-alanine was shown to reduce both cell migration and proliferation without acting in a cytotoxic fashion. Moreover, ß-alanine significantly increased malignant cell sensitivity to doxorubicin, suggesting a potential role as a co-therapeutic agent. CONCLUSION: Taken together, our results suggest that ß-alanine may elicit several anti-tumor effects. Our observations support the need for further investigation into the mechanism(s) of action and specificity of ß-alanine as a co-therapeutic agent in the treatment of breast tumors.

Leucine treatment enhances oxidative capacity through complete carbohydrate oxidation and increased mitochondrial density in skeletal muscle cells.

Leucine has been largely implicated for increasing muscle protein synthesis in addition to stimulating mitochondrial biosynthesis. Limited evidence is currently available on the effects and potential benefits of leucine treatment on skeletal muscle cell glycolytic and oxidative metabolism. This work identified the effects of leucine treatment on oxidative and glycolytic metabolism as well as metabolic rate of human and murine skeletal muscle cells. Human rhabdomyosarcoma cells (RD) and mouse myoblast cells (C2C12) were treated with leucine at either 100 or 500 µM for 24 or 48 h. Glycolytic metabolism was quantified by measuring extracellular acidification rate (ECAR) and oxidative metabolism was quantified by measuring oxygen consumption rate. Peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α), an important stimulator of mitochondrial biosynthesis, was quantified using flow cytometry and verified by immunofluorescent confocal microscopy. Mitochondrial content was quantified using mitochondrial and cytochrome C staining measured by flow cytometry and confirmed with confocal microscopy. Treatment with leucine significantly increased both basal and peak oxidative metabolism in both cell models. Leucine treated cells also exhibited significantly greater mitochondrial proton leak, which is associated with heightened energy expenditure. Basal ECAR was significantly reduced in both cell models following leucine treatment, evidence of reduced lactate export and more complete carbohydrate oxidation. In addition, both PGC-1α and cytochrome C expression were significantly elevated in addition to mitochondrial content following 48 h of leucine treatment. Our observations demonstrated few dose-dependent responses induced by leucine; however, leucine treatment did induce a significant dose-dependent expression of PGC-1α in both cell models. Interestingly, C2C12 cells treated with leucine exhibited dose-dependently reduced ATP content, while RD ATP content remain unchanged. Leucine presents a potent dietary constituent with low lethality with numerous beneficial effects for increasing oxidative preference and capacity in skeletal muscle. Our observations demonstrate that leucine can enhance oxidative capacity and carbohydrate oxidation efficiency, as well as verify previous observations of increased mitochondrial content.

Tumor necrosis factor alpha increases aerobic glycolysis and reduces oxidative metabolism in prostate epithelial cells.

BACKGROUND: Chronic inflammation promotes prostate cancer formation and progression. Furthermore, alterations in energy metabolism are a hallmark of prostate cancer cells. However, the actions of inflammatory factors on the energy metabolism of prostate epithelial cells have not been previously investigated. This is the first study to report on the effect of the inflammatory cytokine tumor necrosis factor alpha (TNFα) on the glycolytic and oxidative metabolism, and the mitochondrial function of widely used prostate epithelial cells. METHODS: Pre-malignant RWPE-1 and cancerous LNCaP and PC-3 cells were treated with low-dose TNFα. Glycolytic and oxidative metabolism was quantified by measuring extracellular acidification and oxygen consumption rates, respectively. ATP content and lactate export were measured by luminescence and fluorescence, respectively. Mitochondrial content and the expression of glucose transporter 1 (GLUT1), peroxisome proliferator-activated receptor co-activator 1 alpha (PGC-1α), and Cytochrome C were measured by flow cytometry. RESULTS: Our data suggest that TNFα increases glycolysis, ATP production, and lactate export, while it reduces oxidative metabolism and mitochondrial function in prostate epithelial cells. The highly aggressive PC-3 cells tend to be less responsive to the actions of TNFα than the pre-malignant RWPE-1 and the non-aggressive LNCaP cells. CONCLUSIONS: Cellular energetics, that is, glycolytic and oxidative metabolism is significantly influenced by low-level inflammation in prostate epithelial cells. In widely used prostate epithelial cell models, the micro-environmental inflammatory cytokine TNFα induces aerobic glycolysis while inhibiting oxidative metabolism. This supports the hypothesis that low-level inflammation can induce Warburg metabolism in prostate epithelial cells, which may promote cancer formation and progression.

Tumor necrosis factor alpha induces Warburg-like metabolism and is reversed by anti-inflammatory curcumin in breast epithelial cells.

The reprogramming of cellular metabolism in cancer cells is a well-documented effect. It has previously been shown that common oncogene expression can induce aerobic glycolysis in cancer cells. However, the direct effect of an inflammatory microenvironment on cancer cell metabolism is not known. Here, we illustrate that treatment of nonmalignant (MCF-10a) and malignant (MCF-7) breast epithelial cells with low-level (10 ng/ml) tumor necrosis factor alpha (TNF-α) significantly increased glycolytic reliance, lactate export and expression of the glucose transporter 1 (GLUT1). TNF-α decreased total mitochondrial content; however, oxygen consumption rate was not significantly altered, suggesting that overall mitochondrial function was increased. Upon glucose starvation, MCF7 cells treated with TNF-α demonstrated significantly lower viability than nontreated cells. Interestingly, these properties can be partially reversed by coincubation with the anti-inflammatory agent curcumin in a dose-dependent manner. This work demonstrates that aerobic glycolysis can be directly induced by an inflammatory microenvironment independent of additional genetic mutations and signals from adjacent cells. Furthermore, we have identified that a natural dietary compound can reverse this effect.

Ubiquinol rescues simvastatin-suppression of mitochondrial content, function and metabolism: implications for statin-induced rhabdomyolysis.

Statin medications diminish cholesterol biosynthesis and are commonly prescribed to reduce cardiovascular disease. Statins also reduce production of ubiquinol, a vital component of mitochondrial energy production; ubiquinol reduction may contribute to rhabdomyolysis. Human rhabdomyosarcoma cells were treated with either ethanol and dimethyl sulfoxide (DMSO) control, or simvastatin at 5 µM or 10 µM, or simvastatin at 5 µM with ubiquinol at 0.5 µM or 1.0 µM for 24 h or 48 h. PGC-1α RNA levels were determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mitochondrial content was determined using flow cytometry and immunocytochemistry. Metabolism was determined by quantification of extracellular acidification rate and oxygen consumption rate. Treatment of human rhabdomyosarcoma cells with simvastatin significantly reduced oxidative, total metabolism, and cellular ATP content in a time- and dose-dependent manner which was rescued by concurrent treatment with ubiquinol. Treatment with simvastatin significantly reduced mitochondrial content as well as cell viability which were both rescued by simultaneous treatment with ubiquinol. This work demonstrates that the addition of ubiquinol to current statin treatment regimens may protect muscle cells from myopathies.

Treatment of human muscle cells with popular dietary supplements increase mitochondrial function and metabolic rate.

BACKGROUND: Obesity is a common pathology with increasing incidence, and is associated with increased mortality and healthcare costs. Several treatment options for obesity are currently available ranging from behavioral modifications to pharmaceutical agents. Many popular dietary supplements claim to enhance weight loss by acting as metabolic stimulators, however direct tests of their effect on metabolism have not been performed. PURPOSE: This work identified the effects popular dietary supplements on metabolic rate and mitochondrial biosynthesis in human skeletal muscle cells. METHODS: Human rhabdomyosarcoma cells were treated with popular dietary supplements at varied doses for 24 hours. Peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α), an important stimulator of mitochondrial biosynthesis, was quantified using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mitochondrial content was measured using flow cytometry confirmed with confocal microscopy. Glycolytic metabolism was quantified by measuring extracellular acidification rate (ECAR) and oxidative metabolism was quantified by measuring oxygen consumption rate (OCR). Total relative metabolism was quantified using WST-1 end point assay. RESULTS: Treatment of human rhabdomyosarcoma cells with dietary supplements OxyElite Pro (OEP) or Cellucore HD (CHD) induced PGC-1α leading to significantly increased mitochondrial content. Glycolytic and oxidative capacities were also significantly increased following treatment with OEP or CHD. CONCLUSION: This is the first work to identify metabolic adaptations in muscle cells following treatment with popular dietary supplements including enhanced mitochondrial biosynthesis, and glycolytic, oxidative and total metabolism.

Conjugated linoleic acid or omega 3 fatty acids increase mitochondrial biosynthesis and metabolism in skeletal muscle cells.

BACKGROUND: Polyunsaturated fatty acids are popular dietary supplements advertised to contribute to weight loss by increasing fat metabolism in liver, but the effects on overall muscle metabolism are less established. We evaluated the effects of conjugated linoleic acid (CLA) or combination omega 3 on metabolic characteristics in muscle cells. METHODS: Human rhabdomyosarcoma cells were treated with either DMSO control, or CLA or combination omega 3 for 24 or 48 hours. RNA was determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mitochondrial content was determined using flow cytometry and immunohistochemistry. Metabolism was quantified by measuring extracellular acidification and oxygen consumption rates. RESULTS: Omega 3 significantly induced metabolic genes as well as oxidative metabolism (oxygen consumption), glycolytic capacity (extracellular acidification), and metabolic rate compared with control. Both treatments significantly increased mitochondrial content. CONCLUSION: Omega 3 fatty acids appear to enhance glycolytic, oxidative, and total metabolism. Moreover, both omega 3 and CLA treatment significantly increase mitochondrial content compared with control.

Effects of caffeine on metabolism and mitochondria biogenesis in rhabdomyosarcoma cells compared with 2,4-dinitrophenol.

PURPOSE: This work investigated if treatment with caffeine or 2,4-dinitrophenol (DNP) induce expression of peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α) and increase both mitochondrial biosynthesis and metabolism in skeletal muscle. METHODS: Human rhabdomyosarcoma cells were treated with either ethanol control (0.1% final concentration) caffeine, or DNP at 250 or 500 µM for 16 or 24 hours. PGC-1α RNA levels were determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). PGC-1α protein and mitochondrial content was determined using flow cytometry and immunohistochemistry. Metabolism was determined by quantification of extracellular acidification rate and oxygen consumption rate. RESULTS: Treatment with either caffeine or DNP induced PGC-1α RNA and protein as well as mitochondrial content compared with control. Treatment with caffeine and DNP also significantly increased oxidative metabolism and total metabolic rate compared with control. Caffeine similarly increased metabolism and mitochondrial content compared with DNP. CONCLUSION: This work identified that both caffeine and DNP significantly induce PGC-1α, and increase both metabolism and mitochondrial content in skeletal muscle.