Energy metabolism in skeletal muscle cells from donors with different body mass index.

Obesity and physical inactivity have a profound impact on skeletal muscle metabolism. In the present work, we have investigated differences in protein expression and energy metabolism in primary human skeletal muscle cells established from lean donors (BMI<25 kg/m2) and individuals with obesity (BMI>30 kg/m2). Furthermore, we have studied the effect of fatty acid pretreatment on energy metabolism in myotubes from these donor groups. Alterations in protein expression were investigated using proteomic analysis, and energy metabolism was studied using radiolabeled substrates. Gene Ontology enrichment analysis showed that glycolytic, apoptotic, and hypoxia pathways were upregulated, whereas the pentose phosphate pathway was downregulated in myotubes from donors with obesity compared to myotubes from lean donors. Moreover, fatty acid, glucose, and amino acid uptake were increased in myotubes from individuals with obesity. However, fatty acid oxidation was reduced, glucose oxidation was increased in myotubes from subjects with obesity compared to cells from lean. Pretreatment of myotubes with palmitic acid (PA) or eicosapentaenoic acid (EPA) for 24 h increased glucose oxidation and oleic acid uptake. EPA pretreatment increased the glucose and fatty acid uptake and reduced leucine fractional oxidation in myotubes from donors with obesity. In conclusion, these results suggest that myotubes from individuals with obesity showed increased fatty acid, glucose, and amino acid uptake compared to cells from lean donors. Furthermore, myotubes from individuals with obesity had reduced fatty acid oxidative capacity, increased glucose oxidation, and a higher glycolytic reserve capacity compared to cells from lean donors. Fatty acid pretreatment enhances glucose metabolism, and EPA reduces oleic acid and leucine fractional oxidation in myotubes from donor with obesity, suggesting increased metabolic flexibility after EPA treatment.

Paricalcitol Attenuates Metabolic Syndrome-Associated Heart Failure through Enhanced Mitochondrial Fusion.

Objectives: Transition from cardiac hypertrophy to failure involves adverse metabolic reprogramming involving mitochondrial dysfunction. We have earlier shown that vitamin D deficiency induces heart failure, at least in part, through insulin resistance. However, whether activation of vitamin D receptor (VDR) can attenuate heart failure and underlying metabolic phenotype requires investigation. Thus, we aimed to assess the cardioprotective potential of paricalcitol, a vitamin D receptor-activator, against cardiac hypertrophy and failure in high-fat high-fructose-fed rats. Methods: Male Sprague Dawley rats were fed control (Con) or high-fat high-fructose (HFHFrD) diet for 20 weeks. After 12 weeks, rats from HFHFrD group were divided into the following: HFHFrD, HFHFrD+P (paricalcitol i.p. 0.08 µg/kg/day) and HFHFrD+E (enalapril maleate i.p. 10 mg/kg/day). Intraperitoneal glucose tolerance test, blood pressure measurement, and 2D echocardiography were performed. Cardiac fibrosis was assessed by Masson's trichrome staining of paraffin-embedded heart sections. Mitochondrial DNA and proteins, and citrate synthase activity were measured in rat hearts. VDR was silenced in H9c2 cardiomyoblasts, and immunoblotting was performed. Results: Paricalcitol improved glucose tolerance, serum lipid profile, and blood pressure in high-fat high-fructose-fed rats. Paricalcitol reduced cardiac wall thickness and increased ejection fraction in high-fat high-fructose-fed rats but had no effect on perivascular fibrosis. PGC1-α was upregulated in the HFHFrD+P group compared to the HFHFrD group, but there was no significant difference in mitochondrial content. Citrate synthase activity was significantly higher in the HFHFrD+P group compared to the HFHFrD group. Rat hearts of the HFHFrD+P group had significantly higher expression of mitofusins. H9c2 cells with VDR knockdown showed significantly lower expression of Mfn2. Improvement in the HFHFrD+P group was comparable with that in the HFHFrD+E group. Conclusions: Paricalcitol reverses cardiac dysfunction in rats with metabolic syndrome by enhancing mitochondrial fusion. We demonstrate repurposing potential of the drug currently used in end-stage kidney disease.

Thrombotic potential during pediatric acute lymphoblastic leukemia induction: Role of cell-free DNA.

BACKGROUND: Thromboembolism affects up to 30% of children undergoing treatment for acute lymphoblastic leukemia (ALL). Increased thrombin generation has been reported in ALL, but the mechanisms remain elusive. OBJECTIVE: We aimed to show that extracellular traps and cell-free DNA (cfDNA) promote thrombin generation in pediatric ALL. METHODS: In a longitudinal single-center study, we recruited 17 consecutive pediatric ALL patients. Serial blood samples were collected at diagnosis and weekly during the 4-week induction phase of antileukemic chemotherapy. Healthy children (n = 14) and children with deep vein thrombosis (DVT; n = 7) or sepsis (n = 5) were recruited as negative and positive controls, respectively. In plasma, we measured endogenous thrombin generation potential (ETP) and components of extracellular traps, including cfDNA. RESULTS: In patients with ALL, ETP was increased at baseline and remained significantly elevated throughout the induction therapy. Plasma levels of cfDNA were increased at baseline and during the first 3 weeks of induction therapy. The extent of enhancement of ETP and plasma cfDNA in patients with ALL was similar to that seen in patients with DVT or sepsis. Treatment of plasma with DNase 1 lowered ETP in patients with ALL at each time point but did not affect ETP in healthy controls. CONCLUSION: We conclude that childhood ALL is associated with a prothrombotic milieu at the time of diagnosis that continues during induction chemotherapy, and cfDNA contributes to increased thrombogenic potential.

Green surfactant-dendrimer aggreplexes: An ingenious way to launch dual attack on arch-enemy cancer.

Combination therapy, which combines anti-cancer drugs with different oligonucleotides, have shown potential in cancer treatment. However, delivering a hydrophobic anti-cancer drug and a hydrophilic oligonucleotide simultaneously is a herculean task. This study takes advantage of interactions between histidine-lauric acid-based green surfactant and poly(amidoamine) dendrimers to achieve this aim. The green surfactant was synthesized by carbodiimide chemistry and characterized by FTIR, 1H-NMR, and mass spectroscopy. Further, green surfactant-dendrimer aggregates encapsulating DTX and complexing SIRT 1 shRNA i.e., "aggreplexes" were developed and characterized. The term "aggreplexes" signifies complexes which are formed between green-surfactant-dendrimer aggregates and SIRT-1 shRNA via electrostatic interaction. The aggreplexes displayed particle size of 262.33 ± 3.87 nm, PDI of 0.25 and entrapment efficiency of 70.56 %. The TEM images revealed spherical shape of aggreplexes with irregular outer surface and corroborated particle size obtained from zetasizer. The in-vitro release study revealed biphasic release patterns of DTX from aggreplexes and were compatible for intravenous administration. Further, aggreplexes augmented cellular uptake in MDA-MB-231 cells by ∼1.87-fold compared to free DTX. Also, EGFP expression revealed significantly higher transfection of aggreplexes compared to naked shRNA and Superfect™ complexes. Further, aggreplexes showed higher cytotoxicity in MDA-MB-231 cells and ∼4.16-fold reduction in IC50 value compared to free DTX. Finally, apoptosis-index observed in case of aggreplexes was ∼3.57-fold higher than free DTX. These novel aggreplexes showed increased drug loading capacity and superior gene transfection potential. Thus, they open new avenues for co-delivery of hydrophobic anti-cancer drugs and hydrophilic therapeutic genes for improving current standards of cancer therapy.

pH sensitive liposomes assisted specific and improved breast cancer therapy using co-delivery of SIRT1 shRNA and Docetaxel.

Combining the bio-therapeutics with chemotherapeutic drugs can assist in augmenting the therapeutic standards by increasing the efficacy and decreasing the toxicity. Hence, in the present investigation Docetaxel (DTX) loaded pH-sensitive SIRT1 shRNA complexed lipoplex (DTX-lipoplex) were developed and explored for their improved breast cancer potential. The DTX-lipoplex were prepared by solvent evaporation and rehydration method and were evaluated for various quality attributes (particle size, % entrapment efficiency, hemotoxicity, DNA stability efficiency etc.), in vitro drug release, cell culture assays, antitumor efficacy and in vivo toxicity. The DTX-lipoplex exhibited a size of ~200 nm and zeta-potential of ~20 mV with ~70% encapsulation. Through systematic in vitro and in vivo examinations, DTX-lipoplex showed ~3 fold higher DTX titre within the tumor cells thereby significantly reducing the tumor burden (~78%) when compared to the marketed non pH sensitive lipid transfection agent and clinical counterpart i.e. Taxotere®. Thus, to conclude it can be said that co-delivering DTX and SIRT1 shRNA in a single tumor-specific nano-platform can improve the therapeutic potential of current therapy.

Memantine Protects From Exacerbation of Ischemic Stroke and Blood Brain Barrier Disruption in Mild But Not Severe Hyperhomocysteinemia.

Background Hyperhomocysteinemia is a risk factor for ischemic stroke; however, a targeted treatment strategy is lacking partly because of limited understanding of the causal role of homocysteine in cerebrovascular pathogenesis. Methods and Results In a genetic model of cystathionine beta synthase (CBS) deficiency, we tested the hypothesis that elevation in plasma total homocysteine exacerbates cerebrovascular injury and that memantine, a N-methyl-D-aspartate receptor antagonist, is protective. Mild or severe elevation in plasma total homocysteine was observed in Cbs+/- (6.1±0.3 µmol/L) or Cbs-/- (309±18 µmol/L) mice versus Cbs+/+ (3.1±0.6 µmol/L) mice. Surprisingly, Cbs-/- and Cbs+/- mice exhibited similar increases in cerebral infarct size following middle cerebral artery ischemia/reperfusion injury, despite the much higher total homocysteine levels in Cbs-/- mice. Likewise, disruption of the blood brain barrier was observed in both Cbs+/- and Cbs-/- mice. Administration of the N-methyl-D-aspartate receptor antagonist memantine protected Cbs+/- but not Cbs-/- mice from cerebral infarction and blood brain barrier disruption. Our data suggest that the differential effect of memantine in Cbs+/- versus Cbs-/- mice may be related to changes in expression of N-methyl-D-aspartate receptor subunits. Cbs-/-, but not Cbs+/- mice had increased expression of NR2B subunit, which is known to be relatively insensitive to homocysteine. Conclusions These data provide experimental evidence that even a mild increase in plasma total homocysteine can exacerbate cerebrovascular injury and suggest that N-methyl-D-aspartate receptor antagonism may represent a strategy to prevent reperfusion injury after acute ischemic stroke in patients with mild hyperhomocysteinemia.

Musa balbisiana Fruit Rich in Polyphenols Attenuates Isoproterenol-Induced Cardiac Hypertrophy in Rats via Inhibition of Inflammation and Oxidative Stress.

Musa balbisiana Colla (Family: Musaceae), commonly known as banana and native to India and other parts of Asia, is very rich in nutritional value and has strong antioxidant potential. In the present study, we have developed Musa balbisiana (MB) fruit pulp powder and evaluated its cardioprotective effect in cardiac hypertrophy, which is often associated with inflammation and oxidative stress. An ultra-high-pressure liquid chromatography-mass spectrometer (UPLC-MS/MS) has been used for the detection and systematic characterization of the phenolic compounds present in Musa balbisiana fruit pulp. The cardioprotective effect of MB was evaluated in a rat model of isoproterenol- (ISO-) induced cardiac hypertrophy by subcutaneous administration of isoproterenol (5 mg/kg-1/day-1), delivered through an alzet minipump for 14 days. Oral administration of MB fruit pulp powder (200 mg/kg/day) significantly (p < 0.001) decreased heart weight/tail length ratio and cardiac hypertrophy markers like ANP, BNP, ß-MHC, and collagen-1 gene expression. MB also attenuated ISO-induced cardiac inflammation and oxidative stress. The in vivo data were further confirmed in vitro in H9c2 cells where the antihypertrophic and anti-inflammatory effect of the aqueous extract of MB was observed in the presence of ISO and lipopolysaccharide (LPS), respectively. This study strongly suggests that supplementation of dried Musa balbisiana fruit powder can be useful for the prevention of cardiac hypertrophy via the inhibition of inflammation and oxidative stress.

Exploring the potential of novel pH sensitive lipoplexes for tumor targeted gene delivery with reduced toxicity.

The present investigation explores the potential of pH sensitive cationic liposomes for its in vivo tumor targeted gene transfection in comparison to its marketed transfecting reagent Lipofectamine® 2000. The lipoplexes were prepared by varying the molar mass ratio of cationic pH-sensitive liposomes with respect to pDNA and were evaluated for optimum size, zeta potential and for complete gel retardation. Similarly, the stability of lipoplexes in the presence of DNase I and serum was evaluated by using gel retardation and heparin displacement assay. The in vitro hemocompatibility assessment of pDNA lipoplexes revealed < 8.5% of hemolysis which was lower than the hemolysis observed for Lipofectamine® lipoplexes (15.9%). The internalization and pH dependent uptake inhibition using ammonium chloride in MCF-7 cells revealed higher internalization and pH sensitive nature of the prepared pH-sensitive system. The pDNA lipoplexes displayed > 80% of cell viability along with 4.42, 5.18 and 5.00 fold higher transfection efficiency than Lipofectamine® lipoplexes in MCF-7, HeLa and HEK-293 cells respectively. Also the in vivo toxicity assessment exhibited no significant change in the levels of biomarkers and no histopathological deformations in case of pDNA lipoplexes treated animals in comparison to control group (PBS). Further, pDNA lipoplexes demonstrated ~1.3 fold higher tumor transfection over Lipofectamine® lipoplexes indicating superior in vivo gene deliverable capabilities. Thus, the developed pH sensitive lipoplexes promises to be a potential tumor targeting and safe delivery system than Lipofectamine® 2000.

SIRT-3 Modulation by Resveratrol Improves Mitochondrial Oxidative Phosphorylation in Diabetic Heart through Deacetylation of TFAM.

BACKGROUND AND PURPOSE: Mitochondrial dysfunction remains the crucial cause for many heart diseases including diabetic cardiomyopathy (DCM). Sirtuin-3 (SIRT-3) is a protein deacetylase localized in the mitochondria and regulates mitochondrial function. Being a noteworthy mitochondrial protein deacetylase enzyme, the role of SIRT-3 in DCM is yet to be explored. EXPERIMENTAL APPROACH: Diabetes mellitus (Type-I, T1DM) was induced using streptozotocin (STZ, 50 mg/kg) in male Sprague Dawley (SD) rats. Rats with >200 mg/dL blood glucose levels were then divided randomly into two groups, DIA and DIA + RESV, where vehicle and resveratrol (25 mg/kg/day) were administered orally in both groups, respectively. Cardiac oxidative stress, fibrosis, and mitochondrial parameters were evaluated. H9c2 cells were transfected with SIRT-3 siRNA and shRNA, and ORF plasmid for silencing and overexpression, respectively. KEY RESULTS: After eight weeks, diabetic rat heart showed reduced cardiac cell size, increased oxidative stress and reduction of the activities of enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS). There was reduced expression and activity of SIRT-3 and mitochondrial transcription factor (TFAM) in diabetic heart. Reduced SIRT-3 expression is also correlated with increased acetylation, decreased mitochondrial DNA (mtDNA) binding activity of TFAM, and reduced transcription of mitochondrial DNA encoded genes. Administration of resveratrol prevented the decrease in SIRT-3 and TFAM activity, which was corresponding to the reduced acetylation status of TFAM. Silencing SIRT-3 using siRNA in H9C2 cells showed increased acetylation of TFAM. CONCLUSION AND IMPLICATIONS: Together our data shows that resveratrol activates SIRT-3, regulates the acetylation status of TFAM and preserves the mitochondrial function along with cellular size in diabetic rat heart.

ε-Poly-l-Lysine/plasmid DNA nanoplexes for efficient gene delivery in vivo.

The present work addresses the development and characterization of ε-Poly-l-Lysine/pDNA polyplexes and evaluation for their improved transfection efficacy and safety as compared to polyplexes prepared using Poly-l-Lysine and SuperFect®. Self-assembling polyplexes were prepared by varying the N/P ratio to obtain the optimum size, a net positive zeta potential and gel retardation. The stability in presence of DNase I and serum was assured using gel retardation assay. Their appreciable uptake in MCF-7 and 3.5, 3.79 and 4.79-fold higher transfection compared to PLL/pDNA polyplexes and 1.60, 1.53 and 1.79-fold higher transfection compared to SuperFect®/pDNA polyplexes in MCF-7, HeLa and HEK-293 cell lines respectively, affirmed the enhanced transfection of ε-PLL/pDNA polyplexes which was well supported with in vivo transfection and gene expression studies. The <8% in vitro hemolysis and >98% viability of MCF-7, HeLa and HEK-293 cells in presence of ε-PLL/pDNA polyplexes addressed their safety, which was also ensured using in vivo toxicity studies, where hemocompatibility, unaltered levels of biochemical markers and histology of vital organs confirmed ε-PLL to be an effective and safer alternative for non-viral genetic vectors.

Toll-Like Receptor 4 Inhibition Improves Oxidative Stress and Mitochondrial Health in Isoproterenol-Induced Cardiac Hypertrophy in Rats.

BACKGROUND: Inflammation remains a crucial factor for progression of cardiac diseases and cardiac hypertrophy remains an important cause of cardiac failure over all age groups. As a key regulator of inflammation, toll-like receptor 4 (TLR4) plays an important role in pathogenesis of cardiac diseases. Being an important regulator of innate immunity, the precise pathway of TLR4-mediated cardiac complications is yet to be established. Therefore, the primary objective of the present study was to find the role of TLR4 in cardiac hypertrophy and the molecular mechanism thereof. METHODS: Cardiac hypertrophy was induced with administration of isoproterenol (5 mg/kg/day, sc). TLR4 receptor inhibitor RS-LPS (lipopolysaccharide from the photosynthetic bacterium Rhodobacter sphaeroides; 5 µg/day) and agonist lipopolysaccharide (LPS) (from Escherichia coli; 3.12 µg/day) were administered through osmotic pump along with isoproterenol. Cardiac hypertrophy as well as oxidative stress and mitochondrial parameters were evaluated. RESULTS: Cardiac hypertrophy was confirmed with increased heart weight/body weight ratio as well as assessment of hypertrophic markers in heart. There was a marked increase in the TLR4 expression and oxidative stress along with mitochondrial dysfunction in ISO group. TLR4 inhibition significantly decreased heart weight/body weight ratio and ANP, collagen, and ß-MHC expression and restored the disturbed cellular antioxidant flux. The mitochondrial perturbations that were observed in hypertrophy heart was normalized after administration of TLR4 inhibitor but not with the agonist. TLR4 agonism further exaggerated the oxidative stress in heart and hence accelerated the disease development and progression. CONCLUSION: Our data show that increased TLR4 ligand pool in cardiac hypertrophy may exaggerate the disease progression. However, inhibition of TLR4 attenuated cardiac hypertrophy through reduced cardiac redox imbalance and mitochondrial dysfunction.

Repositioning of Drugs in Cardiometabolic Disorders: Importance and Current Scenario.

Cardiometabolic disorder (CMD) is a cluster of diseases, including cardiovascular diseases (CVDs), metabolic syndrome (MS) and diabetes mellitus (DM). Cardiometabolic disorders (CMDs) remain the principal cause of death in both developed and developing countries, accounting for nearly 32% of all deaths worldwide per year. In addition, dyslipidemia, angina, arrhythmia, cardiac failure, myocardial infarction (MI), and diabetes mellitus represent the leading killer with an estimated 19 million people died from CMDs in 2012. By 2030 more than 23 million people will die annually from CVDs. Existing drugs are not efficient enough to reduce the disease burden as well as mortality. Therefore, there is an urgent demand for new drugs in this area to reduce the mortality and control the associated disability. Nonetheless, new drug discovery (NDD) in CMDs has become more challenging for last couple of decades due to increased expenses and decreased success rate. In such a scenario, drug repositioning in the CMDs appears promising for introducing existing drugs for new therapeutic indication. Repositioning is quite an old strategy dating back to 1960s and mainly followed by serendipitous observations during clinical use of drugs. A major advantage of repositioning is that the safety profile of the drug is well established thus reducing the chances of failure due to adverse toxic effects. In addition, repositioning requires less time and investment than NDD. Considering these facts, pharmaceutical companies are now becoming increasingly interested in drug repositioning. In this follow-up, we have talked about the concept of repositioning with important examples of repositioned drugs in cardiometabolic disorder.