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This study investigated toxicity of nanocarriers comprised of cationic polymer and lipid components often used in gene and drug delivery, formulated as cationic micelles and liposomes. Rats were injected intravenously with 10, 25 or 100 mg/kg and sacrificed after 24 or 48 h, or 24 h after the last of three intravenous injections of 100 mg/kg every other day. Histological evaluation of liver, lung and spleen, clinical chemistry parameters, and hematology indicated little effect of treatment. DNA strand breaks were increased in the lung and spleen. Further, in the dose response study we found unaltered expression levels of genes in the antioxidant response ( HMOX1 ) and repair of oxidized nucleobases ( OGG1 ), whereas expression levels of cytokines ( IL6 , CXCL2 and CCL2 ) were elevated in lung, spleen or liver. The results indicate that assessment of genotoxicity and gene expression add information on toxicity of nanocarriers, which is not obtained by histology and hematology. From the Clinical Editor This study investigates the toxicity of cationic micelles and liposomes utilized as nanocarriers in gene and drug delivery, demonstrating its effects on the lungs, spleen and liver. [ABSTRACT FROM AUTHOR]

Abstract: It has been suggested that human mitochondrial variants influence maximal oxygen uptake (VO2max). Whether mitochondrial respiratory capacity per mitochondrion (intrinsic activity) in human skeletal muscle is affected by differences in mitochondrial variants is not known. We recruited 54 males and determined their mitochondrial haplogroup, mitochondrial oxidative phosphorylation capacity (OXPHOS), mitochondrial content (citrate synthase (CS)) and VO2max. Intrinsic mitochondrial function is calculated as mitochondrial OXPHOS capacity divided by mitochondrial content (CS). Haplogroup H showed a 30% higher intrinsic mitochondrial function compared with the other haplo group U. There was no relationship between haplogroups and VO2max. In skeletal muscle from men with mitochondrial haplogroup H, an increased intrinsic mitochondrial function is present. [Copyright &y& Elsevier]

Objectives: Glucose tolerance and skeletal muscle coenzyme Q10 (Q10) content, mitochondrial density, and mitochondrial oxidative phosphorylation (OXPHOS) capacity were measured in simvastatin-treated patients (n = 10) and in well-matched control subjects (n = 9). Background: A prevalent side effect of statin therapy is muscle pain, and yet the basic mechanism behind it remains unknown. We hypothesize that a statin-induced reduction in muscle Q10 may attenuate mitochondrial OXPHOS capacity, which may be an underlying mechanism. Methods: Plasma glucose and insulin concentrations were measured during an oral glucose tolerance test. Mitochondrial OXPHOS capacity was measured in permeabilized muscle fibers by high-resolution respirometry in a cross-sectional design. Mitochondrial content (estimated by citrate synthase [CS] activity, cardiolipin content, and voltage-dependent anion channel [VDAC] content) as well as Q10 content was determined. Results: Simvastatin-treated patients had an impaired glucose tolerance and displayed a decreased insulin sensitivity index. Regarding mitochondrial studies, Q10 content was reduced (p = 0.05), whereas mitochondrial content was similar between the groups. OXPHOS capacity was comparable between groups when complex I– and complex II–linked substrates were used alone, but when complex I + II–linked substrates were used (eliciting convergent electron input into the Q intersection [maximal ex vivo OXPHOS capacity]), a decreased (p < 0.01) capacity was observed in the patients compared with the control subjects. Conclusions: These simvastatin-treated patients were glucose intolerant. A decreased Q10 content was accompanied by a decreased maximal OXPHOS capacity in the simvastatin-treated patients. It is plausible that this finding partly explains the muscle pain and exercise intolerance that many patients experience with their statin treatment. [Copyright &y& Elsevier]

NAD+ is a co-factor and substrate for enzymes maintaining energy homeostasis. Nicotinamide phosphoribosyltransferase (NAMPT) controls NAD+ synthesis, and in skeletal muscle, NAD+ is essential for muscle integrity. However, the underlying molecular mechanisms by which NAD+ synthesis affects muscle health remain poorly understood. Thus, the objective of the current study was to delineate the role of NAMPT-mediated NAD+ biosynthesis in skeletal muscle development and function. To determine the role of Nampt in muscle development and function, we generated skeletal muscle-specific Nampt KO (SMNKO) mice. We performed a comprehensive phenotypic characterization of the SMNKO mice, including metabolic measurements, histological examinations, and RNA sequencing analyses of skeletal muscle from SMNKO mice and WT littermates. SMNKO mice were smaller, with phenotypic changes in skeletal muscle, including reduced fiber area and increased number of centralized nuclei. The majority of SMNKO mice died prematurely. Transcriptomic analysis identified that the gene encoding the mitochondrial permeability transition pore (mPTP) regulator Cyclophilin D (Ppif) was upregulated in skeletal muscle of SMNKO mice from 2 weeks of age, with associated increased sensitivity of mitochondria to the Ca2+-stimulated mPTP opening. Treatment of SMNKO mice with the Cyclophilin D inhibitor, Cyclosporine A, increased membrane integrity, decreased the number of centralized nuclei, and increased survival. Our study demonstrates that NAMPT is crucial for maintaining cellular Ca2+ homeostasis and skeletal muscle development, which is vital for juvenile survival. • NAD+ salvage capacity is important for skeletal muscle development and survival. • Skeletal muscle-specific Nampt knockout mice exhibit a dystrophy-like phenotype. • Nampt deletion alters Ca2+ homeostasis and impairs mitochondrial function. • Low NAD+ levels signals mitochondrial permeability transition pore opening. • Cyclosporin A treatment improves sarcolemma integrity and increases survival rate. [Display omitted] [ABSTRACT FROM AUTHOR]

High-resolution respirometry in human permeabilized muscle fibers is extensively used for analysis of mitochondrial adaptions to nutrition and exercise interventions, and is linked to athletic performance. However, the lack of standardization of experimental conditions limits quantitative inter- and intra-laboratory comparisons. In our study, an international team of investigators measured mitochondrial respiration of permeabilized muscle fibers obtained from three biopsies (vastus lateralis) from the same healthy volunteer to avoid inter-individual variability. High-resolution respirometry assays were performed together at the same laboratory to assess whether the heterogenity in published results are due to the effects of respiration media (MiR05 versus Z) with or without the myosin inhibitor blebbistatin at low- and high-oxygen regimes. Our findings reveal significant differences between respiration media for OXPHOS and ETcapacities supported by NADH&succinate-linked substrates at different oxygen concentrations. Respiratory capacities were approximately 1.5-fold higher in MiR05 at high-oxygen regimes compared to medium Z near air saturation. The presence or absence of blebbistatin in human permeabilized muscle fiber preparations was without effect on oxygen flux. Our study constitutes a basis to harmonize and establish optimum experimental conditions for respirometric studies of permeabilized human skeletal muscle fibers to improve reproducibility. [Display omitted] • Understanding muscle energetics' link to athletic performance is crucial. • Harmonizing mitochondrial bioenergetics protocols is critical for inter-study data analysis. • Medium composition and oxygen concentration influence muscle mitochondria respiratory capacity in permeabilized fibers. • Optimizing experimental protocols is pivotal for precise athletic performance evaluation. [ABSTRACT FROM AUTHOR]

Evidence for AMP-activated protein kinase (AMPK)-mediated regulation of skeletal muscle metabolism during exercise is mainly based on transgenic mouse models with chronic (lifelong) disruption of AMPK function. Findings based on such models are potentially biased by secondary effects related to a chronic lack of AMPK function. To study the direct effect(s) of AMPK on muscle metabolism during exercise, we generated a new mouse model with inducible muscle-specific deletion of AMPKα catalytic subunits in adult mice. Tamoxifen-inducible and muscle-specific AMPKα1/α2 double KO mice (AMPKα imdKO) were generated by using the Cre/loxP system, with the Cre under the control of the human skeletal muscle actin (HSA) promoter. During treadmill running at the same relative exercise intensity, AMPKα imdKO mice showed greater depletion of muscle ATP, which was associated with accumulation of the deamination product IMP. Muscle-specific deletion of AMPKα in adult mice promptly reduced maximal running speed and muscle glycogen content and was associated with reduced expression of UGP2, a key component of the glycogen synthesis pathway. Muscle mitochondrial respiration, whole-body substrate utilization, and muscle glucose uptake and fatty acid (FA) oxidation during muscle contractile activity remained unaffected by muscle-specific deletion of AMPKα subunits in adult mice. Inducible deletion of AMPKα subunits in adult mice reveals that AMPK is required for maintaining muscle ATP levels and nucleotide balance during exercise but is dispensable for regulating muscle glucose uptake, FA oxidation, and substrate utilization during exercise. • Inducible deletion of AMPKα in adult mice disturbs nucleotide balance during exercise. • Inducible deletion of AMPKα in adult mice lowers muscle glycogen content and reduces exercise capacity. • Muscle mitochondrial respiration, and glucose uptake and FA oxidation during muscle contractions remain unaffected by AMPKα deletion. [ABSTRACT FROM AUTHOR]

Ageing, a sedentary lifestyle, and obesity are associated with increased oxidative stress, while regular exercise is associated with an increased antioxidant capacity in trained skeletal muscles. Whether a higher aerobic fitness is associated with increased expression of antioxidant enzymes and their regulatory factors in skeletal muscle remains unknown. Although oestrogens could promote a higher antioxidant capacity in females, it remains unknown whether a sex dimorphism exists in humans regarding the antioxidant capacity of skeletal muscle. Thus, the aim was to determine the protein expression levels of the antioxidant enzymes SOD1, SOD2, catalase and glutathione reductase (GR) and their regulatory factors Nrf2 and Keap1 in 189 volunteers (120 males and 69 females) to establish whether sex differences exist and how age, VO 2 max and adiposity influence these. For this purpose, vastus lateralis muscle biopsies were obtained in all participants under resting and unstressed conditions. No significant sex differences in Nrf2, Keap1, SOD1, SOD2, catalase and GR protein expression levels were observed after accounting for VO 2 max, age and adiposity differences. Multiple regression analysis indicates that the VO 2 max in mL.kg LLM−1.min−1can be predicted from the levels of SOD2, Total Nrf2 and Keap1 (R = 0.58, P < 0.001), with SOD2 being the main predictor explaining 28 % of variance in VO 2 max, while Nrf2 and Keap1 explained each around 3 % of the variance. SOD1 protein expression increased with ageing in the whole group after accounting for differences in VO 2 max and body fat percentage. Overweight and obesity were associated with increased pSer40-Nrf2, pSer40-Nrf2/Total Nrf2 ratio and SOD1 protein expression levels after accounting for differences in age and VO 2 max. Overall, at the population level, higher aerobic fitness is associated with increased basal expression of muscle antioxidant enzymes, which may explain some of the benefits of regular exercise. [Display omitted] • It remains unknown how aerobic fitness, adiposity, age and sex may influence antioxidant enzymes in human skeletal muscle. • Here we show that SOD2 is the main antioxidant enzyme associated with VO 2 max. • In young and middle-aged humans, there is no sex-dimorphism in antioxidant enzymes expression in skeletal muscle or their regulatory factors. • SOD1 protein expression is increased with ageing after accounting for differences in VO2max and body fat percentage. • Overweight and obesity are associated with increased pSer40-Nrf2, pSer40-Nrf2/Total Nrf2 ratio and SOD1 protein expression levels. [ABSTRACT FROM AUTHOR]

Nicotinamide phosphoribosyltransferase (NAMPT) converts nicotinamide to NAD+. As low hepatic NAD+ levels have been linked to the development of nonalcoholic fatty liver disease, we hypothesized that ablation of hepatic Nampt would affect susceptibility to liver injury in response to diet-induced metabolic stress. Following 3 weeks on a low-methionine and choline-free 60% high-fat diet, hepatocyte-specific Nampt knockout (HNKO) mice accumulated less triglyceride than WT littermates but had increased histological scores for liver inflammation, necrosis, and fibrosis. Surprisingly, liver injury was also observed in HNKO mice on the purified control diet. This HNKO phenotype was associated with decreased abundance of mitochondrial proteins, especially proteins involved in oxidoreductase activity. High-resolution respirometry revealed lower respiratory capacity in purified control diet-fed HNKO liver. In addition, fibrotic area in HNKO liver sections correlated negatively with hepatic NAD+, and liver injury was prevented by supplementation with NAD+ precursors nicotinamide riboside and nicotinic acid. MS-based proteomic analysis revealed that nicotinamide riboside supplementation rescued hepatic levels of oxidoreductase and OXPHOS proteins. Finally, single-nucleus RNA-Seq showed that transcriptional changes in the HNKO liver mainly occurred in hepatocytes, and changes in the hepatocyte transcriptome were associated with liver necrosis. In conclusion, HNKO livers have reduced respiratory capacity, decreased abundance of mitochondrial proteins, and are susceptible to fibrosis because of low NAD+ levels. Our data suggest a critical threshold level of hepatic NAD+ that determines the predisposition to liver injury and supports that NAD+ precursor supplementation can prevent liver injury and nonalcoholic fatty liver disease progression. [ABSTRACT FROM AUTHOR]

Obesity has been associated with increased production of reactive oxygen species (ROS), which may be involved in the development of cardiovascular disease and type 2 diabetes (T2D). Endurance exercise lowers ROS production and increases antioxidant capacity in muscle cells, but it is currently unknown whether high intensity interval training (HIT) elicits the same effects. Twelve sedentary obese subjects at risk of developing T2D took part in a six-week intervention, performing three HIT sessions per week (five 1-min sets of high-intensity cycling (125% of VO 2 peak), with 90 s recovery in between sets). Muscle biopsies were obtained for assessment of ROS production (H 2 O 2 emission), mitochondrial respiratory capacity, and antioxidant protein levels before and after the intervention. H 2 O 2 emission decreased 60.4% after the intervention (Succinate 3 mmol･l−1), concurrent with a 35.1% increase in protein levels of the antioxidant manganese superoxide dismutase (MnSOD) and a trend towards increased levels of the antioxidant catalase (p = 0.06, 72.9%). These findings were accompanied by a 19% increased mitochondrial respiratory capacity (CI + II), a 6.9% increased VO 2 peak and a 1.7% lower body fat percentage. These effects were achieved after just 15 min of high-intensity work and 40 min of total time spent per week. Overall, this suggests that a relatively small amount of HIT is sufficient to induce beneficial effects on ROS production and antioxidant status in muscle cells, which may lower oxidative stress and potentially protect against the development of cardiovascular disease. [Display omitted] • HIT reduces H 2 O 2 emission in muscle cells of obese adults in risk of developing T2D. • HIT leads to increased protein levels of antioxidant enzymes in skeletal muscle cells. • HIT improves mitochondrial respiratory capacity and general health parameters. • The effects occur after just 40 min of weekly time commitment for 6 weeks. [ABSTRACT FROM AUTHOR]

Supplementation with NAD precursors such as nicotinamide riboside (NR) has been shown to enhance mitochondrial function in the liver and to prevent hepatic lipid accumulation in high-fat diet (HFD)-fed rodents. Hepatocyte-specific knockout of the NAD+-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT) reduces liver NAD+ levels, but the metabolic phenotype of Nampt-deficient hepatocytes in mice is unknown. Here, we assessed Nampt's role in maintaining mitochondrial and metabolic functions in the mouse liver. Using the Cre-LoxP system, we generated hepatocyte-specific Nampt knockout (HNKO) mice, having a 50% reduction of liver NAD+ levels. We screened the HNKO mice for signs of metabolic dysfunction following 60% HFD feeding for 20 weeks ± NR supplementation and found that NR increases hepatic NAD+ levels without affecting fat mass or glucose tolerance in HNKO or WT animals. High-resolution respirometry revealed that NR supplementation of the HNKO mice did not increase state III respiration, which was observed in WT mice following NR supplementation. Mitochondrial oxygen consumption and fatty-acid oxidation were unaltered in primary HNKO hepatocytes. Mitochondria isolated from whole-HNKO livers had only a 20% reduction in NAD+, suggesting that the mitochondrial NAD+ pool is less affected by HNKO than the whole-tissue pool. When stimulated with tryptophan in the presence of [15N]glutamine, HNKO hepatocytes had a higher [15N]NAD+ enrichment than WT hepatocytes, indicating that HNKO mice compensate through de novo NAD+ synthesis. We conclude that NAMPT-deficient hepatocytes can maintain substantial NAD+ levels and that the Nampt knockout has only minor consequences for mitochondrial function in the mouse liver. [ABSTRACT FROM AUTHOR]

Brain aging is accompanied by declining mitochondrial respiration. We hypothesized that mitochondrial morphology and dynamics would reflect this decline. Using hippocampus and frontal cortex of a segmental progeroid mouse model lacking Cockayne syndrome protein B (CSB m/m ) and C57Bl/6 (WT) controls and comparing young (2–5 months) to middle-aged mice (13–14 months), we found that complex I-linked state 3 respiration (CI) was reduced at middle age in CSB m/m hippocampus, but not in CSB m/m cortex or WT brain. In hippocampus of both genotypes, mitochondrial size heterogeneity increased with age. Notably, an inverse correlation between heterogeneity and CI was found in both genotypes, indicating that heterogeneity reflects mitochondrial dysfunction. The ratio between fission and fusion gene expression reflected age-related alterations in mitochondrial morphology but not heterogeneity. Mitochondrial DNA content was lower, and hypoxia-induced factor 1α mRNA was greater at both ages in CSB m/m compared to WT brain. Our findings show that decreased CI and increased mitochondrial size heterogeneity are highly associated and point to declining mitochondrial quality control as an initial event in brain aging. [ABSTRACT FROM AUTHOR]

Abstract: Background: The incretin effect is reduced and the insulinotropic effect of the incretin hormone glucose-dependent insulinotropic polypeptide (GIP) is abolished in patients with type 2 diabetes mellitus (T2DM). Objective and design: To evaluate the causality of this deficiency we investigated 8 patients with chronic pancreatitis (CP) and normal glucose tolerance (NGT) (fasting plasma glucose (FPG): 5.5 (4.5–6.0) mM (mean (range); HbA1c: 5.8 (5.4–6.3) %) and 8 patients with CP and secondary diabetes not requiring insulin (FPG: 7.1 (6.0–8.8) mM; HbA1c: 7.0 (5.8–10.0) %) during three 15-mM hyperglycaemic clamps with continuous iv infusion of saline, glucagon-like peptide-1 (GLP-1) or GIP. Results: The initial (0–20 min) insulin and C-peptide responses were enhanced significantly in both groups by GLP-1 and GIP, respectively, compared to saline (P <0.05). In both groups GLP-1 infusion resulted in significantly greater insulin and C-peptide responses from 20–120 min compared with saline infusion. During GIP infusion the late-phase insulin response (20–120 min) was 3.1±1.0 fold greater than during saline infusion in the group of patients with CP and NGT (P <0.05), whereas there was no significant differences in patients with CP and DM. Conclusions: The lack of GIP amplification of the late insulin response to iv glucose develops alongside the deterioration of glucose tolerance in patients with CP, suggesting that the same may be true for the loss of the GIP effect in patients with T2DM. [Copyright &y& Elsevier]