Assessment of ATP Metabolism to Adenosine by Ecto-Nucleotidases Carried by Tumor-Derived Small Extracellular Vesicles.

Background: Immunosuppression is a hallmark of cancer progression. Tumor-derived small extracellular vesicles (sEV), also known as TEX, produce adenosine (ADO) and can mediate tumor-induced immunosuppression. Methods: Here, the ATP pathway of ADO production (ATP◊ADP◊AMP◊ADO) by ecto-nucleotidases carried in sEV was evaluated by a novel method using N6-etheno-ATP (eATP) and N6-etheno-AMP (eAMP) as substrates. The "downstream" N6-etheno-purines (ePurines) were measured by high performance liquid chromatography with fluorescence detection (HPLC-FL). Results: Human melanoma cell-derived TEX (MTEX) metabolized eATP to N6-etheno-ADP (eADP), eAMP and N6-etheno-Adenosine (eADO) more robustly than control keratinocyte cell-derived sEV (CEX); due to accelerated conversion of eATP to eADP and eADP to eAMP MTEX and CEX similarly metabolized eAMP to eADO. Blocking of the ATP pathway with the selective CD39 inhibitor ARL67156 or pan ecto-nucleotidase inhibitor POM-1 normalized the ATP pathway but neither inhibitor completely abolished it. In contrast, inhibition of CD73 by PSB12379 or AMPCP abolished eADO formation in both MTEX and CEX, suggesting that targeting CD73 is the preferred approach to eliminating ADO produced by sEV. Conclusions: The noninvasive, sensitive, and specific assay assessing ePurine metabolism ± ecto-nucleotidase inhibitors in TEX enables the personalized identification of the ecto-nucleotidase primarily involved in ADO production in patients with cancer. The assay could guide precision medicine by determining which purine is the preferred target for inhibitory therapeutic interventions.

Biochemical pathways of 8-aminoguanine production in Sprague-Dawley and Dahl salt-sensitive rats.

BACKGROUND: 8-Aminoguanine exerts natriuretic and antihypertensive activity. Whether and how "free" 8-aminoguanine exists in vivo is unclear. Because 8-nitroguanosine is naturally occurring, we tested the hypothesis that 8-aminoguanine can arise from: pathway 1, 8-nitroguanosine â†’ 8-aminoguanosine â†’ 8-aminoguanine; and pathway 2, 8-nitroguanosine â†’ 8-nitroguanine â†’ 8-aminoguanine. METHODS: 8-Aminoguanine biosynthesis was explored in rats using renal microdialysis, mass spectrometry and enzyme kinetics. RESULTS: In Sprague-Dawley rats, 8-nitroguanosine infusions increased kidney levels of 8-nitroguanine, 8-aminoguanosine and 8-aminoguanine; 8-nitroguanine infusions increased 8-aminoguanine. Purine nucleoside phosphorylase (PNPase) converted 8-nitroguanosine to 8-nitroguanine and 8-aminoguanosine to 8-aminoguanine. Forodesine (PNPase inhibitor) reduced metabolism of 8-nitroguanosine by pathway 2 and shunted metabolism of 8-nitroguanosine to 8-aminoguanosine. In Dahl salt-sensitive rats, 8-nitroguanosine infusions increased kidney levels of 8-nitroguanine, 8-aminoguanosine and 8-aminoguanine. These results indicate that both pathways 1 and 2 participate in the biosynthesis of 8-aminoguanine in Sprague-Dawley and Dahl rats. Endogenous 8-aminoguanine in kidneys and urine were elevated many-fold in Dahl, compared to Sprague-Dawley, rats. The increased levels of 8-aminoguanine in Dahl rats were not due to alterations in pathways 1 and 2 but were associated with increased urine levels of endogenous 8-nitroguanosine suggesting that the "upstream" production of 8-nitroguanosine was increased in Dahl rats. Dahl rats are known to have high levels of peroxynitrite, and peroxynitrite is known to nitrate guanosine in biomolecules. Here we confirm that a peroxynitrite donor increases kidney levels of 8-aminoguanine. CONCLUSION: 8-Aminoguanine occurs naturally via two distinct pathways and kidney levels of 8-aminoguanine are increased in Dahl rats, likely due to increased production of 8-nitroguanosine, a by-product of peroxynitrite chemistry.

8-Aminoinosine and 8-Aminohypoxanthine Inhibit Purine Nucleoside Phosphorylase and Exert Diuretic and Natriuretic Activity.

8-Aminoguanine and 8-aminoguanosine (via metabolism to 8-aminoguanine) are endogenous 8-aminopurines that induce diuresis, natriuresis, and glucosuria by inhibiting purine nucleoside phosphorylase (PNPase); moreover, both 8-aminopurines cause antikaliuresis by other mechanisms. Because 8-aminoinosine and 8-aminohypoxanthine are structurally similar to 8-aminoguanosine and 8-aminoguanine, respectively, we sought to define their renal excretory effects. First, we compared the ability of 8-aminoguanine, 8-aminohypoxanthine, and 8-aminoinosine to inhibit recombinant PNPase. These compounds inhibited PNPase with a potency order of 8-aminoguanine > 8-aminohypoxanthine = 8-aminoinosine. Additional studies showed that 8-aminoinosine is a competitive substrate that is metabolized to a competitive PNPase inhibitor, namely 8-aminohypoxanthine. Administration of each 8-aminopurine (33.5 µmol/kg) reduced the guanine-to-guanosine and hypoxanthine-to-inosine ratios in urine, a finding confirming their ability to inhibit PNPase in vivo. All three 8-aminopurines induced diuresis, natriuresis, and glucosuria; however, the glucosuric effects of 8-aminohypoxanthine and 8-aminoinosine were less pronounced than those of 8-aminoguanine. Neither 8-aminohypoxanthine nor 8-aminoinosine altered potassium excretion, whereas 8-aminoguanine caused antikaliuresis. In vivo administration of 8-aminoinosine increased 8-aminohypoxanthine excretion, indicating that 8-aminohypoxanthine mediates, in part, the effects of 8-aminoinosine. Finally, 8-aminohypoxanthine was metabolized to 8-aminoxanthine by xanthine oxidase. Using ultraperformance liquid chromatography-tandem mass spectrometry, we identified 8-aminoinosine as an endogenous 8-aminopurine. In conclusion, 8-aminopurines have useful pharmacological profiles. To induce diuresis, natriuresis, glucosuria, and antikaliuresis, 8-aminoguanine (or its prodrug 8-aminoguanosine) would be preferred. If only diuresis and natriuresis, without marked glucosuria or antikaliuresis, is desired, 8-aminohypoxanthine or 8-aminoinosine might be useful. Finally, here we report the in vivo existence of another pharmacologically active 8-aminopurine, namely 8-aminoinosine. SIGNIFICANCE STATEMENT: Here, we report that a family of 8-aminopurines affects renal excretory function: effects that may be useful for treating multiple diseases including hypertension, heart failure, and chronic kidney disease. For diuresis and natriuresis accompanied by glucosuria and antikaliuresis, 8-aminoguanine (or its prodrug 8-aminoguanosine) would be useful; if only diuresis and natriuresis is called for, 8-aminohypoxanthine or 8-aminoinosine would be useful. Previously, we identified 8-aminoguanine and 8-aminoguanosine as endogenous 8-aminopurines; here, we extend the family of endogenous 8-aminopurines to include 8-aminoinosine.

Novel Guidewire Design and Coating for Continuous Delivery of Adenosine During Interventional Procedures.

Background The "no-reflow phenomenon" compromises percutaneous coronary intervention outcomes. There is an unmet need for a device that prevents no-reflow phenomenon. Our goal was to develop a guidewire platform comprising a nondisruptive hydrophilic coating that allows continuous delivery of adenosine throughout a percutaneous coronary intervention. Methods and Results We developed a guidewire with spaced coils to increase surface area for drug loading. Guidewires were plasma treated to attach hydroxyl groups to metal surfaces, and a methoxy-polyethylene glycol-silanol primer layer was covalently linked to hydroxyl groups. Using polyvinyl alcohol, polyvinyl pyrrolidone, and polyvinyl acetate, a drug layer containing jet-milled adenosine was hydrogen-bonded to the polyethylene glycol-silanol layer and coated with an outer diffusive barrier layer. Coatings were processed with a freeze/thaw curing method. In vitro release studies were conducted followed by in vivo evaluation in pigs. Coating quality, performance, and stability with sterilization were also evaluated. Antiplatelet properties of the guidewire were also determined. Elution studies with adenosine-containing guidewires showed curvilinear and complete release of adenosine over 60 minutes. Porcine studies demonstrated that upon insertion into a coronary artery, adenosine-releasing guidewires induced immediate and robust increases (2.6-fold) in coronary blood flow velocity, which were sustained for ≈30 minutes without systemic hemodynamic effects or arrhythmias. Adenosine-loaded wires prevented and reversed coronary vasoconstriction induced by acetylcholine. The wires significantly inhibited platelet aggregation by >80% in vitro. Guidewires passed bench testing for lubricity, adherence, integrity, and tracking. Conclusions Our novel drug-releasing guidewire platform represents a unique approach to prevent/treat no-reflow phenomenon during percutaneous coronary intervention.

Author Correction: Simultaneous Inhibition of Glycolysis and Oxidative Phosphorylation Triggers a Multi-Fold Increase in Secretion of Exosomes: Possible Role of 2',3'-cAMP.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of the N6-etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism.

The goal of this study was to determine the validity of using N6-etheno-bridged adenine nucleotides to evaluate ecto-nucleotidase activity. We observed that the metabolism of N6-etheno-ATP versus ATP was quantitatively similar when incubated with recombinant CD39, ENTPD2, ENTPD3, or ENPP-1, and the quantitative metabolism of N6-etheno-AMP versus AMP was similar when incubated with recombinant CD73. This suggests that ecto-nucleotidases process N6-etheno-bridged adenine nucleotides similarly to endogenous adenine nucleotides. Four cell types rapidly (t1/2, 0.21 to 0.66 h) metabolized N6-etheno-ATP. Applied N6-etheno-ATP was recovered in the medium as N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and surprisingly N6-etheno-adenine; intracellular N6-etheno compounds were undetectable. This suggests minimal cellular uptake, intracellular metabolism, or deamination of these compounds. N6-etheno-ATP, N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and N6-etheno-adenine had little affinity for recombinant A1, A2A, or A2B receptors, for a subset of P2X receptors (3H-α,ß-methylene-ATP binding to rat bladder membranes), or for a subset of P2Y receptors (35S-ATP-αS binding to rat brain membranes), suggesting minimal pharmacological activity. N6-etheno-adenosine was partially converted to N6-etheno-adenine in four different cell types; this was blocked by purine nucleoside phosphorylase (PNPase) inhibition. Intravenous N6-etheno-ATP was quickly metabolized, with N6-etheno-adenine being the main product in naïve rats, but not in rats pretreated with a PNPase inhibitor. PNPase inhibition reduced the urinary excretion of endogenous adenine and attenuated the conversion of exogenous adenosine to adenine in the renal cortex. The N6-etheno-bridge method is a valid technique to assess extracellular metabolism of adenine nucleotides by ecto-nucleotidases. Also, rats express an enzyme with PNPase-like activity that metabolizes N6-etheno-adenosine to N6-etheno-adenine.

Tumor-derived exosomes promote angiogenesis via adenosine A2B receptor signaling.

RATIONALE: One hallmark of tumor-derived exosomes (TEX) is the promotion of cancer progression by stimulating angiogenesis. This study was performed to evaluate the role of adenosine receptors in TEX-induced angiogenesis. METHODS: TEX produced by UMSCC47 head and neck cancer cell line were isolated by mini size exclusion chromatography (mini-SEC). Enzymatic activity of ectonucleotidases CD39/CD73 carried by TEX was measured by HPLC. Adenosine content of TEX was measured by UPLC-MS/MS. Primary human macrophages were co-incubated with TEX or exosomes derived from the plasma of head and neck cancer patients and their marker expression profile was analyzed by flow cytometry. The macrophage secretome was analyzed by angiogenesis arrays. The in vitro angiogenic potential of TEX was evaluated in endothelial growth studies. Results were validated in vivo using basement membrane extract plug assays in A1R-/-, A2AR-/- and A2BR-/- rats. Vascularization was analyzed by hemoglobin quantification and immunohistology with vessel and macrophage markers. RESULTS: TEX carried enzymatically active CD39/CD73 and adenosine. TEX promoted A2BR-mediated polarization of macrophages toward an M2-like phenotype (p < 0.05) and enhanced their secretion of angiogenic factors. Growth of endothelial cells was stimulated directly by TEX and indirectly via macrophage-reprogramming dependent on A2BR signaling (p < 0.01). In vivo, TEX stimulated the formation of defined vascular structures and macrophage infiltration. This response was absent in A2BR-/- rats (p < 0.05). CONCLUSION: This report provides the first evidence for adenosine production by TEX to promote angiogenesis via A2BR. A2BR antagonism emerges as a potential strategy to block TEX-induced angiogenesis.

Simultaneous Inhibition of Glycolysis and Oxidative Phosphorylation Triggers a Multi-Fold Increase in Secretion of Exosomes: Possible Role of 2'3'-cAMP.

Exosome secretion by cells is a complex, poorly understood process. Studies of exosomes would be facilitated by a method for increasing their production and release. Here, we present a method for stimulating the secretion of exosomes. Cultured cells were treated or not with sodium iodoacetate (IAA; glycolysis inhibitor) plus 2,4-dinitrophenol (DNP; oxidative phosphorylation inhibitor). Exosomes were isolated by size-exclusion chromatography and their morphology, size, concentration, cargo components and functional activity were compared. IAA/DNP treatment (up to 10 µM each) was non-toxic and resulted in a 3 to 16-fold increase in exosome secretion. Exosomes from IAA/DNP-treated or untreated cells had similar biological properties and functional effects on endothelial cells (SVEC4-10). IAA/DNP increased exosome secretion from mouse organ cultures, and in vivo injections enhanced the levels of circulating exosomes. IAA/DNP decreased ATP levels (p < 0.05) in cells. A cell membrane-permeable form of 2',3'-cAMP and 3'-AMP mimicked the potentiating effects of IAA/DNP on exosome secretion. In cells lacking 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase; an enzyme that metabolizes 2',3'-cAMP into 2'- and 3'-AMP), effects of IAA/DNP on exosome secretion were enhanced. The IAA/DNP combination is a powerful stimulator of exosome secretion, and these stimulatory effects are, in part, mediated by intracellular 2',3'-cAMP.

Proximal tubule apical endocytosis is modulated by fluid shear stress via an mTOR-dependent pathway.

Cells lining the proximal tubule (PT) have unique membrane specializations that are required to maintain the high-capacity ion transport and endocytic functions of this nephron segment. PT cells in vivo acutely regulate ion transport in response to changes in glomerular filtration rate (GFR) to maintain glomerulotubular balance. PT cells in culture up-regulate endocytic capacity in response to acute changes in fluid shear stress (FSS); however, it is not known whether GFR modulates PT endocytosis to enable maximally efficient uptake of filtered proteins in vivo. Here, we show that cells cultured under continuous FSS develop an expanded apical endocytic pathway and increased endocytic capacity and lysosomal biogenesis. Furthermore, endocytic capacity in fully differentiated cells is rapidly modulated by changes in FSS. PT cells exposed to continuous FSS also acquired an extensive brush border and basolateral membrane invaginations resembling those observed in vivo. Culture under suboptimal levels of FSS led to intermediate phenotypes, suggesting a threshold effect. Cells exposed to FSS expressed higher levels of key proteins necessary for PT function, including ion transporters, receptors, and membrane-trafficking machinery, and increased adenine nucleotide levels. Inhibition of the mechanistic target of rapamycin (mTOR) using rapamycin prevented the increase in cellular energy levels, lysosomal biogenesis, and endocytic uptake, suggesting that these represent a coordinated differentiation program. In contrast, rapamycin did not prevent the FSS-induced increase in Na+/K+-ATPase levels. Our data suggest that rapid tuning of the endocytic response by changes in FSS may contribute to glomerulotubular balance in vivo. Moreover, FSS provides an essential stimulus in the differentiation of PT cells via separate pathways that up-regulate endocytosis and ion transport capacity. Variations in FSS may also contribute to the maturation of PT cells during kidney development and during repair after kidney injury.

Adenosine production by brain cells.

The early release of adenosine following traumatic brain injury (TBI) suppresses seizures and brain inflammation; thus, it is important to elucidate the cellular sources of adenosine following injurious stimuli triggered by TBI so that therapeutics for enhancing the early adenosine-release response can be optimized. Using mass spectrometry with 13 C-labeled standards, we investigated in cultured rat neurons, astrocytes, and microglia the effects of oxygen-glucose deprivation (OGD; models energy failure), H2 O2 (produces oxidative stress), and glutamate (induces excitotoxicity) on intracellular and extracellular levels of 5'-AMP (adenosine precursor), adenosine, and inosine and hypoxanthine (adenosine metabolites). In neurons, OGD triggered increases in intracellular 5'-AMP (2.8-fold), adenosine (2.6-fold), inosine (2.2-fold), and hypoxanthine (5.3-fold) and extracellular 5'-AMP (2.2-fold), adenosine (2.4-fold), and hypoxanthine (2.5-fold). In neurons, H2 O2 did not affect intracellular or extracellular purines; yet, glutamate increased intracellular adenosine, inosine, and hypoxanthine (1.7-fold, 1.7-fold, and 1.6-fold, respectively) and extracellular adenosine, inosine, and hypoxanthine (2.9-fold, 2.1-fold, and 1.6-fold, respectively). In astrocytes, neither H2 O2 nor glutamate affected intracellular or extracellular purines, and OGD only slightly increased intracellular and extracellular hypoxanthine. Microglia were unresponsive to OGD and glutamate, but were remarkably responsive to H2 O2 , which increased intracellular 5'-AMP (1.6-fold), adenosine (1.6-fold), inosine (2.1-fold), and hypoxanthine (1.6-fold) and extracellular 5'-AMP (5.9-fold), adenosine (4.0-fold), inosine (4.3-fold), and hypoxanthine (1.9-fold). CONCLUSION: Under these particular experimental conditions, cultured neurons are the main contributors to adenosine production/release in response to OGD and glutamate, whereas cultured microglia are the main contributors upon oxidative stress. Developing therapeutics that recruit astrocytes to produce/release adenosine could have beneficial effects in TBI.

Renal 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase Is an Important Determinant of AKI Severity after Ischemia-Reperfusion.

A positional isomer of 3',5'-cAMP, 2',3'-cAMP, is produced by kidneys in response to energy depletion, and renal 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) metabolizes 2',3'-cAMP to 2'-AMP; 2',3'-cAMP is a potent opener of mitochondrial permeability transition pores (mPTPs), which can stimulate autophagy. Because autophagy protects against AKI, it is conceivable that inhibition of CNPase protects against ischemia-reperfusion (IR) -induced AKI. Therefore, we investigated renal outcomes, mitochondrial function, number, area, and autophagy in CNPase-knockout (CNPase(-/-)) versus wild-type (WT) mice using a unique two-kidney, hanging-weight model of renal bilateral IR (20 minutes of ischemia followed by 48 hours of reperfusion). Analysis of urinary purines showed attenuated metabolism of 2',3'-cAMP to 2'-AMP in CNPase(-/-) mice. Neither genotype nor IR affected BP, heart rate, urine volume, or albumin excretion. In WT mice, renal IR reduced (14)C-inulin clearance (index of GFR) and increased renal vascular resistance (measured by transit time nanoprobes) and urinary excretion of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. IR did not affect these parameters in CNPase(-/-) mice. Histologic analysis revealed that IR induced severe damage in kidneys from WT mice, whereas histologic changes were minimal after IR in CNPase(-/-) mice. Measurements of renal cardiolipin levels, citrate synthase activity, rotenone-sensitive NADH oxidase activity, and proximal tubular mitochondrial and autophagosome area and number (by transmission electron microscopy) indicted accelerated autophagy/mitophagy in injured CNPase(-/-) mice. We conclude that CNPase deletion attenuates IR-induced AKI, in part by accelerating autophagy with targeted removal of damaged mitochondria.

Exercise and Weight Loss Improve Muscle Mitochondrial Respiration, Lipid Partitioning, and Insulin Sensitivity After Gastric Bypass Surgery.

Both Roux-en-Y gastric bypass (RYGB) surgery and exercise can improve insulin sensitivity in individuals with severe obesity. However, the impact of RYGB with or without exercise on skeletal muscle mitochondria, intramyocellular lipids, and insulin sensitivity index (SI) is unknown. We conducted a randomized exercise trial in patients (n = 101) who underwent RYGB surgery and completed either a 6-month moderate exercise (EX) or a health education control (CON) intervention. SI was determined by intravenous glucose tolerance test. Mitochondrial respiration and intramyocellular triglyceride, sphingolipid, and diacylglycerol content were measured in vastus lateralis biopsy specimens. We found that EX provided additional improvements in SI and that only EX improved cardiorespiratory fitness, mitochondrial respiration and enzyme activities, and cardiolipin profile with no change in mitochondrial content. Muscle triglycerides were reduced in type I fibers in CON, and sphingolipids decreased in both groups, with EX showing a further reduction in a number of ceramide species. In conclusion, exercise superimposed on bariatric surgery-induced weight loss enhances mitochondrial respiration, induces cardiolipin remodeling, reduces specific sphingolipids, and provides additional improvements in insulin sensitivity.

Deficiency of electron transport chain in human skeletal muscle mitochondria in type 2 diabetes mellitus and obesity.

Insulin resistance in skeletal muscle in obesity and T2DM is associated with reduced muscle oxidative capacity, reduced expression in nuclear genes responsible for oxidative metabolism, and reduced activity of mitochondrial electron transport chain. The presented study was undertaken to analyze mitochondrial content and mitochondrial enzyme profile in skeletal muscle of sedentary lean individuals and to compare that with our previous data on obese or obese T2DM group. Frozen skeletal muscle biopsies obtained from lean volunteers were used to estimate cardiolipin content, mtDNA (markers of mitochondrial mass), NADH oxidase activity of mitochondrial electron transport chain (ETC), and activity of citrate synthase and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD), key enzymes of TCA cycle and beta-oxidation pathway, respectively. Frozen biopsies collected from obese or T2DM individuals in our previous studies were used to estimate activity of beta-HAD. The obtained data were complemented by data from our previous studies and statistically analyzed to compare mitochondrial content and mitochondrial enzyme profile in lean, obese, or T2DM cohort. The total activity of NADH oxidase was reduced significantly in obese or T2DM subjects. The cardiolipin content for lean or obese group was similar, and although for T2DM group cardiolipin showed a tendency to decline, it was statistically insignificant. The total activity of citrate synthase for lean and T2DM group was similar; however, it was increased significantly in the obese group. Activity of beta-HAD and mtDNA content was similar for all three groups. We conclude that the total activity of NADH oxidase in biopsy for lean group is significantly higher than corresponding activity for obese or T2DM cohort. The specific activity of NADH oxidase (per mg cardiolipin) and NADH oxidase/citrate synthase and NADH oxidase/beta-HAD ratios are reduced two- to threefold in both T2DM and obesity.

Mitochondrial capacity in skeletal muscle is not stimulated by weight loss despite increases in insulin action and decreases in intramyocellular lipid content.

OBJECTIVE: In obesity and type 2 diabetes, exercise combined with weight loss increases skeletal muscle mitochondrial capacity. It remains unclear whether mitochondrial capacity increases because of weight loss, improvements in insulin resistance, or physical training. In this study, we examined the effects of an intervention of weight loss induced by diet and compared these with those of a similar intervention of weight loss by diet with exercise. Both are known to improve insulin resistance, and we tested the hypothesis that physical activity, rather than improved insulin resistance, is required to increase mitochondrial capacity of muscle. RESEARCH DESIGN AND METHODS: Sixteen sedentary overweight/obese volunteers were randomized to a 16-week intervention of diet (n = 7) or diet plus exercise (n = 9). Insulin sensitivity was measured using euglycemic clamps. Mitochondria were examined in muscle biopsies before and after intervention. We measured mitochondrial content and size by electron microscopy, electron transport chain (ETC) activity, cardiolipin content, and mitochondrial DNA content. Intramyocellular content of lipid (IMCL) and fiber-type distribution were determined by histology. RESULTS: The diet-only and diet plus exercise groups achieved similar weight loss (10.8 and 9.2%, respectively); only the diet plus exercise group improved aerobic capacity. Insulin sensitivity improved similarly in both groups. Mitochondrial content and ETC activity increased following the diet plus exercise intervention but remained unchanged following the diet-only intervention, and mitochondrial size decreased with weight loss despite improvement in insulin resistance. IMCL decreased in the diet-only but not in the diet plus exercise intervention. CONCLUSIONS: Despite similar effects to improve insulin resistance, these interventions had differential effects on mitochondria. Clinically significant weight loss in the absence of increased physical activity ameliorates insulin resistance and IMCL but does not increase muscle mitochondrial capacity in obesity.

Characteristics of skeletal muscle mitochondrial biogenesis induced by moderate-intensity exercise and weight loss in obesity.

There are fewer mitochondria and a reduced oxidative capacity in skeletal muscle in obesity. Moderate-intensity physical activity combined with weight loss increase oxidative enzyme activity in obese sedentary adults; however, this adaptation occurs without a significant increase in mitochondrial DNA (mtDNA), which is unlike the classic pattern of mitochondrial biogenesis induced by vigorous activity. The objective of this study was to examine the hypothesis that the mitochondrial adaptation to moderate-intensity exercise and weight loss in obesity induces increased mitochondrial cristae despite a lack of mtDNA proliferation. Content of cardiolipin and mtDNA and enzymatic activities of the electron transport chain (ETC) and tricarboxylic acid cycle were measured in biopsy samples of vastus lateralis muscle obtained from sedentary obese men and women before and following a 4-mo walking intervention combined with weight loss. Cardiolipin increased by 60% from 47 +/- 4 to 74 +/- 8 microg/mU CK (P < 0.01), but skeletal muscle mtDNA content did not change significantly (1,901 +/- 363 to 2,169 +/- 317 Rc, where Rc is relative copy number of mtDNA per diploid nuclear genome). Enzyme activity of the ETC increased (P < 0.01); that for rotenone-sensitive NADH-oxidase (96 +/- 1%) increased more than for ubiquinol-oxidase (48 +/- 6%). Activities for citrate synthase and succinate dehydrogenase increased by 29 +/- 9% and 40 +/- 6%, respectively. In conclusion, moderate-intensity physical activity combined with weight loss induces skeletal muscle mitochondrial biogenesis in previously sedentary obese men and women, but this response occurs without mtDNA proliferation and may be characterized by an increase in mitochondrial cristae.

Interleukin-6 regulation of AMP-activated protein kinase. Potential role in the systemic response to exercise and prevention of the metabolic syndrome.

Interleukin (IL)-6 is a pleiotropic hormone that has both proinflammatory and anti-inflammatory actions. AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that among its other actions responds to decreases in cellular energy state by enhancing processes that generate ATP and inhibiting others that consume ATP but are not acutely necessary for survival. IL-6 is synthesized and released from skeletal muscle in large amounts during exercise, and in rodents, the resultant increase in its concentration correlates temporally with increases in AMPK activity in multiple tissues. That IL-6 may be responsible in great measure for these increases in AMPK is suggested by the fact it increases AMPK activity both in muscle and adipose tissue in vivo and in incubated muscles and cultured adipocytes. In addition, we have found that AMPK activity is diminished in muscle and adipose tissue of 3-month-old IL-6 knockout (KO) mice at rest and that the absolute increases in AMPK activity in these tissues caused by exercise is diminished compared with control mice. Except for an impaired ability to exercise and to oxidize fatty acids, the IL-6 KO mouse appears normal at 3 months of age. On the other hand, by age 9 months, it manifests many of the abnormalities of the metabolic syndrome including obesity, dyslipidemia, and impaired glucose tolerance. This, plus the association of decreased AMPK activity with similar abnormalities in a number of other rodents, suggests that a decrease in AMPK activity may be a causal factor. Whether increases in IL-6, by virtue of their effects on AMPK, contribute to the reported ability of exercise to diminish the prevalence of type 2 diabetes, coronary heart disease, and other disorders associated with the metabolic syndrome remains to be determined.

Effects of exercise on mitochondrial content and function in aging human skeletal muscle.

Skeletal muscle mitochondria are implicated with age-related loss of function and insulin resistance. We examined the effects of exercise on skeletal muscle mitochondria in older (age = 67.3 +/- 0.6 years) men (n = 5) and women (n = 3). Similar increases in (p <.01) cardiolipin (88.2 +/- 9.0 to 130.6 +/- 7.5 microg/mU creatine kinase activity [CK]) and the total mitochondrial DNA (1264 +/- 170 to 1895 +/- 273 copies per diploid of nuclear genome) reflected increased mitochondria content. Succinate oxidase activity, complexes 2-4 of the electron transport chain (ETC), increased from 0.13 +/- 0.02 to 0.20 +/- 0.02 U/mU CK (p <.01). This improvement was more pronounced (p <.05) in subsarcolemmal (127 +/- 48%) compared to intermyofibrillar (56 +/- 12%) mitochondria. NADH oxidase activity, representing total ETC activity, increased from 0.51 +/- 0.09 to 1.00 +/- 0.09 U/mU CK (p <.01). In conclusion, exercise enhances mitochondria ETC activity in older human skeletal muscle, particularly in subsarcolemmal mitochondria, which is likely related to the concomitant increases in mitochondrial biogenesis.

Analysis of cardiolipin in human muscle biopsy.

Cardiolipin is a phospholipid that is specific to the inner mitochondrial membrane and essential for numerous mitochondrial functions. Accordingly, a quantitative assay for cardiolipin can be a valuable aspect of assessing mitochondrial content and functional capacity. The current study was undertaken to develop a simple and reliable method for direct analysis of the major molecular species of cardiolipin and with particular application for analysis of human skeletal muscle. The method that is presented is based on derivatization of cardiolipin in a total lipid extract with 1-pyrenyldiazomethane (PDAM), to form stable, fluorescent 1-pyrenylmethyl esters. The derivatization reaction takes 30 min on ice in a two-phase system (chloroform:methanol:H(2)O:H(2)SO(4)) containing 0.5-1.0mM PDAM and detergent. The contents of the major cardiolipin species in the derivatization mixture can be estimated by HPLC separation with fluorescent detection during a 20 min run on a reverse phase column and with HPLC grade ethanol/0.5mM H(3)PO(4) as the mobile phase. The recovery is about 80%. The method is specific and sensitive with quantitation limits of 0.5-1 pmol cardiolipin. The response of the fluorescence detector (peak area) is linear across a range 5-40 pmol. The assay is linear over the range between 0.3 and 3.0mg of tissue (R(2)=0.998). The assay provides good reproducibility and accuracy (within 5-10%).

Deficiency of subsarcolemmal mitochondria in obesity and type 2 diabetes.

The current study addresses a novel hypothesis of subcellular distribution of mitochondrial dysfunction in skeletal muscle in type 2 diabetes. Vastus lateralis muscle was obtained by percutaneous biopsy from 11 volunteers with type 2 diabetes; 12 age-, sex-, and weight-matched obese sedentary nondiabetic volunteers; and 8 lean volunteers. Subsarcolemmal and intermyofibrillar mitochondrial fractions were isolated by differential centrifugation and digestion techniques. Overall electron transport chain activity was similar in type 2 diabetic and obese subjects, but subsarcolemmal mitochondria electron transport chain activity was reduced in type 2 diabetic subjects (0.017 +/- 0.003 vs. 0.034 +/- 0.007 units/mU creatine kinase [CK], P = 0.01) and sevenfold reduced compared with lean subjects (P < 0.01). Electron transport chain activity in intermyofibrillar mitochondria was similar in type 2 diabetic and obese subjects, though reduced compared with lean subjects. A reduction in subsarcolemmal mitochondria was confirmed by transmission electron microscopy. Although mtDNA was lower in type 2 diabetic and obese subjects, the decrement in electron transport chain activity was proportionately greater, indicating functional impairment. Because of the potential importance of subsarcolemmal mitochondria for signal transduction and substrate transport, this deficit may contribute to the pathogenesis of muscle insulin resistance in type 2 diabetes.

Effects of weight loss and physical activity on skeletal muscle mitochondrial function in obesity.

The current study was undertaken to address responsiveness of skeletal muscle mitochondrial electron transport chain (ETC) activity to weight loss (WL) and exercise in overweight or obese, sedentary volunteers. Fourteen middle-aged participants (7 male/7 female) had assessments of mitochondrial ETC activity and mitochondrial (mt)DNA in vastus lateralis muscle, obtained by percutaneous biopsy, before and after a 16-wk intervention. Mean WL was 9.7 (1.5%) and the mean increase in Vo(2 max) was [means (SD)] 21.7 (3.7)%. Total ETC activity increased significantly, from 0.13 (0.02) to 0.19 (0.03) U/mU creatine kinase (CK; P < 0.001). ETC activity was also assessed in mitochondria isolated into subsarcolemmal (SSM) and intermyofibrillar (IMF-M) fractions. In response to intervention, there was a robust increase of ETC activity in SSM (0.028 (0.007) to 0.046 (0.011) U/mU CK, P < 0.001), and in IMF-M [0.101 (0.015) to 0.148 (0.018) U/mU CK, P < 0.005]. At baseline, the percentage of ETC activity contained in the SSM fraction was low and remained unchanged following intervention [19 (3) vs. 22 (2)%], despite the increase in ETC activity. Also, muscle mtDNA content did not change significantly [1665 (213) vs. 1874 (214) mtDNA/nuclear DNA], denoting functional improvement rather than proliferation of mitochondria as the principal mechanism of enhanced ETC activity. Increases in ETC activity were correlated with energy expenditure during exercise sessions, and ETC activity in SSM correlated with insulin sensitivity after adjustment for Vo(2 max). In summary, skeletal muscle ETC activity is increased by WL and exercise in previously sedentary obese men and women. We conclude that improved skeletal muscle ETC activity following moderate WL and improved aerobic capacity contributes to associated alleviation of insulin resistance.