Ethan J. Anderson

Induction of Endogenous Uncoupling Protein 3 Suppresses Mitochondrial Oxidant Emission during Fatty Acid-supported Respiration

Uncoupling protein 3 (UCP3) expression increases dramatically in skeletal muscle under metabolic states associated with elevated lipid metabolism, yet the function of UCP3 in a physiological context remains controversial. Here, in situ mitochondrial H2O2 emission and respiration were measured in permeabilized fiber bundles prepared from both rat and mouse (wild-type) gastrocnemius muscle after a single bout of exercise plus 18 h of recovery (Ex/R) that induced a ∼2–4-fold increase in UCP3 protein. Elevated uncoupling activity (i.e. GDP inhibitable) was evident in Ex/R fibers only upon the addition of palmitate (known activator of UCP3) or under substrate conditions eliciting substantial rates of H2O2 production (i.e. respiration supported by succinate or palmitoyl-l-carnitine/malate but not pyruvate/malate), indicative of UCP3 activation by endogenous reactive oxygen species. In mice completely lacking UCP3 (ucp3–/–), Ex/R failed to induce uncoupling activity. Surprisingly, when UCP3 activity was inhibited by GDP (rats) or in the absence of UCP3 (ucp3–/–), H2O2 emission was significantly (p < 0.05) higher in Ex/R versus non-exercised control fibers. Collectively, these findings demonstrate that the oxidant emitting potential of mitochondria is increased in skeletal muscle during recovery from exercise, possibly as a consequence of prolonged reliance on lipid metabolism and/or altered mitochondrial biochemistry/morphology and that induction of UCP3 in vivo mediates an increase in uncoupling activity that restores mitochondrial H2O2 emission to non-exercised, control levels.

Novel role for thioredoxin reductase‐2 in mitochondrial redox adaptations to obesogenic diet and exercise in heart and skeletal muscle

•u2002 For reasons not completely understood, obesogenic high‐fat, high‐sucrose (HFHS) diets and exercise training both increase free fatty acid utilization and chronic oxidative stress, yet the former is deleterious to cardiovascular/metabolic health, whereas the latter is beneficial. •u2002 Here, we report that the heart shows decreased mitochondrial H2O2 (mH2O2) generation following HFHS diet, while skeletal muscle shows increased mH2O2, and uncover a novel role for thioredoxin reductase‐2 (TxnRd2) underlying these differences. •u2002 We also show that TxnRd2 is critical to controlling mH2O2 levels during mitochondrial fatty acid oxidation, especially following exercise training in skeletal muscle. •u2002 These findings are important in that they illustrate how the heart and skeletal muscle have contrasting adaptations in antioxidant capacity in response to HFHS diet, and uncover a new role for TxnRd2 in the overall control of mH2O2 in these organs with HFHS diet and exercise training.

Progesterone increases skeletal muscle mitochondrial H2O2 emission in nonmenopausal women.

The luteal phase of the female menstrual cycle is associated with both 1) elevated serum progesterone (P4) and estradiol (E2), and 2) reduced insulin sensitivity. Recently, we demonstrated a link between skeletal muscle mitochondrial H(2)O(2) emission (mE(H2O2)) and insulin resistance. To determine whether serum levels of P4 and/or E(2) are related to mitochondrial function, mE(H2O2) and respiratory O(2) flux (Jo(2)) were measured in permeabilized myofibers from insulin-sensitive (IS, n = 24) and -resistant (IR, n = 8) nonmenopausal women (IR = HOMA-IR > 3.6). Succinate-supported mE(H2O2) was more than 50% greater in the IR vs. IS women (P < 0.05). Interestingly, serum P4 correlated positively with succinate-supported mE(H2O2) (r = 0. 53, P < 0.01). To determine whether P4 or E2 directly affect mitochondrial function, saponin-permeabilized vastus lateralis myofibers biopsied from five nonmenopausal women in the early follicular phase were incubated in P4 (60 nM), E2 (1.4 nM), or both. P4 alone inhibited state 3 Jo(2), supported by multisubstrate combination (P < 0.01). However, E2 alone or in combination with P4 had no effect on Jo(2). In contrast, during state 4 respiration, supported by succinate and glycerophosphate, mE(H2O2) was increased with P4 alone or in combination with E2 (P < 0.01). The results suggest that 1) P4 increases mE(H2O2) with or without E2; 2) P4 alone inhibits Jo(2) but not when E2 is present; and 3) P4 is related to the mE(H2O2) previously linked to skeletal muscle insulin resistance.

Metformin Improves Insulin Signaling in Obese Rats via Reduced IKK Action in a Fiber-Type Specific Manner

Metformin is a widely used insulin-sensitizing drug, though its mechanisms are not fully understood. Metformin has been shown to activate AMPK in skeletal muscle; however, its effects on the inhibitor of κB kinaseβ (IKKβ) in this same tissue are unknown. The aim of this study was to (1) determine the ability of metformin to attenuate IKKβ action, (2) determine whether changes in AMPK activity are associated with changes in IKKβ action in skeletal muscle, and (3) examine whether changes in AMPK and IKKβ function are consistent with improved insulin signaling. Lean and obese male Zuckers received either vehicle or metformin by oral gavage daily for four weeks (four groups of eight). Proteins were measured in white gastrocnemius (WG), red gastrocnemius (RG), and soleus. AMPK phosphorylation increased (P < .05) in WG in both lean (57%) and obese (106%), and this was supported by an increase in phospho-ACC in WG. Further, metformin increased IκBα levels in both WG (150%) and RG (67%) of obese rats, indicative of reduced IKKβ activity (P < .05), and was associated with reduced IRS1-pSer307 (30%) in the WG of obese rats (P < .02). From these data we conclude that metformin treatment appears to exert an inhibitory influence on skeletal muscle IKKβ activity, as evidenced by elevated IκBα levels and reduced IRS1-Ser307 phosphorylation in a fiber-type specific manner.

A Revised Model for the Structure and Function of the Lactose Permease EVIDENCE THAT A FACE ON TRANSMEMBRANE SEGMENT 2 IS IMPORTANT FOR CONFORMATIONAL CHANGES

The lactose permease is an integral membrane protein that cotransports H+ and lactose into the bacterial cytoplasm. Previous work has shown that bulky substitutions at glycine 64, which is found on the cytoplasmic edge of transmembrane segment 2 (TMS-2), cause a substantial decrease in the maximal velocity of lactose uptake without significantly affecting theK m values (Jessen-Marshall, A. E., Parker, N. J., and Brooker, R. J. (1997) J. Bacteriol.179, 2616–2622). In the current study, mutagenesis was conducted along the face of TMS-2 that contains glycine-64. Single amino acid substitutions that substantially changed side-chain volume at codons 52, 57, 59, 63, and 66 had little or no effect on transport activity, whereas substitutions at codons 49, 53, 56, and 60 were markedly defective and/or had lower levels of expression. According to helical wheel plots, Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64 form a continuous stripe along one face of TMS-2. Several of the TMS-2 mutants (S56Y, S56L, S56Q, Q60A, and Q60V) were used as parental strains to isolate mutants that restore transport activity. These mutations were either first-site mutations or second-site suppressors in TMS-1, TMS-2, TMS-7 or TMS-11. A kinetic analysis showed that the suppressors had a higher rate of lactose transport compared with the corresponding parental strains. Overall, the results of this study are consistent with the notion that a face on TMS-2, containing Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64, plays a critical role in conformational changes associated with lactose transport. We hypothesize that TMS-2 slides across TMS-7 and TMS-11 when the lactose permease interconverts between the C1 and C2 conformations. This idea is discussed within the context of a revised model for the structure of the lactose permease.

Microvascular Endothelial Dysfunction in Sedentary, Obese Humans Is Mediated by NADPH Oxidase: Influence of Exercise Training.

Objective—The objectives of this study were to determine the impact of in vivo reactive oxygen species (ROS) on microvascular endothelial function in obese human subjects and the efficacy of an aerobic exercise intervention on alleviating obesity-associated dysfunctionality. Approach and Results—Young, sedentary men and women were divided into lean (body mass index 18–25; n=14), intermediate (body mass index 28–32.5; n=13), and obese (body mass index 33–40; n=15) groups. A novel microdialysis technique was utilized to detect elevated interstitial hydrogen peroxide (H2O2) and superoxide levels in the vastus lateralis of obese compared with both lean and intermediate subjects. Nutritive blood flow was monitored in the vastus lateralis via the microdialysis-ethanol technique. A decrement in acetylcholine-stimulated blood flow revealed impaired microvascular endothelial function in the obese subjects. Perfusion of apocynin, an NADPH oxidase inhibitor, lowered (normalized) H2O2 and superoxide levels, and reversed microvascular endothelial dysfunction in obese subjects. After 8 weeks of exercise, H2O2 levels were decreased in the obese subjects and microvascular endothelial function in these subjects was restored to levels similar to lean subjects. Skeletal muscle protein expression of the NADPH oxidase subunits p22phox, p47phox, and p67phox was increased in obese relative to lean subjects, where p22phox and p67phox expression was attenuated by exercise training in obese subjects. Conclusions—This study implicates NADPH oxidase as a source of excessive ROS production in skeletal muscle of obese individuals and links excessive NADPH oxidase–derived ROS to microvascular endothelial dysfunction in obesity. Furthermore, aerobic exercise training proved to be an effective strategy for alleviating these maladies.

A carnosine analog mitigates metabolic disorders of obesity by reducing carbonyl stress

Sugar- and lipid-derived aldehydes are reactive carbonyl species (RCS) frequently used as surrogate markers of oxidative stress in obesity. A pathogenic role for RCS in metabolic diseases of obesity remains controversial, however, partly because of their highly diffuse and broad reactivity and the lack of specific RCS-scavenging therapies. Naturally occurring histidine dipeptides (e.g., anserine and carnosine) show RCS reactivity, but their therapeutic potential in humans is limited by serum carnosinases. Here, we present the rational design, characterization, and pharmacological evaluation of carnosinol, i.e., (2S)-2-(3-amino propanoylamino)-3-(1H-imidazol-5-yl)propanol, a derivative of carnosine with high oral bioavailability that is resistant to carnosinases. Carnosinol displayed a suitable ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile and was determined to have the greatest potency and selectivity toward &agr;,&bgr;-unsaturated aldehydes (e.g., 4-hydroxynonenal, HNE, ACR) among all others reported thus far. In rodent models of diet-induced obesity and metabolic syndrome, carnosinol dose-dependently attenuated HNE adduct formation in liver and skeletal muscle, while simultaneously mitigating inflammation, dyslipidemia, insulin resistance, and steatohepatitis. These improvements in metabolic parameters with carnosinol were not due to changes in energy expenditure, physical activity, adiposity, or body weight. Collectively, our findings illustrate a pathogenic role for RCS in obesity-related metabolic disorders and provide validation for a promising new class of carbonyl-scavenging therapeutic compounds rationally derived from carnosine.