A practical perspective on how to develop, implement, execute, and reproduce high-resolution respirometry experiments: The physiologist's guide to an Oroboros O2k.

The development of high-resolution respirometry (HRR) has greatly expanded the analytical scope to study mitochondrial respiratory control relative to specific tissue/cell types across various metabolic states. Specifically, the Oroboros Oxygraph 2000 (O2k) is a common tool for measuring rates of mitochondrial respiration and is the focus of this perspective. The O2k platform is amenable for answering numerous bioenergetic questions. However, inherent variability with HRR-derived data, both within and amongst users, can impede progress in bioenergetics research. Therefore, we advocate for several vital considerations when planning and conducting O2k experiments to ultimately enhance transparency and reproducibility across laboratories. In this perspective, we offer guidance for best practices of mitochondrial preparation, protocol selection, and measures to increase reproducibility. The goal of this perspective is to propagate the use of the O2k, enhance reliability and validity for both new and experienced O2k users, and provide a reference for peer reviewers.

Phytochemical compound PB125 attenuates skeletal muscle mitochondrial dysfunction and impaired proteostasis in a model of musculoskeletal decline.

Impaired mitochondrial function and disrupted proteostasis contribute to musculoskeletal dysfunction. However, few interventions simultaneously target these two drivers to prevent musculoskeletal decline. Nuclear factor erythroid 2-related factor 2 (Nrf2) activates a transcriptional programme promoting cytoprotection, metabolism, and proteostasis. We hypothesized daily treatment with a purported Nrf2 activator, PB125, in Hartley guinea pigs, a model of musculoskeletal decline, would attenuate the progression of skeletal muscle mitochondrial dysfunction and impaired proteostasis and preserve musculoskeletal function. We treated 2- and 5-month-old male and female Hartley guinea pigs for 3 and 10 months, respectively, with the phytochemical compound PB125. Longitudinal assessments of voluntary mobility were measured using Any-MazeTM open-field enclosure monitoring. Cumulative skeletal muscle protein synthesis rates were measured using deuterium oxide over the final 30 days of treatment. Mitochondrial oxygen consumption in soleus muscles was measured using high resolution respirometry. In both sexes, PB125 (1) increased electron transfer system capacity; (2) attenuated the disease/age-related decline in coupled and uncoupled mitochondrial respiration; and (3) attenuated declines in protein synthesis in the myofibrillar, mitochondrial and cytosolic subfractions of the soleus. These effects were not associated with statistically significant prolonged maintenance of voluntary mobility in guinea pigs. Collectively, treatment with PB125 contributed to maintenance of skeletal muscle mitochondrial respiration and proteostasis in a pre-clinical model of musculoskeletal decline. Further investigation is necessary to determine if these documented effects of PB125 are also accompanied by slowed progression of other aspects of musculoskeletal dysfunction. KEY POINTS: Aside from exercise, there are no effective interventions for musculoskeletal decline, which begins in the fifth decade of life and contributes to disability and cardiometabolic diseases. Targeting both mitochondrial dysfunction and impaired protein homeostasis (proteostasis), which contribute to the age and disease process, may mitigate the progressive decline in overall musculoskeletal function (e.g. gait, strength). A potential intervention to target disease drivers is to stimulate nuclear factor erythroid 2-related factor 2 (Nrf2) activation, which leads to the transcription of genes responsible for redox homeostasis, proteome maintenance and mitochondrial energetics. Here, we tested a purported phytochemical Nrf2 activator, PB125, to improve mitochondrial function and proteostasis in male and female Hartley guinea pigs, which are a model for musculoskeletal ageing. PB125 improved mitochondrial respiration and attenuated disease- and age-related declines in skeletal muscle protein synthesis, a component of proteostasis, in both male and female Hartley guinea pigs.

Physiologic Responses to Dietary Sulfur Amino Acid Restriction in Mice Are Influenced by Atf4 Status and Biological Sex.

BACKGROUND: Dietary sulfur amino acid restriction (SAAR) improves body composition and metabolic health across several model organisms in part through induction of the integrated stress response (ISR). OBJECTIVE: We investigate the hypothesis that activating transcription factor 4 (ATF4) acts as a converging point in the ISR during SAAR. METHODS: Using liver-specific or global gene ablation strategies, in both female and male mice, we address the role of ATF4 during dietary SAAR. RESULTS: We show that ATF4 is dispensable in the chronic induction of the hepatokine fibroblast growth factor 21 while being essential for the sustained production of endogenous hydrogen sulfide. We also affirm that biological sex, independent of ATF4 status, is a determinant of the response to dietary SAAR. CONCLUSIONS: Our results suggest that auxiliary components of the ISR, which are independent of ATF4, are critical for SAAR-mediated improvements in metabolic health in mice.

Incorporating Behavior Change Strategies Into an Exercise Trial to Improve Physical Activity Maintenance Among Adults at High Risk for Type II Diabetes.

This study examined the feasibility and effects of a 1-hr physical activity (PA) behavior change (PABC) discussion session on PA, 12 weeks after completing an exercise trial. Adults at high risk of Type II diabetes were randomized to the PABC or a control group. PA was self-reported using the International Physical Activity Questionnaire. Chi-square tests compared the proportion of participants classified as moderately active or greater at the 12-week follow-up. Participants (N = 50) were M = 61.8 ± 5.5 years old and mostly female (80%). All participants completed the PABC discussion session, and compliance with the International Physical Activity Questionnaire at 12-week follow-up was 78%. Barrier self-efficacy increased immediately following the PABC (MΔ0.5 ± 0.9; t(22) = -2.45, p = .023). At 12-week follow-up, 88% in the PABC were moderately active or greater, compared with 50% in the control (p = .015). Incorporating a PABC discussion session as part of an exercise efficacy trial was feasible and may help improve PA maintenance.

Mechanisms of In Vivo Ribosome Maintenance Change in Response to Nutrient Signals.

Control of protein homeostasis is fundamental to the health and longevity of all organisms. Because the rate of protein synthesis by ribosomes is a central control point in this process, regulation, and maintenance of ribosome function could have amplified importance in the overall regulatory circuit. Indeed, ribosomal defects are commonly associated with loss of protein homeostasis, aging, and disease (1-4), whereas improved protein homeostasis, implying optimal ribosomal function, is associated with disease resistance and increased lifespan (5-7). To maintain a high-quality ribosome population within the cell, dysfunctional ribosomes are targeted for autophagic degradation. It is not known if complete degradation is the only mechanism for eukaryotic ribosome maintenance or if they might also be repaired by replacement of defective components. We used stable-isotope feeding and protein mass spectrometry to measure the kinetics of turnover of ribosomal RNA (rRNA) and 71 ribosomal proteins (r-proteins) in mice. The results indicate that exchange of individual proteins and whole ribosome degradation both contribute to ribosome maintenance in vivo In general, peripheral r-proteins and those with more direct roles in peptide-bond formation are replaced multiple times during the lifespan of the assembled structure, presumably by exchange with a free cytoplasmic pool, whereas the majority of r-proteins are stably incorporated for the lifetime of the ribosome. Dietary signals impact the rates of both new ribosome assembly and component exchange. Signal-specific modulation of ribosomal repair and degradation could provide a mechanistic link in the frequently observed associations among diminished rates of protein synthesis, increased autophagy, and greater longevity (5, 6, 8, 9).

Enhanced skeletal muscle regrowth and remodelling in massaged and contralateral non-massaged hindlimb.

KEY POINTS: Muscle fibre cross sectional area is enhanced with massage in the form of cyclic compressive loading during regrowth after atrophy. Massage enhances protein synthesis of the myofibrillar and cytosolic, but not the mitochondrial fraction, in muscle during regrowth. Focal adhesion kinase activation and satellite cell number are elevated in muscles undergoing massage during regrowth. Muscle fibre cross sectional area and protein synthesis of the myofibrillar fraction, but not DNA synthesis, are elevated in muscle of the contralateral non-massaged limb. Massage in the form of cyclic compressive loading is a potential anabolic intervention during muscle regrowth after atrophy. ABSTRACT: Massage, in the form of cyclic compressive loading (CCL), is associated with multiple health benefits, but its potential anabolic effect on atrophied muscle has not been investigated. We hypothesized that the mechanical activity associated with CCL induces an anabolic effect in skeletal muscle undergoing regrowth after a period of atrophy. Fischer-Brown Norway rats at 10 months of age were hindlimb unloaded for a period of 2 weeks. The rats were then allowed reambulation with CCL applied at a 4.5 N load at 0.5 Hz frequency for 30 min every other day for four bouts during a regrowth period of 8 days. Muscle fibre cross sectional area was enhanced by 18% with massage during regrowth compared to reloading alone, and this was accompanied by elevated myofibrillar and cytosolic protein as well as DNA synthesis. Focal adhesion kinase phosphorylation indicated that CCL increased mechanical stimulation, while a higher number of Pax7+ cells likely explains the elevated DNA synthesis. Surprisingly, the contralateral non-massaged limb exhibited a comparable 17% higher muscle fibre size compared to reloading alone, and myofibrillar protein synthesis, but not DNA synthesis, was also elevated. We conclude that massage in the form of CCL induces an anabolic response in muscles regrowing after an atrophy-inducing event. We suggest that massage can be used as an intervention to aid in the regrowth of muscle lost during immobilization.

Short-term changes in diet composition do not affect in vivo hepatic protein synthesis in rats.

Protein synthesis is critical to protein homeostasis (proteostasis), and modifications in protein synthesis influence lifespan and the development of comorbidities associated with obesity. In the present study, we examined the acute response of liver protein synthesis to either high-fat or high-sucrose diets in order to elucidate nutrient-mediated regulation of hepatic protein synthesis in the absence of body fat accumulation. Total and endoplasmic reticulum-associated protein syntheses were assessed by use of the stable isotope, deuterium oxide (2H2O), in rats provided a control diet or diets enriched in polyunsaturated fat, saturated fat, or sucrose for 2, 4, or 7 days. The three experimental diets increased hepatic triglycerides 46-91% on day 7 and fasting insulin levels 83-117% on day 7, but did not result in differences in body weight when compared with control ( n = 6/diet/time). The fraction of newly synthesized proteins in total liver lysates and microsomes was not significantly different among dietary groups ( n = 3/diet/time). To determine whether the experimental diets provoked a transcriptional response to enhance the capacity for protein synthesis, we also measured a panel of genes linked to amino acid transport, synthesis, and processing. There were no significant differences in any of the genes measured among groups. Therefore, dietary treatments that have been linked to impaired proteostasis and that promote hepatic steatosis and insulin resistance, did not result in significant changes in total or ER-associated protein synthesis in the liver over a 7-day period.

Targeting mitochondrial function and proteostasis to mitigate dynapenia.

Traditionally, interventions to treat skeletal muscle aging have largely targeted sarcopenia-the age-related loss of skeletal muscle mass. Dynapenia refers to the age-related loss in skeletal muscle function due to factors outside of muscle mass, which helps to inform treatment strategies for aging skeletal muscle. There is evidence that mechanisms to maintain protein homeostasis and proteostasis, deteriorate with age. One key mechanism to maintain proteostasis is protein turnover, which is an energetically costly process. When there is a mismatch between cellular energy demands and energy provision, inelastic processes related to metabolism are maintained, but there is competition for the remaining energy between the elastic processes of somatic maintenance and growth. With aging, mitochondrial dysfunction reduces ATP generation capacity, constraining the instantaneous supply of energy, thus compromising growth and somatic maintenance processes. Further, with age the need for somatic maintenance increases because of the accumulation of protein damage. In this review, we highlight the significant role mitochondria have in maintaining skeletal muscle proteostasis through increased energy provision, protein turnover, and substrate flux. In addition, we provide evidence that improving mitochondrial function could promote a cellular environment that is conducive to somatic maintenance, and consequently for mitigating dynapenia. Finally, we highlight interventions, such as aerobic exercise, that could be used to improve mitochondrial function and improve outcomes related to dynapenia.

Mitochondrial proteostasis as a shared characteristic of slowed aging: the importance of considering cell proliferation.

Proteostasis is one of the seven "pillars of aging research" identified by the Trans-NIH Geroscience Initiative and loss of proteostasis is associated with aging and age-related chronic disease. Accumulated protein damage and resultant cellular dysfunction are consequences of limited protein repair systems and slowed protein turnover. When relatively high rates of protein turnover are maintained despite advancing age, damaged proteins are more quickly degraded and replaced, maintaining proteome fidelity. Therefore, maintenance of protein turnover represents an important proteostatic mechanism. However, measurement of protein synthesis without consideration for cell proliferation can result in an incomplete picture, devoid of information about how new proteins are being allocated. Simultaneous measurement of protein and DNA synthesis provides necessary mechanistic insight about proteins apportioned for newly proliferating cells versus for somatic maintenance. Using this approach with a number of murine models of slowed aging shows that, compared to controls, energetic resources are directed more toward somatic maintenance and proteostasis, and away from cell growth and proliferation. In particular, slowed aging models are associated with heightened mechanisms of mitochondrial proteostatic maintenance. Taking cell proliferation into account may explain the paradoxical findings that aging itself and slowed aging interventions can both be characterized by slower rates of protein synthesis.

Long-term rates of mitochondrial protein synthesis are increased in mouse skeletal muscle with high-fat feeding regardless of insulin-sensitizing treatment.

Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial, with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 wk of a high-fat compared with a low-fat diet. For 8 additional weeks, diets were enriched with pioglitazone to restore insulin sensitivity compared with nonenriched control low-fat or high-fat diets. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during weeks 10-12 High-resolution respirometry was performed using palmitoyl-l-carnitine, glutamate+malate, and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl- l-carnitine oxidation were increased in mice consuming a high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or H2O2 emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with a high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with a high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance.

Dietary Methionine Restriction Regulates Liver Protein Synthesis and Gene Expression Independently of Eukaryotic Initiation Factor 2 Phosphorylation in Mice.

Background: The phosphorylation of eukaryotic initiation factor 2 (p-eIF2) during dietary amino acid insufficiency reduces protein synthesis and alters gene expression via the integrated stress response (ISR).Objective: We explored whether a Met-restricted (MR) diet activates the ISR to reduce body fat and regulate protein balance.Methods: Male and female mice aged 3-6 mo with either whole-body deletion of general control nonderepressible 2 (Gcn2) or liver-specific deletion of protein kinase R-like endoplasmic reticulum kinase (Perk) alongside wild-type or floxed control mice were fed an obesogenic diet sufficient in Met (0.86%) or an MR (0.12% Met) diet for ≤5 wk. Ala enrichment with deuterium was measured to calculate protein synthesis rates. The guanine nucleotide exchange factor activity of eIF2B was measured alongside p-eIF2 and hepatic mRNA expression levels at 2 d and 5 wk. Metabolic phenotyping was conducted at 4 wk, and body composition was measured throughout. Results were evaluated with the use of ANOVA (P < 0.05).Results: Feeding an MR diet for 2 d did not increase hepatic p-eIF2 or reduce eIF2B activity in wild-type or Gcn2-/- mice, yet many genes transcriptionally regulated by the ISR were altered in both strains in the same direction and amplitude. Feeding an MR diet for 5 wk increased p-eIF2 and reduced eIF2B activity in wild-type but not Gcn2-/- mice, yet ISR-regulated genes altered in both strains similarly. Furthermore, the MR diet reduced mixed and cytosolic but not mitochondrial protein synthesis in both the liver and skeletal muscle regardless of Gcn2 status. Despite the similarities between strains, the MR diet did not increase energy expenditure or reduce body fat in Gcn2-/- mice. Finally, feeding the MR diet to mice with Perk deleted in the liver increased hepatic p-eIF2 and altered body composition similar to floxed controls.Conclusions: Hepatic activation of the ISR resulting from an MR diet does not require p-eIF2. Gcn2 status influences body fat loss but not protein balance when Met is restricted.

The novel combination of theophylline and bambuterol as a potential treatment of hypoxemia in humans.

Hypoxemia can be life-threatening, both acutely and chronically. Because hypoxemia causes vascular dysregulation that further restricts oxygen availability to tissue, it can be pharmacologically addressed. We hypothesized that theophylline can be safely combined with the ß2-adrenergic vasodilator bambuterol to improve oxygen availability in hypoxemic patients. Ergogenicity and hemodynamic effects of bambuterol and theophylline were measured in rats under hypobaric and normobaric hypoxia (12% O2). Feasibility in humans was assessed using randomized, double-blind testing of the influence of combined slow-release theophylline (300 mg) and bambuterol (20 mg) on adverse events (AEs), plasma K+, pulse, blood pressure, and drug interaction. Both drugs and their combination significantly improved hypoxic endurance in rats. In humans, common AEs were low K+ (<3.5 mmol/L; bambuterol: 12, theophylline: 4, combination: 13 episodes) and tremors (10, 0, 14 episodes). No exacerbation or serious AE occurred when drugs were combined. A drop in plasma K+ coincided with peak bambuterol plasma concentrations. Bambuterol increased heart rate by approximately 13 bpm. Drug interaction was present but small. We report promise, feasibility, and relative safety of combined theophylline and bambuterol as a treatment of hypoxemia in humans. Cardiac safety and blood K+ will be important safety endpoints when testing these drugs in hypoxemic subjects.

Ergogenic properties of metformin in simulated high altitude.

Metformin augments glucose/glycogen regulation and may acutely promote fatigue resistance during high-intensity exercise. In hypobaric environments, such as high altitude, the important contribution of carbohydrates to physiological function is accentuated as glucose/glycogen dependence is increased. Because hypoxia/hypobaria decreases insulin sensitivity, replenishing skeletal muscle glycogen in high altitude becomes challenging and subsequent physical performance may be compromised. We hypothesized that in conditions where glycogen repletion was critical to physical outcomes, metformin would attenuate hypoxia-mediated decrements in exercise performance. On three separate randomly ordered occasions, 13 healthy men performed glycogen-depleting exercise and ingested a low-carbohydrate dinner (1200 kcals, <10% carbohydrate). The next morning, in either normoxia or hypoxia (FiO2 =0.15), they ingested a high-carbohydrate breakfast (1225 kcals, 70% carbohydrate). Placebo (719 mg maltodextrin) or metformin (500 mg BID) was consumed 3 days prior to each hypoxia visit. Subjects completed a 12.5 km cycle ergometer time trial 3.5 hours following breakfast. Hypoxia decreased resting and exercise oxyhemoglobin saturation (P<.001). Neither hypoxia nor metformin affected the glucose response to breakfast (P=.977), however, compared with placebo, metformin lowered insulin concentration in hypoxia 45 minutes after breakfast (64.1±6.6 µU/mL vs 48.5±7.8 µU/mL; mean±SE; P<.001). Post-breakfast, pre-exercise vastus lateralis glycogen content increased in normoxia (+33%: P=.025) and in hypoxia with metformin (+81%; P=.006), but not in hypoxia with placebo (+27%; P=.167). Hypoxia decreased time trial performance compared with normoxia (P<.01). This decrement was similar with placebo (+2.6±0.8 minutes) and metformin (+1.6±0.3 minutes). These results indicate that metformin promotes glycogen synthesis but not endurance exercise performance in healthy men exposed to simulated high altitude.

Greater muscle protein synthesis and mitochondrial biogenesis in males compared with females during sprint interval training.

Improved endurance exercise performance in adult humans after sprint interval training (SIT) has been attributed to mitochondrial biogenesis. However, muscle protein synthesis (MPS) and mitochondrial biogenesis during SIT have not been measured, nor have sex-specific differences. We hypothesized that males and females would have similar rates of MPS, mitochondrial biogenesis, and synthesis of individual proteins during SIT. Deuterium oxide (D2O) was orally administered to 21 adults [11 male, 10 female; mean age, 23±1 yr; body mass index (BMI), 22.8±0.6 kg/m(2); mean± SE] for 4 wk, to measure protein synthesis rates while completing 9 sessions of 4-8 bouts of 30 s duration on a cycle ergometer separated by 4 min of active recovery. Samples of the vastus lateralis were taken before and 48 h after SIT. SIT increased maximum oxygen uptake (VO(2max), males 43.4±2.1-44.0±2.3; females 39.5±0.9-42.5±1.3 ml/kg/min; P=0.002). MPS was greater in the males than in the females in the mixed (~150%; P < 0.001), cytosolic (~135%; P=0.038), and mitochondrial (~135%; P=0.056) fractions. The corresponding ontological clusters of individual proteins were significantly greater in the males than in the females (all P<0.00001). For the first time, we document greater MPS and mitochondrial biogenesis during SIT in males than in females and describe the synthetic response of individual proteins in humans during exercise training.

Hypoxia mediated pulmonary edema: potential influence of oxidative stress, sympathetic activation and cerebral blood flow.

BACKGROUND: Neurogenic pulmonary edema (NPE) is a non-cardiogenic form of pulmonary edema that can occur consequent to central neurologic insults including stroke, traumatic brain injury, and seizure. NPE is a public health concern due to high morbidity and mortality, yet the mechanism(s) are unknown. We hypothesized that NPE, evoked by cerebral hypoxia in the presence of systemic normoxia, would be accompanied by sympathetic activation, oxidative stress, and compensatory antioxidant mechanisms. METHODS: Thirteen Walker hounds were assigned to cerebral hypoxia (SaO2 ~ 55 %) with systemic normoxia (SaO2 ~ 90 %) (CH; n = 6), cerebral and systemic (global) hypoxia (SaO2 ~ 60 %) (GH; n = 4), or cerebral and systemic normoxia (SaO2 ~ 90 %) (CON; n = 3). Femoral venous (CH and CON) perfusate was delivered via cardiopulmonary bypass to the brain and GH was induced by FiO2 = 10 % to maintain the SaO2 at ~60 %. Lung wet to lung dry weight ratios (LWW/LDW) were assessed as an index of pulmonary edema in addition to hemodynamic measurements. Plasma catecholamines were measured as markers of sympathetic nervous system (SNS) activity. Total glutathione, protein carbonyls, and malondialdehyde were assessed as indicators of oxidative stress. Brain and lung compensatory antioxidants were measured with immunoblotting. RESULTS: Compared to CON, LWW/LDW and pulmonary artery pressure were greater in CH and GH. Expression of hemeoxygenase-1 in brain was higher in CH compared to GH and CON, despite no group differences in oxidative damage in any tissue. Catecholamines tended to be higher in CH and GH. CONCLUSION: Cerebral hypoxia, with systemic normoxia, is not systematically associated with an increase in oxidative stress and compensatory antioxidant enzymes in lung, suggesting oxidative stress did not contribute to NPE in lung. However, increased SNS activity may play a role in the induction of NPE during hypoxia.

The Effects of Sympathetic Inhibition on Metabolic and Cardiopulmonary Responses to Exercise in Hypoxic Conditions.

OBJECTIVE: Pre-exertion skeletal muscle glycogen content is an important physiological determinant of endurance exercise performance: low glycogen stores contribute to premature fatigue. In low-oxygen environments (hypoxia), the important contribution of carbohydrates to endurance performance is further enhanced as glucose and glycogen dependence is increased; however, the insulin sensitivity of healthy adult humans is decreased. In light of this insulin resistance, maintaining skeletal muscle glycogen in hypoxia becomes difficult, and subsequent endurance performance is impaired. Sympathetic inhibition promotes insulin sensitivity in hypoxia but may impair hypoxic exercise performance, in part due to suppression of cardiac output. Accordingly, we tested the hypothesis that hypoxic exercise performance after intravenous glucose feeding in a low-oxygen environment will be attenuated when feeding occurs during sympathetic inhibition. METHODS: On 2 separate occasions, while breathing a hypoxic gas mixture, 10 healthy men received 1 hour of parenteral carbohydrate infusion (20% glucose solution in saline; 75 g), after which they performed stationary cycle ergometer exercise (~65% maximal oxygen uptake) until exhaustion. Forty-eight hours before 1 visit, chosen randomly, sympathetic inhibition via transdermal clonidine (0.2 mg/d) was initiated. RESULTS: The mean time to exhaustion after glucose feeding both with and without sympathetic inhibition was not different (22.7 ± 5.4 minutes vs 23.5 ± 5.1 minutes; P = .73). CONCLUSIONS: Sympathetic inhibition protects against hypoxia-mediated insulin resistance without influencing subsequent hypoxic endurance performance.

Long-lived crowded-litter mice have an age-dependent increase in protein synthesis to DNA synthesis ratio and mTORC1 substrate phosphorylation.

Increasing mouse litter size [crowded litter (CL)] presumably imposes a transient nutrient stress during suckling and extends lifespan through unknown mechanisms. Chronic calorically restricted and rapamycin-treated mice have decreased DNA synthesis and mTOR complex 1 (mTORC1) signaling but maintained protein synthesis, suggesting maintenance of existing cellular structures. We hypothesized that CL would exhibit similar synthetic and signaling responses to other long-lived models and, by comparing synthesis of new protein to new DNA, that insight may be gained into the potential preservation of existing cellular structures in the CL model. Protein and DNA synthesis was assessed in gastroc complex, heart, and liver of 4- and 7-mo CL mice. We also examined mTORC1 signaling in 3- and 7-mo aged animals. Compared with controls, 4-mo CL had greater DNA synthesis in gastroc complex with no differences in protein synthesis or mTORC1 substrate phosphorylation across tissues. Seven-month CL had less DNA synthesis than controls in heart and greater protein synthesis and mTORC1 substrate phosphorylation across tissues. The increased new protein-to-new DNA synthesis ratio suggests that new proteins are synthesized more so in existing cells at 7 mo, differing from 4 mo, in CL vs. controls. We propose that, in CL, protein synthesis shifts from being directed toward new cells (4 mo) to maintenance of existing cellular structures (7 mo), independently of decreased mTORC1.

Evaluation of quantitative performance of sequential immobilized metal affinity chromatographic enrichment for phosphopeptides.

We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. Although our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semiquantitative method when used in large-scale phosphoproteomics studies in complex backgrounds.

Treatment with PB125® Increases Femoral Long Bone Strength in 15-Month-Old Female Hartley Guinea Pigs.

Nuclear factor-erythroid 2-related factor-2 (Nrf2) is a transcription factor that serves as a master regulator of anti-inflammatory agents, phase I xenobiotic, and phase II antioxidant enzymes, all of which provide a cytoprotective role during disease progression. We hypothesized that oral administration of a purported phytochemical Nrf2-activator, PB125®, would increase long bone strength in aging Hartley guinea pigs, a model prone to musculoskeletal decline. Male (N = 56) and female (N = 56) guinea pigs were randomly assigned to receive daily oral treatment with either PB125® or vehicle control. Animals were treated for a consecutive 3-months (starting at 2-months of age) or 10-months (starting at 5-months of age) and sacrificed at 5-months or 15-months of age, respectively. Outcome measures included: (1) ANY-maze™ enclosure monitoring, (2) quantitative microcomputed tomography, and (3) biomechanical testing. Treatment with PB125® for 10 months resulted in increased long bone strength as determined by ultimate bending stress in female Hartley guinea pigs. In control groups, increasing age resulted in significant effects on geometric and structural properties of long bones, as well as a trending increase in ultimate bending stress. Furthermore, both age and sex had a significant effect on the geometric properties of both cortical and trabecular bone. Collectively, this work suggests that this nutraceutical may serve as a promising target and preventive measure in managing the decline in bone mass and quality documented in aging patients. Auxiliary to this main goal, this work also capitalized upon 5 and 15-month-old male and female animals in the control group to characterize age- and sex-specific differences on long bone geometric, structural, and material properties in this animal model.