Short, frequent, light-intensity walking activity improves postprandial vascular-inflammatory biomarkers in people with type 1 diabetes: The SIT-LESS randomized controlled trial.

AIM: To examine the effect of interrupting prolonged sitting with short, frequent, light-intensity activity on postprandial cardiovascular markers in people with type 1 diabetes (T1D). MATERIALS AND METHODS: In a randomized crossover trial, 32 adults with T1D (mean ± SD age 28 ± 5 years, glycated haemoglobin 67.9 ± 12.6 mmol/mol, 17 women) completed two 7-h laboratory visits separated by >7 days. Participants either remained seated for 7 h (SIT) or interrupted sitting with 3-min bouts of self-paced walking at 30-min intervals commencing 1 h after each meal (SIT-LESS). Physical activity, insulin regimen, experimental start times, and meal consumption were standardized during each arm. Plasma levels of interleukin (IL)-1ß, tumour necrosis factor (TNF)-α, plasminogen activator inhibitor (PAI)-1 and fibrinogen were sampled at baseline, 3.5 and 7 h, and assessed for within- and between-group effects using a repeated measures ANOVA. The estimated glucose disposal rate was used to determine the insulin resistance status. RESULTS: Vascular-inflammatory parameters were comparable between SIT and SIT-LESS at baseline (p > .05). TNF-α, IL-1ß, PAI-1 and fibrinogen increased over time under SIT, whereas these rises were attenuated under SIT-LESS (p < .001). Specifically, over the 7 h under SIT, postprandial increases were detected in TNF-α, IL-1ß, PAI-1 and fibrinogen (+67%, +49%, +49% and +62%, respectively; p < .001 for all). Conversely, the SIT-LESS group showed no change in IL-1ß (-9%; p > .50), whereas reductions were observed in TNF-α, PAI-1 and fibrinogen (-22%, -42% and -44%, respectively; p < .001 for all). The intervention showed enhanced effects in insulin-resistant individuals with T1D. CONCLUSIONS: Interrupting prolonged sitting with light-intensity activity ameliorates postprandial increases in vascular-inflammatory markers in T1D. TRIAL REGISTRATION: The trial was prospectively registered (ISRCTN13641847).

The mitochondrially targeted peptide elamipretide (SS-31) improves ADP sensitivity in aged mitochondria by increasing uptake through the adenine nucleotide translocator (ANT).

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function. ELAM binds directly to ANT and ATP synthase and ELAM treatment improves ADP sensitivity, increases ATP production, and improves physiological function in old muscles. ADP (adenosine diphosphate), ATP (adenosine triphosphate), VDAC (voltage-dependent anion channel), ANT (adenine nucleotide translocator), H+ (proton), ROS (reactive oxygen species), NADH (nicotinamide adenine dinucleotide), FADH2 (flavin adenine dinucleotide), O2 (oxygen), ELAM (elamipretide), -SH (free thiol), -SSG (glutathionylated protein).

Dietary fat intake is associated with insulin resistance and an adverse vascular profile in patients with T1D: a pooled analysis.

BACKGROUND: Insulin resistance (IR) increases vascular risk in individuals with Type 1 Diabetes (T1D). We aimed to investigate the relationship between dietary intake and IR, as well as vascular biomarkers in T1D. METHODS: Baseline data from three randomised controlled trials were pooled. Estimated glucose disposal rate (eGDR) was used as an IR marker. Employing multivariate nutrient density substitution models, we examined the association between macronutrient composition and IR/vascular biomarkers (tumour necrosis factor-α, fibrinogen, tissue factor activity, and plasminogen activator inhibitor-1). RESULTS: Of the 107 patients, 50.5% were male with mean age of 29 ± 6 years. Those with lower eGDR were older with a longer diabetes duration, higher insulin requirements, and an adverse vascular profile (p < 0.05). Patients with higher degrees of IR had higher total energy intake (3192 ± 566 vs. 2772 ± 268 vs. 2626 ± 395 kcal/d for eGDR < 5.1 vs. 5.1-8.6 vs. ≥ 8.7 mg/kg/min, p < 0.001) and consumed a higher absolute and proportional amount of fat (47.6 ± 18.6 vs. 30.4 ± 8.1 vs. 25.8 ± 10.4%, p < 0.001). After adjusting for total energy intake, age, sex, and diabetes duration, increased carbohydrate intake offset by an isoenergetic decrease in fat was associated with higher eGDR (ß = 0.103, 95% CI 0.044-0.163). In contrast, increased dietary fat at the expense of dietary protein intake was associated with lower eGDR (ß = - 0.119, 95% CI - 0.199 to - 0.040). Replacing fat with 5% isoenergetic amount of carbohydrate resulted in decreased vascular biomarkers (p < 0.05). CONCLUSION: Higher fat, but not carbohydrate, intake is associated with increased IR and an adverse vascular profile in patients with T1D.

Glucose variability is associated with an adverse vascular profile but only in the presence of insulin resistance in individuals with type 1 diabetes: An observational study.

AIMS/HYPOTHESIS: We hypothesised that the detrimental effect of high glucose variability (GV) in people with type 1 diabetes is mainly evident in those with concomitant insulin resistance. METHODS: We conducted secondary analyses on continuous glucose monitoring (CGM) using baseline observational data from three randomised controlled trials and assessed the relationship with established vascular markers. We used standard CGM summary statistics and principal component analysis to generate individual glucose variability signatures for each participant. Cluster analysis was then employed to establish three GV clusters (low, intermediate, or high GV, respectively). The relationship with thrombotic biomarkers was then investigated according to insulin resistance, assessed as estimated glucose disposal rate (eGDR). RESULTS: Of 107 patients, 45%, 37%, and 18% of patients were assigned into low, intermediate, and high GV clusters, respectively. Thrombosis biomarkers (including fibrinogen, plasminogen activator inhibitor-1, tissue factor activity, and tumour necrosis factor-alpha) increased in a stepwise fashion across all three GV clusters; this increase in thrombosis markers was evident in the presence of low but not high eGDR and at a threshold of eGDR <5.1 mg/kg/min. CONCLUSION: Higher GV is associated with increased thrombotic biomarkers in type 1 diabetes but only in those with concomitant insulin resistance.

Application of Machine Learning to Assess Interindividual Variability in Rapid-Acting Insulin Responses After Subcutaneous Injection in People With Type 1 Diabetes.

OBJECTIVES: Circulating insulin concentrations mediate vascular-inflammatory and prothrombotic factors. However, it is unknown whether interindividual differences in circulating insulin levels are associated with different inflammatory and prothrombotic profiles in type 1 diabetes (T1D). We applied an unsupervised machine-learning approach to determine whether interindividual differences in rapid-acting insulin levels associate with parameters of vascular health in patients with T1D. METHODS: We re-analyzed baseline pretreatment meal-tolerance test data from 2 randomized controlled trials in which 32 patients consumed a mixed-macronutrient meal and self-administered a single dose of rapid-acting insulin individualized by carbohydrate counting. Postprandial serum insulin, tumour necrosis factor (TNF)-alpha, plasma fibrinogen, human tissue factor (HTF) activity and plasminogen activator inhibitor-1 (PAI-1) were measured. Two-step clustering categorized individuals based on shared clinical characteristics. For analyses, insulin pharmacokinetic summary statistics were normalized, allowing standardized intraindividual comparisons. RESULTS: Despite standardization of insulin dose, individuals exhibited marked interpersonal variability in peak insulin concentrations (48.63%), time to peak (64.95%) and insulin incremental area under the curve (60.34%). Two clusters were computed: cluster 1 (n=14), representing increased serum insulin concentrations; and cluster 2 (n=18), representing reduced serum insulin concentrations (cluster 1: 389.50±177.10 pmol/L/IU h-1; cluster 2: 164.29±41.91 pmol/L/IU h-1; p<0.001). Cluster 2 was characterized by increased levels of fibrinogen, PAI-1, TNF-alpha and HTF activity; higher glycated hemoglobin; increased body mass index; lower estimated glucose disposal rate (increased insulin resistance); older age; and longer diabetes duration (p<0.05 for all analyses). CONCLUSIONS: Reduced serum insulin concentrations are associated with insulin resistance and a prothrombotic milieu in individuals with T1D, and therefore may be a marker of adverse vascular outcome.

Type 1 Diabetes Patients With Different Residual Beta-Cell Function but Similar Age, HBA1c, and Cardiorespiratory Fitness Have Differing Exercise-Induced Angiogenic Cell Mobilisation.

Background: Many individuals with type 1 diabetes retain residual beta-cell function. Sustained endogenous insulin and C-peptide secretion is associated with reduced diabetes related complications, but underlying mechanisms remain unclear. Lower circulating numbers of endothelial and hematopoietic progenitor cells (EPCs and HPCs), and the inability to increase the count of these cells in response to exercise, are also associated with increased diabetes complications and cardiovascular disease. It is unknown whether residual beta-cell function influences HPCs and EPCs. Thus, this study examined the influence of residual beta-cell function in type 1 diabetes upon exercise-induced changes in haematopoietic (HPCs) and endothelial progenitor cells (EPCs). Methods: Participants with undetectable stimulated C-peptide (n=11; Cpepund), 10 high C-peptide (Cpephigh; >200 pmol/L), and 11 non-diabetes controls took part in this observational exercise study, completing 45 minutes of intensive walking at 60% V˙O2peak . Clinically significant HPCs (CD34+) and EPCs (CD34+VEGFR2+) phenotypes for predicting future adverse cardiovascular outcomes, and subsequent cell surface expression of chemokine receptor 4 (CXCR4) and 7 (CXCR7), were enumerated at rest and immediately post-exercise by flow cytometry. Results: Exercise increased HPCs and EPCs phenotypes similarly in the Cpephigh and control groups (+34-121% across phenotypes, p<0.04); but Cpepund group did not significantly increase from rest, even after controlling for diabetes duration. Strikingly, the post-exercise Cpepund counts were still lower than Cpephigh at rest. Conclusions: Residual beta-cell function is associated with an intact exercise-induced HPCs and EPCs mobilisation. As key characteristics (age, fitness, HbA1c) were similar between groups, the mechanisms underpinning the absent mobilisation within those with negative C-peptide, and the vascular implications, require further investigation.

An Analysis of Metabolic Changes in the Retina and Retinal Pigment Epithelium of Aging Mice.

Purpose: The purpose of this study was to present our hypothesis that aging alters metabolic function in ocular tissues. We tested the hypothesis by measuring metabolism in aged murine tissues alongside retinal responses to light. Methods: Scotopic and photopic electroretinogram (ERG) responses in young (3-6 months) and aged (23-26 months) C57Bl/6J mice were recorded. Metabolic flux in retina and eyecup explants was quantified using U-13C-glucose or U-13C-glutamine with gas chromatography-mass spectrometry (GC-MS), O2 consumption rate (OCR) in a perifusion apparatus, and quantifying adenosine triphosphatase (ATP) with a bioluminescence assay. Results: Scotopic and photopic ERG responses were reduced in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected in aged mice. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable. Conclusions: Our examination of metabolism showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The metabolic stability of these tissues ex vivo suggests age-related metabolic alterations may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or structural changes influence ocular metabolism in vivo.

Elamipretide (SS-31) treatment attenuates age-associated post-translational modifications of heart proteins.

It has been demonstrated that elamipretide (SS-31) rescues age-related functional deficits in the heart but the full set of mechanisms behind this have yet to be determined. We investigated the hypothesis that elamipretide influences post-translational modifications to heart proteins. The S-glutathionylation and phosphorylation proteomes of mouse hearts were analyzed using shotgun proteomics to assess the effects of aging on these post-translational modifications and the ability of the mitochondria-targeted drug elamipretide to reverse age-related changes. Aging led to an increase in oxidation of protein thiols demonstrated by increased S-glutathionylation of cysteine residues on proteins from Old (24 months old at the start of the study) mouse hearts compared to Young (5-6 months old). This shift in the oxidation state of the proteome was almost completely reversed by 8 weeks of treatment with elamipretide. Many of the significant changes that occurred were in proteins involved in mitochondrial or cardiac function. We also found changes in the mouse heart phosphoproteome that were associated with age, some of which were partially restored with elamipretide treatment. Parallel reaction monitoring of a subset of phosphorylation sites revealed that the unmodified peptide reporting for Myot S231 increased with age, but not its phosphorylated form and that both phosphorylated and unphosphorylated forms of the peptide covering cMyBP-C S307 increased, but that elamipretide treatment did not affect these changes. These results suggest that changes to thiol redox state and phosphorylation status are two ways in which age may affect mouse heart function, which can be restored by treatment with elamipretide.

Postprandial vascular-inflammatory and thrombotic responses to high-fat feeding are augmented by manipulating the lipid droplet size distribution.

BACKGROUND AND AIMS: Postprandial responses are influenced not only by the type and amount of fat ingested, but also lipid droplet size distribution. However, little research has investigated the impact of differential lipid size distributions within a mixed-macronutrient meal context on postprandial vascular health. Therefore, we examined whether manipulating the lipid droplet size distribution within a mixed-macronutrient meal impacts vascular-inflammatory and thrombotic parameters. METHODS AND RESULTS: In a randomised and counterbalanced fashion, sixteen adults (8 males; age 34 ± 7 years; BMI of 25.3 ± 4.5 kg/m2) completed three separate fasted morning-time feeding challenges, each separated by a minimum washout of 7-days. On each occasion, test-meals matched for carbohydrate and protein content differing only in fat amount and the lipid droplet size distribution were administered, such that participants consumed (1) a low-fat meal (LF) with negligible fat content, (2) an emulsified-high-fat meal with a fine lipid droplet size (FE), or (3) an emulsified-high-fat meal with a coarse lipid droplet size (CE). Periodic blood samples were retrospectively analysed for plasma triglycerides, tumour necrosis factor alpha (TNFα), tissue factor (TF), fibrinogen, and plasminogen activator inhibitor-1 (PAI-1). Triglyceride concentrations increased rapidly overtime under FE (P-time<0.05); this rise was attenuated under CE (P-time>0.05) and was comparable to LF (P-condition>0.05). Similarly, FE induced a significant rise in TNFα, TF, fibrinogen, and PAI-1 (P-time<0.05); these parameters remained unchanged under LF and CE (P-time>0.05). CONCLUSION: A high-fat mixed-macronutrient meal with a larger lipid droplet size distribution ameliorates the associated rise in vascular-inflammatory and thrombotic parameters. TRIAL REGISTRATION: ISRCTN88881254.

Type 1 diabetes patients increase CXCR4+ and CXCR7+ haematopoietic and endothelial progenitor cells with exercise, but the response is attenuated.

Exercise mobilizes angiogenic cells, which stimulate vascular repair. However, limited research suggests exercise-induced increase of endothelial progenitor cell (EPCs) is completely lacking in type 1 diabetes (T1D). Clarification, along with investigating how T1D influences exercise-induced increases of other angiogenic cells (hematopoietic progenitor cells; HPCs) and cell surface expression of chemokine receptor 4 (CXCR4) and 7 (CXCR7), is needed. Thirty T1D patients and 30 matched non-diabetes controls completed 45 min of incline walking. Circulating HPCs (CD34+, CD34+CD45dim) and EPCs (CD34+VEGFR2+, CD34+CD45dimVEGFR2+), and subsequent expression of CXCR4 and CXCR7, were enumerated by flow cytometry at rest and post-exercise. Counts of HPCs, EPCs and expression of CXCR4 and CXCR7 were significantly lower at rest in the T1D group. In both groups, exercise increased circulating angiogenic cells. However, increases was largely attenuated in the T1D group, up to 55% lower, with CD34+ (331 ± 437 Δcells/mL vs. 734 ± 876 Δcells/mL p = 0.048), CD34+VEGFR2+ (171 ± 342 Δcells/mL vs. 303 ± 267 Δcells/mL, p = 0.006) and CD34+VEGFR2+CXCR4+ (126 ± 242 Δcells/mL vs. 218 ± 217 Δcells/mL, p = 0.040) significantly lower. Exercise-induced increases of angiogenic cells is possible in T1D patients, albeit attenuated compared to controls. Decreased mobilization likely results in reduced migration to, and repair of, vascular damage, potentially limiting the cardiovascular benefits of exercise.Trial registration: ISRCTN63739203.

Increased tumour burden alters skeletal muscle properties in the KPC mouse model of pancreatic cancer.

BACKGROUND: Cancer cachexia is a multifactorial wasting syndrome that is characterized by the loss of skeletal muscle mass and weakness, which compromises physical function, reduces quality of life, and ultimately can lead to mortality. Experimental models of cancer cachexia have recapitulated this skeletal muscle atrophy and consequent decline in muscle force generating capacity. We address these issues in a novel transgenic mouse model Kras, Trp53 and Pdx-1-Cre (KPC) of pancreatic ductal adenocarcinoma (PDA) using multi-parametric magnetic resonance (mp-MR) measures. METHODS: KPC mice (n = 10) were divided equally into two groups (n = 5/group) depending on the size of the tumor i.e. tumor size <250 mm3 and >250 mm3. Using mp-MR measures, we demonstrated the changes in the gastrocnemius muscle at the microstructural level. In addition, we evaluated skeletal muscle contractile function in KPC mice using an in vivo approach. RESULTS: Increase in tumor size resulted in decrease in gastrocnemius maximum cross sectional area, decrease in T2 relaxation time, increase in magnetization transfer ratio, decrease in mean diffusivity, and decrease in radial diffusivity of water across the muscle fibers. Finally, we detected significant decrease in absolute and specific force production of gastrocnemius muscle with increase in tumor size. CONCLUSIONS: Our findings indicate that increase in tumor size may cause alterations in structural and functional parameters of skeletal muscles and that MR parameters may be used as sensitive biomarkers to noninvasively detect structural changes in cachectic muscles.

Omega-3 polyunsaturated fatty acid supplementation versus placebo on vascular health, glycaemic control, and metabolic parameters in people with type 1 diabetes: a randomised controlled preliminary trial.

BACKGROUND: The role of omega-3 polyunsaturated fatty acids (n-3PUFA), and the potential impact of n-3PUFA supplementation, in the treatment and management of type 1 diabetes (T1D) remains unclear and controversial. Therefore, this study aimed to examine the efficacy of daily high-dose-bolus n-3PUFA supplementation on vascular health, glycaemic control, and metabolic parameters in subjects with T1D. METHODS: Twenty-seven adults with T1D were recruited to a 6-month randomised, double-blind, placebo-controlled trial. Subjects received either 3.3 g/day of encapsulated n-3PUFA or encapsulated 3.0 g/day corn oil placebo (PLA) for 6-months, with follow-up at 9-months after 3-month washout. Erythrocyte fatty acid composition was determined via gas chromatography. Endpoints included inflammation-associated endothelial biomarkers (vascular cell adhesion molecule-1 [VCAM-1], intercellular adhesion molecule-1 [ICAM-1], E-selectin, P-selectin, pentraxin-3, vascular endothelial growth factor [VEGF]), and their mediator tumor necrosis factor alpha [TNFα] analysed via immunoassay, vascular structure (carotid intima-media thickness [CIMT]) and function (brachial artery flow mediated dilation [FMD]) determined via ultrasound technique, blood pressure, glycosylated haemoglobin (HbA1c), fasting plasma glucose (FPG), and postprandial metabolism. RESULTS: Twenty subjects completed the trial in full. In the n-3PUFA group, the mean ± SD baseline n-3PUFA index of 4.93 ± 0.94% increased to 7.67 ± 1.86% (P < 0.001) after 3-months, and 8.29 ± 1.45% (P < 0.001) after 6-months. Total exposure to n-3PUFA over the 6-months (area under the curve) was 14.27 ± 3.05% per month under n-3PUFA, and 9.11 ± 2.74% per month under PLA (P < 0.001). VCAM-1, ICAM-1, E-selectin, P-selectin, pentraxin-3, VEGF, TNFα, CIMT, FMD, blood pressure, HbA1c, FPG, and postprandial metabolism did not differ between or within groups after treatment (P > 0.05). CONCLUSIONS: This study indicates that daily high-dose-bolus of n-3PUFA supplementation for 6-months does not improve vascular health, glucose homeostasis, or metabolic parameters in subjects with T1D. The findings from this preliminary RCT do not support the use of therapeutic n-3PUFA supplementation in the treatment and management of T1D and its associated complications. Trial Registration ISRCTN, ISRCTN40811115. Registered 27 June 2017, http://www.isrctn.com/ISRCTN40811115 .

Mitochondrial protein interaction landscape of SS-31.

Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer a glimpse of the protein interaction landscape of SS-31 and provide mechanistic insight relevant to SS-31 mitochondrial therapy.

Improving mitochondrial function with SS-31 reverses age-related redox stress and improves exercise tolerance in aged mice.

Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3 mg/kg/day SS-31. Mitochondrial function was assessed in vivo using 31P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased in vivo mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly.

Building strength, endurance, and mobility using an astaxanthin formulation with functional training in elderly.

BACKGROUND: Building both strength and endurance has been a challenge in exercise training in the elderly, but dietary supplements hold promise as agents for improving muscle adaptation. Here, we test a formulation of natural products (AX: astaxanthin, 12 mg and tocotrienol, 10 mg and zinc, 6 mg) with both anti-inflammatory and antioxidant properties in combination with exercise. We conducted a randomized, double-blind, placebo-controlled study of elderly subjects (65-82 years) on a daily oral dose with interval walking exercise on an incline treadmill. METHODS: Forty-two subjects were fed AX or placebo for 4 months and trained 3 months (3×/week for 40-60 min) with increasing intervals of incline walking. Strength was measured as maximal voluntary force (MVC) in ankle dorsiflexion exercise, and tibialis anterior muscle size (cross-sectional area, CSA) was determined from magnetic resonance imaging. RESULTS: Greater endurance (exercise time in incline walking, >50%) and distance in 6 min walk (>8%) accompanied training in both treatments. Increases in MVC by 14.4% (±6.2%, mean ± SEM, P < 0.02, paired t-test), CSA by 2.7% (±1.0%, P < 0.01), and specific force by 11.6% (MVC/CSA, ±6.0%, P = 0.05) were found with AX treatment, but no change was evident in these properties with placebo treatment (MVC, 2.9% ± 5.6%; CSA, 0.6% ± 1.2%; MVC/CSA, 2.4 ± 5.7%; P > 0.6 for all). CONCLUSIONS: The AX formulation improved muscle strength and CSA in healthy elderly in addition to the elevation in endurance and walking distance found with exercise training alone. Thus, the AX formulation in combination with a functional training programme uniquely improved muscle strength, endurance, and mobility in the elderly.

A small dose of whey protein co-ingested with mixed-macronutrient breakfast and lunch meals improves postprandial glycemia and suppresses appetite in men with type 2 diabetes: a randomized controlled trial.

Background: Large doses of whey protein consumed as a preload before single high-glycemic load meals has been shown to improve postprandial glycemia in type 2 diabetes. It is unclear if this effect remains with smaller doses of whey co-ingested at consecutive mixed-macronutrient meals. Moreover, whether hydrolyzed whey offers further benefit under these conditions is unclear. Objective: The aim of this study was to investigate postprandial glycemic and appetite responses after small doses of intact and hydrolyzed whey protein co-ingested with mixed-nutrient breakfast and lunch meals in men with type 2 diabetes. Design: In a randomized, single-blind crossover design, 11 men with type 2 diabetes [mean ± SD age: 54.9 ± 2.3 y; glycated hemoglobin: 6.8% ± 0.3% (51.3 ± 3.4 mmol/mol)] attended the laboratory on 3 mornings and consumed 1) intact whey protein (15 g), 2) hydrolyzed whey protein (15 g), or 3) placebo (control) immediately before mixed-macronutrient breakfast and lunch meals, separated by 3 h. Blood samples were collected periodically and were processed for insulin, intact glucagon-like peptide 1 (GLP-1), gastric inhibitory polypeptide (GIP), leptin, peptide tyrosine tyrosine (PYY3-36), and amino acid concentrations. Interstitial glucose was measured during and for 24 h after each trial. Subjective appetite was assessed with the use of visual analog scales. Results: Total postprandial glycemia area under the curve was reduced by 13% ± 3% after breakfast following the intact whey protein when compared with control (P < 0.05). Hydrolyzed whey attenuated early glucose after breakfast when compared with control (P < 0.05). Glycemia was improved postlunch after the intact whey protein only when compared with control (P < 0.05). Greater satiety was observed after the intact whey protein only after both meals when compared with control (P < 0.05). Insulin concentrations increased after both the intact and hydrolyzed whey protein, showing strong positive correlations with increases in valine and isoleucine (P < 0.05). Incretin and appetite regulatory hormone responses were similar across trials (P > 0.05). Conclusions: The consumption of a small 15-g dose of intact whey protein immediately before consecutive mixed-macronutrient meals improves postprandial glycemia, stimulates insulin release, and increases satiety in men with type 2 diabetes. This trial was registered at www.clinicialtrials.gov as NCT02903199.

An additional bolus of rapid-acting insulin to normalise postprandial cardiovascular risk factors following a high-carbohydrate high-fat meal in patients with type 1 diabetes: A randomised controlled trial.

AIM: To evaluate an additional rapid-acting insulin bolus on postprandial lipaemia, inflammation and pro-coagulation following high-carbohydrate high-fat feeding in people with type 1 diabetes. METHODS: A total of 10 males with type 1 diabetes [HbA1c 52.5 ± 5.9 mmol/mol (7.0% ± 0.5%)] underwent three conditions: (1) a low-fat (LF) meal with normal bolus insulin, (2), a high-fat (HF) meal with normal bolus insulin and (3) a high-fat meal with normal bolus insulin with an additional 30% insulin bolus administered 3-h post-meal (HFA). Meals had identical carbohydrate and protein content and bolus insulin dose determined by carbohydrate-counting. Blood was sampled periodically for 6-h post-meal and analysed for triglyceride, non-esterified-fatty acids, apolipoprotein B48, glucagon, tumour necrosis factor alpha, fibrinogen, human tissue factor activity and plasminogen activator inhibitor-1. Continuous glucose monitoring captured interstitial glucose responses. RESULTS: Triglyceride concentrations following LF remained similar to baseline, whereas triglyceride levels following HF were significantly greater throughout the 6-h observation period. The additional insulin bolus (HFA) normalised triglyceride similarly to low fat 3-6 h following the meal. HF was associated with late postprandial elevations in tumour necrosis factor alpha, whereas LF and HFA was not. Fibrinogen, plasminogen activator inhibitor-1 and tissue factor pathway levels were similar between conditions. CONCLUSION: Additional bolus insulin 3 h following a high-carbohydrate high-fat meal prevents late rises in postprandial triglycerides and tumour necrosis factor alpha, thus improving cardiovascular risk profile.

The mitochondrial-targeted peptide, SS-31, improves glomerular architecture in mice of advanced age.

Although age-associated changes in kidney glomerular architecture have been described in mice and man, the mechanisms are unknown. It is unclear if these changes can be prevented or even reversed by systemic therapies administered at advanced age. Using light microscopy and transmission electron microscopy, our results showed glomerulosclerosis with injury to mitochondria in glomerular epithelial cells in mice aged 26 months (equivalent to a 79-year-old human). To test the hypothesis that reducing mitochondrial damage in late age would result in lowered glomerulosclerosis, we administered the mitochondrial targeted peptide, SS-31, to aged mice. Baseline (24-month-old) mice were randomized to receive 8 weeks of SS-31, or saline, and killed at 26 months of age. SS-31 treatment improved age-related mitochondrial morphology and glomerulosclerosis. Assessment of glomeruli revealed that SS-31 reduced senescence (p16, senescence-associated-ß-Gal) and increased the density of parietal epithelial cells. However, SS-31 treatment reduced markers of parietal epithelial cell activation (Collagen IV, pERK1/2, and α-smooth muscle actin). SS-31 did not impact podocyte density, but it reduced markers of podocyte injury (desmin) and improved cytoskeletal integrity (synaptopodin). This was accompanied by higher glomerular endothelial cell density (CD31). Thus, despite initiating therapy in late-age mice, a short course of SS-31 has protective benefits on glomerular mitochondria, accompanied by temporal changes to the glomerular architecture. This systemic pharmacological intervention in old-aged animals limits glomerulosclerosis and senescence, reduces parietal epithelial cell activation, and improves podocyte and endothelial cell integrity.

NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation.

Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD+) synthesis, consistent with a potential role for the essential cofactor NAD+ in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene's muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5'-diphosphate (ADP)-ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD+ and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD+ levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ biosynthesis. Replenishing NAD+ stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr-/- mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD+ repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD+ may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures.

Evaluation of in vivo mitochondrial bioenergetics in skeletal muscle using NMR and optical methods.

It is now clear that mitochondria are involved as either a cause or consequence of many chronic diseases. This central role of the mitochondria is due to their position in the cell as important integrators of cellular energetics and signaling. Mitochondrial function affects many aspects of the cellular environment such as redox homeostasis and calcium signaling, which then also exert control over mitochondrial function. This complex dynamic between mitochondrial function and the cellular environment highlights the value of examining mitochondria in vivo in the intact physiological environment. This review discusses NMR and optical approaches used to measure mitochondria ATP and oxygen fluxes that provide in vivo measures of mitochondrial capacity and quality in animal and human models. Combining these in vivo measurements with more traditional ex vivo analyses can lead to new insights into the importance of the cellular environment in controlling mitochondrial function under pathological conditions. Interpretation and underlying assumptions for each technique are discussed with the goal of providing an overview of some of the most common approaches used to measure in vivo mitochondrial function encountered in the literature.