Nair, K. Sreekumaran

The effects of different modes of exercise on mixed muscle protein synthesis (MMPS) in young (Y) and older (O) adults are not fully understood. We determined the effects of ET, RT, and CT on MMPS in 33Y (25±1y) and 31O (70±1y) adults who were randomized to one of three 8wk interventions: (i) Control: no exercise, (ii) ET: cycling 1h @ 65% VO 2 peak, 5x/wk, (iii) RT: RT 4x/wk. After completing the control period, the controls completed an 8wk (iv) CT program (ET & RT). Subjects were studied at baseline and 48h following the last training bout. [ 13 C 6 ]Phe was infused for 8h, with serial v. lateralis biopsies. Isotopic enrichments (IE) were measured by LC/MS for free Phe from tissue fluid (TF) and from that incorporated into MMP. MMPS (%/h) = (ΔIE MMP )/(IE TF )/t*100. A rank sum test was used to test baseline differences between Y and O. 2‐way ANCOVA was used to test for differences between groups by treatment, with baseline values serving as the covariate. MMPS (%/h) tended to be higher in Y (0.060±0.003) compared to O (0.053±0.003) (P=0.06). ET [57%, P<0.0001], RT [21%, P=0.042] and CT [42%, P<0.0001] increased MMPS independently of age (P>0.4). The ET‐induced increase in MMPS was greater than the RT‐induced increase (P<0.05), although ET unlike RT does not increase muscle mass suggesting that muscle protein turnover was increased. In conclusion, ET, RT and CT were effective at increasing MMPS independently of age. R01AG09531, KL2RR024151

Altered protein homeostasis may explain the increased risk of degenerative and cardiometabolic disorders with aging. Here, we investigated the impact of chronological age and obesity on the accumulation (ratio of old to de novo synthesized proteins) of haptoglobin, fibrinogen, and apolipoprotein A‐1 (ApoA‐1) in 7 each of elderly lean (69.8 ± 5.0 years, 23.2 ± 1.8 kg/m 2 ), elderly obese (72.6 ± 5.6 years, 30.6 ± 2.3 kg/m 2 ), young lean (23.1 ± 3.2 years, 23.4 ± 1.9 kg/m 2 ), and young obese (25.2 ± 2.4 years, 30.6 ± 2.4 kg/m 2 ). Proteins were labeled with [ 13 C 6 ]‐phenylalanine, separated by 2‐dimentional gel electrophoresis, and identified by mass spectrometry. Insulin sensitivity was lower in the obese groups, while percent body fat was higher, and lean mass was lower, in the elderly. Haptoglobin accumulation was greater in the obese compared to lean (0.512 ± 0.229 vs. 0.272 ± 0.114; p = 0.008) across all ages, with no age effect. Fibrinogen trended toward higher accumulation in the obese compared to lean, with no age effect, but did not reach statistical significance (1.263 ± 2.108 vs. 0.598 ± 0.197; p = 0.094). ApoA‐1 accumulation showed no obesity or age variation. Associations remained unchanged when controlled for gender, age, and BMI. Thus, adiposity, and not chronological age, promotes protein accumulation, potentially contributing to degenerative and cardiometabolic disease risk. Supported by NIH grant RO1‐AG09531.

Omega‐3 polyunsaturated fatty acids (n‐3 PUFAs) protect insulin sensitivity and glucose homeostasis in rodent models of insulin resistance. These effects of n‐3 PUFAs are linked with anti‐inflammatory properties, but emerging data suggests that mechanisms may also converge on mitochondria. We evaluated the influence of dietary n‐3 PUFAs on mitochondrial physiology in the context of high‐fat diet (HFD) in mice. We hypothesized that the insulin‐sensitizing effects of n‐3 PUFAs would be accompanied by enhanced mitochondrial function in skeletal muscle, leading to attenuated levels of lipid metabolites known to interfere with insulin signaling. We fed mice HFD with or without fish oil for 10 weeks. HFD decreased glucose tolerance, but not in the presence of fish oil. Skeletal muscle long chain acyl coenzyme‐As and ceramides were significantly elevated with HFD, but attenuated in the presence of fish oil. Mitochondrial oxidative capacity was similarly increased in mice fed HFD with and without fish oil. These results indicate that n‐3 PUFAs protect insulin sensitivity, in part, by preventing the accumulation of lipid intermediated that interfere with insulin action. Furthermore, the respiratory function of skeletal muscle mitochondria does not appear to be a major factor in ectopic lipid accumulation, diet‐induced insulin resistance, or the protective effects of n‐3 PUFAs on insulin sensitivity.

Mitochondrial function declines with age but increases with caloric restriction (CR). Mitochondria and muscle fibers were prepared from young (8–11mo) and old (22–24mo) AL‐fed and old CR mice. State 3 respiration was lower in OAL vs. YAL, but preserved in OCR (). Normalizing to protein content eliminated the age‐effect in AL mice but amplified the effect of CR, indicating that CR enhances the intrinsic mito function. ROS production in isolated fibers increased with age in AL but not CR mice (). Coupling was reduced in OAL but not OCR. These results indicate that mito capacity declines with age in situ and in vitro, apparently driven largely by decreased mitochondrial protein content. CR increases mito capacity and efficiency independently of mito protein content. YAL OAL OCR Fibers (pmol/s/m g tiss) CI 54.0±4.1 38.9±1.6 50.8±3.0 CII 44.4±3.4 36.9±1.4 45.0±2.3 CI+II 63.4±5.5 51.8±2.5 67.0±3.4 Mito (pmol/s/m g tiss) CI 38.9±1.8 21.5±2.6 30.7±2.0 CII 30.6±0.9 19.6±1.2 24.7±1.4 CI+II 59.0±1.6 34.0±1.9 46.7±2.7 Mito (pmol/s/m g prot) CI 8350±635 7071±1082 10422±863 CII 6536±414 6309±506 8408±676 CI+II 12601±762 11063±1156 15949±1381 H 2 O 2 (pM/s/mg tiss) CI 10.0±1.8 30.8±3.7 15.0±2.0 CI+II 141.4±20.9 187.2±53.8 101.1±13.6 P:O State IV to III 2.94±0.03 2.71±0.02 2.92±0.05 Means±SEM. P<0.05 OAL vs YAL, P<0.05 OCR vs OAL, P<0.05 OCR vs. OAL & YAL.

Insulin has been shown to have major impact on muscle mitochondrial function. Recent data show that ceramide can decrease oxygen consumption and ATP production in isolated mitochondria. The aim of the present study was to determine whether insulin deficiency alters ceramide (CER) and its metabolites ‐ sphinganine (SPA), sphingosine (SPH) and sphingosine‐1‐phosphate (S1P) ‐ in mitochondrial (mt) and sarcoplasmic (src) fractions from quadriceps muscle of streptozotocin‐diabetic C57BL/6 male mice. We studied non‐diabetic controls (C); diabetic, insulin‐treated (D+); and diabetic, insulin‐deprived (D−). D− and D+ group received LinBit insulin implants, whereas C group received blank implants. Implants from D− group were removed 7 days prior to sacrifice. CER and its metabolites were measured in a tandem mass spectrometer connected to a UPLC system. Insulin deprivation increased CER in muscle src although decreased in mt. Insulin treatment normalized CER content in src, but further lowered CER content in mt, which was accompanied by elevation in mitochondrial SPH content. Changes in SPA and S1P levels in both the src and mt were non‐significant. In conclusion, diabetes and insulin have profound effects on CER concentrations in muscle subcellular fractions. C D− D+ src mt src mt src mt SPA Mean 0.34 nd 0.32 nd 0.34 nd SEM ±0.04 ±0.02 ±0.04 SPH Mean 0.44 1.63 0.61 2.15 0.47 2.55 SEM ±0.07 ±0.23 ±0.10 ±0.14 ±0.03 ±0.21 S1P Mean 0.32 1.14 0.27 1.06 0.23 0.91 SEM ±0.03 ±0.2 ±0.05 ±0.14 ±0.01 ±0.1 CER Mean 85.7 326.0 162.5 267.3 93.0 199.7 SEM ±17.4 ±38.4 ±19.8 ±24.0 ±4.4 ±11.2

BACKGROUND Type 1 diabetes (T1D) is a risk factor for dementia and structural brain changes. It remains to be determined whether transient insulin deprivation that frequently occurs in insulin-treated individuals with T1D alters brain function. METHODS We therefore performed functional and structural magnetic resonance imaging, magnetic resonance spectroscopy, and neuropsychological testing at baseline and following 5.4 ± 0.6 hours of insulin deprivation in 14 individuals with T1D and compared results with those from 14 age-, sex-, and BMI-matched nondiabetic (ND) participants with no interventions. RESULTS Insulin deprivation in T1D increased blood glucose, and β-hydroxybutyrate, while reducing bicarbonate levels. Participants with T1D showed lower baseline brain N-acetyl aspartate and myo-inositol levels but higher cortical fractional anisotropy, suggesting unhealthy neurons and brain microstructure. Although cognitive functions did not differ between participants with T1D and ND participants at baseline, significant changes in fine motor speed as well as attention and short-term memory occurred following insulin deprivation in participants with T1D. Insulin deprivation also reduced brain adenosine triphosphate levels and altered the phosphocreatine/adenosine triphosphate ratio. Baseline differences in functional connectivity in brain regions between participants with T1D and ND participants were noted, and on insulin deprivation further alterations in functional connectivity between regions, especially cortical and hippocampus-caudate regions, were observed. These alterations in functional connectivity correlated to brain metabolites and to changes in cognition. CONCLUSION Transient insulin deprivation therefore caused alterations in executive aspects of cognitive function concurrent with functional connectivity between memory regions and the sensory cortex. These findings have important clinical implications, as many patients with T1D inadvertently have periods of transient insulin deprivation. TRIAL REGISTRATION ClinicalTrials.gov NCT03392441. FUNDING Clinical and Translational Science Award (UL1 TR002377) from the National Center for Advancing Translational Science; NIH grants (R21 AG60139 and R01 AG62859); the Mayo Foundation.

Insulin resistance is associated with skeletal muscle mitochondrial (mito) dysfunction in many conditions including type 2 diabetes (T2D). Poor glycemic control in T2D is associated with muscle mito gene transcript alterations. It is unknown whether muscle mito changes are related to reduced insulin action or vice versa. We obtained vastus lateralis muscle biopsies from 7 type 1 diabetic patients (T1D) following 8–12 hrs of insulin deprivation (I−) and replacement (I+) and measured skeletal muscle mito ATP production (MATP) and gene transcripts. I− and I+ changed glucose and glucagon levels (glucose=291±15mg/dl in I− vs 97±3 in I+ and glucagon=79.8±15.3 pg/mL in I− vs 47.3±5.9 in I+, p<0.04). Whole body O 2 consumption increased during I− by 15% (p<0.05) but maximal MATP decreased by 21.3–27.9% (p<0.05). Gene transcript profiling in skeletal muscle analyzed using Affymetrix GeneChip arrays showed alterations in 2355 transcripts. Pathway analysis showed down‐regulation of oxidative phosphorylation and arachidonic acid metabolism, and up‐regulation of VEGF and actin cytoskeleton signaling in I−. We conclude that the reduced insulin action that occurred during I− in T1D resulted in substantial changes in gene transcript levels of muscle mito genes and reduced maximal MATP, supporting that insulin is key regulator of muscle mito function by exerting its effect at various levels. Supported by NIHRO1DK41973.

We report a technique to simultaneously measure fractional synthesis rates (FSR) of several muscle proteins that facilitate further understanding of translational regulation, a key step involved in gene expression in a tissue. Skeletal muscle samples were collected from five rats following bolus injection of [ring 13 C 6 ] phenylalanine. Muscle proteins were fractionated by differential centrifugation followed by separation of individual proteins by high resolution two‐dimensional gel electrophoresis. We identified 60 mitochondrial and 30 non‐mitochondrial proteins by liquid chromatography tandem mass spectrometry. Isotope enrichment of these 90 proteins was measured by gas chromatography tandem mass spectrometry, and FSR was calculated using the muscle tissue fluid isotopic enrichment as the precursor. Approximately 25 fold differences in the FSR of muscle proteins were observed, (e.g. FSR of structural protein such as myosin heavy chain‐1 = 0.19 %/h ± 0.03 and tropomysin‐4 = 0.16 ± 03 and that of mitochondrial proteins such as cytochrome c oxidase Va = 0.64 ± 0.07 and dihydrolipoamide dehydrogenase = 4.3 %/h ± 0.53). The current approach, based on in‐vivo metabolic labeling, to simultaneously measure FSR of multiple muscle proteins offers tremendous opportunity to study the regulation of skeletal muscle mitochondrial and non‐mitochondrial protein synthesis in various physiologic and pathologic states. Funding: NIH RO1 DK41973

Objective In contrast to intentionally restricting energy intake, restricting the eating window may be an option for treating obesity. By comparing time‐restricted eating (TRE) with an unrestricted (non‐TRE) control, it was hypothesized that TRE facilitates weight loss, alters body composition, and improves metabolic measures. Methods Participants (17 women and 3 men; mean [SD]: 45.5 [12.1] years; BMI 34.1 [7.5] kg/m 2 ) with a prolonged eating window (15.4 [0.9] hours) were randomized to TRE ( n = 11: 8‐hour window, unrestricted eating within window) versus non‐TRE ( n = 9: unrestricted eating) for 12 weeks. Weight, body composition (dual x‐ray absorptiometry), lipids, blood pressure, 2‐hour oral glucose tolerance, 2‐week continuous glucose monitoring, and 2‐week physical activity (actigraphy assessed) were measured during the pre‐ and end‐intervention periods. Results The TRE group significantly reduced the eating window (end‐intervention window: 9.9 [2.0] hours) compared with the non‐TRE group (end‐intervention window: 15.1 [1.1] hours) ( P < 0.01). Compared with non‐TRE, TRE decreased the number of eating occasions, weight, lean mass, and visceral fat (all P ≤ 0.05). Compared with preintervention measures, the TRE group reduced the number of eating occasions (−21.9% [30.1%]) and reduced weight (−3.7% [1.8%]), fat mass (−4% [2.9%]), lean mass (−3.0% [2.7%]), and visceral fat (−11.1% [13.4%]) (all P ≤ 0.05). Physical activity and metabolic measures remained unchanged. Conclusions In the setting of a randomized trial, TRE presents a simplified view of food intake that reduces weight.

It has been shown ingestion of whey protein (WP) results in rapid systemic appearance rates of amino acids (AA) but in higher amino acid (AA) oxidation and whole body protein synthesis than in with casein (cas) ingestion which inhibits protein breakdown. To determine whether combined ingestion of both cas and WP in a meal containing carbohydrate has any disparate effects on protein economy in splanchnic and muscle beds we studied 12 healthy volunteers who ingested intrinsically labeled WP and Cas protein fractions. Following ingestion of 0.625 g·kg FFM −1 each of [ 15 N]WP and [ 13 C]Cas and 0.9 g·kg FFM −1 of lactose; or 0.625 g·kg FFM −1 each of [ 13 C]WP and [ 15 N]CAS and 0.9 g·kg FFM −1 of lactose blood samples were collected from femoral artery and vein and hepatic vein at frequent intervals. At 80–120 min, the rate of appearance (PheRa) of Phe at hepatic vein from WP and Cas were similar (5.5 (0.9) vs 5.2 (0.7) μmol, min −1 , but at 220–260 min, PheRa from WP was slower than PheRa from Cas (2.4 (0.5) vs 5.3 (1.2) μmol, min −1 , P<0.05). Retention rates of Phe in muscle (PheRd) absorbed from WP and Cas were similar at 80–120 min (0.8 (0.1) vs 0.9 (0.2) μmol, min −1 , ns) but was significantly lower for WP than Cas at 220–260 min (0.5 (0.1) vs 1.1 (0.3) μmol, min −1 , P<0.05). In conclusion Cas has more sustained systemic effects and muscle protein accretion than WP. The different metabolic effects of co‐ingested protein fractions have potential implications in designing appropriate nutritional support.