Exercise training modifies skeletal muscle clock gene expression but not 24-hour rhythmicity in substrate metabolism of men with insulin resistance.

Twenty-four hour rhythmicity in whole-body substrate metabolism, skeletal muscle clock gene expression and mitochondrial respiration is compromised upon insulin resistance. With exercise training known to ameliorate insulin resistance, our objective was to test if exercise training can reinforce diurnal variation in whole-body and skeletal muscle metabolism in men with insulin resistance. In a single-arm longitudinal design, 10 overweight and obese men with insulin resistance performed 12 weeks of high-intensity interval training recurrently in the afternoon (between 14.00 and 18.00 h) and were tested pre- and post-exercise training, while staying in a metabolic research unit for 2 days under free-living conditions with regular meals. On the second days, indirect calorimetry was performed at 08.00, 13.00, 18.00, 23.00 and 04.00 h, muscle biopsies were taken from the vastus lateralis at 08.30, 13.30 and 23.30 h, and blood was drawn at least bi-hourly over 24 h. Participants did not lose body weight over 12 weeks, but improved body composition and exercise capacity. Exercise training resulted in reduced 24-h plasma glucose levels, but did not modify free fatty acid and triacylglycerol levels. Diurnal variation of muscle clock gene expression was modified by exercise training with period genes showing an interaction (time × exercise) effect and reduced mRNA levels at 13.00 h. Exercise training increased mitochondrial respiration without inducing diurnal variation. Twenty-four-hour substrate metabolism and energy expenditure remained unchanged. Future studies should investigate alternative exercise strategies or types of interventions (e.g. diet or drugs aiming at improving insulin sensitivity) for their capacity to reinforce diurnal variation in substrate metabolism and mitochondrial respiration. KEY POINTS: Insulin resistance is associated with blunted 24-h flexibility in whole-body substrate metabolism and skeletal muscle mitochondrial respiration, and disruptions in the skeletal muscle molecular circadian clock. We hypothesized that exercise training modifies 24-h rhythmicity in whole-body substrate metabolism and diurnal variation in skeletal muscle molecular clock and mitochondrial respiration in men with insulin resistance. We found that metabolic inflexibility over 24 h persisted after exercise training, whereas mitochondrial respiration increased independent of time of day. Gene expression of Per1-3 and Rorα in skeletal muscle changed particularly close to the time of day at which exercise training was performed. These results provide the rationale to further investigate the differential metabolic impact of differently timed exercise to treat metabolic defects of insulin resistance that manifest at a particular time of day.

Divergent remodeling of the skeletal muscle metabolome over 24 h between young, healthy men and older, metabolically compromised men.

24 h whole-body substrate metabolism and the circadian clock within skeletal muscle are both compromised upon metabolic disease in humans. Here, we assessed the 24 h muscle metabolome by serial muscle sampling performed under 24 h real-life conditions in young, healthy (YH) men versus older, metabolically compromised (OMC) men. We find that metabolites associated with the initial steps of glycolysis and hexosamine biosynthesis are higher in OMC men around the clock, whereas metabolites associated with glutamine-alpha-ketoglutarate, ketone, and redox metabolism are lower in OMC men. The night period shows the largest number of differently expressed metabolites. Both groups demonstrate 24 h rhythmicity in half of the metabolome, but rhythmic metabolites only partially overlap. Specific metabolites are only rhythmic in YH men (adenosine), phase shifted in OMC men (cis-aconitate, flavin adenine dinucleotide [FAD], and uridine diphosphate [UDP]), or have a reduced 24 h amplitude in OMC men (hydroxybutyrate and hippuric acid). Our data highlight the plasticity of the skeletal muscle metabolome over 24 h and large divergence across the metabolic health spectrum.

The influence of bright and dim light on substrate metabolism, energy expenditure and thermoregulation in insulin-resistant individuals depends on time of day.

AIMS/HYPOTHESIS: In our modern society, artificial light is available around the clock and most people expose themselves to electrical light and light-emissive screens during the dark period of the natural light/dark cycle. Such suboptimal lighting conditions have been associated with adverse metabolic effects, and redesigning indoor lighting conditions to mimic the natural light/dark cycle more closely holds promise to improve metabolic health. Our objective was to compare metabolic responses to lighting conditions that resemble the natural light/dark cycle in contrast to suboptimal lighting in individuals at risk of developing metabolic diseases. METHODS: Therefore, we here performed a non-blinded, randomised, controlled, crossover trial in which overweight insulin-resistant volunteers (n = 14) were exposed to two 40 h laboratory sessions with different 24 h lighting protocols while staying in a metabolic chamber under real-life conditions. In the Bright day-Dim evening condition, volunteers were exposed to electric bright light (~1250 lx) during the daytime (08:00-18:00 h) and to dim light (~5 lx) during the evening (18:00-23:00 h). Vice versa, in the Dim day-Bright evening condition, volunteers were exposed to dim light during the daytime and bright light during the evening. Randomisation and allocation to light conditions were carried out by sequential numbering. During both lighting protocols, we performed 24 h indirect calorimetry, and continuous core body and skin temperature measurements, and took frequent blood samples. The primary outcome was plasma glucose focusing on the pre- and postprandial periods of the intervention. RESULTS: Spending the day in bright light resulted in a greater increase in postprandial triacylglycerol levels following breakfast, but lower glucose levels preceding the dinner meal at 18:00 h, compared with dim light (5.0 ± 0.2 vs 5.2 ± 0.2 mmol/l, n = 13, p=0.02). Dim day-Bright evening reduced the increase in postprandial glucose after dinner compared with Bright day-Dim evening (incremental AUC: 307 ± 55 vs 394 ± 66 mmol/l × min, n = 13, p=0.009). After the Bright day-Dim evening condition the sleeping metabolic rate was identical compared with the baseline night, whereas it dropped after Dim day-Bright evening. Melatonin secretion in the evening was strongly suppressed for Dim day-Bright evening but not for Bright day-Dim evening. Distal skin temperature for Bright day-Dim evening was lower at 18:00 h (28.8 ± 0.3°C vs 29.9 ± 0.4°C, n = 13, p=0.039) and higher at 23:00 h compared with Dim day-Bright evening (30.1 ± 0.3°C vs 28.8 ± 0.3°C, n = 13, p=0.006). Fasting and postprandial plasma insulin levels and the respiratory exchange ratio were not different between the two lighting protocols at any time. CONCLUSIONS/INTERPRETATION: Together, these findings suggest that the indoor light environment modulates postprandial substrate handling, energy expenditure and thermoregulation of insulin-resistant volunteers in a time-of-day-dependent manner. TRIAL REGISTRATION: ClinicalTrials.gov NCT03829982. FUNDING: We acknowledge the financial support from the Netherlands Cardiovascular Research Initiative: an initiative with support from the Dutch Heart Foundation (CVON2014-02 ENERGISE).

Circadian misalignment disturbs the skeletal muscle lipidome in healthy young men.

Circadian misalignment, as seen in shift work, is associated with an increased risk to develop type 2 diabetes. In an experimental setting, we recently showed that a rapid day-night shift for 3 consecutive nights leads to misalignment of the core molecular clock, induction of the PPAR pathway, and insulin resistance in skeletal muscle of young, healthy men. Here, we investigated if circadian misalignment affects the skeletal muscle lipidome and intramyocellular lipid droplet characteristics, explaining the misalignment-induced insulin resistance. Fourteen healthy men underwent one aligned and one circadian misalignment period, both consisting of ~3.5 days. In the misaligned condition, day and night were rapidly shifted by 12 hours leading to opposite eating, sleep, and activity times compared with the aligned condition. For each condition, two muscle biopsies were taken from the m. vastus lateralis in the morning and evening and subjected to semi-targeted lipidomics and confocal microscopy analysis. We found that only 2% of detected lipids were different between morning and evening in the aligned condition, whereas 12% displayed a morning-evening difference upon misalignment. Triacylglycerols, in particular species of a carbon length ≥55, were the most abundant lipid species changed upon misalignment. Cardiolipins were decreased upon misalignment, whereas phosphatidylcholines consistently followed the same morning-evening pattern, suggesting regulation by the circadian clock. Cholesteryl esters adjusted to the shifted behavior. Lipid droplet characteristics remained unaltered upon misalignment. Together, these findings show that simulated shift work disturbs the skeletal muscle lipidome, which may contribute to misalignment-induced insulin resistance.

H-reflex and M-wave responses after voluntary and electrically evoked muscle cramping.

PURPOSE: Despite the widespread occurrence of muscle cramps, their underlying neurophysiological mechanisms remain unknown. To better understand the etiology of muscle cramps, this study investigated acute effects of muscle cramping induced by maximal voluntary isometric contractions (MVIC) and neuromuscular electrical stimulation (NMES) on the amplitude of Hoffmann reflexes (H-reflex) and compound muscle action potentials (M-wave). METHODS: Healthy men (n = 14) and women (n = 3) participated in two identical sessions separated by 7 days. Calf muscle cramping was induced by performing MVIC of the plantar flexors in a prone position followed by 2.5-s NMES over the plantar flexors with increasing frequency and intensity. H-reflexes and M-waves evoked by tibial nerve stimulation in gastrocnemius medialis (GM) and soleus were recorded at baseline, and after MVIC-induced cramps and the NMES protocol. RESULTS: Six participants cramped after MVIC, and H-reflex amplitude decreased in GM and soleus in Session 1 (- 33 ± 32%, - 34 ± 33%, p = 0.031) with a similar trend in Session 2 (5 cramped, p = 0.063), whereas the maximum M-wave was unchanged. After NMES, 11 (Session 1) and 9 (Session 2) participants cramped. H-reflex and M-wave recruitment curves shifted to the left in both sessions and muscles after NMES independent of cramping (p ≤ 0.001). CONCLUSION: Changes in H-reflexes after a muscle cramp induced by MVIC and NMES were inconsistent. While MVIC-induced muscle cramps reduced H-reflex amplitude, muscle stretch to end cramping was a potential contributing factor. By contrast, NMES may potentiate H-reflexes and obscure cramp-related changes. Thus, the challenge for future studies is to separate the neural consequences of cramping from methodology-based effects.

Neuromuscular Electrical Stimulation Reduces Leg Cramps in Patients With Lumbar Degenerative Disorders: A Randomized Placebo-Controlled Trial.

OBJECTIVES: Lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH) are often accompanied by frequently occurring leg cramps severely affecting patients' life and sleep quality. Recent evidence suggests that neuromuscular electric stimulation (NMES) of cramp-prone muscles may prevent cramps in lumbar disorders. MATERIALS AND METHODS: Thirty-two men and women (63 ± 9 years) with LSS and/or LDH suffering from cramps were randomly allocated to four different groups. Unilateral stimulation of the gastrocnemius was applied twice a week over four weeks (3 × 6 × 5 sec stimulation trains at 30 Hz above the individual cramp threshold frequency [CTF]). Three groups received either 85%, 55%, or 25% of their maximum tolerated stimulation intensity, whereas one group only received pseudo-stimulation. RESULTS: The number of reported leg cramps decreased in the 25% (25 ± 14 to 7 ± 4; p = 0.002), 55% (24 ± 10 to 10 ± 11; p = 0.014) and 85%NMES (23 ± 17 to 1 ± 1; p < 0.001) group, whereas it remained unchanged after pseudo-stimulation (20 ± 32 to 19 ± 33; p > 0.999). In the 25% and 85%NMES group, this improvement was accompanied by an increased CTF (p < 0.001). CONCLUSION: Regularly applied NMES of the calf muscles reduces leg cramps in patients with LSS/LDH even at low stimulation intensity.

Ticking for Metabolic Health: The Skeletal-Muscle Clocks.

To be prepared for alternating metabolic demands occurring over the 24-hour day, the body preserves information on time in skeletal muscle, and in all cells, through a circadian-clock mechanism. Skeletal muscle can be considered the largest collection of peripheral clocks in the body, with a major contribution to whole-body energy metabolism. Comparison of circadian-clock gene expression between skeletal muscle of nocturnal rodents and diurnal humans reveals very common patterns based on rest/active cycles rather than light/dark cycles. Rodent studies in which the circadian clock is disrupted in skeletal muscle demonstrate impaired glucose handling and insulin resistance. Experimental circadian misalignment in humans modifies the skeletal-muscle clocks and leads to disturbed energy metabolism and insulin resistance. Preclinical studies have revealed that timing of exercise over the day can influence the beneficial effects of exercise on skeletal-muscle metabolism, and studies suggest similar applicability in humans. Current strategies to improve metabolic health (e.g., exercise) should be reinvestigated in their capability to modify the skeletal-muscle clocks by taking timing of the intervention into account.

Invasive Assessment of Hemodynamic, Metabolic and Ionic Consequences During Blood Flow Restriction Training.

Purpose: Medically recommended training often faces the dilemma that necessary mechanical intensities for muscle adaptations exceed patients' physical capacity. In this regard, blood flow restriction (BFR) training is becoming increasingly popular because it enables gains in muscle mass and strength despite using low-mechanical loads combined with external venous occlusion. Since the underlying mechanisms are still unknown, we applied invasive measurements during exercise with and without BFR to promote physiological understanding and safety of this popular training technique. Methods: In a randomized cross-over design, ten healthy men (28.1 ± 6.5 years) underwent two trials of unilateral biceps curls either with (BFR) and without BFR (CON). For analysis of changes in intravascular pressures, blood gases, oximetry and electrolytes, an arterial and a venous catheter were placed at the exercising arm before exercise. Arterial and venous blood gases and intravascular pressures were analyzed before, during and 5 min after exercise. Results: Intravascular pressures in the arterial and venous system were more increased during exercise with BFR compared to CON (p < 0.001). Furthermore, arterial and venous blood gas analyses revealed a BFR-induced metabolic acidosis (p < 0.05) with increased lactate production (p < 0.05) and associated elevations in [K+], [Ca2+] and [Na+] (p < 0.001). Conclusion: The present study describes for the first time the local physiological changes during BFR training. While BFR causes greater hypertension in the arterial and venous system of the exercising extremity, observed electrolyte shifts corroborate a local metabolic acidosis with concurrent rises in [K+] and [Na+]. Although BFR could be a promising new training concept for medical application, its execution is associated with comprehensive physiological challenges.

Tensiomyography parameters and serum biomarkers after eccentric exercise of the elbow flexors.

PURPOSE: The tensiomyography (TMG) technique is increasingly used to determine muscle contractile properties in exercise and injury management. The present study investigated the informative value of TMG parameters in correlation with commonly used (creatine kinase, CK; myoglobin, Mb) and novel candidate biomarkers of muscle damage (heart-type fatty acid-binding protein, h-FABP; high-mobility group box 1, HMGB1). METHODS: Ten untrained men performed 6 × 10 eccentric contractions of the elbow flexors at 110% of the concentric one repetition maximum. CK, Mb, h-FABP, HMGB1, arm circumference, pain and TMG data, including maximal displacement (Dm) and temporal outcomes as the contraction time (Tc), sustained time (Ts), delay time (Td), and relaxation time (Tr), were assessed pre-exercise, post-exercise, 20 min, 2 h and on the consecutive 3 days post-exercise. RESULTS: CK and h-FABP significantly increased beginning at 24 h, Mb already increased at 2 h (p < 0.05). HMGB1 was only increased immediately post-exercise (p = 0.02). Tc and Td remained unchanged, whereas Ts and Tr were significantly slower beginning at 24 h (p < 0.05). Dm was decreased within the first 24 h and after 72 h (p < 0.01). The % change from pre-exercise correlated for Dm with CK, Mb, and h-FABP the highest at 48 h (r = - 0.95, - 0.87 and - 0.79; p < 0.01) and for h-FABP with CK and Mb the highest at 24 h (r = 0.96 and 0.94, for all p < 0.001). CONCLUSION: This study supports the correlation of TMG parameters with muscle damage markers after eccentric exercise. Therefore, TMG could represent a non-invasive and cost effective alternative to quantify the degree of muscle damage after exercise interventions.

The role of oxidative and nitrosative stress in the pathology of osteoarthritis: Novel candidate biomarkers for quantification of degenerative changes in the knee joint.

Osteoarthritis (OA) is the most frequently diagnosed joint disorder worldwide with increasing prevalence and crucial impact on the quality of life of affected patients through chronic pain, decreasing mobility and invalidity. Although some risk factors, such as age, obesity and previous joint injury are well established, the exact pathogenesis of OA on a cellular and molecular level remains less understood. Today, the role of nitrosative and oxidative stress has not been investigated conclusively in the pathogenesis of OA yet. Therefore, the objective of this study was to identify biological substances for oxidative and nitrosative stress, which mirror the degenerative processes in an osteoarthritic joint. 69 patients suffering from a diagnosed knee pain participated in this study. Based on the orthopedic diagnosis, patients were classified into an osteoarthritis group (OAG, n=24) or in one of two control groups (meniscopathy, CG1, n=11; anterior cruciate ligament rupture, CG2, n=34). Independently from the study protocol, all patients underwent an invasive surgical intervention which was used to collect samples from the synovial membrane, synovial fluid and human serum. Synovial biopsies were analyzed histopathologically for synovitis (Krenn-Score) and immunohistochemically for detection of end products of oxidative (8-isoprostane F2α) and nitrosative (3-nitrotyrosine) stress. Additionally, the fluid samples were analyzed for 8-isoprostane F2α and 3-nitrotyrosine by competitive ELISA method. The analyzation of inflammation in synovial biopsies revealed a slight synovitis in all three investigated groups. Detectable concentrations of 3-nitrotyrosine were reported in all three investigated groups without showing any significant differences between the synovial biopsies, fluid or human serum. In contrast, significant increased concentrations of 8-isoprostane F2α were detected in OAG compared to both control groups. Furthermore, our data showed a significant correlation between the histopathological synovitis and oxidative stress in OAG (r=0.728, P<0.01). There were no significant differences between the concentrations of 8-isoprostane F2α in synovial fluid and human serum. The findings of the current study support the hypothesis that oxidative and nitrosative stress are components of the multi-factory pathophysiological formation of OA. It seems reasonable that an inflammatory process in the synovial membrane triggers the generation of oxidative and nitrosative acting substances which can lead to a further degradation of the articular cartilage. Based on correlations between the observed degree of inflammation and investigated biomarkers, especially 8-isoprostane F2α seems to be a novel candidate biomarker for OA. However, due to the finding that also both control groups showed increased concentrations of selected biomarkers, future studies have to validate the diagnostic potential of these biomarkers in OA and in related conditions of the knee joint.

Ischemic Preconditioning Blunts Muscle Damage Responses Induced by Eccentric Exercise.

PURPOSE: Ischemic preconditioning (IPC) is known to reduce muscle damage induced by ischemia and reperfusion injury during surgery. Because of similarities between the pathophysiological formation of ischemia and reperfusion injury and eccentric exercise-induced muscle damage (EIMD), as characterized by an intracellular accumulation of Ca, an increased production of reactive oxygen species, and increased proinflammatory signaling, the purpose of the present study was to investigate whether IPC performed before eccentric exercise may also protect against EIMD. METHODS: Nineteen healthy men were matched to an eccentric-only (ECC; n = 9) or eccentric proceeded by IPC group (IPC + ECC; n = 10). The exercise protocol consisted of bilateral biceps curls (3 × 10 repetitions at 80% of the concentric one-repetition maximum). In IPC + ECC, IPC was applied bilaterally at the upper arms by a tourniquet (200 mm Hg) immediately before the exercise (3 × 5 min of occlusion, separated by 5 min of reperfusion). Creatine kinase (CK), arm circumference, subjective pain (visual analog scale score), and radial displacement (tensiomyography, maximal radial displacement) were assessed before IPC, preexercise, postexercise, and 20 min, 2 h, 24 h, 48 h, and 72 h postexercise. RESULTS: CK differed from baseline only in ECC at 48 h (P < 0.001) and 72 h (P < 0.001) postexercise. After 24, 48, and 72 h, CK was increased in ECC compared with IPC + ECC (between groups: 24 h, P = 0.004; 48 h, P < 0.001; 72 h, P < 0.001). The visual analog scale score was significantly higher in ECC at 24-72 h postexercise when compared with IPC + ECC (between groups: all P values < 0.001). The maximal radial displacement was decreased on all postexercise days in ECC (all P values < 0.001) but remained statistically unchanged in IPC + ECC (between groups: P < 0.01). CONCLUSIONS: These findings indicate that IPC performed before a bout of eccentric exercise of the elbow flexors blunts EIMD and exercise-induced pain while maintaining the contractile properties of the muscle.

Effects of Neuromuscular Electrical Stimulation on the Frequency of Skeletal Muscle Cramps: A Prospective Controlled Clinical Trial.

OBJECTIVES: We investigated if neuromuscular electrical stimulation (NMES) of calf muscles prevents spontaneous calf cramps. MATERIALS AND METHODS: In 19 individuals affected by more than or equal to one calf cramp per week the gastrocnemius of the predominantly affected leg was stimulated twice a week (intervention leg, IL) over six weeks (3 × 6 stimulation trains at 30 Hz above the individual cramp threshold frequency). The other leg served as control (CL). The participants were advised to record all spontaneous muscle cramps from two weeks before the intervention until two weeks after the last NMES session. RESULTS: The number of spontaneous calf cramps in the two weeks after the intervention was 78% lower (2.1 ± 6.8 cramps) in the stimulated (p < 0.001) and 63% lower (2.0 ± 6.9 cramps) in the unstimulated calves (p < 0.001), when compared to the two weeks prior to the intervention (IL: 9.6 ± 12.4 cramps; CL: 5.5 ± 12.7 cramps). Only in the IL, this improvement was accompanied by an increase in the cramp threshold frequency from 15.5 ± 8.5 Hz before the NMES intervention to 21.7 ± 12.4 Hz after the intervention. The severity of the remaining calf cramps tended to be lower in both legs after the intervention. CONCLUSIONS: The applied stimulation protocol seems to provide an effective prevention strategy in individuals affected by regular calf cramps.

Soft-tissue damage during total knee arthroplasty: Focus on tourniquet-induced metabolic and ionic muscle impairment.

PURPOSE: Advantages of tourniquet use in TKA include benefits for surgeons and patients, varying from a bloodless operation site to a reduced intervention time. The time under ischemia and the reperfusion period are crucial phases for affected soft-tissue, most commonly the extensor mechanism. CASE REPORTS: documented its impact on soft-tissue, ranging from necrotic muscle damage to systemic inflammation. Recently, research regarding tourniquet application patterns discuss clinical outcome parameters in the context of soft-tissue damage, excluding the underlying pathophysiological mechanisms. METHODS: This review summarizes the molecular aspects of soft-tissue damage occurring during tourniquet application in TKA with special focus on ischemia/reperfusion injury. Recent meta-analyses and original trials were reviewed for data on muscle damage and are presented. CONCLUSION: Although underlying pathomechanisms are well known and presented, clinical orthopedic research has so far not addressed this issue. In context of physical training, positive effects regarding postoperative recovery might be possible if more attention is paid to prepare involved muscle preoperatively to TKA (prehabilitation).