Tertius A. Kohn

CaMK activation during exercise is required for histone hyperacetylation and MEF2A binding at the MEF2 site on the Glut4 gene

The role of CaMK II in regulating GLUT4 expression in response to intermittent exercise was investigated. Wistar rats completed 5 x 17-min bouts of swimming after receiving 5 mg/kg KN93 (a CaMK II inhibitor), KN92 (an analog of KN93 that does not inhibit CaMK II), or an equivalent volume of vehicle. Triceps muscles that were harvested at 0, 6, or 18 h postexercise were assayed for 1) CaMK II phosphorylation by Western blot, 2) acetylation of histone H3 at the Glut4 MEF2 site by chromatin immunoprecipitation (ChIP) assay, 3) bound MEF2A at the Glut4 MEF2 cis-element by ChIP, and 4) GLUT4 expression by RT-PCR and Western blot. Compared with controls, exercise caused a twofold increase in CaMK II phosphorylation. Immunohistochemical stains indicated increased CaMK II phosphorylation in nuclear and perinuclear regions of the muscle fiber. Acetylation of histone H3 in the region surrounding the MEF2 binding site on the Glut4 gene and the amount of MEF2A that bind to the site increased approximately twofold postexercise. GLUT4 mRNA and protein increased approximately 2.2- and 1.8-fold, respectively, after exercise. The exercise-induced increases in CaMK II phosphorylation, histone H3 acetylation, MEF2A binding, and GLUT4 expression were attenuated or abolished when KN93 was administered to rats prior to exercise. KN92 did not affect the increases in pCaMK II and GLUT4. These data support the hypothesis that CaMK II activation by exercise increases GLUT4 expression via increased accessibility of MEF2A to its cis-element on the gene.

Caffeine induces hyperacetylation of histones at the MEF2 site on the Glut4 promoter and increases MEF2A binding to the site via a CaMK-dependent mechanism

This study was conducted to explore the mechanism by which caffeine increases GLUT4 expression in C(2)C(12) myotubes. Myoblasts were differentiated in DMEM containing 2% horse serum for 13 days and the resultant myotubes exposed to 10 mM caffeine in the presence or absence of 25 microM KN93 or 10 mM dantrolene for 2 h. After the treatment, cells were kept in serum-free medium and harvested between 0 and 6 h later, depending on the assay. Chromatin immunoprecipitation (ChIP) assays revealed that caffeine treatment caused hyperacetylation of histone H3 at the myocyte enhancer factor 2 (MEF2) site on the Glut4 promoter (P < 0.05) and increased the amount of MEF2A that was bound to this site approximately 2.2-fold (P < 0.05) 4 h posttreatment compared with controls. These increases were accompanied by an approximately 1.8-fold rise (P < 0.05 vs. control) in GLUT4 mRNA content at 6 h post-caffeine treatment. Both immunoblot and immunocytochemical analyses showed reduced nuclear content of histone deacetylase-5 in caffeine-treated myotubes compared with controls at 0-2 h posttreatment. Inclusion of 10 mM dantrolene in the medium to prevent the increase in cytosolic Ca(2+), or 25 microM KN93 to inhibit Ca(2+)/calmodulin-dependent protein kinase (CaMK II), attenuated all the above caffeine-induced changes. These data indicate that caffeine increases GLUT4 expression by acetylating the MEF2 site to increase MEF2A binding via a mechanism that involves CaMK II.

Gluconeogenesis during endurance exercise in cyclists habituated to a long‐term low carbohydrate high‐fat diet

Blood glucose is an important fuel for endurance exercise. It can be derived from ingested carbohydrate, stored liver glycogen and newly synthesized glucose (gluconeogenesis). We hypothesized that athletes habitually following a low carbohydrate high fat (LCHF) diet would have higher rates of gluconeogenesis during exercise compared to those who follow a mixed macronutrient diet. We used stable isotope tracers to study glucose production kinetics during a 2 h ride in cyclists habituated to either a LCHF or mixed macronutrient diet. The LCHF cyclists had lower rates of total glucose production and hepatic glycogenolysis but similar rates of gluconeogenesis compared to those on the mixed diet. The LCHF cyclists did not compensate for reduced dietary carbohydrate availability by increasing glucose synthesis during exercise but rather adapted by altering whole body substrate utilization.

High oxidative capacity and type IIx fibre content in springbok and fallow deer skeletal muscle suggest fast sprinters with a resistance to fatigue

SUMMARY Some wild antelopes are fast sprinters and more resistant to fatigue than others. This study therefore investigated two wild antelope species to better understand their reported performance capability. Muscle samples collected post mortem from the vastus lateralis and longissimus lumborum of fallow deer (Dama dama) and springbok (Antidorcas marsupialis) were analysed for myosin heavy chain isoform content, citrate synthase, 3-hydroxyacyl CoA dehydrogenase, phosphofructokinase, lactate dehydrogenase and creatine kinase activities. Cross-sectional areas, fibre type and oxidative capacities of each fibre type were determined in the vastus lateralis only. The predominant fibre type in both muscle groups and species were type IIX (>50%), with springbok having more type IIX fibres than fallow deer (P<0.05). Overall cross-sectional area was not different between the two species. The metabolic pathway analyses showed high glycolytic and oxidative capacities for both species, but springbok had significantly higher CS activities than fallow deer. Large variation and overlap in oxidative capacities existed within and between the fibre types. Some type IIX fibres presented with oxidative capacities similar to those from type I and IIA fibres. The data suggest that springbok and fallow deer are able sprint at >90 and 46 km h−1, respectively, partly from having large type IIX fibre contents and high glycolytic capacities. The high oxidative capacities also suggest that these animals may be able to withstand fatigue for long periods of time.

Fiber type and metabolic characteristics of lion (Panthera leo), caracal (Caracal caracal) and human skeletal muscle.

Lion (Panthera leo) and caracal (Caracal caracal) skeletal muscle samples from Vastus lateralis, Longissimus dorsi and Gluteus medius were analyzed for fiber type and citrate synthase (CS; EC 2.3.3.1), 3-hydroxyacyl Co A dehydrogenase (3HAD; EC 1.1.1.35), phosphofructokinase-1 (PFK; EC 2.7.1.11), creatine kinase (CK; EC 2.7.3.2), phosphorylase (PHOS; EC 2.4.1.1) and lactate dehydrogenase (LDH; EC 1.1.1.27) activities and compared to human runners, the latter also serving as validation of methodology. Both felids had predominantly type IIx fibers (range 50-80%), whereas human muscle had more types I and IIa. Oxidative capacity of both felids (CS: 5-9 μmol/min/g ww and 3HAD: 1.4-2.6 μmol/min/g ww) was lower than humans, whereas the glycolytic capacity was elevated. LDH activity of caracal (346 ± 81) was higher than lion (227 ± 62 μmol/min/g ww), with human being the lowest (55 ± 17). CK and PHOS activities were also higher in caracal and lion compared to human, but PFK was lower in both felid species. The current data and past research are illustrated graphically showing a strong relationship between type II fibers and sprinting ability in various species. These data on caracal and lion muscles confirm their sprinting behavior.

Black wildebeest skeletal muscle exhibits high oxidative capacity and a high proportion of type IIx fibres

SUMMARY The aim of the study was to investigate the skeletal muscle characteristics of black wildebeest (Connochaetes gnou) in terms of fibre type and metabolism. Samples were obtained post mortem from the vastus lateralis and longissimus lumborum muscles and analysed for myosin heavy chain (MHC) content. Citrate synthase (CS), 3-hydroxyacyl co A dehydrogenase (3HAD), phosphofructokinase (PFK), lactate dehydrogenase (LDH) and creatine kinase (CK) activities were measured spectrophotometrically to represent the major metabolic pathways in these muscles. Both muscles had less than 20% MHC I, whereas MHC IIa and MHC IIx were expressed in excess of 50% in the vastus lateralis and longissimus lumborum muscles, respectively. Overall fibre size was 2675±1034 μm2, which is small compared with other species. Oxidative capacity (CS and 3HAD) in both muscles was high and did not differ from one another, but the longissimus lumborum had significantly (P<0.05) higher PFK, LDH and CK activities. No relationships were observed between fibre type and the oxidative and oxygen-independent metabolic capacity as measured by specific enzyme activities. This study confirms the presence of both fast-twitch fibres and high oxidative capacity in black wildebeest, indicating an animal that can run very fast but is also fatigue resistant.

Lion (Panthera leo) and caracal (Caracal caracal) type IIx single muscle fibre force and power exceed that of trained humans

SUMMARY This study investigated for the first time maximum force production, shortening velocity (Vmax) and power output in permeabilised single muscle fibres at 12°C from lion, Panthera leo (Linnaeus 1758), and caracal, Caracal caracal (Schreber 1776), and compared the values with those from human cyclists. Additionally, the use and validation of previously frozen tissue for contractile experiments is reported. Only type IIx muscle fibres were identified in the caracal sample, whereas type IIx and only two type I fibres were found in the lion sample. Only pure type I and IIa, and hybrid type IIax fibres were identified in the human samples – there were no pure type IIx fibres. Nevertheless, compared with all the human fibre types, the lion and caracal fibres were smaller (P<0.01) in cross-sectional area (human: 6194±230 μm2, lion: 3008±151 μm2, caracal: 2583±221 μm2). On average, the felid type IIx fibres produced significantly greater force (191–211 kN m−2) and ~3 times more power (29.0–30.3 kN m−2 fibre lengths s−1) than the human IIax fibres (100–150 kN m−2, 4–11 kN m−2 fibre lengths s−1). Vmax values of the lion type IIx fibres were also higher than those of human type IIax fibres. The findings suggest that the same fibre type may differ substantially between species and potential explanations are discussed.

McArdle disease does not affect skeletal muscle fibre type profiles in humans

ABSTRACT Patients suffering from glycogen storage disease V (McArdle disease) were shown to have higher surface electrical activity in their skeletal muscles when exercising at the same intensity as their healthy counterparts, indicating more muscle fibre recruitment. To explain this phenomenon, this study investigated whether muscle fibre type is shifted towards a predominance in type I fibres as a consequence of the disease. Muscle biopsies from the Biceps brachii (BB) (n = 9) or Vastus lateralis (VL) (n = 8) were collected over a 13-year period from male and female patients diagnosed with McArdle disease, analysed for myosin heavy chain (MHC) isoform content using SDS-PAGE, and compared to healthy controls (BB: n = 3; VL: n = 10). All three isoforms were expressed and no difference in isoform expression in VL was found between the McArdle patients and healthy controls (MHC I: 33±19% vs. 43±7%; MHC IIa: 52±9% vs. 40±7%; MHC IIx: 15±18% vs. 17±9%). Similarly, the BB isoform content was also not different between the two groups (MHC I: 33±14% vs. 30±11%; MHC IIa: 46±17% vs. 39±5%; MHC IIx: 21±13% vs. 31±14%). In conclusion, fibre type distribution does not seem to explain the higher surface EMG in McArdle patients. Future studies need to investigate muscle fibre size and contractility of McArdle patients.

Suppression of the GLUT4 adaptive response to exercise in fructose-fed rats

Exercise-induced increase in skeletal muscle GLUT4 expression is associated with hyperacetylation of histone H3 within a 350-bp DNA region surrounding the myocyte enhancer factor 2 (MEF2) element on the Glut4 promoter and increased binding of MEF2A. Previous studies have hypothesized that the increase in MEF2A binding is a result of improved accessibility of this DNA segment. Here, we investigated the impact of fructose consumption on exercise-induced GLUT4 adaptive response and directly measured the accessibility of the above segment to nucleases. Male Wistar rats (n = 30) were fed standard chow or chow + 10% fructose or maltodextrin drinks ad libitum for 13 days. In the last 6 days five animals per group performed 3 × 17-min bouts of intermittent swimming daily and five remained untrained. Triceps muscles were harvested and used to measure 1) GLUT4, pAMPK, and HDAC5 contents by Western blot, 2) accessibility of the DNA segment from intact nuclei using nuclease accessibility assays, 3) acetylation level of histone H3 and bound MEF2A by ChIP assays, and 4) glycogen content. Swim training increased GLUT4 content by ∼66% (P < 0.05) but fructose and maltodextrin feeding suppressed the adaptation. Accessibility of the DNA region to MNase and DNase I was significantly increased by swimming (∼2.75- and 5.75-fold, respectively) but was also suppressed in trained rats that consumed fructose or maltodextrin. Histone H3 acetylation and MEF2A binding paralleled the accessibility pattern. These findings indicate that both fructose and maltodextrin modulate the GLUT4 adaptive response to exercise by mechanisms involving chromatin remodeling at the Glut4 promoter.

Metabolic cost of running is greater on a treadmill with a stiffer running platform

ABSTRACT Exercise testing on motorised treadmills provides valuable information about running performance and metabolism; however, the impact of treadmill type on these tests has not been investigated. This study compared the energy demand of running on two laboratory treadmills: an HP Cosmos (C) and a Quinton (Q) model, with the latter having a 4.5 times stiffer running platform. Twelve experienced runners ran identical bouts on these treadmills at a range of four submaximal velocities (reported data is for the velocity that approximated 75–81% VO2max). The stiffer treadmill elicited higher oxygen consumption (C: 46.7 ± 3.8; Q: 50.1 ± 4.3 ml·kg−1 · min−1), energy expenditure (C: 16.0 ± 2.5; Q: 17.7 ± 2.9 kcal · min−1), carbohydrate oxidation (C: 9.6 ± 3.1; Q: 13.0 ± 3.9 kcal · min−1), heart rate (C: 155 ± 16; Q: 163 ± 16 beats · min−1) and rating of perceived exertion (C: 13.8 ± 1.2; Q: 14.7 ± 1.2), but lower fat oxidation (C: 6.4 ± 2.3; Q: 4.6 ± 2.5 kcal · min−1) (all analysis of variance treadmill comparisons P < 0.01). This study confirms that caution is required when comparing performance and metabolic results between different treadmills and suggests that treadmills will vary in their comparability to over-ground running depending on the running platform stiffness.