Effect of different blood-guided conditioning programmes on skeletal muscle ultrastructure and histochemistry of sport horses.

The effects of three different blood-guided conditioning programmes on ultrastructural and histochemical features of the gluteus medius muscle of 2-year-old sport horses were examined. Six non-trained Haflinger horses performed three consecutive conditioning programmes of varying lactate-guided intensities [velocities eliciting blood lactate concentrations of 1.5 (v1.5 ), 2.5 (v2.5 ) and 4 (v4 ) mm respectively] and durations (25 and 45 min). Each conditioning programme lasted 6 weeks and was followed by a 5-week resting period. Pre-, post- and deconditioning muscle biopsies were analysed. Although training and detraining adaptations were similar in nature, they varied significantly in magnitude among the three different conditioning programmes. Overall, the adaptations consisted in significant increases in size of mitochondria and myofibrils, as well as a hypertrophy of myofibrillar ATPase type IIA muscle fibres and a reduction in number of type IIx low-oxidative fibres. Together, these changes are compatible with a significant improvement in both muscle aerobic capacity and muscle strength. The use of v1.5 and v2.5 as the exercise intensities for 45 min elicited more significant adaptations in muscle, whereas conditioning horses at v4 for 25 min evoked minimal changes. Most of these muscular adaptations returned towards the pre-conditioning status after 5 weeks of inactivity. It is concluded that exercises of low or moderate intensities (in the range between v1.5 and v2.5 ) and long duration (45 min) are more effective for improving muscle features associated with stamina and power in sport horses than exercises of higher intensity (equivalent to v4 ) and shorter duration (25 min).

Relation between cardiac dimensions and peak oxygen uptake.

BACKGROUND: Long term endurance training is known to increase peak oxygen uptake (VO2peak) and induce morphological changes of the heart such as increased left ventricular mass (LVM). However, the relationship between and the total heart volume (THV), considering both the left and right ventricular dimensions in both males and females, is not completely described. Therefore, the aim of this study was to test the hypothesis that THV is an independent predictor of VO2peak and to determine if the left and right ventricles enlarge in the same order of magnitude in males and females with a presumed wide range of THV. METHODS AND RESULTS: The study population consisted of 131 subjects of whom 71 were athletes (30 female) and 60 healthy controls (20 female). All subjects underwent cardiovascular MR and maximal incremental exercise test. Total heart volume, LVM and left- and right ventricular end-diastolic volumes (LVEDV, RVEDV) were calculated from short-axis images. was significantly correlated to THV, LVM, LVEDV and RVEDV in both males and females. Multivariable analysis showed that THV was a strong, independent predictor of (R2 = 0.74, p < 0.001). As LVEDV increased, RVEDV increased in the same order of magnitude in both males and females (R2 = 0.87, p < 0.001). CONCLUSION: Total heart volume is a strong, independent predictor of maximal work capacity for both males and females. Long term endurance training is associated with a physiologically enlarged heart with a balance between the left and right ventricular dimensions in both genders.

Effect of blood lactate-guided conditioning of horses with exercises of differing durations and intensities on heart rate and biochemical blood variables.

The velocity at which blood lactate concentration ([LA]) of 4 mmol/L is reached (v(4)) is widely used to determine fitness, but there are few published data on using [LA] as a guide for the exercise speed for training in horses. In this study, the effect of 3 conditioning programs with [LA] guided exercise speeds on v(4), v(200) (speed at a heart rate of 200 beats/min), blood [LA], plasma FFA ([FFA]), and alanine concentrations ([alanine]), before and after exercise, as well as heart rate during exercise, of horses was examined. Six 2-yr-old Haflinger stallions underwent an initial treadmill-based standard exercise test (SET). A regression analysis [LA]-speed relationship was used to calculate v(1.5), v(2.5), and v(4). Horses were then randomly assigned to 1 of 3 conditioning programs according to a 3 x 6 Latin square design. During 6 wk, horses exercised on the treadmill every other day for 45 min at their calculated v(1.5) or v(2.5) or 25 min at their v(4). Each conditioning period (CDP) was followed by 5 wk without conditioning. At 2 and 9 d, and 5 wk, after the end of the CDP, the horses performed another SET to evaluate again the v(4) and v(200). Blood [LA], plasma [FFA], and [alanine] were measured before and after heart rate during exercise sessions 1, 11, and 21 in each CDP. None of the exercise programs had an effect on v(4) and v(200) (P > 0.05). The increase of the mean [LA] after exercise decreased during CDP (P < 0.05), and the increase of mean heart rate during exercise tended to decrease as well (P = 0.07). There was no difference among the conditioning programs. Plasma [FFA] before exercise was not influenced by the CDP (P > 0.05). The plasma [FFA] was always greater after exercise (P < 0.05), but there was no difference among conditioning programs. Overall, the increase was greatest after the 21st exercise session compared with the 1st and 11th exercise sessions (P < 0.05). The mean plasma [alanine] before exercise remained similar during all CDP (P > 0.05). Mean plasma [alanine] of the horses was increased after all exercise sessions measured (P < 0.05). There was no difference among conditioning programs (P > 0.05). It is concluded that conditioning with the exercise types used had small effects. This could have been because the exercise stress was too small, but also because the workload was not increased during the CDP.

Imidazoline-induced amplification of glucose- and carbachol-stimulated insulin release includes a marked suppression of islet nitric oxide generation in the mouse.

AIM: The role of islet nitric oxide (NO) production in insulin-releasing mechanisms is unclear. We examined whether the beneficial effects of the imidazoline derivative RX 871024 (RX) on beta-cell function might be related to perturbations of islet NO production. METHODS: Experiments were performed with isolated islets or intact mice challenged with glucose or carbachol with or without RX treatment. Insulin was determined with radioimmunoassay, NO generation with high-performance liquid chromatography and expression of inducible NO synthase (iNOS) with confocal microscopy. RESULTS: RX treatment, in doses lacking effects on basal insulin, greatly amplified insulin release stimulated by the NO-generating secretagogues glucose and carbachol both in vitro and in vivo. RX also improved the glucose tolerance curve. Islets incubated at high glucose levels (20 mmol L(-1)) displayed increased NO production derived from both neuronal constitutive NO synthase (ncNOS) and iNOS. RX abrogated this glucose-induced NO production concomitant with amplification of insulin release. Confocal microscopy revealed abundant iNOS expression in beta cells after incubation of islets at high but not low glucose levels. This was abolished after RX treatment. Similarly, islets cultured for 24 h at high glucose levels showed intense iNOS expression in beta cells. This was abrogated with RX and followed by an amplified glucose-induced insulin release. CONCLUSION: RX effectively counteracts the negative impact of beta-cell NO generation on insulin release stimulated by glucose and carbachol suggesting imidazoline compounds by virtue of NOS inhibitory properties being of potential therapeutic value for treatment of beta-cell dysfunction in hyperglycaemia and type 2 diabetes.

Nitric oxide, islet acid glucan-1,4-alpha-glucosidase activity and nutrient-stimulated insulin secretion.

The mechanism of nutrient-evoked insulin release is clearly complex. One part of that mechanism is postulated to be the activation of the glycogenolytic enzyme acid glucan-1,4-alpha-glucosidase. As nitric oxide (NO) has been found to be a potent inhibitor of glucose-stimulated insulin secretion, we have now investigated a possible influence of exogenous NO and inhibition of endogenous NO production on islet acid glucan-1,4-alpha-glucosidase activity in relation to insulin release stimulated by glucose and l-arginine. In isolated islets, NO derived from the intracellular NO donor hydroxylamine inhibited the activation of acid glucan-1, 4-alpha-glucosidase and its isoform acid alpha-glucosidase in parallel with inhibition of glucose-stimulated insulin release. In comparison, other lysosomal enzymes were largely unaffected. Similarly, the spontaneous NO donor sodium nitroprusside, as well as NO gas, when added to islet homogenates, suppressed the activities of these acid alpha-glucosidehydrolases and, to a lesser extent, the activities of other lysosomal enzymes. Finally, in the presence of the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester, insulin release from isolated islets stimulated by glucose or l-arginine was markedly potentiated in parallel with an accompanying increase in the activities of acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase. Other lysosomal enzymes and neutral alpha-glucosidase were not influenced. We propose that an important inhibitory effect of NO on the insulin secretory processes stimulated by glucose and l-arginine is exerted via inactivation of islet acid glucan-1,4-alpha-glucosidase, a putative key enzyme in nutrient-stimulated insulin release.

Dysfunction of the islet lysosomal system conveys impairment of glucose-induced insulin release in the diabetic GK rat.

Accumulated evidence links an important signal involved in glucose-stimulated insulin release to the activation of the islet lysosomal glycogenolytic enzyme acid glucan-1,4-alpha-glucosidase. We have analyzed the function of the lysosomal system/lysosomal enzyme activities in pancreatic islets of young (6-8 weeks), spontaneously diabetic, GK (Goto-Kakizaki) rats and Wistar control rats in relation to glucose-induced insulin release. The insulin secretory response to glucose was markedly impaired in the GK rat, but was restored by the adenylate cyclase activator forskolin. Islet activities of classical lysosomal enzymes, e.g.. acid phosphatase, N-acetyl-beta-D-glucosaminidase, beta-glucuronidase, and cathepsin D, were reduced by 20-35% in the GK rat compared with those in Wistar controls. In contrast, the activities of the lysosomal alpha-glucosidehydrolases, i.e.. acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase, were increased by 40-50%. Neutral alpha-glucosidase (endoplasmic reticulum) was unaffected. Comparative analysis of liver tissue showed that lysosomal enzyme activities were of the same magnitude in GK and Wistar rats. Notably, in Wistar rats, the activities of acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase were approximately 15-fold higher in islets than in liver. Other lysosomal enzymes did not display such a difference. Normalization of glycemia in GK rats by phlorizin administered for 9 days did not influence either the lysosomal alpha-glucosidehydrolase activities or other lysosomal enzyme activities in GK islets. Finally, the pseudotetrasaccharide acarbose, which accumulates in the lysosomal system, inhibited acid glucan-1,4-alpha-glucosidase activity in parallel with its inhibitory action on glucose-induced insulin release in intact Wistar islets, whereas no effect was recorded for either parameter in intact GK islets. In contrast, acarbose inhibited the enzyme activity equally in islet homogenates from both GK and Wistar rats, showing that the catalytic activity of the enzyme itself in disrupted cells was unaffected. We propose that dysfunction of the islet lysosomal/vacuolar system is an important defect impairing the transduction mechanisms for glucose-induced insulin release in the GK rat.

Effects of conditioning horses with lactate-guided exercise on muscle glycogen content.

The effects of 3 different conditioning programmes on muscle glycogen concentration in horses were examined. Speed of exercise was selected according to the blood lactate values for each horse derived from a standardised exercise test before beginning a conditioning programme. Six 2-year-old Haflinger stallions were assigned randomly to one of 3 conditioning programmes according to a 6 x 3 latin square design: 45 min at their individual v1.5 or v2.5 and 25 min at v4. Each conditioning programme lasted 6 weeks (21 exercise sessions), followed by 5 weeks without conditioning (resting period). All exercise was carried out on a treadmill inclined at 17%. Muscle biopsies were taken 5 times from the gluteus medius muscle at 2 cm and 6 cm depth: before the start and in the middle of the conditioning period, then at Days 2, 9 and 35 after the last exercise session. It was found that glycogen concentration was not affected by conditioning until 9 days after finishing conditioning at v1.5 and v2.5 for 45 min (P < 0.05). By this time glycogen concentration in the muscle samples taken at 6 cm depth increased by 47 and 48%, respectively, and remained elevated until the end of the resting period. It was concluded that conditioning at lower intensity and for longer duration seemed to increase glycogen stores in the muscle while faster intensity but shorter duration exercise did not. To increase the likelihood of measuring effects of conditioning programmes on muscle variables, sampling should be done at different depths of a muscle and at several days after finishing a conditioning programme.

Insulin release transduction mechanism through acid glucan 1,4-alpha-glucosidase activation is Ca2+ regulated.

An important signal involved in glucose-stimulated insulin secretion is transduced through the action of a lysosomal acid, glucan 1,4-alpha-glucosidase. We investigated the Ca2+ dependency of this enzyme activity in relation to insulin release. In isolated islets, increased levels of extracellular Ca2+ induced a large increase in acid glucan 1,4-alpha-glucosidase activity accompanied by a similar increase in insulin release at both substimulatory and stimulatory concentrations of glucose. At low glucose the Ca2+ "inflow" blocker nifedipine unexpectedly stimulated enzyme activity without affecting insulin release. However, nifedipine suppressed 45Ca2+ outflow from perifused islets at low glucose and at Ca2+ deficiency when intracellular Ca2+ was mobilized by carbachol. This nifedipine-induced retention of Ca2+ was reflected in increased acid glucan 1,4-alpha-glucosidase activity. Adding different physiological Ca2+ concentrations or nifedipine to islet homogenates did not increase enzyme activity. Neither selective glucan 1,4-alpha-glucosidase inhibition nor the ensuing suppression of glucose-induced insulin release was overcome by a maximal Ca2+ concentration. Hence, Ca(2+)-induced changes in acid glucan 1,4-alpha-glucosidase activity were intimately coupled to similar changes in Ca(2+)-glucose-induced insulin release. Ca2+ did not affect the enzyme itself but presumably activated either glucan 1,4-alpha-glucosidase-containing organelles or closely interconnected messengers.

Interaction of the islet nitric oxide system with L-arginine-induced secretion of insulin and glucagon in mice.

1. Several recent in vitro studies have suggested that production of nitric oxide (NO) from the islet NO system may have an important regulatory influence on the secretion of insulin and glucagon. In the present paper we have investigated, mainly with an in vivo approach, the influence and specificity of the NO synthase (NOS) blocker NG-nitro-L-arginine methyl ester (L-NAME) on L-arginine-induced secretion of insulin and glucagon. 2. In freely fed mice, L-NAME pretreatment (1.2 mmol kg-1) influenced the dynamics of insulin and glucagon release following an equimolar dose of L-arginine, the specific substrate for NOS activity, in that the NOS inhibitor enhanced the insulin response but suppressed the glucagon responses. This was reflected in a large decrease in the plasma glucose levels of the L-NAME pretreated animals. 3. L-NAME pretreatment did not influence the insulin and glucagon secretory responses to the L-arginine-enantiomer D-arginine, which cannot serve as a substrate for NOS activity. 4. Replacing L-NAME pretreatment by pretreatment with D-arginine or L-arginine itself, which both carry the same cationic change and are devoid of NOS inhibitory properties, did not mimic the effects of L-NAME on L-arginine-induced hormone release. 5. Fasting the animals for 24 h totally abolished the L-NAME-induced potentiation of L-arginine stimulated insulin release suggesting that the sensitivity of the beta-cell secretory machinery to NO-production is greatly changed in the fasting state. However, the L-NAME-induced suppression of L-arginine stimulated glucagon release was unaffected by starvation. 6. In isolated islets from freely fed mice, L-arginine (5 mM) stimulated insulin release was greatly enhanced and glucagon release markedly suppressed by the presence of the NOS inhibitor L-NAME in the incubation medium. These effects were abolished in isolated islets taken from 24 h fasted mice. 7. Our present results, which showed that the NOS inhibitor L-NAME markedly enhances insulin release but suppresses glucagon release induced by L-arginine in the intact animal, give strong support to our previous hypothesis that the islet NO system is a negative modulator of insulin secretion and a positive modulator of glucagon secretion. Additionally, we observed that the importance of the beta-cell NO-production for secretory mechanisms, as evaluated by the effect of L-NAME on L-arginine-induced insulin release, was greatly changed after starvation, an effect less prominent with regard to glucagon release.

Three-dimensional electron microscopy of the internal nucleolus-associated chromatin and of the nucleolar vacuoles during early germination of Sinapis alba.

Spatial relationships between the internal nucleolus-associated chromatin (NAC) and the numerous nucleolar vacuoles that appear during early germination have been studied in nucleoli of quiescent (non-germinated) and early germinating embryos of Sinapis using serial sections. In quiescent non-vacuolated nucleoli, the transcriptionally inactive internal NAC is a short strand about 900 nm thick that in cross-section appears as heterogeneous fibrillar centres (FCs). At 4 and 6 h after germination one or several large networks of interconnected nucleolar vacuoles develop around the dispersing internal NAC. Clumps of dense chromatin are still present within the nucleolar vacuoles and are probably unfolding into deoxyribonucleoprotein (DNP) fibres (about 110 nm thick), which rapidly intrude within the nucleolar body and form thin chromatin threads. At 24 h after germination the internal NAC is more dispersed and forms, for its greatest part, a long thread (about 240 nm in diameter) wrapped up with a few dense fibrillar component, the whole forming the first outline of a nucleolonema. In cross-section most of the internal NAC appears as homogeneous FCs but short portions remain more condensed and appear as heterogeneous FCs always associated with a nucleolar vacuole. From 48 h the internal NAC is a longer thinner strand (about 160 nm in diameter), probably continuous and surrounded entirely by a homogeneous muff of dense fibrillar component, the whole forming a typical nucleolonema (about 950 nm thick) meandering throughout the nucleolus. Small amounts of the internal NAC still remain undispersed in the form of heterogeneous FCs associated with a nucleolar vacuole. The repeated association of nucleolar vacuoles and dispersing internal NAC suggests that they could play a role in chromatin dispersion and, or, activation by creating a favourable microenvironment.