Effect of cold-acclimation on oxygen uptake and glucose production of perfused duckling liver

The control of hepatic metabolism by substrates and hormones was assessed in perfused liver from young Muscovy ducklings. Studies were performed in fed or 24-h fasted 5-week-old thermoneutral (25 degrees C; TN) or cold-acclimated ducklings (4 degrees C; CA) and results were compared with those obtained in rats. Basal oxygen uptake of perfused liver (LVO2) was higher after cold acclimation both in fed (+65%) and 24-h fasted (+29%) ducklings and in 24-h fasted rats (+34%). Lactate (2 mM), the main gluconeogenic substrate in birds, similarly increased LVO2 in both TN and CA ducklings and the effect was larger after fasting. Both glucagon and norepinephrine dose-dependently increased LVO2 in ducklings and rats, but cold acclimation did not improve liver response and liver sensitivity to norepinephrine in ducklings was even reduced in CA animals. Liver contribution to glucagon-induced thermogenesis in vivo was estimated to be 22% in TN and 12% in CA ducklings. Glucagon stimulated gluconeogenesis from lactate in duckling liver and the stimulation was 2.2-fold higher in CA than in TN fasted birds. These results indicate a stimulated hepatic oxidative metabolism in CA ducklings but hepatic glucagon-induced thermogenesis (as measured by LVO2) was not improved. A role of the liver is suggested in duckling metabolic acclimation to cold through an enhanced hepatic gluconeogenesis under glucagon control.     

Increased lipogenesis in isolated hepatocytes from cold-acclimated ducklings

Thermogenic endurance and development of metabolic cold adaptation in birds may critically depend on their ability to synthesize and use fatty acids (FA) as fuel substrates. Hepatic lipogenesis and the capacity to oxidize FA in thermogenic tissues were measured in cold-acclimated (CA) ducklings (Cairina moschata) showing original mechanisms of metabolic cold adaptation in the absence of brown adipose tissue, the specialized thermogenic tissue of rodents. The rate of FA synthesis from [U-(14)C]glucose and from [1-(14)C]acetate, measured in incubated hepatocytes isolated from 5-wk-old thermoneutral (TN; 25 degrees C) or CA (4 degrees C) fed ducklings, was higher than in other species. Hepatic de novo lipogenesis was further increased by cold acclimation with both glucose (+194%) and acetate (+111%) as precursor. Insulin slightly increased (+11-14%) hepatic lipogenesis from both precursors in CA ducklings, whereas glucagon was clearly inhibitory (-29 to -51%). Enhanced de novo lipogenesis was associated with higher (+171%) hepatocyte activity of glucose oxidation and larger capacity (+50 to +100%) of key lipogenic enzymes. The potential for FA oxidation was higher in liver (+61%) and skeletal muscle (+29 to +81%) homogenates from CA than from TN ducklings, suggesting that the higher hepatic lipogenesis may fuel oxidation in thermogenic tissues. Present data underline the high capacity to synthesize lipids from glucose in species like muscovy ducks susceptible to hepatic steatosis. Lipogenic capacity can be further increased in the cold and may represent an important step in the metabolic adaptation to cold of growing ducklings.     

Increased respiration in skeletal muscle mitochondria from cold-acclimated ducklings: uncoupling effects of free fatty acids

Intermyofibrillar mitochondria from skeletal muscle (m. gastrocnemius) and liver mitochondria were isolated from cold-acclimated (4 degrees C) or control (30 degrees C) 4-week old ducklings. The respiratory rate of isolated mitochondria, with Na-succinate as substrate, was followed polarographically at 25 degrees C in order to determine the basal respiratory rate, the rate of respiration in the presence of free fatty acids (FFA) (Na-palmitate), and the fully uncoupled rate, after addition of FCCP. The basal respiration (which in liver mitochondria was unaffected by acclimation to cold) was higher (+53%) in intermyofibrillar mitochondria from cold-acclimated ducklings than from controls, and the maximal FCCP-stimulated respiration was also increased (+98%) by acclimation to cold. FFA-stimulated respiration increased as a function of FFA concentration in both types of mitochondria. The increase in respiration due to FFA was about double in intermyofibrillar mitochondria from cold-acclimated ducklings than that of controls, but in liver mitochondria there was no increase due to cold. The membrane potential was estimated by the dye safranine in the absence or in the presence of FFA in the incubation medium. There were no significant differences in the basal membrane potential in the two groups and the addition of FFA led to the same depolarization in both groups. The significance of these alterations for acclimation to cold is discussed.     

Increased lipoprotein lipase activity of skeletal muscle in cold-acclimated rats

The activity of lipoprotein lipase (LPL) in the heart, diaphragm, and soleus muscles was markedly increased in cold-acclimated rats and it was even greater in rats treated with oxytetracycline (OTC) while exposed to cold. Other skeletal muscles studied had low and variable activities which were not significantly increased by cold acclimation or by cold plus OTC treatment. It appears therefore that, apart from the heart and the muscles involved in respiratory movements, LPL activity is primarily associated with those muscles which contain a predominance of slow-twitch oxidative fibers, and that the enzyme in muscle, heart, and diaphragm responds to cold acclimation and cold plus OTC treatment in a parallel fashion in these tissues.     

The effects of reduced temperature and photoperiod on body composition in hibernator and non-hibernator rodents

We examined the differential change in body composition in response to a gradual reduction in both environmental temperature and photoperiod to mimic seasonal fluctuations in the wild (summer–winter transition), from ambient to 5°C and 1:23 light:dark for 8 weeks. In contrast to acute cold exposure used in previous studies, cold-acclimated rats showed an initial increase in growth rate relative to normothermic controls, possibly due to cold-stimulated hyperphagia. In hamsters, maintenance of growth rate during initial cold exposure reflects the intrinsic high oxidative capacity, while subsequent cessation of growth is consistent with the preparation for hibernation. Cold-induced atrophy of skeletal muscles coincided with increased capacity for non-shivering thermogenesis (NST) associated with a greater mass of brown adipose tissue (BAT). Cardiac hypertrophy may compensate for an increase in total peripheral resistance and/or work load of heart in both species (40% and 20%, respectively), while hypertrophy of lung (20% and 40%) and diaphragm muscle (7% and 40%) was consistent with increased ventilation associated with a cold-induced increase in basal metabolic rate. Gonadal atrophy in hamsters (160%) may be an energy saving strategy during the non-reproductive season, while maintenance of other endocrine (thyroid, adrenal, pineal) gland masses reflects the continued importance of hormonal regulation of homeostasis. The interspecific differences appear to accommodate the increased demands of shivering thermogenesis (skeletal muscle hypertrophy) or NST (BAT, diaphragm) in rats and hamsters, respectively. Those systems representing cardiovascular and metabolic control completed their adaptation quickly (within 4-week cold acclimation), while the respiratory and reproductive systems continued to respond to a further 4-week exposure. This differential time course may reflect the relative strength of selection pressure on these systems for the process of cold acclimation.     

Effect of cold acclimatization of grass frogs on the heat resistance of their somatic muscles in summer]

During cold acclimation (+5 degrees C) of the grass frog Rana temporaria L. changes in heat resistance were observed for six of seven skeletal muscles examined. Changes in the resistance of different muscles were directed differently. Low-resistant muscles demonstrated an increased resistance, wheras high-resistant muscles showed a decrease resistance. Within 24 hours of acclimation, all the muscles demonstrated totally decreased heat resistance. By the end of acclimation (30 days), the resistance of muscles increased to approach the initial level. On the background of those situation changes, seasonal changes in resistance of muscles proceeded.     

Triglyceride Uptake in Muscles in Rats

Exogenous lipid is assimilated with different priorities in adipose tissue regions and varies in the fasting and fed conditions. The quantitative role of uptake of lipid in muscle has not been evaluated. In order to examine the uptake in other than adipose tissues, U¹⁴C-oleic acid in sesame oil was administered orally to conscious rats, and lipid label measured after different times in serum, heart, liver, mesenteric, retroperitoneal, inguinal and epididymal fat pads, as well as in red and white parts of gastrocnemius, extensor digitorum longus and soleus muscles. Lipid uptake in total adipose tissue was calculated from dissected adipose tissues plus lipids extracted from the eviscerated, skinned carcass. Lipid uptake in total muscle tissue was estimated from label in dissected muscles plus that in the carcass, assuming similar intracellular lipid contents and radioactivity as that averaged from dissected muscles. Lipid uptake in the liver was calculated from directly extracted lipid. Four hours after lipid administration to fed rats lipid radioactivity in heart and serum was minimal and had essentially disappeared at 8 hours. Liver label declined rapidly from peak values at or before 4 hours. Adipose tissue radioactivity increased gradually up to 16 hours and then decreased. Label in muscles was highest at 4 hours in the red gastrocnemius, and then decreased, while the other muscles showed a constant radioactivity over the observation period (24 hours). Radioactivity expressed per unit muscle mass seemed to be proportional to the oxidative capacity of muscles. In comparisons between fed and fasted rats at 16 hours, when adipose tissue label peaked, liver, individual muscles and carcass did not show any significant differences while adipose tissue label was fivefold higher in fed than fasted rats. The distribution of total measured lipid radioactivity between total adipose tissue, total muscle tissue and liver in fed rats at this time-point was 76. 8, 14. 4 and 8. 8% respectively, and in the fasted state 26. 4, 51. 6 and 22. 0%. These estimations suggest that lipid uptake in the fed state is dominated by adipose tissue, while in the fasted state the lipid uptake is higher in muscles than adipose tissues. It was concluded that uptake of absorbed, exogenous triglyceride in muscle is of significance, particularly in the fasted state. This lipid has a half life of several days. It is suggested that this lipid is oxidized in situ, contributing with a hidden fraction to lipid energy needs, or partially transferred to adipose tissue. Lipid uptake in muscle probably constitutes a significant fraction of assimilated exogenous lipid, particularly in the fasting state.     

Effects of chronic cold exposure on the activities of cytochrome c oxidase, glutathione peroxidase and glutathione reductase in rat tissues (Rattus norvegicus)

The effects of cold acclimation on the activity levels of cytochrome c oxidase, glutathione peroxidase and glutathione reductase in various tissues of the rat (Rattus norvegicus) were investigated. One group was individually housed at 4 +/- 1 degrees C and the other at 24 +/- 1 degrees C for 6 months. Chronic cold acclimation resulted in significantly (P < 0.05) increased cytochrome c oxidase activity levels in liver, kidney, heart, interscapular brown adipose tissue and gastrocnemius muscle. The activity of glutathione peroxidase was significantly (P < 0.05) elevated in liver, interscapular brown adipose tissue, lung and muscle, whereas glutathione reductase was only significantly (P < 0.05) elevated in interscapular brown adipose tissue as a result of chronic cold exposure. The results obtained are possibly indicative of a positive compensatory response against the increased production of oxygen derived radicals as a result of chronic cold exposure.     

Anatomic capillarization is maintained in relative excess of fiber oxidative capacity in some skeletal muscles of late middle-aged rats.

The anatomic size of the capillary-to-fiber (C/F) interface plays an important role in O(2) flux from blood to tissue by determining the surface area available for diffusion and is maintained in relative proportion to fiber mitochondrial volume across a wide range of muscle aerobic capacity. In the present study, we examined an estimate of the anatomic size of the C/F interface [the quotient of the individual C/F ratio and fiber perimeter, C/F perimeter exchange (CFPE) index] and fiber oxidative capacity in different skeletal muscles, or muscle regions, to test the hypothesis that capillarization would be maintained in relative excess of reduced fiber oxidative capacity in aged muscles. The right gastrocnemius, plantaris, and soleus muscles from young adult (8 mo old) and late middle-aged (28-30 mo old) Fischer 344 x Brown Norway F1 hybrid rats were excised for evaluation of flux through electron transport chain complexes I-III and/or morphometric estimation of capillarization. Muscle mass was lower in the gastrocnemius muscles of the older animals (2,076 +/- 32 vs. 1,825 +/- 47 mg in young adult vs. late middle-aged, respectively; mean +/- SE) but not the plantaris or soleus muscles. Fibers were smaller in the white region of gastrocnemius muscles but larger in the red region of gastrocnemius muscles of the older animals. There was no difference in the number of capillaries around a fiber, the individual C/F ratio, or the CFPE index between groups for any muscle/region, whereas flux through complexes I-III was reduced by 29-43% in late middle-aged animals. Thus the greater quotient of indexes of anatomic capillarity (individual C/F ratio or CFPE index) and fiber oxidative capacity in soleus and the white region of gastrocnemius muscles, but not in the red region of gastrocnemius muscles of the older animals, shows that anatomic capillarity is maintained in relative excess of oxidative capacity in some muscle regions in late middle-aged rats.     

Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training

The purposes of this study were to determine whether exercise training induces increases in skeletal muscle antioxidant enzymes and to further characterize the relationship between oxidative capacity and antioxidant enzyme levels in skeletal muscle. Male Sprague-Dawley rats were exercise trained (ET) on a treadmill 2 h/day at 32 m/min (8% incline) 5 days/wk or were cage confined (sedentary control, S) for 12 wk. In both S and ET rats, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were directly correlated with the percentages of oxidative fibers in the six skeletal muscle samples studied. Muscles of ET rats had increased oxidative capacity and increased GPX activity compared with the same muscles of S rats. However, SOD activities were not different between ET and S rats, but CAT activities were lower in skeletal muscles of ET rats than in S rats. Exposure to 60 min of ischemia and 60 min of reperfusion (I/R) resulted in decreased GPX and increased CAT activities but had little or no effect on SOD activities in muscles from both S and ET rats. The I/R-induced increase in CAT activity was greater in muscles of ET than in muscles of S rats. Xanthine oxidase (XO), xanthine dehydrogenase (XD), and XO + XD activities after I/R were not related to muscle oxidative capacity and were similar in muscles of ET and S rats. It is concluded that although antioxidant enzyme activities are related to skeletal muscle oxidative capacity, the effects of exercise training on antioxidant enzymes in skeletal muscle cannot be predicted by measured changes in oxidative capacity.     

Changes in myosin ATPase activity in skeletal muscles of rat during cold stress

Three skeletal muscles viz., gastrocnemius, pectoralis and diaphragm from rats acclimated to a low temperature (4 +/- 1 degrees C; 16 hr daily; maximum for 8 weeks) exhibit an increased myosin ATPase activity. An analysis of native myosin from these muscles under non-dissociating conditions reveals two myosin isozymes instead of a single isozyme expressed in control muscles. Isoelectric focusing (IEF) coupled with two dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) confirms an increased phosphorylation of myosin light chain 2 (MLC2) in muscles from cold acclimated rats.     

The consequences of acute cold exposure on protein oxidation and proteasome activity in short-tailed field voles,microtus agrestis

During cold exposure, animals upregulate their metabolism and food intake, potentially exposing them to elevated reactive oxygen species (ROS) production and oxidative damage. We investigated whether acute cold (7 +/- 3 degrees C) exposure (1, 10, or 100 h duration) affected protein oxidation and proteasome activity, when compared to warm controls (22 +/- 3 degrees C), in a small mammal model, the short-tailed field vole Microtus agrestis. Protein carbonyls and the chymotrypsin-like proteasome activity were measured in plasma, heart, liver, kidney, small intestine (duodenum), skeletal muscle (gastrocnemius), and brown adipose tissue (BAT). Trypsin-like and peptidyl-glutamyl-like proteasome activities were determined in BAT, liver, and skeletal muscle. Resting metabolic rate increased significantly with duration of cold exposure. In skeletal muscle (SM) and liver, protein carbonyl levels also increased with duration of cold exposure, but this pattern was not repeated in BAT where protein carbonyls were not significantly elevated. Chymotrpsin-like proteasome activity did not differ significantly in any tissue. However, trypsin-like activity in SM and peptidyl-glutamyl-like activity in both skeletal muscle and liver, were reduced during the early phase of cold exposure (1-10 h), correlated with the increased carbonyl levels in these tissues. In contrast there was no reduction in proteasome activity in BAT during the early phase of cold exposure and peptidyl-glutamyl-like activity was significantly increased, correlated with the lack of accumulation of protein carbonyls in this tissue. The upregulation of proteasome activity in BAT may protect this tissue from accumulated oxidative damage to proteins. This protection may be a very important factor in sustaining uncoupled respiration, which underpins nonshivering thermogenesis at cold temperatures.     

Cold-induced mitochondrial uncoupling and expression of chicken UCP and ANT mRNA in chicken skeletal muscle

Although bird species studied thus far have no distinct brown adipose tissue (BAT) or a related thermogenic tissue, there is now strong evidence that non-shivering mechanisms in birds may play an important role during cold exposure. Recently, increased expression of the duckling homolog of the avian uncoupling protein (avUCP) was demonstrated in cold-acclimated ducklings [Raimbault et al., Biochem. J. 353 (2001) 441-444]. Among the mitochondrial anion carriers, roles for the ATP/ADP antiporter (ANT) as well as UCP variants in thermogenesis are proposed. The present experiments were conducted (i) to examine the effects of cold acclimation on the fatty acid-induced uncoupling of oxidative phosphorylation in skeletal muscle mitochondria and (ii) to clone the cDNA of UCP and ANT homologs from chicken skeletal muscle and study differences compared to controls in expression levels of their mRNAs in the skeletal muscle of cold-acclimated chickens. The results obtained here show that suppression of palmitate-induced uncoupling by carboxyatractylate was greater in the subsarcolemmal skeletal muscle mitochondria from cold-acclimated chickens than that for control birds. An increase in mRNA levels of avANT and, to lesser degree, of avUCP in the skeletal muscle of cold-acclimated chickens was also found. Taken together, the present studies on cold-acclimated chickens suggest that the simultaneous increments in levels of avANT and avUCP mRNA expression may be involved in the regulation of thermogenesis in skeletal muscle.     

冷暴露对中缅树鼩适应性产热特征的影响

在冷暴露(5 ± 1℃)1 d,7 d,14 d 和21d 对中缅树鼩的肝脏、心脏、膈肌、腓肠肌和褐色脂肪组织(BAT)的总蛋白和线粒体蛋白含量、线粒体的状态Ⅳ呼吸能力、细胞色素C 氧化酶活力及血清中甲状腺激素的水平等指标进行了测定。结果表明:冷暴露过程中肝脏线粒体蛋白含量对低温的反应比总蛋白的反应剧烈,心脏和BAT 组织的线粒体蛋白含量也随着冷驯化时间的延长而显著增加,但腓肠肌的反应较为温和;各组织线粒体的状态Ⅳ呼吸能力均显著增强,除腓肠肌外其它各组织细胞色素C 氧化酶活性也随冷暴露时间的延长而急剧增加。说明在低温条件下,肝脏、腓肠肌、心脏和BAT 等组织都参与了体温调节的过程,在自然生境中,低温是重要的刺激产热的调节因子。     

The significance of lipoprotein lipase in rat skeletal muscles.

Lipoprotein lipase was assayed in extracts of acetone-ether powders of rat skeletal muscles. Enzyme activity in soleus had typical characteristics of lipoprotein lipase in other tissues: inhibition by molar NaCl and protamine sulfate and activation by the human apolipoprotein, R-glutamic acid. Activity in muscles with predominantly red fibers (soleus, diaphragm, lateral head of gastrocnemius and anterior band of semitendinosus) was higher than in those with predominantly white fibers (body of gastrocnemius and posterior band of semitendinosus). No effect of a 24 hour fast upon enzyme activity was observed in ten skeletal muscles, but activity decreased substantially in four adipose tissue depots and increased slightly in heart muscle with fasting. Four minutes after intravenous injection of labeled lymph chylomicrons, skeletal muscles with predominantly red fibers incorporated several times more chylomicron triglyceride fatty acids than thos with predominantly white fibers. Estimated lipoprotein lipase activity in total skeletal muscle was about two-thirds that in total adipose tissue of rats fed ad libitum. After a 24 hour fast, total activity in skeletal muscle was about twice that in adipose tissue. These data suggest that a substantial fraction of lipoprotein lipase is in skeletal muscle of rats and that this tissue, especially its red fibers, is an important site of removal of triglycerides from the blood.     

Thermal acclimation,mitochondrial capacities and organ metabolic profiles in a reptile (<Emphasis Type="Italic">Alligator mississippiensis</Emphasis>)

Reptiles thermoregulate behaviourally, but change their preferred temperature and the optimal temperature for performance seasonally. We evaluated whether the digestive and locomotor systems of the alligator show parallel metabolic adjustments during thermal acclimation. To this end, we allowed juvenile alligators to grow under thermal conditions typical of winter and summer, providing them with seasonally appropriate basking opportunities. Although mean body temperatures of alligators in these groups differed by approximately 10°C, their growth and final anatomic status was equivalent. While hepatic mitochondria isolated from cold-acclimated alligators had higher oxidative capacities at 30°C than those from warm-acclimated alligators, the capacities did not differ at 20°C. Cold acclimation decreased maximal oxidative capacities of muscle mitochondria. For mitochondria from both organs and acclimation groups, palmitate increased oligomycin-inhibited respiration. GDP addition reduced palmitate-uncoupled rates more in liver mitochondria from warm- than cold-acclimated alligators. In muscle mitochondria, carboxyatractyloside significantly reduced palmitate-uncoupled rates. This effect was not changed by thermal acclimation. The aerobic capacity of liver, skeletal muscle and duodenum, as estimated by activities of cytochrome c oxidase (COX), increased with cold acclimation. At acclimation temperatures, the activities of COX and citrate synthase (CS) in these organs were equivalent. By measuring COX and CS in isolated mitochondria and tissue extracts, we estimated that cold acclimation did not change the mitochondrial content in liver, but increased that of muscle. The thermal compensation of growth rates and of the aerobic capacity of the locomotor and digestive systems suggests that alligators optimised metabolic processes for the seasonally altered, preferred body temperature. The precision of this compensatory response exceeds that typically shown by aquatic ectotherms whose body temperatures are at the mercy of their habitat.     

Skeletal and cardiac muscle protein turnover during cold acclimation in young rats

The effect of long-term cold exposure on skeletal and cardiac muscle protein turnover was investigated in young growing animals. Two groups of 36 male 28-day-old rats were maintained at either 5 degrees C (cold) or 25 degrees C (control). Rates of protein synthesis and degradation were measured in vivo on days 5, 10, 15, and 20. Protein mass by day 20 was approximately 28% lower in skeletal muscle (gastrocnemius and soleus) and approximately 24% higher in heart in cold compared with control rats (P < 0.05). In skeletal muscle, the fractional rates of protein synthesis (k(syn)) and degradation (k(deg)) were not significantly different between cold and control rats, although k(syn) was lower (approximately -26%) in cold rats on day 5; consequent to the lower protein mass, the absolute rates of protein synthesis (approximately -21%; P < 0. 05) and degradation (approximately -13%; P < 0.1) were lower in cold compared with control rats. In heart, overall, k(syn) (approximately +12%; P < 0.1) and k(deg) (approximately +22%; P < 0.05) were higher in cold compared with control rats; consequently, the absolute rates of synthesis (approximately +44%) and degradation (approximately +54%) were higher in cold compared with control rats (P < 0.05). Plasma triiodothyronine concentration was higher (P < 0.05) in cold compared with control rats. These data indicate that long-term cold acclimation in skeletal muscle is associated with the establishment of a new homeostasis in protein turnover with decreased protein mass and normal fractional rates of protein turnover. In heart, unlike skeletal muscle, rates of protein turnover did not appear to immediately return to normal as increased rates of protein turnover were observed beyond day 5. These data also indicate that increased rates of protein turnover in skeletal muscle are unlikely to contribute to increased metabolic heat production during cold acclimation.