Cold-acclimated Guinea Pigs Research Articles

Thermogenic response of guinea pig adipocytes to noradrenaline and β3-AR agonists

Brown adipocytes isolated from warm acclimated guinea pig do not respond to noradrenaline (NA) in spite of their marked thermogenic response in vivo. In contrast, in cold-acclimated guinea pigs, isolated brown adipocytes show substantial increase in capacity for a thermogenic response to NA. Chronic stimulation with a β3-AR agonist increased insulin-sensitivity of brown adipose tissue (BAT) in the guinea pig. To investigate the responsiveness of glucose transport to insulin, to NA, and to β3-AR agonists, we used BAT of guinea pigs as an animal known to be insulin-resistant. Results of this study showed that no β3-adrenergic agonist used, such as CL 316,243, BRL 37344, was able to stimulate oxygen uptake in BAT cells from cold-acclimated or new born guinea pigs. However, noradrenaline (NA), adrenaline (A) and isoproterenol (ISO) had a marked thermogenic effect on these cells. In contrast, in a comparative study in warm-acclimated rat BAT cells, CL 316,243 was even more potent than NA in stimulating oxygen uptake. We concluded that guinea pigs lack β3-ARs in their BAT. These results were interesting and noteworthy, suggesting that guinea pigs can be a natural model for β3-ARs knockout.

Cold acclimation of guinea pig depressed contraction of cardiac papillary muscle

Guinea pigs were exposed to 5 degrees C for 3 wk, and the contractions of myocardial papillary muscle were compared with preparations dissected from control animals kept at approximately 25 degrees C. Developed tension of the papillary muscle per cross-sectional area was significantly (t-test, P < 0.05) decreased after cold exposure (19,200 +/- 8,160 vs. 3,020 +/- 2,890 dyne/cm2; 1 Hz). Time to peak tension was significantly faster in cold-exposed guinea pigs (126.4 +/- 11.1 ms; 1 Hz) than in controls (162.7 +/- 8. 7 ms). The magnitude of the developed tension after application of ryanodine (2 mM) to muscles from cold-exposed animals was decreased to 37.5 +/- 8.3% of control at 1 Hz, whereas in muscles from control animals, tension was decreased to 82.4 +/- 7.7%. The ryanodine-sensitive component of contraction was not significantly changed in control guinea pigs at frequencies >0.5 Hz, whereas in muscles from cold-acclimated guinea pigs, there was a "positive staircase." These results suggested that reversal of the Na+/Ca2+ exchanger is predominantly involved in the positive staircase in control guinea pigs, whereas rate-dependent increases in the Ca2+ store in the sarcoplasmic reticulum may be involved in the staircase after cold acclimation.

Apparent lack of beta 3-adrenoceptors and of insulin regulation of glucose transport in brown adipose tissue of guinea pigs

Norepinephrine-induced thermogenesis was substantial in adipocytes from brown adipose tissue (BAT) of cold-acclimated guinea pigs but absent in adipocytes from BAT of warm-acclimated guinea pigs. There was no thermogenic response to any beta 3-adrenergic agonist (CL-316,243, ZD-7114, BRL-28410, CGP-12177). The receptor was characterized as a beta 1-adrenoceptor. Adrenergic agonists stimulated adenylate cyclase in membranes from BAT of both warm- and cold-acclimated guinea pigs also via a beta 1-adrenoceptor; beta 3-adrenergic agonists had no effect. Glucose transport by brown adipocytes from warm-acclimated guinea pigs was not stimulated by either norepinephrine or insulin. Cold acclimation induced the appearance of stimulation of glucose transport by norepinephrine in association with the appearance of a large capacity for thermogenesis, but there was little improvement in response to insulin. GLUT4 was present in membranes from BAT of both warm- and cold-acclimated guinea pigs. Insulin is known to have an antilipolytic effect on both BAT and white adipose tissue of guinea pigs. Thus there is a selective lack of insulin-regulated glucose transport that is not improved by cold acclimation. Guinea pigs may have a mutated component of the translocation mechanism for GLUT4. beta 3-Adrenoceptors appear to be absent in brown adipocytes of adult guinea pigs, as in white adipocytes of guinea pigs, yet are known to be present in the gut. Tissue-specific expression of beta 3-adrenergic receptors in guinea pigs may differ from that in rats, in which receptors are expressed in the adipose tissues and gut.

Comparison of in vitro thermogenesis of brown adipose tissue in cold-acclimated rats and guinea pigs

1. 1. Effects of noradrenaline (NA) and glucagon (G) were investigated on in vitro brown adipose tissue (BAT) thermogenesis by measuring oxygen consumption with an oxygen electrode on finely minced tissue blocks incubated in Krebs-Ringer phosphate buffer at 37°C. 2. 2. The interscapular BAT weight increased in rats, while it rather decreased in guinea pigs by cold acclimation. Fat percentage in BAT decreased in the cold-acclimated rats and guinea pigs. 3. 3. Na- and G-stimulated oxygen consumptions of BAT expressed per mg tissue weight were suppressed in the cold-acclimated rats, but were enhanced in the cold-acclimated guinea pigs as compared with those in the warm control animals. Adenosine deaminase did not affect the result in the cold-acclimated rats However, the oxygen consumption per BAT pad did not differ between the cold-acclimated and warm control animals. 4. 4. These findings reveal species differences in the in vitro responsiveness of BAT of cold-acclimated animals to the thermogenic factors. It is further suggested that some compensatory mechanisms such as hyperplasia and/or increased blood flow induce an enhanced thermogenesis in BAT during cold acclimation.

Influence of cold and warm acclimation on neuronal responses in the lower brain stem.

Neurons in two lower brain stem areas, the nucleus raphe magnus and the subcoeruleus region, have been shown to be part of the thermoafferent system. It is concluded from microcut experiments in unanaesthetized guinea pigs that inhibition of shivering caused by nucleus raphe magnus stimulation is mediated partly by ascending and partly by descending efferents of the nucleus raphe magnus. Electrical stimulation of the subcoeruleus area caused excitatory metabolic responses. Interruption of the ascending efferents of the subcoeruleus area did not prevent the metabolic activation. It is concluded that the excitatory responses are partly mediated by descending efferents of the subcoeruleus area. The descending pathways project mainly to motoneurone pools and to dorsal horn cells. In cold-acclimated guinea pigs, the average maximum activity of bell-shaped subcoeruleus cold-responsive units was reduced significantly in comparison with cold-responsive neurons in animals acclimated to normal room temperature. Furthermore, peak activity of warm-responsive units in the nucleus raphe magnus was larger in cold-acclimated animals than in animals acclimated to normal room temperature. These neuronal changes may contribute via descending lower loops and via ascending upper loops to long-term slope reduction of metabolic cold defence and shivering threshold displacements.

Effects of cold and hypoxia on ventilation and oxygen consumption in awake guinea pigs

Ventilation and oxygen consumption were measured in awake, unrestrained and unintubated guinea pigs during chronic and acute exposure to cold or hypoixa. Specific V̇e and V̇ O 2 in acute and chronic exposure to cold were more than twice that of animals in normal environmental temperatures. Increased ventilation was mainly due to a 70% greater Vt in cold. Cold-acclimated guinea pigs returned acutely to normal temperatures, maintained higher V̇e and V̇ O 2 than that of control animals. Acelimation to cold did not result in respiratory advantages over that of control animals acutely exposed to cold. In Hypoxia-acclimated guinea pigs, specific V̇e was 30% higher than that of control animals due to an elevation in Vt; however, V̇ O 2 was similar in both group of animals. In contrast, acute hypoxia did not increase V̇e in control animals. This lack of ventilatory response to acute hypoxia apparently causes the marked erythropoiesis and the severe increase in hematocrit observed throughout chronic exposure to hypoxia. The high blood viscosity resulting from the increased hematocrit contributes to the right ventricular hypertrophy and cardiac failure in guinea pigs chronically exposed to hypoxia.

Microperoxisomes and mitochondria of brown adipose tissue Hydrodynamic parameters, isolation and capacity of long-chain fatty acid oxidation

1. 1. Analytical differential centrifugation of brown adipose tissue homogenates from cold-acclimated guinea pigs revealed a polydispersity of both mitochondria and peroxisomes, with at least two populations of each organelle. The estimated values were s ̄ H = 16 685 ±4220 S and s ̄ L = 4792±951 S (mitochondria), and s ̄ H = 3 364 ±1706 S and s ̄ L = 889±177 S (peroxisomes). Based on these s ̄ values, an optimal procedure is described for the isolation of subcellular fractions enriched in mitochondria and peroxisomes, respectively. 2. 2. When the mitochondrial and peroxisomal fractions were subjected to isopycnic gradient centrifugation on a self-generating gradient of poly(vinylpyrrolidone)-coated colloidal silica particles (Percoll) in 0.25 M sucrose medium, buoyant densities of about 1.11 g/cm 3 (main fraction of mitochondria) and 1.07 g/cm 3 (main fraction of peroxisomes) were obtained. A value of 1.06 g/cm 3 was found for the microsomal fraction. 3. 3. The main peroxisomal fraction, isolated by gradient centrifugation, did not reveal any significant oxidation of palmitoyl-CoA as measured by conventional polarographic technique, whereas a small rate of oxidation (about 2.7 ±0.2 nmol/ min per mg peroxisomal protein) was observed when measured as NAD + reduction. This rate contributes no more than 1% of the mitochondrial oxidation of this fatty acid and it is, therefore, concluded that peroxisomal oxidation of the predominant long-chain fatty acids found in this tissue does not make a quantitatively significant contribution to fatty acid oxidation.

Lung morphometry in guinea pigs acclimated to hypoxia during growth

The effect of chronic hypoxic exposure on lung development has been assessed in growing guinea pigs ( Cavia porcellus). Weanling males of initial W = 229 g were acclimated to a P O 2 of 80 Torr for 2–14 weeks before sacrifice (range of W = 244–965 g). Growth was the same in hypoxic animals as in controls maintained at a P O 2 of 133 Torr (range of W in controls = 89–1274 g). Lungs were fixed by tracheal installation of glutaraldehyde and examined morphometrically with the electron microscope. Within 3 weeks of exposure, lung volume ( vl) and alveolar surface area (S a) were significantly increased by 32% and 27% respectively in the hypoxia acclimated animals compared to controls of similar W. However, these differences were progressively reduced with increasing time of exposure, and mean values of vl and S a were not different between groups when W > 900 g. Chronic hypoxia accelerated lung development towards normal adult dimensions to a degree remarkably similar to that reported in cold acclimated guinea pigs. These findings are compatable with the theory of adaptive lung growth mediated by increased pulmonary blood flow, and suggest anatomical limitations to such growth related to an animal's age.

Acyl-CoA synthetase activity of brown adipose tissue mitochondria substrate specificity and its relation to the endogenous pool of long-chain fatty acids

1. 1. A new assay of acyl-CoA synthetase (acid:CoA ligase (AMP-forming), EC 6.2.1.3) activity was developed for application to measurements on isolated intact mitochondria. The assay was based on the formation of the product AMP as determined by high-performance liquid chromatography (HPLC), making corrections for the partial conversion of AMP to ADP by the adenylate kinase reaction. 2. 2. The substrate specificity of the long-chain acyl-CoA synthetase was measured in brown adipose tissue mitochondria, isolated from cold-acclimated guinea-pigs. Using mitochondria largely deficient of endogenous fatty acids, the synthetase activity ( V') revealed an optimum at 12: 0 and 13: 0 with saturated fatty acids as the substrate. In contrast to other tissues, the highest V' values were observed with unsaturated fatty acids 18: 1 (9) ( cis) and 18: 2 (9, 12) (all cis). The apparent Michaelis constant varied within a narrow range and revealed no systematic dependence on the chain-length as was the case for V', Since the mitochondria have a high capacity of fatty acid binding, with unknown affinity, the estimated K' m values are, however, of questionable significance. 3. 3. The pattern of chain-length specificity in the synthetase reaction of the mitochondria compares well with the pattern of endogenous long-chain fatty acids in which mainly four species contribute i.e. 18: 1 (42 mol%) and 18: 2 (18 mol%), 16: 0 (26 mol%) and 18: 0 (14 mol%).

Analysis of the shape changes of muscle fiber cross sections in guinea pigs raised at 22 degrees C and 5 degrees C.

Seven control and nine cold-acclimated guinea pigs were selected over a wide range of body weights (256–960 g and 239–1.074 g, respectively) from two larger groups of animals chronically exposed to 22°C or 5°C. Thin cross sections of the frozen soleus muscles were treated by the ATPase method to visualize fibers and capillaries. Photomicrographs of the sections were magnified and a total of 1,067 fiber profiles were cut out and weighed to determine individual fiber cross sectional areas (FCSA). Since most fibers are assumed to be cylinders with a circular cross section, the area of the fiber was compared to the area of a circle with the same diameter as the longest axis of the fiber. The longest axis within the fiber was measured and divided by two (LD/2). The LD/2 of each fiber was treated as the radius of a circle and the calculated area of the circle compared to the real area of the fiber. LD/2 and FCSA were positively correlated in both groups of animals by power functions FCSA=a(LD/2) b , wherea is the intercept andb is the regression coefficient. The values forb (1.58 for the 22°C and 1.59 for the 5°C animals) were both significantly less than 2 (b in the formula for the area of a circle), and the regression lines moved from a line representing a circle towards a line representing an ellipse, indicating that as the fibers grow they tend to become more and more elliptical. The adjusted mean of the 5°C regression line was significantly lower than that for the control line, indicating that the fibers of the cold-acclimated animals were more elliptical at the same FCSA than were the fibers of the control animals.

Hematology in developing guinea pigs—II chronic exposure to 5°C

1. 1. Weanling male guinea pigs were exposed for up to 18 weeks in environmental chambers to 5°C and an ambient P o 2 of 133 torr before sacrifice (range of BW at 5° C = 239−1,074 g). Growth rates of animals raised at 5°C were not significantly different from animals maintained at 22°C. Hematological measurements were made on each animal, including hematocrit, [Hb], RBC indices, WBC differentials, and the molar ratio of 2,3-diphosphoglycerate (2,3-DPG) to Hb. 2. 2. Cold exposure results in significant ( P < 0.001) increases of 6–12% in Hct and [Hb], due to greater No. RBC's, compared to controls of similar BW. Within 2 weeks of exposure to 5°C, the 2,3-DPG/Hb ratio rose transiently to 1.84 M/M, and then was constant at about 1.40 M/M in animals exposed to 5°C for longer times. When compared to values for adult controls of 0.92 M/M, such an increase in the 2,3-DPG/Hb ratio would increase the P 50 of blood from cold acclimated guinea pigs by 5–6 torr. 3. 3. The significant increases over control values in circulating [Hb] and in 2,3-DPG would substantially improve tissue O 2 delivery in cold acclimated guinea pigs, by facilitating the transport and unloading of O 2.

Effects of reserpine and propranolol on urinary excretion of histamine and 5-hydroxytryptamine in severe cold exposure in normal and cold-acclimated Guinea-pigs.

The effects of cold-acclimation, reserpine and propranolol were investigated on the survival time, rectal temperature and urinary excretion of histamine and 5-HT in guinea-pigs at -20 degrees C. Both reserpine and propranolol shortened survival time by 3 hours and 1.5 hours respectively, the shortest time being in the cold-acclimated reserpine-treated animals. There was a trend in severe cold exposure to increased excretion of histamine both in the non-acclimated and in cold-acclimated animals. Reserpine did not change the excretion but increased the concentration of histamine from 0.08 to 0.25 microgram/ml. Propranolol proved to be a histamine liberator by increasing the excretion in non-acclimated from 0.10 to 1.40 microgram/h and concentration from 0.10 to 4.52 microgram/ml and in cold-acclimated animals the excretion from 0.20 to 2.85 microgram/h and the concentration from 0.08 to 3.23 microgram/ml. Severe cold increased the excretion of 5-HT in the non-acclimated animals from 0.08 to 0.21 microgram/h and cold acclimation increased this to 0.17 microgram/h. Reserpine diminished the excretion from 0.08 to 0.03 microgram/h in the non-acclimated animals, but propranolol had no effect. The results showed that the excretion of histamine and 5-HT into urine are changed in cold and can be modified with drugs. The application of the findings in proving a cold stress deserves further study.

On the rate-limiting step in the transfer of long-chain acyl groups across the inner membrane of brown adipose tissue mitochondria

Brown adipose tissue mitochondria predominantly oxidize fatty acids in order to generate heat for non-shivering thermogenesis, and have an unusually high capacity for net transfer of long-chain fatty acyl groups from the outer to the inner (matrix) compartment. The activities of the “outer” and “inner” carnitine long-chain acyltransferases have been estimated in isolated mitochondria of cold-acclimated guinea pigs by the continuous spectrophotometric recording of the redox level of flavoproteins in the acyl-CoA dehydrogenase pathway. This redox level is determined by the intramitochondrial content of acyl-CoA under the selected experimental conditions. The apparent initial rate of the “inner” acyltransferase (palmitoyl- l-carnitine added) is three order of magnitudes higher than the “outer” acyltransferase (palmitoyl-CoA added), and this difference is not influenced by the substrate concentration, pH and reaction temperature. Thus, the “outer” acyltransferase reaction is rate limiting in the transfer of long-chain acyl groups across the inner membrane of these mitochondria and catalyzes a non-equilibrium reaction in the intact organelle. Estimates of the absolute rate of the “outer” long-chain acyltransferase indicate that it exceeds that of rat liver mitochondria by a factor of 20.

Effect of propranolol on endotoxin-induced pyrogenesis in newborn and adult guinea pigs

Previous studied have shown that nonshivering thermogenesis (NST) is the prevailing mechanism of fever production in both newborn (during the first 2-3 wk of life) and adult cold-acclimated guinea pigs. This study was undertaken to determine whether this process may be mediated by noradrenergic sympathetics. The temperatures in the interscapular brown fat pad (Tbat) and the colon (Tre), the rate of oxygen consumption (VO2), and shivering activity were measured continuously for 5 h at 27 degrees C ambient temperature in 8- and 16-day-old and adult cold-acclimated guinea pigs following 2 mug/kg iv of Salmonella enteritidis endotoxin, with and without 6.0 mg/kg of propranolol (a beta-adrenergic blocker) injected ip 2 min before the endotoxin. In the older animals, the increase in Tbat normally produced by endotoxin was reduced by propranolol administration, but shivering set in, maintaining both VO2 and Tre at their febrile levels. In the newborn animals, Tbat also was decreased by propranolol, but shivering did not set in, so that VO2 and Tre fell below their febrile values. It is concluded that endotoxin-induced NST is controlled by noradrenergic sympathetics. The failure of NST to be replaced in the present newborn guinea pigs by visible shivering might be related to other observations that the onset of shivering at this age occurs only when Tre is significantly reduced.