Hormonal control of lipolysis from the white adipose tissue of hibernating jerboa (Jaculus orientalis)

1. Plasma glucose, glycerol, free fatty acids and total lipid content of the white adipose tissue were measured in euthermic and hibernating jerboa. 2. During hibernation, plasma glucose and glycerol were low compared to the euthermic animals, whereas there was no obvious difference in plasma free fatty acids. The white adipose tissue lipid content was strongly reduced in the hibernating state. 3. The effect of lipolytic hormones (norepinephrine and glucagon) and antilipolytic hormone (insulin) on in vitro glycerol release by adipose tissue isolated from hibernating or euthermic jerboa has been studied. 4. The white adipose tissue from hibernating jerboa presented a higher sensitivity to norepinephrine and glucagon than that of euthermic jerboa; insulin did not modify either basal glycerol release or lipolysis induced by the two lipolytic hormones at low temperatures (7 degrees C) and during the rewarming (from 7 degrees C to 37 degrees C) of the tissue slices. 5. These results suggested that white adipose tissue constitutes an important source of substrates derived from lipolysis during hibernation.     

Insulin secretion in the hibernating edible dormouse (Glis glis): in vivo and in vitro studies

Plasma glucose and insulin have been studied during lethargy and spontaneous arousal of hibernating edible dormouse. During lethargy blood glucose was low while plasma insulin remained at the same level as in other seasons. Plasma glucose and insulin did not fluctuate along the phase of lethargy. During spontaneous arousal plasma insulin rose strongly from the 17 degrees C stage, reaching the higher values at 26 degrees C while blood glucose was only 85 mg/100 ml, then decreased at 37 degrees C. The effect of glucose and temperature on insulin secretion was studied using perfused pancreas preparation from hibernating edible dormice. During the rewarming of the edible dormouse pancreas the insulin release did not occur in response to the absolute extracellular glucose level but occurred in response to a B cell membrane phenomenon which was dependent on the changing rate of glucose level. The effect of glucose and temperature on insulin secretion from perfused pancreas was compared between edible dormouse and homeotherm permanent, the rat. The B cell response to glucose of the dormouse pancreas increased up to 15 degrees C whereas that of the rat only from 25 degrees C. The dormouse insulin secretion reached a peak value at the 30 degrees C of temperature, whereas that of the rat progressively increased until 37 degrees C. These results showed that some biochemical adjustment or process of acclimatization took place in the B cells of the hibernators.     

Glucagon secretion in the hibernating edible dormouse (Glis glis)

Plasma glucose and glucagon concentrations were measured in edible dormice during the bout of hibernation, arousal and active periods. During lethargy, plasma glucose and glucagon were low, compared to active values and did not fluctuate throughout the phase. During rewarming, plasma glucose regularly increased from 17 degrees to 37 degrees C while plasma glucagon rose after the 17 degrees C stage and reached the higher values at 26 degrees C, then slightly decreased at 37 degrees C. During arousal, plasma levels of free amino acids progressively increased. The effect of temperature and secretagogue (glucose and arginine) on glucagon secretion was studied using perfused pancreas from hibernating edible dormouse. In vitro rewarming of pancreas induced an increase in glucagon secretion. Glucagon secretion was regulated by glucose (inhibitory effect) and by arginine (stimulating effect) up to 25 degrees C. The effect of temperature and glucagon on oxygen uptake of hibernating edible dormouse brown fat was studied using an in vitro technique. Rewarming strongly increased oxygen consumption from 10 to 37 degrees C. Glucagon enhanced oxygen consumption up to 20 degrees C.     

Liver and brown fat mitochondrial response to cold in the garden dormouse (Eliomys quercinus)

The involvement of two organs, i.e. the liver and the brown adipose tissue (BAT) in response to cold in a hibernating species such as the garden dormouse has been studied. 2. In animals living in the cold, mitochondrial respiratory rates significantly increased (with respect to those living at 28 degrees C) in both organs with a larger increase in the BAT (+152% in the BAT and 67% in the liver). 3. The increase in BAT activity was obtained by a concomitant increase in: (a) the BAT mass (+30%), (b) the total mitochondrial mass (+20%), and (c) the mitochondrial respiratory rate (+64%). In the liver the increase was due only to an augmentation in mitochondrial mass and activity. 4. These results indicate that: (a) the BAT exerts a pre-eminent role in the physiological response to cold of garden dormouse, (b) a certain non-shivering thermogenesis (NST) is present in the liver of such species. In addition we suggest that a local thermoregulatory response would take place in a metabolically important organ such as the liver.     

Increased GDP binding and thermogenic activity in brown adipose tissue mitochondria during arousal of the hibernating garden dormouse (Eliomys quercinus L.).

1. The thermogenic activity of brown adipose tissue in hibernating garden dormice during hypothermic torpor and at different states of arousal were studied. High levels of GDP binding were observed on isolated brown fat mitochondria, indicating that the thermogenic proton conductance pathway is very active in brown fat during arousal. 2. In order to investigate this phenomenon, the uncoupling protein was assessed by immunological assay and the mRNA for UCP was analysed. 3. Animals during arousal exhibited neither increase in UCPmRNA nor an increase in UCP. 4. Our results suggest that during the rewarming of garden dormice there is an acute unmasking of GDP binding sites on the protein.     

Evaluation of the role of AMP-activated protein kinase and its downstream targets in mammalian hibernation

Mammalian hibernation requires an extensive reorganization of metabolism that typically includes a greater than 95% reduction in metabolic rate, selective inhibition of many ATP-consuming metabolic activities and a change in fuel use to a primary dependence on the oxidation of lipid reserves. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in this reorganization. AMPK activity and the phosphorylation state of multiple downstream targets were assessed in five organs of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) comparing euthermic animals with squirrels in deep torpor. AMPK activity was increased 3-fold in white adipose tissue from hibernating ground squirrels compared with euthermic controls, but activation was not seen in liver, skeletal muscle, brown adipose tissue or brain. Immunoblotting with phospho-specific antibodies revealed an increase in phosphorylation of eukaryotic elongation factor-2 at the inactivating Thr56 site in white adipose tissue, liver and brain of hibernators, but not in other tissues. Acetyl-CoA carboxylase phosphorylation at the inactivating Ser79 site was markedly increased in brown adipose tissue from hibernators, but no change was seen in white adipose tissue. No change was seen in the level of phosphorylation of the Ser565 AMPK site of hormone-sensitive lipase in adipose tissues of hibernating animals. In conclusion, AMPK does not appear to participate in the metabolic re-organization and/or the metabolic rate depression that occurs during ground squirrel hibernation.     

Activation of pyruvate dehydrogenase in adipose tissue by insulin. Evidence for an effect of insulin on pyruvate dehydrogenase phosphate phosphatase.

1. The mechanism by which insulin activates pyruvate dehydrogenase in rat epididymal adipose tissue was further investigated. 2. When crude extracts, prepared from tissue segments previously exposed to insulin (2m-i.u/ml) for 2min, were supplemented with Mg-2+, Ca-2+, glucose and hexokinase and incubated at 30 degrees C, they displayed an enhanced rate of increase in pyruvate dehydrogenase activity compared with control extracts. 3. When similar extracts were instead supplemented with fluoride, ADP, creatine phosphate and creatine kinase, the rate of decrease in pyruvate dehydrogenase activity observed during incubation at 30 degrees C was unaffected by insulin treatment. 4. It is suggested that insulin increases the fraction of pyruvate dehydrogenase present in the tissue in the active dephospho form by increasing the activity of pyruvate dehydrogenase phosphate phosphatase.     

The effect of insulin and glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP) in rat adipose tissue cultured in vitro.

1. Epididymal adipose tissue from the rat was maintained in culture for periods of up to 96h. 2. After an initial decrease in protein synthesis during the first 24h of culture, the adipose tissue recovered its capacity to synthesize and accumulate proteins of a relatively large size. 3. The activity of phosphoenolpyruvate carboxykinase decreased in a parallel manner, but increased again after 24h of incubation of the tissue in culture, to a value twice that noted in the tissue in vivo. This increase in enzyme activity was due to an increase in its rate of synthesis. 4. Both insulin and dexamethasone (9alpha-fluoro-16alpha-methyl-11beta,17,-21-trihydroxypregna-1,4-diene-3,20-dione) inhibited phosphoenolpyruvate carboxykinase synthesis, but dexamethasone also decreased total protein synthesis. 5. The half-life of phosphoenolpyruvate carboxykinase in adipose tissue cultured in vitro was 5--7h and was not altered by insulin or dexamethasone. 6. It is concluded that both insulin and glucocroticoids lower the activity of phosphoenolpyruvate carboxykinase in rat adipose tissue by decreasing its rate of synthesis.     

Effects of insulin on CO2 fixation in adipose tissue. Evidence for regulation of pyruvate transport

Insulin was found to double the rate of incorporation of H14CO3- into protein by segments of rat epididymal adipose tissue provided the incubation medium contained a suitable energy substrate such as fructose. Overall protein synthesis was increased by insulin to a lesser extent, one-third as measured by tritiated water indicating that insulin also increased CO2 fixation into amino acids. The latter could be demonstrated only when the tissue amino acid pools were expanded by the addition of aspartate to the incubation medium. The pattern of labeling observed in the amino acids indicated that CO2 fixation occurred primarily at the pyruvate carboxylase step. Addition of pyruvate to the incubation medium also increased CO2 fixation and this effect was not additive with that of insulin, suggesting that insulin acted by increasing the availability of pyruvate to the carboxylase. No change in carboxylase activity could be measured. Mitochondria isolated from tissue exposed to insulin retained a higher capacity to fix CO2 into acid-soluble products provided they were not freeze-thawed or sonicated. Uptake of pyruvate by mitochondria incubated 1 min at 2 degrees C or 5 s at 15 degrees C was doubled by prior insulin treatment of the tissue. It is concluded that insulin increases the flux through pyruvate carboxylase in adipose tissue in part by increasing the transport of pyruvate through the inner mitochondrial membrane.     

L-Leucine activates branched chain alpha-keto acid dehydrogenase in rat adipose tissue

The activity of branched chain alpha-keto acid dehydrogenase in extracts of adipose tissue was elevated after homogenization of tissue segments which had been incubated in buffer containing 0.3 mM leucine. A maximum increase (4-fold) was observed in extracts of tissues incubated in buffer containing 2.5 mM leucine, alpha-Ketoisocaproate and leucine caused maximum increases which were of similar magnitude and which required the same length of incubation of the tissue segments (5 to 15 min). The effect of leucine on branched chain alpha-keto acid dehydrogenase activity was observed both in the presence and absence of insulin, which also increased the activity of the enzyme in tissue extracts. Intact adipose tissue segments oxidized [I-14C]leucine at a maximum rate approximately 4 times that of [1-(14)C]valine. The rate of valine oxidation by intact tissue segments was doubled by addition of 0.2 to 0.5 mM unlabeled leucine, but not isoleucine, to medium containing 2 mM [1-(14)C]valine. Leucine, but not valine, also stimulated the rate of oxidation of 2 mM [U-14C]isoleucine by intact tissue segments. These results suggest that branched chain alpha-keto acid dehydrogenase activity, which is thought to limit the rate of branched chain amino acid oxidation in adipose tissue, may be sensitive to changes in the concentration of leucine in rat blood.     

Lactogenic and somatogenic binding sites in intact and detergent-solubilized membrane preparations of female rat liver.

1. Lactation results in decreased glucose and acetate utilization and increased lactate output by sheep adipose tissue. 2. The ability of insulin to stimulate acetate uptake was lost in adipose tissue from lactating sheep, whereas both the response and the sensitivity (ED50) for insulin for stimulation of glucose conversion into products other than lactate were decreased. These impairments were partly restored by prolonged incubation of adipose tissue for 48 h. 3. The ability of insulin to stimulate lactate output was not altered by lactation. 4. Dexamethasone inhibited glucose uptake, lactate output and glycerol output in adipose tissue from both non-lactating and lactating sheep, with an ED50 of about 1 nM. Dexamethasone inhibited acetate uptake by adipose tissue from non-lactating sheep, but this effect was not observed with adipose tissue from lactating sheep. 5. Dexamethasone inhibited the stimulation of glucose uptake at all concentrations of insulin used; the effect varied with insulin concentration and resulted in an accentuation of the insulin dose-response curve. The insulin dose-response curve in the presence of dexamethasone was muted during lactation. 6. The overall effect of these adaptations is to ensure that glucose and acetate utilization by adipose tissue after an insulin surge is diminished during lactation.     

Some biochemical effects of the growth hormone analogue produced by plerocercoids of the tapeworm Spirometra mansonoides on carbohydrate metabolism of adipose tissue from normal, diabetic, and hypophysectomized rats

Plerocercoids of Spirometra mansonoides produce a functional analogue of mammalian growth hormone (GH). Plerocercoid growth factor (PGF) mimics the growth-promoting actions of GH, but has not been shown to duplicate all of the actions reported for GH. The purpose of this study was to determine the effects of plerocercoid infection (chronic PGF treatment) on glucose metabolism of adipose tissue and to compare the effects to those elicited by insulin and GH in intact, diabetic, and hypophysectomized male rats. Groups of rats were constantly exposed to PGF (via plerocercoid infection) or injected twice daily with bovine GH, insulin, or saline for 10 days. Basal oxidation rates of [U-14C]glucose to 14CO2 in adipose tissue segments were measured in vitro immediately after tissue removal. Other aliquots of adipose tissue were preincubated in hormone-free medium for 3 hr prior to testing the ability of the tissue to respond to insulin or human GH (hGH) added in vitro. Adipose tissue from PGF-treated intact and hypophysectomized rats had significantly elevated basal glucose oxidation rates, and the tissue was sensitive to further stimulation by insulin or hGH. The results obtained with intact and hypophysectomized rats were essentially the same, indicating that the effects of PGF were not due to suppression of endogenous GH. The basal glucose oxidation rate in adipose tissue from diabetic rats was stimulated (P less than 0.01) by PGF, but the tissue was not sensitive to insulin added in vitro. Furthermore, PGF had no effect on body growth or blood glucose concentrations of diabetic rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

No apparent suppression by insulin of in vivo skeletal muscle lipolysis in nonobese women

To investigate the antilipolytic effect of insulin in skeletal muscle and adipose tissue in vivo, the rates of glycerol release from the two tissues were compared in 10 nonobese women during a two-step euglycemic hyperinsulinemic clamp. Tissue interstitial glycerol levels were determined by microdialysis, and tissue blood flow was assessed with the (133)Xe clearance technique. Absolute rates of glycerol release were estimated according to Fick's principle. In both adipose tissue and muscle, glycerol levels decreased significantly already during the low insulin infusion rate. The fractional release of glycerol (difference between interstitial glycerol and arterialized venous plasma glycerol) was reduced by more than one-half in adipose tissue (P < 0.0001) in response to insulin, whereas it remained unaltered in skeletal muscle. Muscle blood flow rates increased by 60% (P < 0.02) during insulin infusion; in adipose tissue, blood flow rates did not change significantly in response to insulin. The basal rate of glycerol release from skeletal muscle amounted to approximately 15% of that from adipose tissue. After insulin infusion, the rate of adipose tissue glycerol release was markedly suppressed, whereas in skeletal muscle the rate of glycerol mobilization did not change significantly in response to insulin. It is concluded that insulin does not inhibit the rate of lipolysis in skeletal muscle of nonobese women.     

Effects of insulin and norepinephrine on glucose transport and metabolism in rat brown adipocytes. Potentiation by insulin of norepinephrine-induced glucose oxidation

Glucose is an important fuel for rat brown adipose tissue in vivo and its utilization is highly sensitive to insulin. In this study, the different glucose metabolic pathways and their regulation by insulin and norepinephrine were examined in isolated rat brown adipocytes, using [6-14C]glucose as a tracer. Glucose utilization was stimulated for insulin concentrations in the range of 40-1000 microU/ml. Furthermore, the addition of adenosine deaminase (200 mU/ml) or adenosine (10 microM) did not alter insulin sensitivity of glucose metabolism. The major effect of insulin (1 mU/ml) was a respective 7-fold and 5-fold stimulation of lipogenesis and lactate synthesis, whereas glucose oxidation remained very low. The 5-fold stimulation of total glucose metabolism by 1 mU/ml of insulin was accompanied by an 8-fold increase in glucose transport. In the presence of norepinephrine (8 microM), total glucose metabolism was increased 2-fold. This was linked to a 7-fold increase of glucose oxidation, whereas lipogenesis was greatly inhibited (by 72%). In addition, norepinephrine alone did not modify glucose transport. The addition of insulin to adipocytes incubated with norepinephrine, induced a potentiation of glucose oxidation, while lipogenesis remained very low. In conclusion, in the presence of insulin and norepinephrine glucose is a oxidative substrate for brown adipose tissue. However the quantitative importance of glucose as oxidative fuel remains to be determined.     

Is oxygen supply a limiting factor for survival during rewarming from profound hypothermia?

It has been postulated that unsuccessful resuscitation of victims of accidental hypothermia is caused by insufficient tissue oxygenation. The aim of this study was to test whether inadequate O2 supply and/or malfunctioning O2 extraction occur during rewarming from deep/profound hypothermia of different duration. Three groups of rats (n = 7 each) were used: group 1 served as normothermic control for 5 h; groups 2 and 3 were core cooled to 15 degrees C, kept at 15 degrees C for 1 and 5 h, respectively, and then rewarmed. In both hypothermic groups, cardiac output (CO) decreased spontaneously by > 50% in response to cooling. O2 consumption fell to less than one-third during cooling but recovered completely in both groups during rewarming. During hypothermia, circulating blood volume in both groups was reduced to approximately one-third of baseline, indicating that some vascular beds were critically perfused during hypothermia. CO recovered completely in animals rewarmed after 1 h (group 2) but recovered to only 60% in those rewarmed after 5 h (group 3), whereas blood volume increased to approximately three-fourths of baseline in both groups. Metabolic acidosis was observed only after 5 h of hypothermia (15 degrees C). A significant increase in myocardial tissue heat shock protein 70 after rewarming in group 3, but not in group 2, indicates an association with the duration of hypothermia. Thus mechanisms facilitating O2 extraction function well during deep/profound hypothermia, and, despite low CO, O2 supply was not a limiting factor for survival in the present experiments.     

Glucose oxidation in white adipose tissue from BIO 14.6 dystrophic hamsters

In studies of glucose oxidation in white retroperitoneal adipose tissue of BIO 14.6 dystrophic and F1B normal hamsters aged 55-67 and 368-379 days, no difference was found in the basal state of radiolabelled 14CO2 production using either D-[6-14C]glucose or D-[1-14C]glucose. When C6-labelled glucose was used, insulin induced a slightly greater increase in glucose oxidation in dystrophic adipose tissue at both ages. When C1-labelled glucose was used, insulin enhanced glucose oxidation in dystrophic tissue more than twice normal in tissues from young animals and five times normal in tissues from the old ones. The increase in oxidation with D-[1-14C]glucose likely represents enhanced activity of the pentose phosphate pathway, which has also been observed in certain tissues of other animals with inherited skeletal-muscle degeneration. The change can probably be classified as being compensatory, an attempt by tissues to maintain functional integrity.