Hormonal counterregulation failure in rats is related to previous hyperglycaemia-hyperinsulinaemia.

Hyperglycaemia and hyperinsulinaemia were induced in rats by a continuous 48-h infusion with glucose. Discontinuation of glucose infusion resulted in marked, persistent hypoglycaemia. To further delineate the mechanism underlying this condition, we measured counterregulatory hormone levels, in vivo glucose kinetics (glucose production = rate of appearance = Ra; glucose utilization = rate of disappearance = Rd), and in vitro gluconeogenesis during the 48-h postinfusion period. Prior to cessation of glucose infusion, Rd was increased 6-fold when compared to control rats, whereas Ra was totally abolished. During the first hour after the end of glucose infusion, Ra increased and Rd decreased (but was still higher than Ra), inducing hypoglycaemia which stabilized after 1 h at ¿¿126¿¿3.5 mmol/l when both Ra and Rd became equal. Despite hypoglycaemia, plasma glucagon and catecholamine levels did not increase during the 3-to 36-h time interval. The increase in Ra during the first hour post-infusion was not related to changes in counterregulatory hormone response. The increase in glucose production was accounted for by glycogenolysis, as shown by total depletion in liver glycogen within 6 h and thereafter by gluconeogenesis. In vitro experiments using isolated hepatocytes suggested that gluconeogenesis was supported during the first 24 h by substrates entering the pathway beyond the step catalysed by the PEPCK enzyme. Thereafter, lactate became the major substrate, and this condition was associated with a progressive rise in glucagon concentration. It is concluded that 48 h of hyperglycaemia/hyperinsulinaemia resulted in a failure of counterregulatory hormonal response to hypoglycaemia. Yet, despite this lack of counterregulatory response, hepatic gluconeogenesis was stimulated in response to hypoglycaemia.

Increased pancreatic islet blood flow in 48-hour glucose-infused rats: involvement of central and autonomic nervous systems.

The pancreatic islet blood flow of rats 24 h after a prolonged (48-h) glucose infusion was investigated using a nonradioactive microsphere technique. In the basal state, islet blood flow was significantly increased in previously hyperglycemic rats (HG) compared to that in controls (C). During an i.v. glucose challenge, both plasma insulin and islet blood flow were increased in the two groups, but these increases were significantly higher in HG than in C rats. Although less pronounced, the results were similar when glucose was injected into the carotid artery toward the brain at a dose that did not modify the peripheral glucose level. The effect of this intracarotid injection was abolished after bilateral subdiaphragmatic vagotomy in both C and HG rats. Furthermore, in the latter group, both plasma insulin concentration and islet blood flow returned to values similar to those observed in the basal state in C rats. After pretreatment with the alpha2-adrenoceptor agonist clonidine, the insulin response to the intracarotid glucose load was totally blunted in the two groups of rats. By contrast, whereas such a pretreatment lowered the glucose-induced increase in islet blood flow in C rats, it was without effect in HG rats. These data suggest that a period of hyperglycemia and/or hyperinsulinemia is sufficient to induce a perturbation of pancreatic islet blood flow, which appears to be mainly due to an increased parasympathetic activity, whereas the decrease in sympathetic tone does not play a role. These modifications in autonomic nervous system activity could be due to alterations in some brain areas involved in "glucose sensing."

In vivo effects of glucose and insulin on secretion and gene expression of glucagon in rats.

We investigated the effects of insulin and glucose on the control of secretion and gene expression of glucagon in vivo in rats. Animals were studied during 1) a 48-h period of either glucose infusion (hyperglycemia plus hyperinsulinemia; HG-HI rats) or insulin infusion (euglycemia plus hyperinsulinemia; EG-HI rats), and 2) a prolonged postinfusion period in both groups. In HG-HI rats, elevation of plasma insulin and glucose concentrations by about 7 and 5 times, respectively, resulted in a decline in glucagon levels, which fell significantly within 6 h and remained low thereafter, whereas these levels were unchanged in EG-HI rats. Glucagon messenger RNA levels and pancreatic glucagon content were not significantly affected in either HG-HI or EG-HI rats. After cessation of infusions, hypoglycemia occurred in both group of rats. In HG-HI rats, hypoglycemia lasted for about 36 h without any surge in the plasma glucagon level, whereas in EG-HI rats it was transient (approximately 1 h) and stimulated glucagon secretion. In both groups the pancreatic alpha-cell was unresponsive to arginine during the postinfusion period. In conclusion, although a role of intraislet insulin cannot be excluded, glucagon gene expression is insensitive to changes in plasma glucose and insulin concentrations. In contrast, hyperglycemia/hyperinsulinemia, not hyperinsulinemia alone, lowers glucagon secretion and affects the alpha-cell responsiveness to hypoglycemia.

Exercise-training reduces BAT thermogenesis in rats.

In the energy balance equation, physical activity represents one component of energy expenditure. From various studies it appears that exercise-training does not affect clearly thermogenesis which depends on brown adipose tissue (BAT) activity. In the present work we examine how exercise-training can influence food intake and body weight regulation in relation to BAT thermogenesis. The proton conductance of the uncoupling protein of BAT was examined in male adult Wistar trained 2 h/day for 20 days and compared to that of sedentary (2 h of fasting instead of exercise) or control animals. All animals were provided with separate sources of the 3 macronutrients (protein, fat and carbohydrate) containing an identical percentage of vitamins, salt mixture and cellulose powder. At the end of training, rats were placed at 5 degrees C during 10 days, then during 4 days at 28 degrees C. This condition has been demonstrated to favour and amplify BAT responsiveness to moderate modifications of stimulation. The body weight of trained rats became significantly lower than that of the control and sedentary rats and this difference persisted all throughout the experiment. When placed at 5 degrees C, all rats increased their total ingestion: control rats enhanced fat intake, while sedentary and trained rats enhanced carbohydrate ingestion. When placed at 28 degrees C, all rats had identical total energy and that of the 3 items intakes. BAT proton conductance was about 40% lower in the trained compared with the sedentary plus the control rats. This indicated a lower BAT thermogenic activity in the trained animals. It could be concluded that exercise-training in rats induces negative energy balance; the reduced BAT activity could restrain weight loss and overeating.

Involvement of the autonomic nervous system in the in vivo memory to glucose of pancreatic beta cell in rats.

The fact that the potentiating effect of prolonged hyperglycemia on the subsequent insulin secretion is observed in vivo but not in vitro suggests the involvement of extrapancreatic factors in the in vivo memory of pancreatic beta cells to glucose. We have investigated the possible role of the autonomic nervous system. Rats were made hyperglycemic by a 48-h infusion with glucose (HG rats). At the end of glucose infusion as well as 6 h postinfusion, both parasympathetic and sympathetic nerve activities were profoundly altered: parasympathetic and sympathetic activities, assessed by the firing rate either of the thoracic vagus nerve or the superior cervical ganglion, were dramatically increased and decreased, respectively. Moreover, 6 h after the end of glucose infusion, insulin secretion in response to a glucose load was dramatically increased in HG rats compared to controls. To determine whether these changes could be responsible for the increased sensitivity of the beta cell to glucose, insulin release in response to glucose was measured in HG and control rats, either under subdiaphragmatic vagotomy or after administration of the alpha 2A-adrenergic agonist oxymetazoline. Both treatments partially abolished the hyperresponsiveness of the beta cell to glucose in HG rats. Therefore chronic hyperglycemia brings about changes in the activity of the autonomic nervous system, which in turn are responsible, at least in part, for the generation of enhanced beta cell responsiveness to glucose in vivo.

Energy balance in an inbred strain of rats: comparison with the Wistar strain.

Food intake and body weight gain were examined in two groups of male rats (7 weeks): an inbred strain, Dark Agouti (DA, n = 12) and a noninbred strain, Wistar (n = 13). The animals were allowed to select their diet from separate sources of the three macronutrients protein, fat, and carbohydrate. After 10 days of adaptation to the diets, body weights and food intakes were measured for 3 weeks. During this period, meal patterns were recorded for at least 5 days in each rat. Then, rats were switched to a chow diet (UAR, A.O4) for 10 days. The total caloric intake of DA rats was 60% that of Wistar rats, while their body weight gain was 25% that of Wistar rats (1.3 g/day in DA vs. 5.3 g in Wistar). However, when energy intake was related to total body weight, there was no difference in energy ingestion. It was observed that DA rats ingested mainly proteins (45%) and fats (41%), while Wistar rats ingested an identical proportion of proteins and carbohydrates (40%). The percent of total white adipose tissue to total body weight was identical in both strains (6% on average). Brown adipose tissue thermogenic activity of DA rats was threefold higher than in Wistar rats. This could be one of the elements responsible for the lower body weight gain of this group of rats. Self-selected food intake of the inbred DA strain of rats, in contrast to what was expected, was greatly variable.

In vivo beta-adrenergic induction of the unmasking of the uncoupling protein in rat brown fat.

1. In 28 degrees C adapted rats (WA) both cold stress and norepinephrine (NE) led to a 4-fold increase of uncoupling protein dependent proton conductance which was abolished by propranolol (PRO). 2. In 4-day warm re-exposed rats (after 10 days at 5 degrees C) (WR) the same uncoupling by cold stress was observed but the NE effect was lower. Uncoupling by cold stress was not abolished by PRO. 3. In WR rats, uncoupling was not due to the involvement of an alpha-adrenergic pathway. 4. Both beta-agonist isoproterenol and beta 3-agonists BRL 35135A and ICI D7114 led to high levels of unmasking. 5. Interscapular brown adipose tissue surgical denervation, which abolished cold stress unmasking both in WA and, WR rats, indicates a mediation by direct sympathetic innervation. 6. Depending on the thermal history of the rat, the possibility that unmasking by cold stress could be mediated by different types of beta-receptors is discussed.

In vivo and in vitro increased pancreatic beta-cell sensitivity to glucose in normal rats submitted to a 48-h hyperglycaemic period.

We investigated the importance of the level and the duration of glucose stimulation on the in vivo and in vitro insulin response to glucose in normal rats previously submitted to hyperglycaemia. Rats were made hyperglycaemic by a 48-h glucose infusion. Glucose-induced insulin secretion was investigated in vivo by a 20-min hyperglycaemic clamp and in vitro by the isolated perfused pancreas technique, 3 h after the end of the in vivo glucose infusion. In glucose-infused rats, as compared to controls, in vivo incremental plasma insulin values above baseline integrated over the 20-min hyperglycaemic clamp (delta I) were five times higher during 8 mmol/l glucose clamp, only two times higher in 11 mmol/l glucose clamp and no different in 16.5 mmol/l. Compared to the controls, in vitro incremental plasma insulin concentration above baseline integrated over a 20-min period (delta I) in glucose-infused rats was 16 times higher in response to 2.8 mmol/l glucose, two times higher in response to 5.5 mmol/l, similar in response to 8.3 mmol/l and significantly lower in response to 16.5 mmol/l. In conclusion, our data suggest that a 48-h hyperglycaemic period results in an increased response of the pancreatic beta cell to low glucose. The response is immediately maximal and can not be increased with higher glucose concentrations. This situation could explain the apparent minimal effect of high concentrations on in vitro insulin secretion in previously hyperglycaemic rats and may provide insights into the sequence of events leading to the impairment of beta-cell function in Type 2 (non-insulin-dependent) diabetes mellitus.

Effect of sympathetic de-activation on thermogenic function and membrane lipid composition in mitochondria of brown adipose tissue.

Male Long-Evans rats (9 weeks of age) were exposed to cold (5 degrees C) for 10 days. Then, sympathetic de-activation of brown adipose tissue (BAT) was performed either by BAT surgical denervation (Sy) or by warm re-exposure at 28 degrees C (WE) for 4 days. The incidence of the two treatments on thermogenic activity of BAT mitochondrial membranes and their lipid composition was investigated. Sy and WE induced a large decrease in GDP binding on the uncoupling protein (UCP) (43% and 82%, respectively). Several parameters of mitochondrial energization were investigated. Sy and WE substantially decreased UCP-dependent proton conductance (CmH+) over the whole range of protonmotive force. CmH+ showed greater variation than GDP binding. The low basal UCP-independent CmH+ was the same in all groups. Comparison of GDP binding and CmH+ with UCP content which is not modified revealed a masking of both the nucleotide binding site and the proton channel. Sy and WE induced the same increase of phosphatidylcholine to phosphatidylethanolamine ratio (16%) but had opposite effects on fatty acid unsaturation. The results were discussed with reference to functional significance of these variations in BAT mitochondrial thermogenic activity and lipid composition.

Effect of prolonged in vivo glucose infusion on insulin secretion in rats with previous glucose intolerance.

This work was designed to investigate the effect of an additional hyperglycemia on the subsequent in vivo insulin secretion in rats with various degrees of glucose intolerance. Four groups of rats received a unique injection of a low concentration of streptozotocin (STZ): 20, 27, 30, or 35 mg/kg corresponding, respectively, to STZ 20, STZ 27, STZ 30, or STZ 35 rats. Control rats were injected with citrate buffer. In all STZ groups, impaired glucose tolerance and insulin secretion were observed. These defects were roughly proportional to STZ concentration. Three weeks after STZ administration, hyperglycemia (17 mM) was produced by a 48-h glucose infusion via an indwelling catheter. Insulin secretion in response to glucose was investigated 3 h after stopping glucose infusion, by performing iv glucose tolerance tests. Insulin secretion in response to glucose doubled in control rats previously submitted to glucose infusion, and still more in rats with mild glucose intolerance (three and four times higher in STZ 20 and STZ 27 rats, respectively). By contrast, glucose infusion increased insulin secretion only slightly in STZ 30 rats and it was unchanged in STZ 35 rats. These data show that prolonged hyperglycemia has an improving effect on insulin secretion in rats with mild glucose intolerance, whereas the potentiating effect of previous hyperglycemia is lost in rats with more severe glucose intolerance.

Are adrenal catecholamines involved in the enhancement of aldosterone production in cold acclimated rats?

In order to determine the possible role of adrenal catecholamines in the enhancement of aldosterone production in cold acclimated rats, dopamine (DA), noradrenaline (NA) and adrenaline (A) contents of adrenals were investigated in cold acclimated (or not), hypophysectomized (or not) rats. The DA content, DA being an inhibitor of aldosterone synthesis, increased in hypophysectomized rats and remained unchanged in sham-operated ones following cold exposure. The NA and A contents, these being, in vitro, stimulators of aldosterone production did not increase, in either group of cold acclimated rats. It is concluded that adrenal catecholamines do not seem to be implicated in the enhancement of aldosterone production.

Differential effects of prolonged hyperglycemia on in vivo and in vitro insulin secretion in rats.

The purpose of this study was to investigate the effects of a 48-h glucose (30% wt/vol) infusion in unrestrained catheterized healthy rats (HG) on subsequent in vivo and in vitro insulin response to glucose. High hyperglycemia (400-450 mg/dl) and resulting hyperinsulinemia (1.2 +/- 0.1 mU/ml vs. 0.15 +/- 0.03 mU/ml in controls) were maintained throughout the infusion period. Glucose-induced insulin secretion was examined in vivo 3 h after the end of infusion by performing either a glucose tolerance test or a hyperglycemic clamp (225 mg/dl for 60 min). In addition, in vivo insulin secretion was studied on day 1, 3, 5, and 7 after the end of glucose infusion by performing glucose tolerance tests. Insulin secretion was also investigated in vitro, using the isolated perfused pancreas technique, 3 h and 1 day post glucose infusion. During glucose tolerance tests and hyperglycemic clamps performed at 3 h, insulin secretion was much greater in HG rats than in controls, and remained increased until day 5. By contrast, when studied in vitro 3 h after the end of the infusion, glucose-induced insulin release from isolated perfused pancreases was impaired in HG rats as compared with controls, and the insulin response to arginine was dramatically increased. However, insulin secretion in vitro returned partially to normal after day 1. These data indicate that prolonged hyperglycemia has quite different effects on the subsequent insulin secretion in vivo or in vitro. It impairs, but reversibly, glucose-induced insulin secretion in vitro, whereas it increases it durably in vivo. This suggests that humoral and/or nervous interferences can counterbalance the possible perturbing effects of prolonged hyperglycemia on the normal B cell responsiveness to glucose.

Hyperinsulinaemia increases insulin action in vivo in white adipose tissue but not in muscles.

The effect of 4 days of stable hyperglycaemia and resulting hyperinsulinaemia on insulin-induced glucose utilization by individual rat tissues was studied in vivo. The treatment produced a net increase in the glucose utilization index under both basal and insulin-stimulated (euglycaemic/hyperinsulinaemic clamp) conditions in white adipose tissue. On the contrary, glucose utilization was unchanged in aerobic muscles but was decreased in glycolytic skeletal muscles during the clamp.

In vivo insulin secretion and action in hyperglycemic rat.

This work was designed to study the effects of insulin secretion and action in vivo of moderate hyperglycemia induced by glucose infusion during 4 days in unrestrained rats. The maintenance of a glycemia around 170 mg/dl throughout the infusion time necessitated a gradual increase of glucose infusion rate from 11.5 to 19 g/day. Throughout the infusion period, plasma insulin-to-glucose ratio remained much higher in hyperglycemic rats (HG) than in controls. Glucose tolerance and insulin secretion tests were performed 2 h after the end of the infusion, when glycemia and insulinemia were back to basal values. Incremental plasma glucose values were significantly lower in HG than in control rats without significant changes in incremental plasma insulin concentrations, suggesting an increased insulin efficiency. At the same insulin level, glucose utilization was higher in HG than in control rats during euglycemic-hyperinsulinemic clamps. These data show that short-term hyperglycemia and hyperinsulinemia do not induce a defect in insulin secretion in vivo and do increase tissue sensitivity to insulin.

Evidence for a cold-induced aldosterone stimulation in the rat.

This study was undertaken to determine the secretion of aldosterone by male Long-Evans rats acclimated for six weeks to moderate cold (15 C), in comparison with rats maintained at thermo-neutral temperature (28 C). The following determinations were made: corticosteroids in plasma and adrenals, PRA, and hydromineral balance. Cold acclimation highly increased the plasma and adrenal levels of aldosterone and corticosterone. The cold stimulation of aldosterone was induced neither by the renin-angiotensin system, nor by alterations of hydromineral balance: PRA, plasma sodium and potassium concentrations, blood hematocrit, and hydromineral balance at 15 C and 28 C did not differ. Moreover this stimulation was induced neither by ACTH, nor by any other hypophyseal factors, since plasma aldosterone levels remained high in hypophysectomized rats. This study provides evidence of an aldosterone stimulation which appeared during moderate cold acclimation; the origin of this stimulation must be investigated.

Brown adipose tissue activity in hypophysectomized rats: involvement of sympathetic system.

At thermal neutrality, hypophysectomy enhanced interscapular brown adipose tissue (IBAT) activity (increase of purine nucleotide binding) in the rat. This stimulation is dependent on sympathetic system integrity since surgical denervation of IBAT impairs its thermogenic response.

Effect of environmental temperature on dietary obesity in Osborne-Mendel rats.

The effects of cold acclimation on cellularity, lipoprotein lipase (LPL) activity and lipolysis were studied in white adipose tissue of rats fed a high fat diet. Male Osborne-Mendel rats (7 weeks old) were exposed at either 28 or 5 degrees C for 10 weeks. The rats were fed a semipurified diet (normal fat (NL): 5% lard, high fat (HL): 54% lard) for the last 9 weeks. Caloric intake with NL and HL diets were comparable and cold exposure led to the same increase with both diets. At 28 degrees C, HL diet initiated both hypertrophy and hyperplasia; however, at 5 degrees C only hyperplasia was observed. Total LPL activity showed high stimulation both in 28 and 5 degrees C HL rats. In vitro lipolytic stimulation by norepinephrine was lowered at 5 degrees C and abolished at 28 degrees C in HL-fed rats. HL diet resulted in enhanced lipid deposition without an increase in caloric intake. Even in cold-adapted Osborne-Mendel rats a relative obesity could be produced by a HL diet.

Concomitant food intake and adipose tissue responses under chronic insulin infusion in rats.

Body weight (BW), food intake (FI), and activity of white adipose tissue (WAT) and brown adipose tissue (BAT) were studied in adult male rats under chronic insulin infusion. Insulin was infused for 4, 7 or 10 days via implanted minipumps. Insulin-treated rats gained more BW than control rats until 7th day of infusion. At 10 days, the difference in BW decreased. The average cumulative FI was significantly higher after 4, 7 and 10 days of insulin infusion. Feed efficiency (FE) was increased in insulin-treated rats after 4 and 7 days. An increase in WAT weight was observed in insulin-treated rats together with an increased activity of lipogenic enzymes. BAT weight was augmented after 4 days of insulin infusion. This was due mainly to lipid accumulation. Specific mitochondrial guanosine diphosphate (GDP) binding was significantly decreased by 58% in insulin-treated rats after 4 days of infusion. This reduced thermogenic activity, along with the increased FI and FE were responsible for the rapid BW gain observed during the first 7 days of insulin infusion.

Thermogenesis and brown adipose tissue activity in high fat fed Osborne-Mendel rats.

The consequences of hyperlipidic feeding on interscapular brown adipose tissue (IBAT) activity were examined in the cold-acclimated rat. Male Osborne-Mendel rats (7 weeks old) were exposed for 10 weeks at either 28 or 5 degrees C. The rats were fed a semipurified diet (normal fat (NL): 5% lard; high fat (HL): 54% lard) for the last 9 weeks. IBAT weight was greater in HL than in NL rats. This increase was entirely due to accumulation of neutral lipids. Among different IBAT components (proteins, DNA, phospholipids) no variations were observed in HL 28 degrees C rats. HL diet did not modify lipoprotein lipase and cytochrome oxidase activities, but an increase in purine nucleotide binding (taken as an index of thermogenic activity) was observed in HL 28 degrees C rats. Cold acclimation led to comparable stimulation in NL and HL rats. The calorigenic effect of norepinephrine in vivo was not modified by HL diet. Study of arteriovenous differences showed that IBAT fatty acid and glycerol release by norepinephrine was slightly increased in HL 28 degrees C rats. No effect of HL diet was observed at 5 degrees C. These results indicate that HL feeding leads to a moderate increase in the IBAT thermogenic capacity of Osborne-Mendel rats. HL diet does not modify the normal development of nonshivering thermogenesis when rats are kept in a cold environment.

Involvement of the sympathetic nervous system in lipolytic activity in brown adipose tissue of cold acclimated rats.

In cold acclimated rats, in vitro, NE led to a significant increase in release of FFA and glycerol in denervated IBAT. In vivo, study of arteriovenous differences showed that the denervated BAT loses its full capacity to utilize FFA and glycerol released by NE. After denervation an increase of blood flow in Sulzer's vein was observed. This effect appeared immediately after intervention whereas the effect on fat metabolism appeared later. In cold acclimated rats, the sympathetic nervous system appears to be an important regulator of fatty acid metabolism in BAT.