Pinealectomy causes glucose intolerance and decreases adipose cell responsiveness to insulin in rats.

Although the pineal gland influences several physiological systems, only a few studies have investigated its role in the intermediary metabolism. In the present study, male Wistar rats, pinealectomized or sham-operated 6 wk before the experiment, were submitted to both intravenous glucose tolerance tests (IVGTT) and insulin binding as well as glucose transport assays in isolated adipocytes. The insulin receptor tyrosine kinase activity was assessed in liver and muscle. The insulin secretory response during the IVGTT was impaired, particularly in the afternoon, and the glucose transport responsiveness was 33% lower in pinealectomized rats. However, no difference was observed in the insulin receptor number of adipocytes between groups as well as in insulin-stimulated tyrosine kinase activity, indicating that the initial steps in the insulin signaling were well conserved. Conversely, a 40% reduction in adipose tissue GLUT-4 content was detected. In conclusion, pinealectomy is responsible for both impaired insulin secretion and action, emphasizing the influence of the pineal gland on glucose metabolism.

Responsiveness of glycogen catabolism to adrenergic agonists during insulin-induced hypoglycemia in rat livers.

1. Insulin-induced hypoglycemia (IIH) promoted decreased responsiveness of hepatic glycogen catabolism to phenylephrine and isoproterenol, but not to glucagon and cyanide. 2. In addition, glycogen phosphorylase activity and glycogen levels were not affected by IIH. 3. It was concluded that hypoglycemia promoted changes in hepatic responsiveness to adrenergic agonists. 4. However, the ability of the liver to mobilize glycogen was not influenced by hypoglycemia.

Chronic salt overload increases blood pressure and improves glucose metabolism without changing insulin sensitivity.

The effect of sodium chloride salt restriction and overload on insulin sensitivity is still an open question. Some authors have shown that NaCl salt restriction increases insulin resistance, whereas others have reported the opposite. In the present study, the objective was to get some more insight on this issue by studying the influence of dietary salt content on glucose uptake in isolated adipocytes. Male Wistar rats were fed from weaning either low (0.15%) or high (7.94%) salt diets. On the 12th week of age, weight and tail-cuff blood pressure were measured, followed 10 days later by an intravenous glucose tolerance test with concomitant insulin determinations. One week later, the rats were killed by decapitation and epididymal adipocytes were obtained for glucose metabolism evaluation. No weight differences were observed between both groups of animals. Blood pressure was significantly higher (P < .001) on salt overloaded rats (146 +/- 11 mm Hg) than on salt restricted ones (115 +/- 5 mm Hg). Dietary salt content did not influence the area under the curve of plasma glucose. Area under the curve of insulin levels was lower (P = .023) on the high than on the low salt diet. A higher (P < .001) glucose uptake in the absence and in the presence of insulin was observed in adipocytes from rats on the high salt diet. The median effective concentration (EC50) from the dose-response curves of glucose uptake was the same on both groups of animals. Glucose oxidation and incorporation into lipids was also enhanced by salt overload. High salt increased insulin receptor density (P < .001). In conclusion, salt overload increased blood pressure, and high and low salt dietary content did not influence insulin sensitivity based on the unchanged EC50 from the in vitro studies. However, insulin-independent glucose uptake, oxidation, and incorporation into lipids were enhanced in adipocytes from rats on the high salt diet. The lower levels of insulin during the glucose tolerance test on salt-loaded animals may be a consequence of the higher insulin-independent glucose uptake in that group.

Synergistic effect of counterregulatory hormones during insulin-induced hypoglycemia in rats: participation of lipolysis and gluconeogenesis to hyperglycemia.

AIM: To study the synergistic effect of G (glucagon, 0.02 mg.kg-1), H (hydrocortisone, 20 mg.kg-1) and E (phenylephrine + isoproterenol, both 1 mg.kg-1) during insulin-induced hypoglycemia (IIH) in rats with 6 h of food deprivation (F6 group). METHODS: I (insulin, 1 U.kg-1) was injected i.p. and 30 min later saline (F6 + I group), H, G and E individually or combined (G + H, G + E, H + E and G + H + E) were all injected i.p. and all experiments started 1 h after I injection. RESULTS: The rise in glycemia with H + G + E was greater than the sum of the responses to i.p. H, G and E individually or in double combination plus any single hormone. This effect was reproduced by G + H + Iso (isoproterenol, 1 mg.kg-1), G + H + Iso + Met (metoprolol, 1 mg.kg-1) and G + H + Sal (salbutamol, 1 mg.kg-1). A clear relationship was shown between glycemia and free fatty acids levels. Liver gluconeogenesis from glycerol (2 mmol.L-1) was higher in the group which received G + H + beta-adrenergic agonist vs control rats (F6 or F6 + I groups). CONCLUSION: (a) Acute hyperglycemia is obtained from a condition of IIH by combined i.p. of G + H + beta-adrenergic agonists; (b) This effect cannot be ascribed to a single hormone, but is a consequence of the combined effects of these substances; (c) Blood insulin levels and liver glycogen have no participation; (d) Lipolysis mediated by a beta-adrenergic mechanism and gluconeogenesis from glycerol contribute to the hyperglycemia.

Effect of combined administration of counterregulatory hormones during insulin-induced hypoglycemia in rats: lipolysis mediated by a beta-adrenergic mechanism contributes to hyperglycemia.

The synergistic effect of combined injection of glucagon (G), cortisol (C) and phenylephrine+isoproterenol (E) during hypoglycemia in male adult Wistar rats was investigated. For this purpose we injected insulin (1 mg/kg) and 30 min later saline (controls), C (20 mg/kg), G (0.02 mg/kg), or E (1 mg/kg), individually or combined (G+C, G+E, C+E and C+G+E). All drugs were injected i.p. and all rats were killed 60 min after insulin injection. The rise in glycemia with C+G+E was greater (delta = 107 mg/dl) than the sum of the responses to injection of C, G and E individually, or in double combination plus any single hormone injection. This synergistic effect was reproduced by G + C + isoproterenol (Iso) but not by G + C + phenylephrine (delta = 0 mg/dl). The results also showed a clear relationship between hyperglycemia and lipolysis. Thus, lipolysis mediated by a beta-adrenergic mechanism played a significant role in promoting hyperglycemia when Iso was combined with G and C.

Effect of conbined administration of counterregulatory hormones during insulin-induced hypoglycemia in rats: lipolysis mediated by a Beta-adrenergic mechanism contributes to hyperglycemia

The synergistic effect of combined injection of glucagon (G), cortisol (C) and phenylephrine + isoproterenol (E) during hypoglycemia in male adult Wistar rats was investigated. For this purpose we injected insulin (1 mg/kg), individually or combined (G+C, G+E, C+E and C+G+E). All drugs were injected ip and all rats were killed 60 min after insulin injection. The rise in glycemia with C+G+E was greater (delta = 107 mg/dl) than the sum of the responses to injection of C, G and E individually, or in double combination plus any single hormone injection. This synergistic effect was reproduced by G + C + isoproterenol (Iso) but not by G + C + phenylephrine (delta = 0 mg/dl). The results also showed a clear relationship between hyperglycemia and lipolysis. Thus, lipolysis mediated by a ß-adrenergic mechanism played a significant role in promoting hyperglycemia when Iso was combined with G and C

Neonatal monosodium glutamate treatment increases epididymal adipose tissue sensitivity to insulin in three-month old rats.

We determined the response of glucose transport to insulin in isolated adipocytes and the lipogenic activity of insulin in fragments of epididymal adipose tissue obtained from male MSG-obese rats. Basal glucose transport rates (pmol 3 min-1 10(5) cells-1) were 100% higher in MSG than in control cells (3-month old male Wistar rats) pre-incubated for 30 min (P < 0.01). Nevertheless, when expressed as fmol 3 min-1 microns 2 cell surface area-1, transport rates were similar for the two groups (31.2 +/- 2.6 for MSG and 26.5 +/- 3.2 for controls, N = 7). No differences were observed in maximally insulin-stimulated glucose transport rates between groups (72.6 +/- 10.6 for MSG and 101.0 +/- 12.0 for controls, N = 7). In contrast, for adipocytes pre-incubated for 2 h, the basal uptake rates were 3.7 times higher and the maximal response to insulin was 103% higher in cells from MSG rats compared to control cells. These alterations in MSG rat adipocytes were accompanied by changes in cell sensitivity to insulin (EC50, 0.13 +/- 0.02 ng/ml for MSG vs 0.46 +/- 0.10 ng/ml for controls, P < 0.01). The rates of incorporation of labelled substrates (3H2O and 14C-glucose) into total lipids showed that in vitro lipogenesis was also 79% (3H2O) and 250% (14C-glucose) higher in MSG adipose tissue fragments. The MSG animals were consistently hyperinsulinemic. These data suggest that the obesity of 3-month old MSG rats is a metabolic alteration characterized by an enhanced adipocyte capacity to transport glucose and to synthetize lipids resulting in increased insulin sensitivity.

Neonatal monosodium glutamate treatment increases epididymal adipose tissue sensitivity to insulin in three-month old rats

We determined the response of glucose transport to insulin in isolated adipocytes and the lipogenic activity of insulin in fragments of epididymal adipose tissue obtained from male MSG-obese rats. Basal glucose transport rates (pmol 3 min-1 10(5) cells-1) were 100 higher in MSG than in control cells (3-month old male Wistar rats) pre-incubated for 30 min (P < 0.01). Nevertheless, when expressed as fmol 3 min-1 microns 2 cell surface area-1, transport rates were similar for the two groups (31.2 +/- 2.6 for MSG and 26.5 +/- 3.2 for controls, N = 7). No differences were observed in maximally insulin-stimulated glucose transport rates between groups (72.6 +/- 10.6 for MSG and 101.0 +/- 12.0 for controls, N = 7). In contrast, for adipocytes pre-incubated for 2 h, the basal uptake rates were 3.7 times higher and the maximal response to insulin was 103 higher in cells from MSG rats compared to control cells. These alterations in MSG rat adipocytes were accompanied by changes in cell sensitivity to insulin (EC50, 0.13 +/- 0.02 ng/ml for MSG vs 0.46 +/- 0.10 ng/ml for controls, P < 0.01). The rates of incorporation of labelled substrates (3H2O and 14C-glucose) into total lipids showed that in vitro lipogenesis was also 79 (3H2O) and 250 (14C-glucose) higher in MSG adipose tissue fragments. The MSG animals were consistently hyperinsulinemic. These data suggest that the obesity of 3-month old MSG rats is a metabolic alteration characterized by an enhanced adipocyte capacity to transport glucose and to synthetize lipids resulting in increased insulin sensitivity.

The regulation of insulin action in isolated adipocytes. Role of the periodicity of food intake, time of day and melatonin.

Isolated adipocytes from rats submitted to four weeks of ad libitum feeding (AL) or meal feeding (MF, 2 h/22 h, feeding/fasting, meal time: 8:00-10:00 a.m.) schedules or pre-incubated with or without melatonin (0, 1 nM, 10 nM, 100 nM) for 5 h were submitted to insulin-stimulated [3H]-2-deoxyglucose (0.1 mM, 0.12 microCi) uptake rate measurements and insulin binding assays. Insulin sensitivity was defined as the hormone concentration capable of producing the half-maximal transport rate. Insulin sensitivity varied depending on the previous conditions of the adipocytes. In MF animals, adipose cells were more sensitive (EC50 = 0.175 ng/ml) just at the moment of the expected meal. In AL rats, sensitivity was lower (EC50 = 0.678 ng/ml) at 8:00 a.m. and increased (EC50 = 0.398 ng/ml) at 4:00 p.m. These data clearly implicate the expectation of food and period of the day with the regulation of insulin action. All these modifications in sensitivity occurred without any change in insulin receptor number or affinity. Melatonin, a secretory product of the pineal gland, induced an increase in cell sensitivity to insulin in adipocytes incubated with the highest hormone concentration (100 nM). We conclude that factors related to feeding training and circadian rhythmicity modulate cell sensitivity to insulin.

The regulation of insulin action in isolated adipocytes. Role of the periodicity of food intake, time of day and melatonin

Isolated adipocytes from rats submitted to four weeks of ad libitum feeding (AL) or meal feeding (MF, 2 h/22 h, feeding/fasting, meal time: 8:00-10:00 a.m.) schedules or pre-incubated with or without melatonin (0, 1 nM, 10 nM, 100 nM) for 5 h were submitted to insulin-stimulated [3H]-2-deoxyglucose (0.1 mM, 0.12 microCi) uptake rate measurements and insulin binding assays. Insulin sensitivity was defined as the hormone concentration capable of producing the half-maximal transport rate. Insulin sensitivity varied depending on the previous conditions of the adipocytes. In MF animals, adipose cells were more sensitive (EC50 = 0.175 ng/ml) just at the moment of the expected meal. In AL rats, sensitivity was lower (EC50 = 0.678 ng/ml) at 8:00 a.m. and increased (EC50 = 0.398 ng/ml) at 4:00 p.m. These data clearly implicate the expectation of food and period of the day with the regulation of insulin action. All these modifications in sensitivity occurred without any change in insulin receptor number or affinity. Melatonin, a secretory product of the pineal gland, induced an increase in cell sensitivity to insulin in adipocytes incubated with the highest hormone concentration (100 nM). We conclude that factors related to feeding training and circadian rhythmicity modulate cell sensitivity to insulin.

[Gastrin changes in the gastric antrum G-cells and in serum gastrin levels after 80% jejunoileal resection in rats]. / Alterações da gastrina nas células G do antro gástrico e da gastrinemia após ressecção de 80% de jejunoíleo rem ratos.

A study was made of the changes in the cell population producing gastrin of the gastric antrum in rats submitted to resection of 80% of jejunum-ileum. Ninety days after surgery, the animals were killed after a 12 hour nightly fast and the gastric antrum was removed with the objective of specific histological preparations (PAP method) in order to count the G-cells and the blood was taken for serum doses of gastrin. An optical microscope was used to count the cells using a histometric integraded ocular of 42 points and the counting of 10 fields of each histological cut, and the radioimmunoassay method of double antibody was used for the seric dosing of gastrin. Histometry showed a significant drop in the G-cell population of the antrum of enterectomized animals when compared to the control group. Average percentage of G-cells found were 17.55% in the control group and 7.99% in the enterectomized ones. Blood dosing of hormone showed a significant increase of gastrin in the enterectomized animals when compared to controls. Average value of gastrin dosing the control group was 110 Pg/ml and 170 Pg/ml in enterectomized animals. Therefore, the present study permits to conclude that after resection of 80% of jejunum-ileum, there was a decrease in the G-cell population with gastrin in the gastric antrum even in the presence of increased serum gastrin.

Hepatic metabolism of meal-fed rats: studies in vivo and in the isolated perfused liver.

Hepatic metabolic fluxes (glycolysis, glucose release, glycogenolysis, oxygen consumption, ketogenesis and gluconeogenesis), hepatic glycogen and food ingestion in meal-fed rats were measured and compared to appropriate controls. The following results were obtained: 1) in livers from meal-fed rats a higher fraction of glucosyl units derived from glycogen is used in glycolysis instead of being released in the form of glucose; 2) the rate of glycogen catabolism in livers from meal-fed rats is less than expected when one compares their glycogen levels with those of the appropriate controls; 3) the livers from meal-fed rats become much less ketotic than the livers from rats which were not trained to eat a single meal daily. It was concluded that the liver of meal-fed rats is well adapted to the main characteristics of those animals, e.g., increased lipogenesis from glycolysis products and a reduced need for carbon units from the liver (glucose and ketone bodies) as a consequence of enhanced food intake.

Meal-feeding and physical effort. 1. Metabolic changes induced by exercise training.

To evaluate the consequences of the combination of meal-feeding (which causes in the long term several adaptations that lead to saving stored energetic substrates), rats subjected to a 2-hr feeding/22-hr fasting schedule were forced to swim 30 min everyday at a fixed hour during four weeks. The results indicate that meal-fed exercised rats: 1) increase food intake above that found in the nonexercising and the corresponding (nonfood-restricted) controls; 2) did not lose weight (in contrast to the controls); 3) initially had a high glycogen mobilization but at the end of the fourth week started to save hepatic glycogen again, despite the intense energy demanding exercise; 4) maintained a slight hyperglycemia; 5) mobilized less free fatty acids than the nonexercising meal-fed rats, probably due to higher insulinemia; 6) had a lower content of ascorbic acid in the adrenal glands in comparison to the control exercising rats; this suggests that the exercise was less stressful in the latter group.

Meal-feeding and physical effort. 2. Metabolic changes induced by an acute exercise.

In this study, rats under meal-feeding up to four weeks were submitted to a sudden exercise (swimming) to evaluate the effects of a behavior that requires mobilization of a large amount of energy on some physiological parameters already changed due to the chronic food-restriction. During exercise meal-fed rats: 1) increase the rate of gastric emptying; 2) maintain glycemia more steadily than the controls even during a long-lasting exercise; 3) maintain high liver glycogen concentration and its mobilization starts later on; 4) free fatty acid mobilization is lower than in the controls but during exercise do use much more; 5) keep more glycogen in the muscles (including the heart) than the controls but during the exercise do utilize much more; 6) are slightly less stressful (mainly after a longer exercise) than the controls as suggested by the adrenal ascorbic acid content.

Metabolic changes caused by irregular-feeding schedule as compared with meal-feeding.

In this study rats eating 50% of the quantity of daily food intake observed for free-fed rats were restricted to regular (MF) and irregular intermeal (IF) intervals. Rats which had free access to food (FF) were also included. The experiments were carried out for 1, 2, 3 and 4 weeks. Body weight, daily food intake, stomach fresh weight, blood glucose and free fatty acids (FFA) levels, liver glycogen content and adrenal ascorbic acid were evaluated. The results showed that adaptative metabolic pattern depends on the discipline of the intermeal intervals. Meal-fed rats with a fixed meal time showed better blood glucose maintenance, slower gastric emptying, increased liver glycogen content and lower FFA mobilization during 22-hr fast than the free-fed group. The same amount of food eating by meal-fed rats given randomly in time (IF) promoted a different adaptative metabolic pattern. The results suggest that the regular intermeal period is an important factor for the establishment of the metabolic changes. Therefore, the meal-feeding schedule has to be considered as a particular modality of food restriction.

The sensitivity of glycogenolysis to glucagon, epinephrine and cyanide in livers from rats in different metabolic conditions.

The action of glucagon, epinephrine and cyanide on hepatic glycogen catabolism in meal-fed rats and the corresponding controls was investigated using the isolated perfused liver. The amounts of glycogen catabolites (glucose, L-lactate and pyruvate) released were correlated with the glycogen content of the livers at the different times after feeding. Irrespective of the metabolic condition, the sensitivity of glycogenolysis to glucagon was roughly proportional to the glycogen content of the livers. However, glycogenolysis in livers from meal-fed rats was less sensitive to epinephrine and cyanide. The difference between meal-fed and controls (ad libitum-fed) was particularly pronounced at 22 hours after feeding. It was concluded that resistance to hepatic glycogen depletion in meal-fed rats during the fasting period may be, partly at least, the consequence of a reduced sensitivity of glycogenolysis to epinephrine and to situations of reduced rates of oxidative phosphorylation.

Reversibility of metabolic changes induced by feeding schedule in rats.

This study aimed to investigate the effect of free-feeding on rats kept on meal-feeding schedule for a prolonged period. Thus, rats meal-fed for 4 and 20 weeks were given free access to food for subsequent 5 weeks. The metabolic adaptation of higher hepatic glycogen content, low plasma FFA values and sustained glycemia during 22-hr fast, reported for rats subjected to meal-feeding, completely disappeared after free-eating period. The rate of body weight gain increased as a consequence of the free access to food in both groups but the control values (group with food ad lib all the time) were attained only in rats previously submitted to meal-feeding for the shorter period of time (4 weeks). The findings of this study suggest that the recovery of body weight by meal-fed rats, for the control values, seems to depend on the duration of the meal-feeding schedule and the age when it is imposed.

The relation between inhibition of glycolysis and stimulation of oxygen uptake due to glucagon in livers from rats in different metabolic conditions.

The relation between the effects of glucagon on oxygen consumption and glycolysis in livers from rats under different metabolic conditions was examined. Respiration of substrate-free perfused livers with different glycolytic fluxes, induced by changes in the pattern of food intake, responds differently to the infusion of 1 nM glucagon. The increases in oxygen uptake caused by 1 nM glucagon correlate reasonably well with the absolute decreases in glycolysis. The degree of inhibition of glycolysis is approximately constant (58 per cent) for all metabolic conditions. When no recovery of glycolysis occurs upon cessation of glucagon infusion, the same happens with oxygen consumption, which remains stimulated. It is concluded that in livers with no appreciable biosynthetic activities, the action of glucagon on respiration and glycolysis may be interpreted in terms of an interaction of interpreted in terms of an interaction of cytosolic and mitochondrial ATP generating processes.

Liver phosphorylase activation during early phase of feeding behavior: a neurohumoral regulation.

The mechanisms of liver glycogen depletion during the early phase of spontaneous feeding were examined in adult rats. A 40% increase in the activity of phosphorylase a, the active form of a key glycogenolytic enzyme, was detected 5 min after the onset of feeding, with a concomitant decrease of glycogen in the liver after 5 and 15 min. Within 60 min after the onset of feeding, the enzyme activity returned to the basal level and the glycogen content was restored. These metabolic responses were impaired by hepatic sympathetic denervation or by bilateral adrenodemedullation. It was concluded that the onset of meals may evoke an arousal of the sympathoadrenal system including the hepatic innervation, and that activation of this system is important in stimulating liver phosphorylase activity and glycogenolysis during the early phase of spontaneous feeding.