Ethanol stimulates glycogenolysis and inhibits both glycogenesis via gluconeogenesis and from exogenous glucose in perfused rat liver.

AIMS: Although it is commonly recognized that ethanol suppresses gluconeogenesis, the influence of alcohol intake on blood glucose levels remains controversial. Ethanol may act on both glucose production and glucose consumption in the liver. Thus, we studied each effect of ethanol on glucose oxidation, gluconeogenesis, glycogenesis and glycogenolysis in the liver. METHODS: The rat liver was isolated and cyclically perfused with a medium containing 50 mmol/l ethanol. RESULTS: Ethanol enhanced 14C-glucose oxidation in the liver from 1.09 +/- 0.11 to 1.41 +/- 0.14 micromol for 20 min (p < 0.05). Gluconeogenesis from 14C-lactate was markedly reduced by ethanol from 8.0 +/- 1.3 to 1.5 +/- 0.6 micromol for 12 min (p < 0.01). Ethanol increased glycogenolysis (net hepatic glucose output, 0.47 +/- 0.10 vs. 0.22 +/- 0.04 mmol/30 min, p < 0.01), and then decreased hepatic glycogen content (179 +/- 38 vs. 273 +/- 39 mg in the presence of 1 mU/ml insulin after 30 min of perfusion, p < 0.05). Ethanol decreased the direct glycogenesis from 14C-glucose from 0.55 +/- 0.08 to 0.33 +/- 0.05 micromol per 100 mg glycogen for 30 min (p < 0.01). Ethanol inhibited the indirect glycogenesis from 14C-lactate from 0.21 +/- 0.04 to 0.09 +/- 0.01 micromol per 100 mg glycogen for 30 min (p < 0.01). DISCUSSION: The influence of ethanol on the blood glucose regulation by the liver seems to be different between fasted and fed states. Namely, ethanol has both the hypoglycemic effects through decreased gluconeogenesis and increased glucose oxidation and the hyperglycemic effects through decreased glycogenesis and increased glycogenolysis.

Gliclazide at a lower concentration than therapeutic dose increases the sensitivity of insulin secretion to glucose in perfused rat pancreas.

OBJECTIVES: We studied the difference between effects of therapeutic dose and sub-therapeutic dose of gliclazide on the glucose-induced insulin secretion. METHODS: The normal rat pancreas was isolated and perfused with Krebs-Ringer buffer containing 1-14 mmol/l glucose. Influcences of 0.25 and 2.5 microg/ml gliclazide on the glucose concentration-insulin secretion curve was examined. RESULTS: Gliclazide at 0.25 microg/ml significantly potentiated 5-8 mmol/l glucose-induced insulin secretion (2.5 +/- 0.5 vs 1.0 +/- 0.3 mU for 15 min at 6.5 mmol/l glucose, P<0.01), but did not give influence on either 1-3 or 10-14 mmol/l glucose-induced insulin secretion. The glucose concentration, at which half-maximal insulin secretion was observed, was lower with gliclazide (5.9 mmol/l) than in the control (7.5 mmol/l). Gliclazide at 2.5 microg/ml markedly increased the maximally glucose-stimulated insulin secretion from 3.9 +/- 0.5 mU for 15 min in the control to 6.6 +/- 0.7 mU for 15 min (P<0.01). The half-maximal insulin secretion was observed at a lower glucose concentration (5.0 mmol/l) than in the absence of gliclazide. CONCLUSION: Gliclazide in sub-therapeutically low dose has different effects on insulin secretion from in therapeutic dose, namely sharpens the insulin secretion sensitivity to glucose with no influence on the maximal insulin secretion. It is possible that low doses of gliclazide might be of interest in some type 2 diabetics whose main pathophysiology is the blunting of insulin secretion response to hyperglycemia.

Electroencephalogram is activated by addition of pentobarbital in the isolated perfused head of the rat.

To evaluate the direct effects of a barbiturate on cerebral functions without its influence on brain perfusion pressure, circulatory hormones and metabolites, the electroencephalogram (EEG) was studied in the isolated rat head. Male Wistar rats were anesthetized, and EEG electrodes were inserted into the cranium. A Krebs-Ringer bicarbonate buffer solution containing heparinized rat whole blood, 20 mmol/l glucose, 200 mmol/l mannitol and 0.1 mg/ml dexamethasone was used for the perfusate. The bilateral common carotid arteries were cannulated, pumped at a rate of 6 ml/min and the head was isolated. The venous effluent was reoxygenated and recirculated into the brain. Twenty-five min after isolation of the heads pentobarbital was added to the perfusate at concentrations of 0.1, 0.5 and 2.5 mg/ml. EEG was recorded before and during perfusion. EEG activity could be recorded for more than 25 min after the beginning of perfusion. EEG activity gradually declined from 42+/-5 microV before perfusion (in vivo) to 4+/-1 microV at 25 min after the beginning of perfusion. Then, 3 min after the addition of pentobarbital, the EEG activity became significantly higher in the high dose groups; 12+/-3 microV in the 0.5 mg/ml group (p<0.05) and 12+/-1 microV in 2.5 mg/ml group (p<0.05) compared with the group without pentobarbital (2+/-2 microV). The present study suggests that a barbiturate has mitigating effects on the brain damage induced by the in vitro brain perfusion.

Glucagon is paradoxically secreted at high concentrations of glucose in rat pancreas perfused with diazoxide.

To study the role of B-cells in the regulation of glucagon secretion by glucose, the rat pancreas was perfused with 0.4 mmol/l diazoxide. Perfusate glucose was 5 mmol/l of a basal concentration, and then was decreased to 1 mmol/l, or was increased to 15 mmol/l. Insulin secretion was suppressed by diazoxide below the detectable level at each glucose concentration. Glucagon secretion was increased two-fold during the glucopenic perfusion without diazoxide, but was not changed at a low glucose concentration in the presence of diazoxide. During the glucose-excessive perfusion for 15 min, glucagon secretion was lowered from 0.69 +/- 0.17 pmol at 5 mmol/l glucose to 0.36 +/- 0.10 pmol at 15 mmol/l glucose (p < 0.05) without diazoxide, whereas that was inversely increased from 0.55 +/- 0.14 at 5 mmol/l glucose to 0.85 +/- 0.13 pmol at 15 mmol/l glucose (p < 0.05) in the presence of diazoxide. These results suggest that appropriate insulin secretion is necessary for the normal responses of glucagon secretion to hypoglycemia and hyperglycemia in the non-diabetic rat pancreas.

The early phase of calcipenia-induced parathyroid hormone secretion is blunted in vasculary perfused parathyroid glands of streptozotocin-diabetic rats.

OBJECTIVES: To study effects of diabetes mellitus on parathyroid hormone (PTH) secretion, the rat parathyroid glands were perfused via the bilateral carotic arteries. MATERIAL AND METHODS: Diabetic rats showed plasma glucose levels above 30 mmol/l, but no change in plasma PTH concentration, 14 days after an administration of 70 mg/kg of streptozotocin. Krebs-Ringer bicarbonate buffer containing 0.1% bovine serum albumin was used for perfusate. Perfusate calcium was lowered from 2.5 mmol/l to 0.5 mmol/l. RESULTS: In the normal rat parathyroid glands, PTH secretion was promptly evoked by calcipenia (from below 0.25 ng for 2.5 min in the perfusion with normal calcium to 0.73 +/- 0.17 ng during calcipenia time 0-2.5 min), and slightly decreased (0.51 +/- 0.12 ng during calcipenia time 2.5-5 min), and then increased (0.75 +/- 0.16 ng during calcipenia time 7.5-10 min). Diabetes abolished the early phase of PTH secretion (below 0.25 ng during calcipenia time 0-2.5 min), but did not affect the late phase of PTH secretion (0.64 +/- 0.14 ng during calcipenia time 7.5-10 min). Insulin treatment with 25 U/kg daily for 14 days completely normalized the early phase of PTH secretion. CONCLUSION: It is suggested that an insulin-deficient diabetes, not quantitatively but qualitatively, impaires the PTH secretion.

Acidotic pH augments glucagon secretion and gluconeogenesis in the isolated perfused rat pancreas and liver.

To study effects of acidosis on glucagon secretion and gluconeogenetic action of glucagon, rat pancreas and liver were perfused with media of pH 6.4, 6.9, 7.4 and 7.9. Glucagon secretion from the pancreas during glucopenic perfusion (1 mmol/l) was blunted at alkalotic pH, and was augmented at acidotic pH; 0.28+/-0.18 at pH 7.9 (P<0.01), 3.57+/-0.94 at pH 6.9 (P<0.01) and 1.72+/-0.36 at pH 6.4 (P<0.01), vs. 0.66+/-0.25 pmol for 15 min at pH 7.4. Incorporation rate of 14C of lactate-U-14C into glucose carbon one was decreased at pH 7.9 (1.2+/-0.2% for 15 min, P<0.05) and was increased at pH 6.9 (2.8+/-0.5%, P<0.05) compared to that at pH 7.4 (1.9+/-0.3%). Percent increasing rate of lactate gluconeogenesis by 1 nmol/l glucagon was not different within a range of pH 6.4-7.9. Thus, glucagon-stimulated gluconeogenesis from lactate was smaller at pH 7.9 (2.2+/-0.6%) and was significantly greater at pH 6.9 (4.9+/-0.9%, P<0.05) than that at pH 7.4 (3.2+/-0.6%). These results suggest that the pancreatic glucagon secretion and the glucagon-stimulated hepatic gluconeogenesis play more important roles in the maintainance of blood glucose level in the stress states associated with acidosis than without acidosis.

Streptozotocin-induced diabetic hyperglycaemia produces more EEG activity than normoglycaemia and hypoglycaemia during and after anoxia loading in rats.

The brain requires oxygen and glucose for energy metabolism. Electroencephalogram (EEG) was recorded to determine the effect of glucose concentration on the cerebral function in hypoxic episode. Rats were divided into 3 groups: a streptozotocin-induced diabetic hyperglycaemic group, a normoglycaemic group, and an insulin-induced hypoglycaemic group. Hypoxia was induced by ventilating with 100% N2 for 3 min. EEG amplitude both 5 and 10 min after anoxia loading was higher in the diabetic hyperglycaemic than in the normoglycaemic group, though not significantly. Time for decreasing the EEG amplitude during anoxia loading was significantly longer in the hyperglycaemic than in the normoglycaemic group. Time for recovering the EEG amplitude after anoxia loading was significantly shorter in the hyperglycaemic group and was longer, though not significantly, in the hypoglycaemic group than in the normoglycaemic one. These results suggest the brain is more tolerant to hypoxia during diabetic hyperglycaemia than during normoglycaemia.

Impaired parathyroid hormone action on urinary phosphorus excretion in isolated perfused kidney of streptozotocin-induced diabetic rats.

To study the effects of diabetes on the renal actions of parathyroid hormone (PTH), we observed urinary excretion of cyclic adenosine monophosphate (cAMP) and phosphorus in isolated perfused rat kidney. Diabetic rats were kept for 7 days after an intraperitoneal injection of 70 mg/kg streptozotocin (STZ). STZ-induced diabetic rats were treated with a daily injection of 20 U/kg lente-type insulin for 7 days. Plasma albumin, calcium, phosphorus, and PTH levels were not different among normal control, diabetic and insulin-treated diabetic groups. In the control rat kidney, the addition of PTH increased urinary cAMP excretion from 8 +/- 3 to 190 +/- 49 pmol/5 min and urinary phosphorus excretion from 11.3 +/- 4.4 to 33.6 +/- 10.8 microg/5 min. In the STZ-diabetic rat kidney, basal urinary cAMP was impaired, and PTH altered neither urinary cAMP nor phosphorus excretion (from below 0.7 to below 0.7 pmol/5 min, and from 15.5 +/-4.5 to 13.6 +/- 8.1 microg/5 min, respectively). Insulin treatment completely recovered the PTH actions. These results show that insulinopenic diabetes induces PTH resistance in the kidney.

Peripheral insulin sensitivity is decreased by elevated non-esterified fatty acid level in dexamethasone-treated rats.

The pathogenesis of glucocorticoid-induced insulin resistance in the peripheral tissue was studied by perfusion experiments of the isolated rat hindquarter and insulin tolerance tests in the rat with the cutting off operation of blood supplies to the liver, kidneys, intestines and pancreas. The rat was injected with 0.5 mg/kg dexamethasone for 7 days. Just after the multiple functional organectomies, insulin was loaded in doses of 0.1 and 0.5 U/kg. Plasma insulin levels during 0-10 min were approximately 100-20 and 500-100 microU/ml, respectively. The decreasing rates in plasma glucose level after injections of saline and 0.1 U/kg insulin were much smaller in dexamethasone-treated than control rats (0.0+/-7.2 vs 13.3+/-3.4%/10 min, p<0.05, and 8.0+/-5.8 vs 39.4+/-3.7%/10 min, p<0.01 respectively). The decreasing rate after 0.5 U/kg insulin loads was similar between both groups (40.2+/-10.0 in dexamethasone-treated and 52.3+/-7.3%/10 min in control rats). Plasma non-esterified fatty acids were raised in dexamethasone-treated compared to control rats (1.23+/-0.23 vs 0.73+/-0.08 mM, p<0.01). In the hindquarter perfusion study, glucose uptake in the dexamethasone-treated rat leg was no less than in the normal rat leg under a palmitate-free condition, and was decreased by the addition of 1.0 mM palmitate without and together with 100 and 500 microU/ml insulin. These results suggest that the glucocorticoid-induced peripheral insulin resistance is characterised by the decreased sensitivity and the preserved responsiveness to insulin, and is caused mainly by an elevated non-esterified fatty acid level.

Sodium at high concentrations increases glucose-induced insulin secretion in the perfused rat pancreas.

To study the effects of supraphysiologically high sodium concentrations on insulin secretion, the rat pancreas was perfused with normal Krebs-Ringer bicarbonate buffer (KRBB) solution and with KRBB solutions containing an additional 25 and 50 mmol/l NaCl. The first phase of insulin secretion in response to 18 mmol/l glucose was enhanced in a dose-dependent manner, from 20.8+/-4.0 pmol for 4 min in normal KRBB to 36.2+/-10.3 pmol (P<0.05) and 60.3+/-14.6 pmol (P<0.01) with addition of 25 and 50 mmol/l NaCl, respectively. The second phase secretion was significantly increased by the addition of 50 mmol/l NaCl (259.4+/-38.6 vs 170.9+/-47.4 pmol for 16 min P<0.05). The addition of 50 mmol/l isethionate Na to normal KRBB solution also increased the first and second phases of insulin secretion in response to glucose. The increase in osmolarity by the addition of mannitol to normal KRBB solution did not affect the glucose-induced insulin secretion. A high sodium concentration affected neither tolbutamide-induced nor arginine-induced insulin secretion. It is suggested that sodium concentrations have an important role in insulin secretion in response to glucose, but not to other secretagogues.

Troglitazone reduces free fatty acid-induced insulin resistance in perfused rat hindquarter.

In order to study the effects of troglitazone on insulin resistance associated with elevated plasma free fatty acid (FFA), the hindquarters of rats treated with troglitazone for 14 days were perfused with a medium containing 15 mmol/l glucose, 0-1,000 microU/ml insulin, and 0 or 1.0 mmol/l palmitate. In the absence of palmitate, net glucose uptake was similar between control and troglitazone-treated rat hindquarters at each insulin concentration: 47 +/- 10 and 45 +/- 9 mumol for 30 min at 0 microU/ml insulin; and 94 +/- 14 and 99 +/- 13 mumol for 30 min at 1,000 microU/ml insulin respectively. Addition of palmitate abolished insulin stimulation of net glucose uptake in control rat hindquarter but did not decrease it in troglitazone-treated rat hindquarter (60 +/- 11 vs 99 +/- 12 mumol for 30 min at 1,000 microU/ml insulin, p < 0.01). It is suggested that the "FFA-tolerant effect" is one mechanism by which troglitazone reduces insulin resistance.

Ay-4166 increases the sensitivity of insulin secretion to glucose in isolated perfused rat pancreas.

To study the effect of a therapeutic dose (3 microM) of AY-4166 on the glucose-stimulated insulin secretion, the rat pancreas was perfused with 5 to 15 mM glucose. AY-4166 did not affect the basal insulin secretion at 5 mM glucose, and increased the first and second phases of insulin secretion stimulated by 7.5 to 15 mM glucose. The dose-response curve of the insulin secretion to glucose was shifted to the left side by AY-4166. These results suggest that AY-4166 ist not a stimulator releasing insulin independently of glucose concentrations, but a potentiator of insulin secretion.

Glucocorticoids potentiate effect of ACTH on cortisol secretion in isolated perfused guinea pig adrenal glands.

To clearly understand the autoregulation of glucocorticoid secretion by the glucocorticoid level, we studied the effects of cortisol and dexamethasone on the cortisol secretion in the perfused guinea pig adrenal glands. The basal cortisol secretion was suppressed by cortisol infusion at 0.1 microgram/ml, but not affected by dexamethasone infusion at 1 microgram/ml. The cortisol response to ACTH after pre-perfusion for 35 min was much quicker and larger in the presence of 0.1 microgram/ml cortisol, and was quicker in the presence of 1 microgram/ml dexamethasone than in the absence of glucocorticoid. It is considered that glucocorticoids play a permissive role in the action of ACTH on the adrenal gland.

Increased glucagon action on lactate gluconeogenesis in perfused liver of dexamethasone-treated rats.

To clearly understand the hyperglycemic action of glucocorticoids, we studied the action of glucagon on lactate gluconeogenesis in the liver of rats 7 days after adrenalectomy and after treatment with 1 mg/kg dexamethasone for 7 days. The liver was isolated and cyclically perfused at 20 ml/min with 25 ml of perfusion medium containing 5 mM lactate, [U-14C]lactate, and 0-100 ng/ml glucagon. In the absence of glucagon, incorporation of [14C]lactate into glucose carbon 1 did not change significantly in the adrenalectomized rat liver (1.66 +/- 0.12% of total radioactivity for 5 min) and increased in the dexamethasone-treated rat liver (3. 61 +/- 0.54%, P < 0.01) compared to the normal rat liver (1.99 +/- 0. 28%). The response of lactate gluconeogenesis to glucagon was extremely blunted in the adrenalectomized rat liver and was much larger in the dexamethasone-treated rat than in the normal rat liver (at a glucagon concentration of 100 ng/ml, 2.13 +/- 0.33, 8.55 +/- 1. 06, and 4.61 +/- 0.53% for 5 min, respectively). Glucagon binding to liver plasma membrane was not changed by adrenalectomy and was decreased by dexamethasone treatment. These results suggest that glucocorticoids induce hyperglycemia by increasing the response to glucagon, together with the high basal activity of hepatic gluconeogenesis. In addition, these effects do not occur through changes in glucagon binding to receptors.

Effect of various glucagon/insulin molar ratios on blood ketone body levels in rats by use of osmotic minipumps.

The bihormonal control by insulin and glucagon of blood ketone body level was studied. Mixed solutions with various molar ratios of glucagon and insulin (G/I) were subcutaneously infused continuously for five days by use of the osmotic minipump in the normal rats. The concentrations of insulin and glucagon solution were set at the high G/I molar ratio, the moderate G/I molar ratio and the low G/I molar ratio. In addition, the moderate G/I molar ratio group was divided into three sub-groups: low glucagon and low insulin, moderate glucagon and moderate insulin, and high glucagon and high insulin. After five days, the rats were decapitated to measure plasma ketone body, free fatty acid (FFA), glucose, insulin and glucagon. The FFA level was not significantly different among three groups. The glucose level was not different between the high and moderate G/I molar ratio groups, and decreased in the low G/I molar ratio group. 3-beta-hydroxybutyrate (3-OHBA) and acetoacetate (AcAc) levels in the high G/I molar ratio group were elevated, and 3-OHBA level in the low G/I molar ratio group was lowered compared to those in the moderate G/I molar ratio group. Among three moderate G/I molar ratio sub-groups, there was no difference in 3-OHBA and AcAc levels. These results demonstrate that plasma ketone body levels are controlled by the plasma G/I molar ratio.

Captopril increases renin release stimulated by furosemide and hypotension in isolated perfused guinea pig kidneys.

To clearly understand the feedback mechanism of renin secretion by intrarenal angiotensin II synthesis, we studied the stimulatory effect of captopril, an angiotensin converting enzyme inhibitor, on renin release from the isolated perfused guinea pig kidneys. Captopril did not affect the basal renin secretion. Captopril increased on a dose dependent basis renin release induced by 0.1 mg/ml furosemide; 5.8 +/- 1.3 ng/ml/hr at 0 mg/ml of captopril vs. 8.8 +/- 1.6 ng/ml/hr at 0.01 mg/ml (p < 0.05) and 11.8 +/- 2.4 ng/ml/hr at 0.1 mg/ml (p < 0.01), 15-20 min after furosemide infusion. After lowering the flow rates from 10 to 5 ml/min, renin secretion was not altered during the first 12 minutes. However, after adding 0.1 mg/ml captopril, renin secretion was enhanced within the following 8 minutes (8.3 +/- 1.5 vs. 3.7 +/- 0.5 ng/ml/hr at the end of low flow rate period, p < 0.01). Lowering the perfusion flow of the guinea pig kidneys decreases the NaCl flux at the macula densa and also seems to have a similar effect to furosemide. Therefore, these effects of captopril suggest that intrarenal angiotensin II production has an important role in the regulation of renin secretion, probably via effects on the macula densa function.

Impaired glucose uptake and intact gluconeogenesis in perfused rat liver after carbon tetrachloride injury.

The dynamics of glucose movement across perfused livers were assessed in carbon tetrachloride (CCl4)-injured rats. Rats were given CCl4 for 8 weeks and became glucose intolerant and hyperinsulinemic. The fasted rat liver was cyclically perfused with 4 mM lactate and various concentrations (0-20 mM) of glucose for 20 min. In the CCl4-injured liver, net glucose output was less suppressed at high glucose levels than in the normal liver (147 +/- 70 vs 18 +/- 10 mumol at 20 mM glucose, P < 0.05). Deposition of the carbon from [14C] glucose into glycogen was stimulated at high glucose levels and was markedly reduced in the CCl4-injured liver compared to the normal liver (0.58 +/- 0.33 mumol vs 1.44 +/- 0.20 mumol at 20 mM, P < 0.01). Conversion of [14C] lactate to [14C] glucose was not different between the CCl4-injured and the normal liver at each glucose level. Deposition of the carbon from [14C] acetate into glycogen in the CCl4-injured liver was larger than that in the normal liver at 0 mM glucose (0.81 +/- 0.15 mumol vs 0.32 +/- 0.06 mumol, P < 0.01), but was similar to the normal at 20 mM glucose. In the CCl4-injured liver, utilization of exogenous glucose was impaired at high glucose levels, and gluconeogenetic activity was not impaired at low glucose levels. These changes in the hepatic glucose metabolism seem to account for postprandial hyperglycemia without fasting hypoglycemia associated with liver diseases.

Insulin-induced hypoglycemia stimulates gluconeogenesis from 14C-lactate independently of glucagon and adrenaline releases in rats.

Correlation between blood glucose, glucagon and adrenaline levels and gluconeogenesis was studied during the recovery from insulin-induced hypoglycemia in rats. Rats, overnight fasted, were intravenously injected with 40 microCi/kg of [U-14C]-lactate and 1 U/kg of porcine insulin under an anesthesia with pentobarbital sodium. Blood samples were drawn via the peripheral vein at 0, 5, 10 and 20 min. Plasma insulin level was 617 +/- 115 microU/ml at 5 min. Plasma glucose level was significantly decreased at 5 min (2.7 +/- 0.3 mM at 5 min v.s. 4.3 +/- 0.2 mM at 0 min, P < 0.01). Plasma glucagon and adrenaline did not significantly respond at 5 min, and then rised. Specific radioactivity of plasma [1-14C]-glucose was significantly higher at 5 and 10 min in the insulin-injected rat than the saline-injected rat (204 +/- 34 v.s. 130 +/- 14 d.p.m./mumol at 5 min, P < 0.01; 275 +/- 32 v.s. 186 +/- 16 d.p.m./mumol at 10 minm P < 0.01). These results suggest that lowering of blood glucose level stimulates gluconeogenesis independently of the release of counter-regulatory hormones in insulin-induced hypoglycemia.