Impaired sensitivity to insulin of rat livers perfused with blood of diminished haematocrit.

1. In livers from fed rats perfused with homologous whole blood of a haematocrit value of 37%, insulin decreased the perfusate concentrations of glucose and amino acids, production of ketone bodies (3-hydroxybutyrate + acetoacetate) and increased bile flow. 2. Perfusion with blood diluted with buffer to a haematocrit value of 17% decreased hepatic O2 consumption by 40-50%. Perfusate concentrations of glucose and lactate, the rate of ketogenesis and the ratios [lactate]/[pyruvate] and [3-hydroxybutyrate]/[acetoacetate] were all increased. 3. In livers perfused with blood of diminished haematocrit, effects of insulin on perfusate glucose an amino acids, ketogenesis and bile flow were abolished.     

Lack of effect of somatostatin on the glucagon-induced alterations of hepatic metabolism of [1-14C]oleate

Livers from normal fed male rats were perfused in a recycling system in vitro. Glucagon was infused in varying quantities to give final concentration in the cell-free perfusate of 4.9 . 10(-10)-4.9 . 10(-7) M after 3 h of perfusion, assuming no degradation of the hormone. Where indicated, cyclic somatostatin was infused simultaneously to give a final concentration of 3.0 . 10(-6) M. In the absence of somatostatin, glucagon at a concentration as low as 4.9 . 10(-10) M increased the release of glucose and increased ketogenesis, but impaired the synthesis and release of perfusate triacylglycerol and very low density lipoprotein lipids. Somatostatin did not affect these actions of glucagon. Somatostatin alone, however, did reduce the output of very low density lipoprotein. It is suggested that the alteration of fatty acid metabolism by somatostatin in vivo results from modulation of pancreatic glucagon secretion, not from interference by somatostatin of the action of glucagon on the liver.     

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.     

Glucagon-like peptide-1 secretion is influenced by perfusate glucose concentration and by a feedback mechanism involving somatostatin in isolated perfused porcine ileum

Glucagon-like peptide-1 (GLP-1) is released from intestinal L-cells in response to ingestion of meals. The mechanisms regulating its secretion are not clear, but local somatostatin (SS) restrains GLP-1 secretion. We investigated feedback and substrate regulation of GLP-1 and SS secretion, using isolated perfused porcine ileum (n=17). Effluents were measured for GLP-1 and SS. Perfusion pressure and motility were recorded. Investigated parameters included spontaneous fluctuations, changes in perfusate glucose concentrations (3.5, 5, 11 mM) and addition of insulin (1 nM). We also investigated the effect of proglucagon products, glucagon (10 nM), GLP-1 and GLP-2 (0.1, 1, and 10 nM) on GLP-1 and SS secretion, as well as on glucagon-like peptide-2 (GLP-2), peptide YY (PYY) and GIP secretion, all possible product of L-cells or neighbour cells. Perfusate glucose concentration dose-dependently stimulated GLP-1 secretion (p=0.011). Insulin had no effect. Glucagon weakly stimulated GIP secretion. GLP-1 stimulated SS secretion and motor activity, but inhibited GLP-2, GIP and PYY secretion and perfusion pressure. GLP-2 weakly stimulated SS secretion. We conclude (a) that GLP-1 secretion is influenced by perfusate glucose concentration and (b) that L-cell secretion is feedback regulated by GLP-1 itself, probably via paracrine SS activity.     

The influence of hepatic venous oxygen saturation on the liver's synthetic response to metabolic stress.

Hepatic oxygen consumption (HVO2) and hepatic venous oxygen saturation (ShvO2) were assessed in the isolated perfused rat liver under conditions that mimic critical illness in an effort to assess their utility in predicting the functional status of the liver. Flow rates were adjusted over the physiologic range of oxygen transport as indicated by the hepatic venous O2 saturation range of 10%-75%. HVO2 was found to be transport (HDO2) dependent only when perfusate conditions contained an increased counterregulatory hormone (glucagon, epinephrine, dexamethasone) stimulus or a high lactate concentration. In the absence of a metabolic load, (substrate and hormone-free perfusate), HVO2 was transport independent even at an ShvO2 as low as 10%. Although transport dependency of HVO2 is frequently used to infer tissue ischemia, hepatic oxygen consumption was poorly correlated with synthetic function under all conditions. In contrast, hepatic albumin production was directly related to ShvO2 at all levels of HDO2 and under all perfusion conditions indicating that this metabolic process is particularly sensitive to reductions in oxygen availability, which is more reliably predicted by venous saturation measurements. However, glucose and urea synthesis were almost independent of ShvO2. These findings indicate that various hepatic processes are affected differentially by stress conditions and flow alterations that may exist during critical illness, and protein synthesis is particularly sensitive to oxygen deprivation. Additionally, hepatic venous oxygen saturation measurement, but not HVO2, serves as a useful surrogate marker for hepatic albumin production suggesting that regional venous oximetry may play an important role in the detection of hepatic functional impairment.     

O2 dependence of insulin stimulation of glucose uptake by perfused rat liver: effects of carboxyhaemoglobin and haematocrit

Livers from fed male rats were perfused in a non-recirculating system with undiluted rat blood containing 14 mM glucose. In these experiments there was a substantial uptake of glucose which was stimulated by insulin. Perfusion with blood containing carboxyhaemoglobin at a concentration of 40% of total haemoglobin lowered O2 consumption and abolished hepatic glucose uptake in control and insulin-infused livers, respectively. In experiments with rat erythrocytes resuspended in buffer to haematocrit values of 38 and 22%, O2 consumption and control and insulin-stimulated rates of glucose uptake were similar to corresponding perfusions with undiluted blood and blood containing carboxyhaemoglobin. It is concluded that serum factors are of relatively small importance and that hepatic glucose uptake is dictated by O2 supply.     

Metabolic responses of perfused rat livers to alpha- and beta-adrenergic agonists, glucagon and cyclic AMP.

1. The mechanism of action of glucagon and epinephrine was studied in perfused rat livers. Hormone-induced transitions from one metabolic steady state to another were followed in a non-recirculating perfusion system. Glucose and lactate production rates, oxygen uptake and K+ redistribution were measured. 2. Glucagon (3 nM), cyclic AMP (0.2 mM) and epinephrine (0.5 muM) had similar effects on K+ concentrations in the perfusate. Glycogenolysis responded more rapidly and O2 uptake was enhanced to a larger extent with epinephrine than with the other agents. alpha- and beta-receptor responses were differentiated by the use of phenylephrine (0.5 muM), isoproterenol (0.5 muM) and adrenergic blocking agents (phentolamine and beta-blocker Ro 3-4787 at 0.1 mM). 3. alpha-receptors mediated an activation of glucose production that was very rapid and was paralleled by a transient decrease of K+ concentrations in the effluent from the liver, lactate production rose gradually. Respiration was also enhanced, but fell again as lactate production increased. 4. beta-receptor stimulation was followed by an increase of glucose production that was less drastic and was paralleled by a K+ release, lactate production and respiration were only slightly enhanced. beta stimulation and glucagon both resulted in an inhibition of the alpha-adrenergic effect on lactate release and simultaneously increased O2 uptake. 5. We concluded that in perfused rat livers alpha- as well as beta-adrenergic receptor stimulation resulted in an activation of glycogenolysis, possibly by two different mechanisms.     

Characterization of the hormone-sensitive Ca2+ uptake activity of the hepatic endoplasmic reticulum

The characteristics and kinetics of calcium uptake activity were studied in isolated hepatic microsomes. The sustained accumulation of calcium was ATP- and oxalate-dependent. Glucagon increased microsomal Ca2+ uptake upon either in vivo injection, or in vitro perfusion of the hormone in the liver. In contrast, the effect of insulin depended on the route of administration. Calcium accumulation by subsequently isolated hepatic microsomes increased when insulin was injected intraperitoneally whereas it decreased when the hormone was perfused directly into the liver. These effects of glucagon and insulin were dose dependent. When insulin was added to the perfusate prior to the addition of glucagon, insulin blocked the glucagon-stimulated increase in microsomal Ca2+ uptake. Cyclic AMP mimicked the effect of glucagon on microsomal Ca2+ accumulation when the cyclic nucleotide was perfused into the liver. The effects of glucagon and insulin on the kinetics of hepatic microsomal Ca2+ uptake were investigated. In microsomes isolated from perfused rat livers treated with glucagon the V of the uptake was significantly increased over the control values (12.2 vs. 8.6 nmol Ca2+ per min per mg protein, P less than 0.02). In contrast, the addition of insulin to the perfusate significantly decreased the V of Ca2+ uptake by subsequently isolated microsomes (6.8 vs. 8.3 nmol Ca2+ per min per mg protein, P less than 0.05). However, neither hormone had an effect on the apparent Km for Ca2+ (4.1 +/- 0.5 microM) of the reaction. The effect of these hormones on the activity of Ca2+-stimulated ATPase was also studied. No significant changes in either V or Km for Ca2+ of the enzymatic reaction were detected.     

Effect of glucagon antiserum on somatostatin,glucagon and insulin release from the isolated perfused rat pancreas

In order to elucidate the effect of glucagon antiserum on the endocrine pancreas, the release of somatostatin, glucagon, and insulin from the isolated perfused rat pancreas was studied following the infusion of arginine both with and without pretreatment by glucagon antiserum. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated both somatostatin and glucagon secretion. However, the responses of somatostatin and glucagon were different at different doses of arginine. The infusion of glucagon antiserum strongly stimulated basal secretion in the perfusate total glucagon (free + antibody bound glucagon) and also enhanced its response to arginine, but free glucagon was undetectable in the perfusate during the infusion. On the other hand, the glucagon antiserum had no significant effect on either insulin or somatostatin secretion. Moreover, electron microscopic study revealed degrannulation and vacuolization in the cytoplasm of the A cells after exposure to glucagon antiserum, suggesting a hypersecretion of glucagon, but no significant change was found in the B cells or the D cells. We conclude that in a single pass perfusion system glucagon antiserum does not affect somatostatin or insulin secretion, although it enhances glucagon secretion.     

Glucagon stimulation of hepatic gluconeogenesis in neonatal pigs.

The effect of an infusion of glucagon on gluconeogenesis was studied in 5-day-old piglets. Glucagon stimulated hepatic new glucose formation from lactate, but did not significantly change blood glucose or plasma free fatty acid levels. The data suggest that glucagon enhances substrate flow in the gluconeogenic pathway in neonatal animals.     

Rapid stimulation of calcium uptake into rat liver by L-tri-iodothyronine.

The short-term effect of L-tri-iodothyronine (T3) on hepatic Ca2+ uptake from perfusate was compared with changes induced by T3 on cellular respiration and glucose output in isolated perfused livers from fasted and fed rats. The same parameters were also studied after the addition of glucagon or vasopressin. T3 (1 microM) induced Ca2+ uptake from the perfusate into the liver within minutes, and the time course was similar to that for stimulation of respiration and gluconeogenesis in livers from fasted rats, and for the stimulation of respiration and glucose output in livers from fed rats. The effects were dose-dependent in the range 1 microM-0.1 nM. Similar changes in the same parameters could be observed with glucagon and vasopressin, but with a completely different time course. Also, the influence of the T3 analogues L-thyroxine (L-T4), 3,5-di-iodo-L-thyronine (L-T2) and 3,3',5-tri-iodo-D-thyronine (D-T3) on hepatic energy metabolism was examined. Whereas D-T3 had practically no effect, L-T4 and L-T2 caused changes in Ca2+ uptake, O2 consumption and gluconeogenesis in livers from fasted rats similar to those with T3. It is concluded that changes in mitochondrial and cytosolic Ca2+ concentrations are involved in the stimulation of respiration and glucose metabolism observed with T3, glucagon and vasopressin.     

Regulation of the hepatic response to glucagon: role of insulin, growth hormone and cortisol

The hepatic response to glucagon was investigated in five groups of animals: (1) controls; (2) excess growth hormone (GH; tumor-bearing); (3) streptozotocin-induced diabetic; (4) cortisol-treated, and (5) insulin-treated animals. Blood samples were collected from the animal models and hepatocytes were prepared and used for glucagon-binding studies and studies of total glucose production, gluconeogenesis and glycogen determinations. Glucagon binding was elevated in GH-tumor-bearing and cortisol-treated hepatocytes but lower in hepatocytes from diabetic animals. Basal total glucose production wash higher in hepatocytes from diabetic rats but not changed in hepatocytes from GH-tumor-bearing, insulin-treated or cortisol-treated animals. Glucagon significantly stimulated total glucose production in hepatocytes from control, insulin-treated and cortisol-treated but not diabetic and GH tumor models. Gluconeogenesis as evaluated by alanine conversion to glucose was significantly increased in hepatocytes from diabetic and cortisol-treated animals and was significantly lower in hepatocytes from GH-tumor-bearing animals. Glucagon failed to significantly stimulate gluconeogenesis in hepatocytes from diabetic and tumor-bearing animals. Hepatic glycogen content was significantly decreased in diabetic and GH-tumor-bearing animals but not changed in insulin-treated and cortisol-treated animals. We conclude that increased glucagon binding was not always correlated with an increase in glucagon-stimulated glycogenolysis, gluconeogenesis or increased sensitivity to glucagon. Persistent hyperinsulinism may effectively suppress glucagon- or cortisol-stimulated pathways.     

Effects of vanadate on glucose production in cultured hepatocytes isolated from rats on high saturated fat diet

Insulin resistance is a common phenomenon in obesity and Type 2 diabetes. Common factor important for development of diabetes and insulin resistance is intake of saturated fat. Vanadate treatment improves glucose homeostasis in vivo. The aim of this study was to find out changing of hepatic glucose output in dependence of saturated fat diet and possible direct action of vanadate in cultured hepatocytes. Hepatocytes were isolated by a collagenase perfusion technique and cultured for 24 h in M 199 serum-free medium. The glucose production in hepatocytes isolated from rats on high saturated fat diet was significantly 139% higher comparable to standard controls. Glucagon 100% increased glucose production in hepatocytes from rats on standard diet and 200% in hepatocytes on saturated high fat diet. The addition vanadate significantly decreased basic glucose production and did not influence glucagon stimulated glucose production. Presence of insulin did not influence either glucagon or vanadate effect. High saturated fat diet not only increases insulin resistance but also decreases chances of successful therapy of diabetes.     

Lead inhibits oxidative metabolism of macrophages exposed to macrophage-activating factor.

1. An improved rat spleen perfusion is described incorporating a method of defibrination which avoids the use of heparin and enables the spleen to be perfused with rat blood for several hours at a haematocrit of 40% and for 12 h or more at a haematocrit of 20%. 2. Glucose oxidation accounted for 11.6% of the total oxygen consumption but this represented only 8% of total glucose uptake, which was largely converted to lactate and released into the perfusate. However, significant amounts of lactate were oxidized. These results can be explained by the presence of at least two cell populations, one emphasizing the anaerobic oxidation of glucose and the other aerobic metabolism, particularly of lactate. 3. Non-esterified fatty acid and 3-hydroxybutyrate, when available at physiological concentrations, were shown to be major oxidative fuels of the spleen. 4. Chylomicron triacylglycerol was hydrolysed readily and taken up, but not oxidized extensively.     

Urinary and plasma cyclic AMP levels during short term starvation in obese man: response to glucagon stimulation.

Glucagon is known to elevate the intracellular concentration of cyclic AMP in the hepatocyte. The increase in intracellular cyclic AMP is reflected by an increase in the plasma concentration of the nucleotide. Intravenous glucagon stimulation was performed on six obese non-diabetic human subjects before and after a three day fast. All patients responded to starvation by a lowering of plasma immunoreactive insulin and blood glucose. Whereas the plasma immunoreactive glucagon concentration increased over the three day period, the plasma and urinary cyclic AMP did not significantly change. Intravenous glucagon promoted qualitatively similar increases in the blood glucose and plasma concentrations of insulin and cyclic AMP before and after three days starvation.     

Insulin and non-esterified fatty acids. Acute regulators of lipogenesis in perfused rat liver.

Livers from fed rats were perfused with whole rat blood and infused with oleate to maintain constant concentrations of serum non-esterified fatty acids over a wide physiological range. Infusion of insulin opposed the antilipogenic effects of increasing concentrations of serum non-esterified fatty acids. Secretion of very-low-density-lipoprotein triacylglycerols was directly proportional to the concentration of serum non-esterified fatty acids and was increased by insulin. The secretion of newly-synthesized fatty acids in very-low-density-lipoprotein triacylglycerols decreased with increasing concentrations of serum non-esterified fatty acid. Insulin opposed this change. Cholesterol biosynthesis was unaffected by alterations in concentration of serum non-esterified fatty acid but was increased by insulin. Equilibrium concentrations of perfusate lactate and glucose were increased by serum non-esterified fatty acids but steady-state rates of hepatic glucose output and lactate uptake were unchanged. Insulin decreased perfusate glucose concentrations and abolished the increase in its concentration that resulted from increases in non-esterified fatty acid concentrations.     

Effects of hormones and of ethanol on the fructose 6-P-fructose 1,6-P2 futile cycle during gluconeogenesis in the liver

Carbon-14 was incorporated into C-6 of glucose from [1-¹⁴C]galactose during gluconeogenesis from dihydroxyacetone in liver cells from fasted rats, proving the existence of a futile cycle between fructose-6-P and fructose-1,6-P₂ under the conditions used. Using a steady-state model and assumed values for the rates of aldolase and glucose-6-P isomerase, the rates of phosphofructokinase were estimated, ranging from about 15% to nearly 40% of the net rate of gluconeogenesis. Glucagon depressed the rate of phosphofructokinase by as much as 85% and increased the rate of gluconeogenesis by up to 45%. l-epinephrine in the range from 10 to 100 μm also depressed phosphofructokinase, being nearly as effective as glucagon only at high concentrations. The effect of epinephrine was only partially reversed by 10 μm dl-propranolol. Ethanol (10 mm) depressed phosphofructokinase flux nearly as well as glucagon, but had no significant effect on the rate of gluconeogenesis from dihydroxyacetone.     

Pancreatic glucagon secretion during a short period of hemorrhage in anesthetized dogs

Glucagon has been implicated in the hormonal metabolic response to hemorrhage. However, evidence for this has been obtained largely from observations of circulating plasma glucagon concentration. A clear increase in the pancreatic glucagon secretion remains to be demonstrated. Plasma concentrations of pancreatic immunoreactive glucagon (IRG) and insulin (IRI) were determined in portal venous and aortic blood, and plasma glucose in aortic blood. Dogs were bled (approximately 15 mL/kg) until aortic systolic blood pressure dropped to approximately 50% (70.5 +/- 8.1 mmHg, n = 7) (1 mmHg = 133.32 Pa) of its control value (135 +/- 7.1 mmHg, n = 7), and the hemorrhagic hypotension was maintained for 10 min. The net portal venous IRG delivery rate rose significantly and continued to increase during the hemorrhagic hypotension despite a significant fall in the portal venous blood flow. Aortic IRG increased significantly along with the increase in portal venous IRG delivery rate (r = 0.838, n = 42, p less than 0.01). The portal venous delivery rate of IRI decreased significantly in response to hemorrhage. The aortic IRG/IRI concentration ratio increased significantly during the hemorrhage-induced hypotension. Aortic glucose concentration increased significantly 5 min after hemorrhage and continued to rise until the end of the hemorrhagic hypotension. The present study demonstrates that the secretion of pancreatic glucagon actually increases during the early phase of hemorrhage. The results also indicate that the increase in aortic IRG during the hemorrhagic hypotension is due to the increased pancreatic glucagon secretion. It is suggested that the pancreatic glucagon may be involved in the early hyperglycemic response to hemorrhage.     

Rabbit kidney function in vitro: the effect of colloids, energy substrate, a vasodilator, perfusion pressure, and bovine serum albumin

An in vitro perfusion system at 37 degrees C for the assessment of rabbit kidney function is described. The purpose of this assay system is to evaluate the effects of cryobiological manipulation on kidney function. The effect of the colloids dextran (MW = 70,000, 80,000, and 180,000) in the perfusate at 110 mm Hg were compared to a reduced perfusion pressure, colloid-free perfusate. Better function was obtained at lower perfusion pressure with the colloid-free perfusate. Less damage was noted histologically on light and electron microscopy. Investigation of energy substrates on rabbit kidney function demonstrated that butyrate, or lactate, in addition to glucose resulted in increased sodium and glucose reabsorption over glucose alone. Substrate-free perfused kidneys exhibited depressed Na transport. Lactate, and to some extent butyrate, decreased net glucose utilization. An alpha-adrenergic blocking agent, isoxsuprine, in the initial flush solution did not appear to be beneficial. An increase of perfusion pressure from 50 to 75 mm Hg resulted in an increase in GFR. Tubular function was enhanced by inclusion of small amounts of BSA in the perfusate.