In vitro effects of glycosylated insulin and glucagon in perfused liver of the rat.

Using perfused liver of the rat, the hepatic uptake of glycosylated insulin (GI) and glucagon (GG) and its effects on hepatic glucose output were investigated. Insulin and glucagon were glycosylated in ambient high glucose concentration, and GI80 or GG80 (insulin or glucagon incubated with 0.08% glucose), GI350 or GG350 (incubated with 0.35% glucose), and GI1000 or GG1000 (incubated with 1% glucose) were prepared. The liver was perfused with the medium containing 1000 microU/ml insulin and 200 pg/ml glucagon or 200 microU/ml insulin and 1000 pg/ml glucagon. The fractional uptake of insulin or glucagon by perfused liver was not significantly altered by the glycosylation. In the liver perfused with 1000 microU/ml insulin and 200 pg/ml glucagon, glucose output was not changed by the glycosylation of the hormones, while in the liver perfused with 200 microU/ml insulin and 1000 pg/ml glucagon, GI1000 reduced its biological activity, as reflected by insulin-mediated decrease in glucose output. These results suggest that in the liver insulin incubated with markedly high concentration of glucose reduces its biological activity at a physiological concentration in the presence of high concentration of glucagon.     

Levonorgestrel and norethindrone alter insulin action on skeletal muscle of the female rat

Prospective studies of women receiving oral contraceptives suggest that the progestin component may induce insulin resistance and variable deterioration of glucose tolerance. Because the tissue sites and nature of this insulin antagonism are not well-defined, we studied the effects of two parenterally administered progestins, levonorgestrel (NG) and norethindrone (NE), on insulin-regulated glucose uptake and phenylalanine release by the perfused rat hindquarter. Female rats were injected sc for 14 days with NG or NE (10 or 30 micrograms/kg/day). Low-dose NG and high-dose NE approximate the per kg dose received by women taking a high-dose progestin oral contraceptive. Phenylalanine release and glucose uptake (nmole/min/g) by the perfused hindquarters were calculated from the A-V difference for each. Progestin treatment (30 micrograms/kg/d) significantly reduced phenylalanine release from hindquarters perfused without exogenous insulin. Hindquarters from the high dose NG and low and high dose NE rats perfused with insulin (100 microU/ml) released 22% less phenylalanine than control rats perfused with the same insulin concentration (P less than 0.01) but the net suppression below baseline was similar in the control and steroid-treated groups. High-dose progestin treatment did not alter glucose uptake by hindquarters perfused without exogenous insulin. Insulin (100 microU/ml) increased glucose uptake by hindquarters of control and progestin-treated rats as compared to animals in the same treatment group perfused without exogenous insulin (P less than 0.01). High dose NE impaired insulin-stimulated glucose uptake 24% below values of the control group (P less than 0.01). The other NE and NG doses had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitivity and responsiveness of glucose output to insulin in isolated perfused liver from dexamethasone-treated rats

To elucidate insulin action on hepatic glucose output (glycogenolysis) in the state exposed to an excess glucocorticoid, the fed rat liver was isolated and cyclically perfused with a medium containing 5 mM glucose and various concentrations of insulin. The rat was subcutaneously injected with 1 mg/kg of dexamethasone (Dex) for 7 days. Dex-treated rats showed marked increases of serum insulin and plasma glucose level compared with those in control rats. Hepatic glycogen contents in Dex group were markedly increased compared with those in control (115 +/- 5 and 28 +/- 4 mg/g, respectively). Insulin extraction rate in the perfused liver was not different between control and Dex group. Perfusate glucose level after 60 min perfusion was much higher in the Dex-treated rat liver than that of the control at 0 microU/ml insulin (34.5 +/- 2.5 vs 23.0 +/- 2.0 mM, P less than 0.01), and reduced to the nadir level (19.0 +/- 3.0 and 13.0 +/- 1.5 mM, respectively) at 100 microU/ml insulin in both groups, i.e., the decreasing rate in perfusate glucose level was not different between Dex and control group (43% and 44%, respectively). These results suggest that Dex-treatment augments hepatic glucose output, but does not affect the sensitivity and responsiveness of that to insulin.     

Glucose and insulin improve cardiac efficiency and postischemic functional recovery in perfused hearts from type 2 diabetic (db/db) mice

Hearts from type 2 diabetic (db/db) mice demonstrate altered substrate utilization with high rates of fatty acid oxidation, decreased functional recovery following ischemia, and reduced cardiac efficiency. Although db/db mice show overall insulin resistance in vivo, we recently reported that insulin induces a marked shift toward glucose oxidation in isolated perfused db/db hearts. We hypothesize that such a shift in metabolism should improve cardiac efficiency and consequently increase functional recovery following low-flow ischemia. Hearts from db/db and nondiabetic (db/+) mice were perfused with 0.7 mM palmitate plus either 5 mM glucose (G), 5 mM glucose and 300 microU/ml insulin (GI), or 33 mM glucose and 900 microU/ml insulin (HGHI). Substrate oxidation and postischemic recovery were only moderately affected by GI and HGHI in db/+ hearts. In contrast, GI and particularly HGHI markedly increased glucose oxidation and improved postischemic functional recovery in db/db hearts. Cardiac efficiency was significantly improved in db/db, but not in db/+ hearts, in the presence of HGHI. In conclusion, insulin and glucose normalize cardiac metabolism, restore efficiency, and improve postischemic recovery in type 2 diabetic mouse hearts. These findings may in part explain the beneficial effect of glucose-insulin-potassium therapy in diabetic patients with cardiac complications.     

The determinants of insulin extraction in the isolated perfused rat liver.

The effect of hepatic blood flow and portal insulin concentration on insulin extraction during one passage through the isolated perfused rat liver was studied. The percentage of insulin extracted was constant over the physiological range of blood flows (4 to 28 ml/min). The total amount of insulin extracted increased as the input concentration was raised from 48 to 4860 microU/ml with the highest level of extraction being approximately 700 microU of insulin per gram of liver per minute. When square wave input pulses of 243 to 4860 microU/ml were presented, about 5% of this insulin was retained and then released by the liver for periods up to 15 minutes after the cessation of the input. The possible roles of glucose and glucagon as regulators of insulin extraction were studied. Glucose (300 mg/dl), as compared with no glucose, led to a significant reduction of insulin extraction (22% vs. 38%, p less than 0.001). Glucagon had no effect on insulin extraction in the presence of constant levels of glucose. It is concluded, therefore, that glucose may increase circulating insulin levels not only by its well known stimulation of insulin secretion by the pancreas, but also by inhibiting insulin extraction by the liver.     

Effect of prior exercise and insulin on potential thermogenic systems in rat skeletal muscle.

We previously reported that insulin stimulates oxygen consumption by the perfused rat hindquarter after high-intensity exercise. The purpose of the present study was to examine whether fructose 6-phosphate-fructose 1,6-bisphosphate cycling or an uncoupling of mitochondrial respiration contributes to this phenomenon. Hindquarter skeletal muscle was analyzed after perfusion in the absence or presence of insulin (150-200 microU/ml) for high-energy phosphate content, fructose 6-phosphate-fructose 1,6-bisphosphate cycling of glucose before incorporation into glycogen, and mitochondrial respiratory control. Muscle from exercised rats perfused with insulin did not display greater rates of glucose cycling or mitochondrial uncoupling; in fact, insulin decreased the rate of fructose 6-phosphate cycling and tended to increase respiratory control in skeletal muscle mitochondria. In addition, the concentrations of ATP and creatine phosphate and the calculated free ADP level in muscle of previously exercised rats perfused with insulin were similar to those of control rats. The results do not exclude the possibility that localized subcellular changes in ADP occurred, however. In conclusion, the results suggest that insulin-induced increases in other substrate cycles, ion transport systems, and/or as yet unidentified energy-requiring processes account for the 25-30% increase in hindquarter oxygen consumption after intense exercise.     

In vitro simulation of calorie restriction-induced decline in glucose and insulin leads to increased insulin-stimulated glucose transport in rat skeletal muscle

In vivo calorie restriction [CR; consuming 60% of ad libitum (AL) intake] induces elevated insulin-stimulated glucose transport (GT) in skeletal muscle. The mechanisms triggering this adaptation are unknown. The aim of this study was to determine whether physiological reductions in extracellular glucose and/or insulin, similar to those found with in vivo CR, were sufficient to elevate GT in isolated muscles. Epitrochlearis muscles dissected from rats were incubated for 24 h in media with glucose (8 mM) and insulin (80 microU/ml) at levels similar to plasma values of AL-fed rats and compared with muscles incubated with glucose (5.5 mM) and/or insulin (20 microU/ml) at levels similar to plasma values of CR rats. Muscles incubated with CR levels of glucose and insulin for 24 h had a subsequently greater (P < 0.005) GT with 80 microU/ml insulin and 8 mM [(3)H]-3-O-methylglucose but unchanged GT without insulin. Reducing only glucose or insulin for 24 h or both glucose and insulin for 6 h did not induce altered GT. Increased GT after 24-h incubation with CR levels of glucose and insulin was not attributable to increased insulin receptor tyrosine phosphorylation, Akt serine phosphorylation, or Akt substrate of 160 kDa phosphorylation. Nor did 24-h incubation with CR levels of glucose and insulin alter the abundance of insulin receptor, insulin receptor substrate-1, GLUT1, or GLUT4 proteins. These results provide the proof of principle that reductions in extracellular glucose and insulin, similar to in vivo CR, are sufficient to induce an increase in insulin-stimulated glucose transport comparable to the increase found with in vivo CR.     

The B2 receptor of bradykinin is not essential for the post-exercise increase in glucose uptake by insulin-stimulated mouse skeletal muscle

Bradykinin can enhance skeletal muscle glucose uptake (GU), and exercise increases both bradykinin production and muscle insulin sensitivity, but bradykinin's relationship with post-exercise insulin action is uncertain. Our primary aim was to determine if the B2 receptor of bradykinin (B2R) is essential for the post-exercise increase in GU by insulin-stimulated mouse soleus muscles. Wildtype (WT) and B2R knockout (B2RKO) mice were sedentary or performed 60 minutes of treadmill exercise. Isolated soleus muscles were incubated with [3H]-2-deoxyglucose +/-insulin (60 or 100 microU/ml). GU tended to be greater for WT vs. B2RKO soleus with 60 microU/ml insulin (P=0.166) and was significantly greater for muscles with 100 microU/ml insulin (P<0.05). Both genotypes had significant exercise-induced reductions (P<0.05) in glycemia and insulinemia, and the decrements for glucose (approximately 14 %) and insulin (approximately 55 %) were similar between genotypes. GU tended to be greater for exercised vs. sedentary soleus with 60 microU/ml insulin (P=0.063) and was significantly greater for muscles with 100 microU/ml insulin (P<0.05). There were no significant interactions between genotype and exercise for blood glucose, plasma insulin or GU. These results indicate that the B2R is not essential for the exercise-induced decrements in blood glucose or plasma insulin or for the post-exercise increase in GU by insulin-stimulated mouse soleus muscle.     

Kupffer cells play a major role in insulin-mediated hepatic glucose uptake in vivo.

The effect of insulin on the in vivo glucose utilization by different hepatic cells was investigated using the euglycemic, hyperinsulinemic clamp, combined with the 2-deoxyglucose tracer technique. Rats were infused with insulin at a rate of 2.8 or 9.0 mU/min/kg for 220 min, resulting in plasma concentrations of the hormone of about 80 microU/ml and 340 microU/ml, respectively. Glucose use by the whole liver was elevated by more than 200% following insulin. However, glucose uptake by the parenchymal cells was only elevated by 50-60%. By contrast nonparenchymal cells were more responsive to insulin. Glucose uptake by endothelial cells was increased 100% and Kupffer cells displayed the most marked response to insulin showing a 3- to 6-fold increase in glucose uptake. These data indicate that the sinusoidal nonparenchymal cells are the major sites of the insulin-mediated increased glucose utilization by the liver.     

Phosphorylation of cardiac protein kinase B is regulated by palmitate

In this study isolated perfused working rat hearts were used to investigate the role of palmitate-regulated protein kinase B (PKB) phosphorylation on glucose metabolism. Rat hearts were perfused aerobically in working mode with 11 mM glucose and either 100 microU/ml insulin or 100 microU/ml insulin and 1.2 mM palmitate. PKB activity and phosphorylation state were reduced in the presence of 1.2 mM palmitate, which correlates with a decrease in glycolysis (47%), glucose oxidation (84%), and glucose uptake (43%). In contrast to skeletal muscle, neither p38 nor ERK underwent changes in their phosphorylation states in response to insulin or insulin and palmitate. Moreover, pharmacological restoration of glucose oxidation rates in hearts perfused with 1.2 mM palmitate demonstrated no increase in PKB phosphorylation state. In cultured mouse cardiac muscle HL-1 cells, insulin markedly increased PKB phosphorylation, which was blunted by pre- and cotreatment with 1.2 mM palmitate. However, neither palmitate nor C(2)-ceramide treatment of insulin-stimulated cells was able to accelerate PKB dephosphorylation beyond that observed following the removal of insulin alone. Taken together, these experiments show the control of PKB phosphorylation by palmitate is independent of ceramide and suggest that this signaling event may be an important regulator of myocardial glucose uptake and oxidation.     

Epinephrine inhibits insulin-stimulated muscle glucose transport.

We recently demonstrated that epinephrine could inhibit the activation by insulin of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase (PI3-kinase) in skeletal muscle (Hunt DG, Zhenping D, and Ivy JL. J Appl Physiol 92: 1285-1292, 2002). Activation of PI3-kinase is recognized as an essential step in the activation of muscle glucose transport by insulin. We therefore investigated the effect of epinephrine on insulin-stimulated glucose transport in both fast-twitch (epitrochlearis) and slow-twitch (soleus) muscle of the rat by using an isolated muscle preparation. Glucose transport was significantly increased in the epitrochlearis and soleus when incubated in 50 and 100 microU/ml insulin, respectively. Activation of glucose transport by 50 microU/ml insulin was inhibited by 24 nM epinephrine in both muscle types. This inhibition of glucose transport by epinephrine was accompanied by suppression of IRS-1-associated PI3-kinase activation. However, when muscles were incubated in 100 microU/ml insulin, 24 nM epinephrine was unable to inhibit IRS-1-associated PI3-kinase activation or glucose transport. Even when epinephrine concentration was increased to 500 nM, no attenuating effect was observed on glucose transport. Results of this study indicate that epinephrine is capable of inhibiting glucose transport activated by a moderate, but not a high, physiological insulin concentration. The inhibition of glucose transport by epinephrine appears to involve the inhibition of IRS-1-associated PI3-kinase activation.     

Stimulation of glucose uptake in skeletal muscle using bolus or continuous insulin delivery

We investigated glucose uptake in the non-cyclically perfused rat hindlimb in response to continuous infusion (CI) or bolus injection (BI) of insulin. Ten mM glucose was infused at 3 ml/min, venous glucose was monitored at two minute intervals, and glucose uptake was calculated on the basis of arteriovenous-difference and expressed as micron/min/100 g body wt. Insulin BI given every ten minutes equaled the amount of insulin given by CI for ten minutes. Insulin doses of 1500, 3000, 6000, and 45,000 microU/30 min showed no significant difference between the two modes of delivery in either onset of stimulation or maximal stimulation of glucose uptake. At the lowest insulin dose tested (1500 microU/30 min) neither BI nor CI stimulated glucose uptake above the control of 1.849 micron/min/100 g. A dose response curve for glucose uptake was obtained using insulin boluses ranging from 2000 to 20,000 microU. Insulin uptake by the muscle was always greater when insulin was administered CI. Net disappearance of immunoreactive insulin over the entire 30 minutes of perfusion was 29.4 +/- 2.6% for CI but only 7.1 +/- 1.6% for BI. Thus in the perfused rat hindlimb, stimulation of glucose uptake in skeletal muscle is comparable with BI and CI delivery of insulin but insulin uptake by the muscle is several-fold greater with CI delivery.     

Somatostatin-induced inhibition of insulin secretion from isolated islets of rat pancreas in presence of glucagon.

In order to study the oeffect of somatostatin on the endocrine pancreas directly, islets isolated from rat pancreas by collagenase were incubated for 2 hrs 1) at 50 and 200 mg/100 ml glucose in the absence and presence of somatostatin (1, 10 and 100 mg/ml) and2) at 200 mg/100 ml glucose together with glucagon (5 mug/ml), with or without somatostatin (100 ng/ml). Immunologically measurable insulin was determined in the incubation media at 0, 1 and 2 hrs. Insulin release was not statistically affected by any concentration stomatostatin. On the other hand, somatostatin exerted a significant inhibitory action on glucagon-potentiated insulin secretion (mean +/- SEM, mu1/2 hrs/10 islets: glucose and glucagon: 1253 +/- 92; glucose, glucagon and somatostatin: 786 +/- 76). The insulin output in th epresence of glucose, glucagon and somatostatin was also significantly smaller than in thepresence of glucose alone (1104 +/- 126) or of glucose and somatostatin (1061 +/- 122). The failure of somatostatin to affect glucose-stimulated release of insulin from isolated islets contrasts its inhibitory action on insulin secretion as observed in the isolated perfused pancreas and in vivo. This discrepancy might be ascribed to the isolation procedure using collagenase. However, somatostatin inhibited glucagon-potentiated insulin secretion in isolated islets which resulted in even lower insulin levels than obtained in the parallel experiments without glucagon. It is concluded that the hormone of the alpha cells, or the cyclic AMP system, might play a part in the machanism of somatostatin-induced inhibition of insulin release from the beta-cell.     

Evaluation of the isolated perfused rat hindquarter for the study of muscle metabolism

1. The metabolic integrity of a new isolated rat hindquarter preparation was studied. The hindquarter was perfused with a semi-synthetic medium containing aged human erythrocytes. More than 95% of the oxidative metabolism of the preparation was due to muscle, the remainder being due to bone, adipose tissue and, where present, skin. 2. Consumption of O(2), glucose utilization, glycerol release and lactate production were similar in the presence and in the absence of the skin, indicating that the latter contributed little to the overall metabolism of the preparation. 3. After 40min of perfusion, tissue concentrations of creatine phosphate, ATP and ADP were similar to those found in muscle taken directly from intact animals. The muscle also appeared normal under the electron microscope. 4. The hindquarter did not lose K(+) to the medium during a 30min perfusion. In the presence of insulin it had a net K(+) uptake. 5. Insulin caused a sixfold increase in glucose uptake, stimulated O(2) consumption by nearly 40% and depressed glycerol release to less than half the control value. 6. Bilateral sciatic-nerve stimulation caused severalfold increases in O(2) consumption and lactate production. In the absence of insulin nerve stimulation also enhanced glucose uptake; in the presence of insulin it did not further increase the already high rate of glucose uptake. 7. Rates of lactate production and O(2) consumption of the rat hindquarter in vivo and the isolated perfused hindquarter were very similar. 8. Ketone bodies were a major oxidative fuel in vivo of the hindquarter of a rat starved for 2 days. If the acetoacetate and 3-hydroxybutyrate removed by the tissue were completely oxidized, they would have accounted for 77% of the O(2) consumption. 9. Acetoacetate accounted for 84% of the ketone bodies removed by the hindquarter in vivo even though its arterial concentration was half that of 3-hydroxybutyrate. 10. Similar rates of acetoacetate and 3-hydroxybutyrate utilization were observed in the perfused hindquarter. 11. Acetoacetate utilization by the perfused hindquarter was not diminished by the addition of either oleate or insulin to the perfusate. 12. Oxidation of glucose to CO(2) accounted for less than 4% of the O(2) consumed by the perfused hindquarter in both the presence and the absence of insulin. 13. The results indicate that the isolated perfused hindquarter is a useful tool for studying muscle metabolism. They also suggest that ketone bodies, if present in sufficient concentration, are the preferred oxidative fuel of resting muscle.     

How muscle insulin sensitivity is regulated: testing of a hypothesis

Muscle contractions induce an increase in glucose transport. The acute effect of muscle contractions on glucose transport is independent of insulin and reverses rapidly after cessation of exercise. As the acute increase in glucose transport reverses, a marked increase in the sensitivity of muscle to insulin occurs. The mechanism for this phenomenon is unknown. We hypothesize that an increase in insulin sensitivity is a general phenomenon that occurs during reversal of an increase in cell surface GLUT4 induced by any stimulus, not just exercise. To test this hypothesis, epitrochlearis, rat soleus, and flexor digitorum brevis muscles were incubated for 30 min with a maximally effective insulin concentration (1.0 mU/ml). Muscles were allowed to recover for 3 h in the absence of insulin. Muscles were then exposed to 60 microU/ml insulin for 30 min followed by measurement of glucose transport. Preincubation with 1.0 mU/ml insulin resulted in an approximately 2-fold greater increase in glucose transport 3.5 h later in response to 60 microU/ml insulin than that which occurred in control muscles treated with 60 microU/ml insulin. Pretreatment of muscles with combined maximal insulin and exercise stimuli greatly amplified the increase in insulin sensitivity. The increases in glucose transport were paralleled by increases in cell surface GLUT4. We conclude that stimulation of glucose transport by any agent is followed by an increase in sensitivity of glucose transport to activation that is mediated by translocation of more GLUT4 to the cell surface.     

Effect of insulin on glucose uptake in near-term fetal lambs

Glucose clamp experiments were performed in 27 chronically catheterized, late-gestation fetal lambs in order to measure the effect of fetal insulin concentration on fetal glucose uptake at a constant glucose concentration. Fetal arterial blood glucose concentration was measured over a 30-min control period and then maintained at the control value by a variable glucose infusion into the fetus while insulin was infused at a constant rate into the fetus. Plasma insulin concentration increased from 21 +/- 10 (SD) to 294 +/- 179 (SD) microU X ml-1. The exogenous glucose infusion rate necessary to maintain constant glycemia during the plateau hyperinsulinemia averaged 4.3 +/- 1.6 (SD) mg X min-1 X kg-1. In a subset of 13 animals, total fetal exogenous glucose uptake (FGU; sum of glucose uptake from the placenta via the umbilical circulation plus the steady-state exogenous glucose infusion rate) was measured during the control and hyperinsulinemia period. FGU was directly related to insulin concentration (y = 4.24 + 0.07x) at insulin levels less than 100 microU/ml and increased 132% above control at insulin levels above 100 microU/ml. Hyperinsulinemia did not affect fetal glucose uptake from the placenta via the umbilical circulation. These studies demonstrate that insulin concentration is a major factor controlling glucose uptake in the near-term fetal lamb, and that an increase of fetal insulin does not affect the transport of glucose to the fetus from the placenta.     

The effects of amylin on carbohydrate metabolism in skeletal muscle in vitro and in vivo.

1. The effects of synthetic human amylin on basal and insulin-stimulated (100 and 1000 microunits/ml) rates of lactate formation, glucose oxidation and glycogen synthesis were measured in the isolated rat soleus muscle preparation incubated in the presence of various concentrations of glucose (5, 11 and 22 mM). 2. The rate of glucose utilization was increased by about 2-fold by increasing the glucose concentration from 5 to 22 mM. 3. Synthetic human amylin (10 nM) significantly inhibited (by 46-56%) glycogen synthesis, irrespective of the concentration of insulin or glucose present in the incubation medium. 4. Amylin (10 nM) did not affect insulin-stimulated rates of 2-deoxy[3H]glucose transport and phosphorylation. 5. Intraperitoneal administration of insulin (100 micrograms/kg) to rats in vivo stimulated the rate of [U-14C]glucose incorporation into glycogen in the diaphragm by about 80-fold. This rate was decreased (by 28%) by co-administration of amylin (66 micrograms/kg).     

Insulin and exercise stimulate muscle alpha-aminoisobutyric acid transport by a Na+-K+-ATPase independent pathway

Sodium ions are required for the active transport of amino acids such as alpha-aminoisobutyric acid (AIB) into skeletal muscle. To examine the role of Na+-K+-ATPase in this phenomenon, studies were carried out using the isolated perfused rat hindquarter preparation. Perfusion for 30 min with ouabain at a dose sufficient to inhibit the Na+-K+ pump (10(-4) M) inhibited the basal rate of AIB uptake in all muscles studied by up to 80%. However, it failed to inhibit the stimulation of AIB uptake, either by insulin (200 microU/ml) or electrically-induced muscle contractions. The increase in K+ release by the hindquarter in the presence of ouabain was the same under all conditions suggesting comparable inhibition of the Na+-K+ pump. These studies suggest that the basal, but not insulin or exercise-stimulated AIB transport into muscle is acutely dependent on a functional Na+-K+ pump. They also suggest that stimulated and basal uptake of AIB involve different mechanisms.     

Inhibition of glucose uptake and glycogenolysis by availability of oleate in well-oxygenated perfused skeletal muscle

The effects of exogenous oleate on glucose uptake, lactate production and glycogen concentration in resting and contracting skeletal muscle were studied in the perfused rat hindquarter. In preliminary studies with aged erythrocytes at a haemoglobin concentration of 8g/100ml in the perfusion medium, 1.8mm-oleate had no effect on glucose uptake or lactate production. During these studies it became evident that O(2) delivery was inadequate with aged erythrocytes. Perfusion with rejuvenated human erythrocytes at a haemoglobin concentration of 12g/100ml resulted in a 2-fold higher O(2) uptake at rest and a 4-fold higher O(2) uptake during muscle contraction than was obtained with aged erythrocytes. Rejuvenated erythrocytes were therefore used in subsequent experiments. Glucose uptake and lactate production by the well-oxygenated hindquarter were inhibited by one-third, both at rest and during muscle contraction, when 1.8mm-oleate was added to the perfusion medium. Addition of oleate also significantly protected against glycogen depletion in the fast-twitch red and slow-twitch red types of muscle, but not in white muscle, during sciatic-nerve stimulation. In the absence of added oleate, glucose was confined to the extracellular space in resting muscle. Addition of oleate resulted in intracellular glucose accumulation in red muscle. Contractile activity resulted in accumulation of intracellular glucose in all three muscle types, and this effect was significantly augmented in the red types of muscle by perfusion with oleate. The concentrations of citrate and glucose 6-phosphate were also increased in red muscle perfused with oleate. We conclude that, as in the heart, availability of fatty acids has an inhibitory effect on glucose uptake and glycogen utilization in well-oxygenated red skeletal muscle.     

Effects of insulin and glucose on the production and utilization of glucose in sheep (Ovis aries)

1. The rates of appearance (RA) and metabolic clearance rates (MCR) of glucose were determined during the infusion of insulin plus glucose in sheep. 2. During euglycaemia 50% inhibition of RA of endogenous glucose occurred at insulin concentrations of 60 microU/ml in arterial plasma. 3. During euglycaemia the MCR of glucose doubled with an increment of about 100 microU/ml of insulin. 4. Hypoglycaemia was associated with a reduction in insulin's suppression of RA of endogenous glucose and hyperglycaemia significantly reduced the increase in MCR due to insulin.