Duration of Improved Muscle Glucose Uptake After Acute Exercise in Obese Zucker Rats

Skeletal muscle is insulin resistant in the obese Zucker rat. Endurance training reduces muscle insulin resistance, but the effects of a single acute exercise session on muscle insulin resistance in the obese Zucker rat are unknown. Therefore, insulin responsiveness of muscle glucose uptake was measured in 15-week-old obese rats either 1, 48, or 72 hours after two hours of intermittent exercise (3030 min; work:rest). Hindlimbs of sedentary lean (LS) and obese (OS) rats and exercised obese (OE) rats were perfused after a 10-hour fast under both basal (0 mU.ml^?1) and maximal (20 mU.ml^?1) insulin concentrations to measure net glucose uptake. Insulin responsiveness of net glucose uptake was significantly reduced in OS compared to LS (8.5 ± 1.6 vs 15.3 ± 2.0 μmol.g^?1.h^?1, respectively). Compared to OS, insulin responsiveness of net glucose uptake was significantly increased by 56% and 80% at 1 hour and 48 hours after acute exercise. However, 72 hours after acute exercise, the increased insulin responsiveness of net glucose uptake was no longer evident. These results indicate that improved responsiveness of muscle glucose uptake persists for at least 48 hours after two hours of acute intermittent exercise in 15-week-old obese Zucker rats. (OBESITY RESEARCH 1993; 1:295–302)     

Effects of glucagon, insulin and cyclic-AMP on mitochondrial calcium uptake in the liver.

The effects of glucagon and insulin administration $\text{in vivo}$ on hepatic mitochondrial Ca²⁺ uptake were compared with the effects of these hormones when they were added directly to the perfused liver. Glucagon administration increased mitochondrial calcium uptake both $\text{in vivo}$ and in the perfused liver. In contrast, while injection of insulin into rats stimulated, addition of insulin to the perfusate, inhibited Ca²⁺ uptake. Cyclic AMP, when added to the perfusate, also increased the uptake of Ca²⁺ by mitochondria, subsequently isolated. The possible implications of the results are discussed.     

The Distribution of Tissue Insulin Receptors in the Mouse by Whole – Body Autoradiography

Abstract

The distribution of insulin binding sites in the mouse was investigated by in vivo whole – body autoradiography. Male mice were injected intravenousely with ¹²⁵ I – insulin in the absence of and, in the presence of, excess unlabeled insulin. Three, 6, 15, 30 and 60 minutes after injection, the animals were perfused and subjected to autoradiographic procedures.

Specific insulin binding was observed in the choroid plexus, liver, gastrointestinal tract, spleen, pancreas, deferent duct, and Harderian gland.

In the liver and spleen, the distribution of binding sites was heterogeneous. In the liver, the density of the binding was higher around the branches of the portal vein than around the central vein. In the spleen, the marginal zone exhibited a higher density than the white and red pulp.

The kidney cortex, and the thyroid gland showed a high degree of insulin binding, but the binding was nonspecific. The binding of insulin to other tissues and organs, including the skeletal muscle and fat, was weak, and most of the binding was nonspecific.     

Ketogenesis evaluation in perfused liver of diabetic rats submitted to short‐term insulin‐induced hypoglycemia

Ketogenesis, inferred by the production of acetoacetate plus ß‐hydroxybutyrate, in isolated perfused livers from 24‐h fasted diabetic rats submitted to short‐term insulin‐induced hypoglycemia (IIH) was investigated. For this purpose, alloxan‐diabetic rats that received intraperitoneal regular insulin (IIH group) or saline (COG group) injection were compared. An additional group of diabetic rats which received oral glucose (gavage) (100 mg kg^?1) 15 min after insulin administration (IIH + glucose group) was included. The studies were performed 30 min after insulin (1.0 U kg^?1) or saline injection. The ketogenesis before octanoate infusion was diminished (p < 0.05) in livers from rats which received insulin (COG vs. IIH group) or insulin plus glucose (COG vs. IIH + glucose group). However, the liver ketogenic capacity during the infusion of octanoate (0.3 mM) was maintained (COG vs. IIH group and COG vs. IIH + glucose group). In addition, the blood concentration of ketone bodies was not influenced by the administration of insulin or insulin plus glucose. Taken together, the results showed that inspite the fact that insulin and glucose inhibits ketogenesis, livers from diabetic rats submitted to short‐term IIH which received insulin or insulin plus glucose showed maintained capacity to produce acetoacetate and ß‐hydroxybutyrate from octanoate. Copyright © 2009 John Wiley & Sons, Ltd.     

Regulation of rat liver phosphofructokinase by glucagon-induced phosphorylation

In perfused rat liver, the effects of various hormones on the stimulation of phosphorylation and allosteric properties of purified phosphorfructokinase were investigated. Rat livers were perfused with [³²P]phosphate followed with various hormones or cyclicAMP, and ³²P-labeled phosphofructokinase was isolated. ³²P incorporation into the enzyme and enzyme inhibition by ATP or citrate were determined. Only glucagon increased the ³²P incorporation into phosphofructokinase and this increase was approximately threefold. The cyclicAMP level was increased simultaneously approximately four- to fivefold compared to the control perfused liver. Similar results were obtained by perfusing the liver with cyclicAMP (0.1 mm). The phosphorylated phosphofructokinase showed a decrease in the K_i values for ATP (from 0.4 to 0.2 mm) and citrate (from 2 to 0.6 mm). Neither epinephrine nor insulin affected the extent of phosphorylation or the allosteric properties of the enzyme. The half-maximal concentration of glucagon required for phosphorylation of phosphofructokinase and modification of its allosteric properties was approximately 6 × 10^?11m. It is concluded that glucagon increases the inhibition of liver phosphofructokinase by ATP and citrate through phosphorylation of the enzyme involving a β-receptor-mediated cyclicAMP-dependent mechanism.     

Activation of the Protein Synthetic Capacity of Rat Liver Cell Sap by Amino-acids

CHANGES in the availability of amino-acids have marked effects on the rate of protein synthesis in rat liver^1–4. A high amino-acid concentration in the perfusion⁵ or incubation⁶ medium is needed to observe an effect of growth hormone in vitroon incorporation of precursors into protein and RNA of isolated hepatic tissue. We report here changes in the ability of cell-free systems to incorporate amino-acids into acid-insoluble material in vitrowhen they were prepared from slices incubated in various concentrations of amino-acids.     

The Effect of Insulin on Myofibrillar Protein Degradation in Normal and Streptozotocin-induced Diabetic Rats Measured by the Rate of Nτ-Methylhistidine Release from Perfused Hindquarters

Myofibrillar protein degradation was measured by the rate of N^τ-methylhistidine (MeHis) release from the perfused hindquarters in normal and streptozotocin-induced diabetic rats. In diabetic rats, the rate of MeHis release to the perfusate was elevated 2-fold compared with normal rats. The daily excretion of MeHis into urine was also increased 2-fold in the diabetic rats.

Insulin in the perfusate did not suppress the release of MeHis from the perfused muscle in normal rats. On the other hand, in diabetic rats, MeHis release was suppressed by insulin. The high concentration of free MeHis in the diabetic muscle was decreased to the normal level with insulin added to the perfusate. These results give further evidence to show that myofibrillar protein degradation is controlled by insulin.     

Active Absorption of Amino-acids in the Rectum of the Desert Locust (<Emphasis Type="Italic">Schistocerca gregaria</Emphasis>)

ALTHOUGH the concentration of amino-acids in the haemolymph of insects is high compared with that in most other groups of animals, there has been no rigorous demonstration that amino-acids are actively transported across any membrane system within the class Insecta. Treherne¹ established that ingested amino-acids are absorbed largely in the midgut of the desert locust by simple diffusion down a concentration gradient created by absorption of water and ions and Ramsay² concluded that the high concentrations of amino-acids in the secretion of the Malpighian tubules of the stick insect were the result of passive diffusion across the tubular epithelium. Because most of the water and essential solutes secreted by the Malpighian tubules are reabsorbed in the rectum of most insects³, selective retention and regulation of haemolymph amino-acids seem likely^2,4–6. Evidence for active reabsorption of amino-acids in the rectum has been sparse and inconclusive^1,2,5.     

The control by vasopressin of carbohydrate and lipid metabolism in the perfused rat liver

Vasopressin-induced glucose release from the perfused livers of fed rats is diminished in the presence of insulin or following adrenal ablation. The reduced rate of glucose release following vasopressin treatment in the perfused livers of adrenalectomized rats was restored towards the control value by cortisol treatment in vivo.Vaspressin did not influence the total rate of fatty acid synthesis in the livers of fed rats perfused with medium containing glucose and two concentrations of lactate. The contribution of these precursors to hepatic fatty acid synthesis and CO₂ production was similarly uninfluenced by vasopressin.Vasopressin caused a transient increase in the release of K⁺ by the perfused liver which was observed within 2 min of hormone administration.These results are discussed in relation to the possible mode of action of vasopressin in the liver.     

Chronic treatment with myo-inositol reduces white adipose tissue accretion and improves insulin sensitivity in female mice

Type 2 diabetes is a complex disease characterized by a state of insulin resistance in peripheral tissues such as skeletal muscle, adipose tissue or liver. Some inositol isomers have been reported to possess insulin-mimetic activity and to be efficient in lowering blood glucose level. The aim of the present study was to assess in mice the metabolic effects of a chronic treatment with myo-inositol, the most common stereoisomer of inositol. Mice given myo-inositol treatment (0.9 or 1.2 mg g^?1 day^?1, 15 days, orally or intraperitoneally) exhibited an improved glucose tolerance due to a greater insulin sensitivity. Mice treated with myo-inositol exhibited a decreased white adipose tissue accretion (?33%, P<.005) compared with controls. The decrease in white adipose tissue deposition was due to a decrease in adipose cell volume (?33%, P<.05), while no change was noticed in total adipocyte number. In skeletal muscle, in vivo as well as ex vivo myo-inositol treatment increased protein kinase B/Akt phosphorylation under baseline and insulin-stimulated conditions, suggesting a synergistic action of myo-inositol treatment and insulin on proteins of the insulin signalling pathway. Myo-inositol could therefore constitute a viable nutritional strategy for the prevention and/or treatment of insulin resistance and type 2 diabetes.     

Oxidation of branched-chain amino acids in skeletal muscle and liver of rat Effects of octanoate and energy state

The effect of octanoate on the oxidative decarboxylation of ¹⁴C-labeled amino acids has been studied in perfused hindquarter and liver of rat. Regulation of the branched-chain α-keto acid dehydrogenase has been further studied with α-[¹⁴C-1]ketoisovalerate in isolated rat muscle and liver mitochondria. (1) Octanoate has a stimulatory effect on the oxidation of branched-chain amino acids in perfused hindquarter. The oxidative decarboxylation of other amino acids are inhibited. Octanoate inhibits the oxidative decarboxylation of all amino acids in perfused liver. (2) The oxidation of valine is stimulated by octanoate and hexanoate also in isolated muscle mitochondria. The stimulatory effect is probably related to activation of the fatty acids since acyl-carnitines inhibit the oxidation. (3) The oxidation of α-ketoisovalerate in mitochondria is inhibited by competing substrates (pyruvate, α-ketoglutarate and succinate). This inhibition is counteracted by octanoate and ADP. (4) Low concentrations (1–5 μM) of 2,4-dinitrophenol (DNP) activates wheras higher concentrations inactivates the branched-chain α-keto acid dehydrogenase in intact but not in solubilized muscle mitochondria. The inactivation is counteracted by ATP, but is increased by octanoate. (5) The observations seem to suggest that the activation (like the inactivation) of branched-chain α-keto acid dehydrogenase in skeletal muscle is dependent on the mitochondrial energy state which therefore may regulate both activation and inactivation of the dehydrogenase.     

Absence of direct action of insulin on metabolism of the isolated perfused rat brain

The ability of insulin to influence directly the metabolism of the mammalian brain has been evaluated with an isolated, perfused rat brain preparation. Insulin was added to the perfusion fluid or was injected into the rat from which the isolated brain preparation was subsequently made. The spontaneous electrical activity of the brain, the rate of cerebral glucose consumption and the rate of efflux of K⁺ from the brain were not affected by insulin. We conclude that insulin either does not act directly on the brain or that its action is very small and/or very slow in comparison with its action on other tissues. We suggest that the effects on brain metabolism reported to occur after administering insulin and glucose to the intact animal may be secondary to the large stimulation of the metabolism of the liver and/or other organs.     

Glycogen synthesis in the perfused liver of the starved rat

1. In the isolated perfused liver from 48h-starved rats, glycogen synthesis was followed by sequential sampling of the two major lobes. 2. The fastest observed rates of glycogen deposition (0.68–0.82μmol of glucose/min per g fresh liver) were obtained in the left lateral lobe, when glucose in the medium was 25–30mm and when gluconeogenic substrates were present (pyruvate, glycerol and serine: each initially 5mm). In this situation there was no net disappearance of glucose from the perfusion medium, although ¹⁴C from [U-¹⁴C]glucose was incorporated into glycogen. There was no requirement for added hormones. 3. In the absence of gluconeogenic precursors, glycogen synthesis from glucose (30mm) was 0–0.4μmol/min per g. 4. When livers were perfused with gluconeogenic precursors alone, no glycogen was deposited. The total amount of glucose formed was similar to the amount converted into glycogen when 30mm-glucose was also present. 5. The time-course, maximal rates and glucose dependence of hepatic glycogen deposition in the perfused liver resembled those found in vivo in 48h-starved rats, during infusion of glucose. 6. In the perfused liver, added insulin or sodium oleate did not significantly affect glycogen synthesis in optimum conditions. In suboptimum conditions (i.e. glucose less than 25mm, or with gluconeogenic precursors absent) insulin caused a moderate acceleration of glycogen deposition. 7. These results suggest that on re-feeding after starvation in the rat, hepatic glycogen deposition could be initially the result of continued gluconeogenesis, even after the ingestion of glucose. This conclusion is discussed, particularly in connexion with the role of hepatic glucokinase, and the involvement of the liver in the glucose intolerance of starvation.     

The Structure of Calf Liver Cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript> at 2.8 Å Resolution

CYTOCHROME b₅ is a haem-containing protein in the microsomes of liver tissue. It interacts specifically with a flavo-protein, cytochrome b₅ reductase, which catalyses the transfer of electrons from NADH to the haem iron of the cytochrome¹. The microsomal cytochrome b₅ system has been implicated in fatty acid desaturation reactions² and a similar system in erythrocytes may catalyse the reduction of methaemoglobin³. Calf liver cytochrome b₅, solubilized by pancreatic lipase, has a molecular weight of 11,000 and consists of ninety-three amino-acids in the sequence shown in Fig. 1 (refs. 4 and 5). The haem group is non-covalently bound to the protein and can be removed reversibly by acid acetone treatment⁶.     

Transport and metabolism of pyridoxine in rat liver

Evidence, obtained with in situ perfused rat liver, indicated that pyridoxine is taken up from the perfusate by a non-concentrative process, followed by metabolic trapping. These conclusions were reached on the basis of the fact that at low concentrations (0.125 μM), the ³H of [³H]pyridoxine accumulated against a concentration gradient, but high concentrations (333 μM) of pyridoxine or 4-deoxypyridoxine prevented this apparent concentrative uptake. Under no conditions did the tissue water : perfusate concentration ratio of [³H]pyridoxine exceed unity.The perfused liver rapidly converted the labeled pyridoxine to pyridoxine phosphate, pyridoxal phosphate and pyridoxamine phosphate and released a substantial amount of pyridoxal and some pyridoxal phosphate into the perfusate. Since muscle and erythrocytes failed to oxidize pyridoxine phosphate to pyridoxal phosphate, it is suggested that the liver plays a major role in oxidizing dietary pyridoxine and pyridoxamine as their phosphate esters to supply pyridoxal phosphate which then reaches to other organs chiefly as circulating pyridoxal.     

Inhibitory effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport and On 125I-labelled insulin binding by rat soleus muscle

These experiments examined the effects of N-ethylmaleimide on insullin- and oxidant-stimulated sugar transport in soleus muscle in terms of the Thiol-Redox model for insulin-stimulated adipocyte sugar transport (Czech, M.P. (1976) J. Cell. Physiol. 89, 661–668). Brief exposure (1 min) to N-ethylmaleimide (0.3?10 nM) inhibited the stimulatory effect of insulin (0.1 U/ml) on D-[U-¹⁴C]xylose uptake by rat soleus muscle. N-Ethylmaleimide also inhibited the stimulatory effects of H₂O₂ (5 mM), diamide (0.2 mM) and vitamin K-5 (0.05 mM). This effect of N-ethylmaleimide on insulin was paralleled by the inhibition of ¹²⁵I-labelled insulin binding by the muscle. N-ethylmaleimide lowered muscle ATP; however, its effects on sugar transport and ¹²⁵I-labelled insulin binding could be dissociated from its effect on ATP. Exposing muscles to insulin prior to N-ethylmaleimide did not abolish the inhibitory effect of sulphydryl blockae on insulin-stimulated sugar transport, but did reduce the effect of the inhibitor by 20–30%. Conversely, when muscles were first allowed to bind ¹²⁵I-labelled insulin and then exposed to the inhibitor, there was no effect of N-ethylmaleimide on pre-bound insulin. Exposure to diamide or vitamin K-5 before N-ethylmaleimide (1 mM) attenuated the inhibitory effet of sulphydryl blockade but no protective effect was observed with H₂O₂. None of the oxidants protected against the inhibitory effect of 3 nM N-ethylmaleimide. It is concluded that there are two N-ethylmaleimide-sensitive sites involved in the activation of muscle sugar transport at the post-receptor level. One of these would appear to be similar to the Thiol-Redox site described in the adipocyte; the other site appears to be an essential sulphydryl group whose function does not involve oxidation to a disulphide.     

An extract of Artemisia dracunculus L. enhances insulin receptor signaling and modulates gene expression in skeletal muscle in KK-A mice

An ethanolic extract of Artemisia dracunculus L. (PMI 5011) has been observed to decrease glucose and insulin levels in animal models, but the cellular mechanisms by which insulin action is enhanced in vivo are not precisely known. In this study, we evaluated the effects of PMI 5011 to modulate gene expression and cellular signaling through the insulin receptor in skeletal muscle of KK-A^y mice. Eighteen male KK-A^y mice were randomized to a diet (w/w) mixed with PMI 5011 (1%) or diet alone for 8 weeks. Food intake, adiposity, glucose and insulin were assessed over the study, and at study completion, vastus lateralis muscle was obtained to assess insulin signaling parameters and gene expression. Animals randomized to PMI 5011 were shown to have enhanced insulin sensitivity and increased insulin receptor signaling, i.e., IRS-associated PI-3 kinase activity, Akt-1 activity and Akt phosphorylation, in skeletal muscle when compared to control animals (P<.01, P<.01 and P<.001, respectively). Gene expression for insulin signaling proteins, i.e., IRS-1, PI-3 kinase and Glut-4, was not increased, although a relative increase in protein abundance was noted with PMI 5011 treatment. Gene expression for specific ubiquitin proteins and specific 20S proteasome activity, in addition to skeletal muscle phosphatase activity, i.e., PTP1B activity, was significantly decreased in mice randomized to PMI 5011 relative to control. Thus, the data demonstrate that PMI 5011 increases insulin sensitivity and enhances insulin receptor signaling in an animal model of insulin resistance. PMI 5011 may modulate skeletal muscle protein degradation and phosphatase activity as a possible mode of action.     

Fat Oxidation,Fitness and Skeletal Muscle Expression of Oxidative/Lipid Metabolism Genes in South Asians: Implications for Insulin Resistance?

Background

South Asians are more insulin resistant than Europeans, which cannot be fully explained by differences in adiposity. We investigated whether differences in oxidative capacity and capacity for fatty acid utilisation in South Asians might contribute, using a range of whole-body and skeletal muscle measures.

Methodology/Principal Findings

Twenty men of South Asian ethnic origin and 20 age and BMI-matched men of white European descent underwent exercise and metabolic testing and provided a muscle biopsy to determine expression of oxidative and lipid metabolism genes and of insulin signalling proteins. In analyses adjusted for age, BMI, fat mass and physical activity, South Asians, compared to Europeans, exhibited; reduced insulin sensitivity by 26% (p = 0.010); lower VO_2max (40.6±6.6 vs 52.4±5.7 ml.kg⁻¹.min⁻¹, p = 0.001); and reduced fat oxidation during submaximal exercise at the same relative (3.77±2.02 vs 6.55±2.60 mg.kg⁻¹.min⁻¹ at 55% VO_2max, p = 0.013), and absolute (3.46±2.20 vs 6.00±1.93 mg.kg⁻¹.min⁻¹ at 25 ml O₂.kg⁻¹.min⁻¹, p = 0.021), exercise intensities. South Asians exhibited significantly higher skeletal muscle gene expression of CPT1A and FASN and significantly lower skeletal muscle protein expression of PI3K and PKB Ser473 phosphorylation. Fat oxidation during submaximal exercise and VO_2max both correlated significantly with insulin sensitivity index and PKB Ser473 phosphorylation, with VO_2max or fat oxidation during exercise explaining 10–13% of the variance in insulin sensitivity index, independent of age, body composition and physical activity.

Conclusions/Significance

These data indicate that reduced oxidative capacity and capacity for fatty acid utilisation at the whole body level are key features of the insulin resistant phenotype observed in South Asians, but that this is not the consequence of reduced skeletal muscle expression of oxidative and lipid metabolism genes.     

Amino acid--induced insulin release from the perfused rat pancreas. The influence of phenylalanine and tyrosine

The effects of L or D phenylalanine and L tyrosine on insulin release from the perfused rat pancreas were investigated. It was found that in the presence of D-glucose, all three amino-acids stimulate insulin secretion. After L-Phe had been removed from perfusate in the presence or absence of L-Tyr, the secondary rise of insulin release (an "off response") was noticed. This phenomenon did not follow to either D-Phe or L-Tyr.     

Effect of Insulin on Potassium Flux and Water and Electrolyte Content of Muscles from Normal and from Hypophysectomized Rats

It was reported previously that insulin hyperpolarized rat skeletal muscle and decreased K⁺ flux in both directions. The observations on K⁺ flux are now extended to take advantage of the greater sensitivity to insulin of hyperphysectomized rats. Insulin caused a shift of water from extracellular to intracellular space if glucose was present, but not in its absence. Insulin caused net gain of muscle fiber K⁺, though not necessarily an increase in K⁺ concentration in fiber water. It probably also decreased intrafiber Na⁺ and Cl^-. Insulin decreased K⁺ efflux. The effect was dose-dependent. Muscles from hypophysectomized rats were more sensitive to the action of insulin on K⁺ flux than were those from normal rats. The effect was demonstrable within the time resolution of the system, suggesting that insulin's action is on cell surfaces. K⁺ influx was also decreased by insulin. Bookkeeping suggests that some K⁺ influx be called active. Insulin seemed to decrease active K⁺ influx and passive K⁺ efflux. It is not resolved whether insulin has a true dual effect or whether it acts only on passive fluxes in both directions (the apparent action on active K⁺ influx being an artefact of incomplete definition of passive flux) or whether a single alteration in the membrane may affect both active and passive fluxes.