Insulin-like growth factor I supports differentiation of cultured osteoblast-like cells

Rat calvaria cells grown in culture for one week had properties of osteoblasts: a high content in alkaline phosphatase and a marked cyclic AMP response to parathyroid hormone (PTH). In short-term experiments, insulin-like growth factor I (IGF I) stimulated the incorporation of [14C] glucose into glycogen. When IGF I was present in the medium during 6 days the cell number increased slightly and there was a substantial, disproportionate rise in alkaline phosphatase activity of the cultures. Thus, IGF I stimulates growth, and in addition, and in contrast to other growth factors, mainly enhances differentiation of osteoblasts.     

Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling

Insulin-stimulated glucose uptake and incorporation of glucose into skeletal muscle glycogen contribute to physiological regulation of blood glucose concentration. In the present study, glucose handling and insulin signaling in isolated rat muscles with low glycogen (LG, 24-h fasting) and high glycogen (HG, refed for 24 h) content were compared with muscles with normal glycogen (NG, rats kept on their normal diet). In LG, basal and insulin-stimulated glycogen synthesis and glycogen synthase activation were higher and glycogen synthase phosphorylation (Ser(645), Ser(649), Ser(653), Ser(657)) lower than in NG. GLUT4 expression, insulin-stimulated glucose uptake, and PKB phosphorylation were higher in LG than in NG, whereas insulin receptor tyrosyl phosphorylation, insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity, and GSK-3 phosphorylation were unchanged. Muscles with HG showed lower insulin-stimulated glycogen synthesis and glycogen synthase activation than NG despite similar dephosphorylation. Insulin signaling, glucose uptake, and GLUT4 expression were similar in HG and NG. This discordant regulation of glucose uptake and glycogen synthesis in HG resulted in higher insulin-stimulated glucose 6-phosphate concentration, higher glycolytic flux, and intracellular accumulation of nonphosphorylated 2-deoxyglucose. In conclusion, elevated glycogen synthase activation, glucose uptake, and GLUT4 expression enhance glycogen resynthesis in muscles with low glycogen. High glycogen concentration per se does not impair proximal insulin signaling or glucose uptake. "Insulin resistance" is observed at the level of glycogen synthase, and the reduced glycogen synthesis leads to increased levels of glucose 6-phosphate, glycolytic flux, and accumulation of nonphosphorylated 2-deoxyglucose.     

Glucose uptake in vivo in skeletal muscles of insulin-injected chicks

Glucose uptake across the plasma membrane in animal cells plays a crucial role in whole-body glucose homeostasis. Insulin-stimulated glucose transport activity in vivo in several tissues was estimated using the 2-deoxy-D-[1-(3)H]glucose ([(3)H]2DG) uptake determination method. A tracer dose of [(3)H]2DG was injected intravenously into 8-day-old chicks (Gallus gallus) administered simultaneously or previously with porcine insulin (40 microg/kg BW). After 10 or 20 min, several major tissues, including skeletal and cardiac muscle, were sampled and their 2-deoxy-D-[1-(3)H]glucose 6-phosphate content analyzed. Plasma glucose concentration and [(3)H]2DG radioactivity were lowered by insulin within 20 min of [(3)H]2DG administration, while the plasma [(3)H]2DG/glucose ratio was not significantly different between chicks injected with insulin and their control counterparts. A marked uptake of 2DG was observed in cardiac tissue and brain, followed by kidney and skeletal muscles. In skeletal muscles, insulin increased the 2DG uptake in soleus, extensor digitorum longus and pectoralis superficialis muscles. On the other hand, no significant increases in insulin-induced 2DG uptake were detected in cardiac muscle or adipose tissue compared to controls. The results show that glucose transport across the plasma membrane in vivo in most skeletal muscles tested, but not cardiac muscle, was increased by insulin administration to chicks. These findings suggest that an insulin-responsive glucose transport mechanism is present in chickens, even though they intrinsically lack GLUT4 homologous gene, the insulin-responsive glucose transporter in mammals.     

Effects of epidermal growth factor (urogastrone) on gluconeogenesis, glucose oxidation, and glycogen synthesis in isolated rat hepatocytes

Using isolated rat hepatocytes, we studied the effect of epidermal growth factor (urogastrone) (EGF-URO) on the incorporation of [3-14C]pyruvate into glucose and glycogen, on the incorporation of [U-14C]glucose into glycogen, and on the oxidation of [U-14C]glucose to 14CO2. The effects of EGF-URO were compared with those of glucagon and insulin. EGF-URO, with an EC50 of 0.2 nM, enhanced by 34% (maximal stimulation) the conversion of [3-14C]pyruvate into glucose; no effect was observed on the oxidation of glucose to CO2 and on the incorporation of either pyruvate or glucose into glycogen. The effect of EGF-URO on pyruvate conversion to glucose was observed only when hepatocytes were preincubated with EGF-URO for 40 min prior to the addition of substrate. Glucagon (10 nM) increased the incorporation of [3-14C]pyruvate into glucose (44% above control); however, unlike EGF-URO, glucagon stimulated gluconeogenesis better without than with a preincubation period. Neither insulin nor EGF-URO (both 10 nM) affected the incorporation of [U-14C]glucose into glycogen during a 20-min incubation period. However, at longer time periods of incubation with the substrate (60 instead 20 min), insulin (but not EGF-URO) increased the incorporation of [14C]glucose into glycogen; EGF-URO counteracted this stimulatory effect of insulin. In contrast with previous data, our work indicates that EGF-URO can, under certain conditions, counteract the effects of insulin and, like glucagon, promote gluconeogenesis in isolated rat hepatocytes.     

Receptor binding and biological potency of despentapeptide insulin

The receptor binding and biological potency of despentapeptide insulin (DPI) was assessed in human adipocytes, rat adipocytes and rat hepatocytes. DPI displayed a lower affinity for binding to both human adipocytes (half-maximum displacement at 0.89 +/- 0.04 and 0.20 +/- 0.02 nmol/l for DPI and insulin respectively; P less than 0.001) and rat adipocytes (half-maximum displacement at 7.12 +/- 1.06 and 1.14 +/- 0.18 nmol/l respectively, P less than 0.05). However, although DPI was less potent than unmodified insulin in stimulating glucose uptake in rat adipocytes (half-maximal stimulation at 2.0 +/- 0.67 and 0.47 +/- 0.18 nmol/l respectively; P less than 0.05), DPI was equipotent with insulin in human adipocytes (half-maximal stimulation at 0.034 +/- 0.001 and 0.027 +/- 0.001 nmol/l respectively; P greater than 0.2). In rat hepatocytes, DPI was twofold less potent in binding displacement activity (half-maximum displacement at 3.8 +/- 0.9 and 1.7 +/- 0.3 nmol/l respectively; P less than 0.01) but appeared to be equivalent in stimulating amino butyric acid uptake (half-maximum stimulation at 0.98 +/- 0.12 and 0.95 +/- 0.26 nmol/l respectively). The difference in affinity of DPI binding to rat liver membranes was less marked (1.3 fold decreased compared with insulin: 5.3 +/- 0.7 and 4.2 +/- 0.6 nmol/l respectively; P less than 0.001). Thus, the decreased receptor affinity of DPI was reflected in decreased biological potency in rat adipocytes, but not in human adipocytes nor rat hepatocytes. These data suggest differences in the binding-action linking in the cells of different tissues and different species.     

Increased beta-oxidation in muscle cells enhances insulin-stimulated glucose metabolism and protects against fatty acid-induced insulin resistance despite intramyocellular lipid accumulation

Skeletal muscle insulin resistance may be aggravated by intramyocellular accumulation of fatty acid-derived metabolites that inhibit insulin signaling. We tested the hypothesis that enhanced fatty acid oxidation in myocytes should protect against fatty acid-induced insulin resistance by limiting lipid accumulation. L6 myotubes were transduced with adenoviruses encoding carnitine palmitoyltransferase I (CPT I) isoforms or beta-galactosidase (control). Two to 3-fold overexpression of L-CPT I, the endogenous isoform in L6 cells, proportionally increased oxidation of the long-chain fatty acids palmitate and oleate and increased insulin stimulation of [(14)C]glucose incorporation into glycogen by 60% while enhancing insulin-stimulated phosphorylation of p38MAPK. Incubation of control cells with 0.2 mm palmitate for 18 h caused accumulation of triacylglycerol, diacylglycerol, and ceramide (but not long-chain acyl-CoA) and decreased insulin-stimulated [(14)C]glucose incorporation into glycogen (60%), [(3)H]deoxyglucose uptake (60%), and protein kinase B phosphorylation (20%). In the context of L-CPT I overexpression, palmitate preincubation produced a relative decrease in insulin-stimulated incorporation of [(14)C]glucose into glycogen (60%) and [(3)H]deoxyglucose uptake (40%) but did not inhibit phosphorylation of protein kinase B. Due to the enhancement of insulin-stimulated glucose metabolism induced by L-CPT I overexpression itself, net insulin-stimulated incorporation of [(14)C]glucose into glycogen and [(3)H]deoxyglucose uptake in L-CPT I-transduced, palmitate-treated cells were significantly greater than in palmitate-treated control cells (71 and 75% greater, respectively). However, L-CPT I overexpression failed to decrease intracellular triacylglycerol, diacylglycerol, ceramide, or long-chain acyl-CoA. We propose that accelerated beta-oxidation in muscle cells exerts an insulin-sensitizing effect independently of changes in intracellular lipid content.     

Beta-D-Galactoside transport in Escherichia coli: Mr determination of the transport protein in organic solvent

Parathyroid hormone stimulates [U-14C]glucose incorporation into glycogen of cultured osteoblast-like calvaria cells. This effect is detectable only several hours after the addition of PTH and it is mimicked by dibutyryl cyclic AMP. In contrast to insulin (in pharmacological concentrations), PTH enhances glycogen formation only in calvaria cells, but not in fibroblasts. Insulin-like growth factor I in physiological concentrations promotes glycogen-synthesis shortly after addition.     

The effect of human placental lactogen upon the metabolism of rat and human adipose tissue.

The metabolic effects of human placental lactogen (HPL) on rat and human white fat were tested in vitro. When tested against rat tissue, HPL resembled insulin in stimulating uptake of glucose and incorporation of [14C] glucose into CO2, triglyceride and glycogen, but differed from insulin in stimulating glycerol release and in failing to stimulate the incorporation of [14C] The stimulation of [14C] glucose incorporation and the inhibition of glycerol release by insulin were antagonized by HPL. The effects of HPL on human white fat resembled those on rat white fat,except that glycerol release was not stimulated in human tissue. The possible role of HPL in causing the diabetogenic stress of pregnancy is discussed in the light of these findings.     

Isolation and Identification of l-Deoxy-l-(L-asparagino)-D-fructose Formed in the Autoclaved Culture Medium

To elucidate the effect of nutrition during induction on peripheral muscle responsiveness to insulin, the incorporation of radiolabeled glucose to glycogen and the uptake of radiolabeled deoxyglucose were studied in isolated diaphragms from the fetuses of normal and diabetic pregnant rats in vitro. Basal- and insulin-stimulated incorporation of [1-¹⁴C]glucose into diaphragm glycogen were greater in the fetuses of diabetic mothers (IDM) than in normal fetuses, but there was no difference in the degree of stimulation by insulin of labeled glucose into glycogen between normal fetuses and IDM. Diaphragms from normal fetuses and IDM had the same basal uptake of 2-deoxy-[1-³H]glucose as well as insulin-stimulated uptake. Consequently the sensitivity of glucose uptake to insulin was similar both in normal fetuses and IDM. These data indicate that glucose utilization (incorporation of labeled glucose into glycogen) was increased in IDM, but that the response of glucose uptake and glycogenesis to insulin was not altered.     

Adaptations of glycogen metabolism in rat epididymal adipose tissue during fasting and refeeding.

It is well documented that adipose tissue glycogen content decreases during fasting and increases above control during refeeding. We now present evidence that these fluctuations result from adaptations intrinsic to adipose tissue glycogen metabolism that persist in vitro: in response to insulin (1 milliunit/ml), [3H]glucose incorporation into rat fat pad glycogen was reduced to 10% of control after a 3-day fast; incorporation increased 6-fold over fed control on the 4th day of refeeding following a 3-day fast. We have characterized this adaptation with regard to alterations in glycogen synthase and phosphorylase activity. In addition, we found that incubation of fat pads from fasted rats with insulin (1 milliunit/ml) increased glucose-6-P content, indicating that glucose transport was not the rate-limiting step for glucose incorporation into glycogen in the presence of insulin. In contrast, feeding a fat-free diet resulted in dramatic increases in glycogen content of fat pads without a concomitant increase in glucose incorporation into glycogen in response to insulin (1 milliunit/ml). Thus, fasting and refeeding appeared to alter insulin action on adipose tissue glycogen metabolism more than this dietary manipulation.     

L-Glutamate and Insulin Enhance Glycogen Synthesis in Cultured Astrocytes from the Rat Brain Through Different Intracellular Mechanisms

The effects of L-glutamate and insulin on glycogen synthesis in astrocytes were examined. L-Glutamate and insulin both stimulated glycogen synthesis in primary cultures of rat astrocytes in a dose-dependent manner, as measured by the incorporation of 14C from [14C]glucose into glycogen. D-Aspartate also increased the incorporation of 14C into glycogen. When insulin and L-glutamate were added together, the glycogen synthesis as well as glycogen content of the cells was additively increased. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, had little effect on glycogen synthesis induced by L-glutamate, whereas it suppressed the insulin-induced glycogen synthesis. These results suggest that the insulin- and L-glutamate-induced glycogen syntheses are mediated by different intracellular mechanisms. In fact, insulin stimulated the conversion of glycogen synthase b to glycogen synthase a, which was suppressed by wortmannin. L-Glutamate and D-aspartate, however, did not increase the level of glycogen synthase a activity. By contrast, L-glutamate increased 2-deoxy-D-[3H]glucose uptake by the astrocytes, whereas insulin did not affect the uptake. These results suggest that insulin stimulates glycogen synthesis in astrocytes by activating glycogen synthase, which is dependent on a wortmannin-sensitive signaling pathway. L-Glutamate, however, enhances the glucose uptake, which contributes to the increase in glycogen synthesis in the cells.     

An antibody that blocks insulin-like growth factor (IGF) binding to the type II IGF receptor is neither an agonist nor an inhibitor of IGF-stimulated biologic responses in L6 myoblasts

To better define the biologic function of the type II insulin-like growth factor (IGF) receptor, we raised a blocking antiserum in a rabbit by immunizing with highly purified rat type II IGF receptor. On immunoblots of crude type II receptor preparations, only bands corresponding to the type II IGF receptor were seen with IgG 3637, indicating that the antiserum was specific for the type II receptor. Competitive binding and chemical cross-linking experiments showed that IgG 3637 blocked binding of 125I-IGF-II to the rat type II IGF receptor, but did not block binding of 125I-IGF-I to the type I IGF receptor, nor did IgG 3637 block binding of 125I-insulin to the insulin receptor. In addition, IgG 3637 did not inhibit the binding of 125I-IGF-II to partially purified 150- and 40-kDa IGF carrier proteins from adult and fetal rat serum. L6 myoblasts have both type I and type II IGF receptors. IGF-I was more potent than IGF-II in stimulating N-methyl-alpha-[14C]aminoisobutyric acid uptake, 2-[3H]deoxyglucose uptake, and [3H]leucine incorporation into cellular proteins. IgG 3637 did not stimulate either 2-[3H]deoxyglucose uptake, N-methyl-alpha-[14C]aminoisobutyric acid uptake, or [3H]leucine incorporation into protein when tested alone. Furthermore, IgG 3637 at concentrations sufficient to block type II receptors under conditions of the uptake and incorporation experiments did not cause a shift to the right of the dose-response curve for stimulation of these biologic functions by IGF-II. We conclude that the type II IGF receptor does not mediate IGF stimulation of N-methyl-alpha-[14C]aminoisobutyric acid and 2-[3H]deoxyglucose uptake and protein synthesis in L6 myoblasts; presumably, the type I receptor mediates these biologic responses. The anti-type II receptor antibody inhibited IGF-II degradation in the media by greater than 90%, suggesting that the major degradative pathway for IGF-II in L6 myoblasts utilizes the type II IGF receptor.     

Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions

Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms.     

Effects of insulin on the pattern of glucose metabolism in the perfused working and Lagendorff heart of normal and insulin-deficient rats

1. The metabolic pattern of [U-(14)C]glucose in the isolated rat heart has been studied, with both retrograde aortic (Langendorff) and atrially (working) perfused preparations in the presence and absence of insulin, in normal animals, animals rendered insulin-deficient (by injection of anti-insulin serum 1hr. before excision of the heart) and animals rendered diabetic by streptozotocin injection 7 days before use. 2. Radioautochromatograms of heart extracts show that the pattern of glucose metabolism in heart muscle is more complex than in diaphragm muscle. In addition to (14)CO(2), glycogen, oligosaccharides, phosphorylated sugars and lactate (the main metabolites formed from [(14)C]glucose in diaphragm muscle), (14)C label from [(14)C]glucose appears in heart muscle in glutamate, glutamine, aspartate and alanine, and in tricarboxylic acid-cycle intermediates. 3. By a quantitative scanning technique of two-dimensional chromatograms it was found that a mechanical work load stimulates glucose metabolism, increasing by a factor of 2-3 incorporation of (14)C into all the metabolites mentioned above except lactate and phosphorylated sugars, into which (14)C incorporation is in fact diminished; (14)CO(2) production is equally stimulated. 4. Addition of insulin to the perfusion fluid of the working heart causes increases in (14)C incorporation, by a factor of about 1.5 into (14)CO(2), by a factor of about 3-5 into glycogen, lactate and phosphorylated sugars, by a factor of about 2-3 into glutamate and tricarboxylic acid-cycle intermediates and by a factor of about 0.5 into aspartate, whereas incorporation into alanine and glutamine is not affected. The effect of a work load on the pattern of glucose metabolism is thus different from that of insulin. 5. Increasing the concentration of glucose in the perfusion fluid from 1 to 20mm leads to changes of the pattern of glucose metabolism different from that brought about by insulin. (14)CO(2) production steadily increases whereas [(14)C]lactate and glycogen production levels off at 10mm-glucose, at values well below those reached in the presence of insulin. 6. In Langendorff hearts of animals rendered insulin-deficient by anti-insulin serum or streptozotocin, glucose uptake, formation of (14)CO(2) and [(14)C]lactate, and (14)C incorporation into glycogen and oligosaccharides are decreased. In insulin-deficient working hearts, however, glucose uptake and (14)CO(2) production are normal, whereas incorporation of (14)C into glycogen and [(14)C]lactate production are greatly decreased. 7. Insulin added to the perfusion fluid restores (14)C incorporation from glucose into (14)CO(2), glycogen and lactate in the Langendorff heart from animals rendered insulin-deficient by anti-insulin serum; in hearts from streptozotocin-diabetic animals addition of insulin restores (14)C incorporation into glycogen and lactate, but (14)CO(2) production remains about 50% below normal. 8. The bearing of these results on the problem of the mode of action of insulin is discussed.     

Dehydroepiandrosterone (DHEA) stimulates glucose uptake in rat adipocytes: activation of phospholipase D

We examined the effect of dehydroepiandrosterone (DHEA) on glucose uptake and phospholipase D (PLD) activation in rat adipocytes. DHEA (1 microM) provoked a twofold increase in [3H]2-deoxyglucose (DG) uptake for 30 min. Incorporation of [3H]glycerol into diacylglycerol was increased 150% above basal level for 20 min after stimulation with 1 microM DHEA. DHEA increased PLD activity, measured by the incorporation into [3H]phosphatidylethanol in [3H]palmitate labelled rat adipocytes, or by [3H]choline release in [methyl-(3)H]choline labeled rat adipocytes. Our results suggest that DHEA stimulates glucose uptake with activation of PLD in rat adipocytes.     

Accumulation of 2-deoxy-D-glucose-6-phosphate as a measure of glucose uptake in the isolated perfused heart: a 31P NMR study

The accumulation of 2-deoxy-D-glucose-6-phosphate (2DG6P), detected using 31P NMR spectroscopy, has been used as a measure of the rate of glucose uptake, yet the accuracy of this measurement has not been verified. In this study, isolated rat hearts were perfused with different substrates or isoproterenol for 30 min before measurement of either 2DG6P accumulation or [2-3H]glucose uptake, without and with insulin. Basal contractile function and metabolite concentrations were the same for all hearts. The basal rates of 2DG6P accumulation differed significantly, depending on the preceding perfusion protocol, and were 38-60% of the [2-3H]glucose uptake rates, whereas insulin-stimulated 2DG6P accumulation was the same or 71% higher than the [2-3H]glucose uptake rates. Therefore the ratio of 2DG6P accumulation/[2-3H]glucose uptake rates varied from 0.38 to 1.71, depending on the prior perfusion conditions or the presence of insulin. The rates of 2DG6P hydrolysis were found to be proportional to the intracellular 2DG6P concentrations, with a K(m) of 17.5mM and V(max) of 1.4 micromol/g dry weight/min. We conclude that the rates of 2DG6P accumulation do not accurately reflect glucose uptake rates under all physiological conditions in the isolated heart and should be used with caution.     

Structural analogs of human insulin-like growth factor (IGF) I with altered affinity for type 2 IGF receptors

We have used site-directed mutagenesis of a synthetic gene for insulin-like growth factor (IGF) I to prepare three analogs in which specific residues in the A region are replaced with the corresponding residues in the A chain of insulin. The analogs are [Ile41, Glu45, Gln46, Thr49, Ser50, Ile51, Ser53, Tyr55, Gln56]IGF I (A chain mutant), in which residue 41 is changed from threonine to isoleucine and residues 42 to 56 of the A region are replaced, [Thr49, Ser50, Ile51]IGF I, and [Tyr55, Gln56]IGF I. These analogs are all equipotent to IGF I at the type 1 IGF receptor in human placental membranes, and in stimulating the incorporation of [3H]thymidine into DNA in the rat vascular smooth muscle cell line A10. However, the A chain mutant and [Thr49, Ser50, Ile51]IGF I have greater than 20-fold lower relative affinity for the type 2 IGF receptor of rat liver membranes, respectively. In contrast, [Tyr55, Gln56]IGF I has 7-fold higher affinity than IGF I for the type 2 IGF receptor. Residues 49, 50, and 51 in IGF I are Phe-Arg-Ser and are strictly conserved in IGF II. Residues 55 and 56 of IGF I and the corresponding residues in IGF II are Arg-Arg and Ala-Leu, respectively. Thus, the presence of the charged residues at these positions in IGF I appears to be responsible, in part, for the lower affinity of IGF I for the type 2 IGF receptor. In addition to the alterations in affinity for the type 2 IGF receptor, the A chain mutant has a 7-fold increase in affinity for insulin receptors, and [Thr49, Ser50, Ile51]IGF I has a 4-fold lower affinity for acid-stable human serum binding protein. These data strongly suggest that specific determinants in the A region of IGF I are important for maintaining binding to the type 2 IGF receptor, and that these determinants are different from those required for maintaining high affinity for the type 1 IGF receptor.     

Biological effects of sulphated insulin in adipocytes and hepatocytes

Summary The binding affinity of sulphated insulin compared with unmodified, neutral insulin has been reported to be approximately four times lower in human and rat adipocytes but over twenty times lower in rat hepatocytes. In the present study the biological action of sulphated insulin was assesed in rat hepatocytes and human and rat adipocytes. To achieve half-maximal stimulation of fatty acid synthesis in rat hepatocytes about twenty one times higher concentrations of sulphated than neutral insulin were required (15.07±5.50 vs 0.71±0.34 nmol/l), this ratio being similar to the ratio of binding affinity in rat hepatocytes. In human adipocytes, half-maximal stimulation of initial rates of glucose uptake was observed at 11.6±5.1 vs 2.9±1.3 pmol/l for sulphated and neutral insulin respectively, and half-maximal inhibition of lipolysis at 31.0±13.5 vs 7.3+2.5 pmol/I respectively. These data are consistent with the four-fold lower binding affinity of sulphated insulin to human adipocytes. However, in rat adipocytes the biological potency of sulphated insulin was found to be much lower than anticipated from the binding data, half-maximal stimulation of initial rates of glucose uptake being observed at 757±299 vs 35±13 pmol/l respectively and half-maximal inhibition of lipolysis at 35.9±12.1 vs 1.5±0.5 pmol/l respectively. Thus, in rat adipocytes, approximately 22 times the concentration of sulphated insulin was required to achieve equivalent biological effect. A discrepancy between binding affinity and biological action with respect to sulphated insulin was identified in rat adipocytes but not human adipocytes nor rat hepatocytes suggesting differences in the binding-action linkage in these cells.     

Biologic activities of naturally occurring human insulin receptor mutations. Evidence that metabolic effects of insulin can be mediated by a kinase-deficient insulin receptor mutant.

We have studied insulin receptor-mediated signaling in Chinese hamster ovary (CHO) cell transfectants that expressed either of two naturally occurring mutant human insulin receptors: Trp1200----Ser1200 and Ala1134----Thr1134. Compared with overexpressed normal human insulin receptors, both mutant receptors displayed normal processing and normal binding affinity; however, neither was capable of detectable insulin-stimulated autophosphorylation or tyrosine kinase activity toward endogenous (pp185) or exogenous substrates. Several biologic actions of insulin were evaluated in transfected cells. Compared with neomycin-only transfected CHO cells (CHO-NEO), cells expressing normal receptors demonstrated increased insulin sensitivity for 2-deoxyglucose uptake, [14C]glucose incorporation into glycogen, [3H]thymidine incorporation into DNA, and specific gene expression (accumulation of glucose transporter GLUT-1 mRNA). Cells expressing either Ser1200 or Thr1134 receptors showed no increase in insulin-stimulated thymidine incorporation or GLUT-1 mRNA accumulation compared with CHO-NEO. Surprisingly, cells expressing Ser1200 receptors showed increased insulin stimulation of 2-deoxyglucose uptake and glucose incorporation into glycogen compared with CHO-NEO, whereas Thr1134 receptors failed to signal these metabolic responses. We conclude that 1) transfected kinase-deficient insulin receptor mutants derived from insulin-resistant patients have distinct defects in the ability to mediate insulin action in vitro; 2) divergence of insulin signaling pathways may occur at the level of the receptor; and 3) normal activation of the receptor tyrosine kinase by insulin is not necessarily required for signaling of certain important biologic actions.     

Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice.

To determine the role of GLUT4 on postexercise glucose transport and glycogen resynthesis in skeletal muscle, GLUT4-deficient and wild-type mice were studied after a 3 h swim exercise. In wild-type mice, insulin and swimming each increased 2-deoxyglucose uptake by twofold in extensor digitorum longus muscle. In contrast, insulin did not increase 2-deoxyglucose glucose uptake in muscle from GLUT4-null mice. Swimming increased glucose transport twofold in muscle from fed GLUT4-null mice, with no effect noted in fasted GLUT4-null mice. This exercise-associated 2-deoxyglucose glucose uptake was not accompanied by increased cell surface GLUT1 content. Glucose transport in GLUT4-null muscle was increased 1.6-fold over basal levels after electrical stimulation. Contraction-induced glucose transport activity was fourfold greater in wild-type vs. GLUT4-null muscle. Glycogen content in gastrocnemius muscle was similar between wild-type and GLUT4-null mice and was reduced approximately 50% after exercise. After 5 h carbohydrate refeeding, muscle glycogen content was fully restored in wild-type, with no change in GLUT4-null mice. After 24 h carbohydrate refeeding, muscle glycogen in GLUT4-null mice was restored to fed levels. In conclusion, GLUT4 is the major transporter responsible for exercise-induced glucose transport. Also, postexercise glycogen resynthesis in muscle was greatly delayed; unlike wild-type mice, glycogen supercompensation was not found. GLUT4 it is not essential for glycogen repletion since muscle glycogen levels in previously exercised GLUT4-null mice were totally restored after 24 h carbohydrate refeeding.-Ryder, J. W., Kawano, Y., Galuska, D., Fahlman, R., Wallberg-Henriksson, H., Charron, M. J., Zierath, J. R. Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice.