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To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3-O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.

In muscle, insulin enhances influx of glucose and its conversion to glucose 6-phosphate (G6P) by hexokinase (HK). While effects of insulin on glucose transport have been demonstrated, its effect on the activity of HK of cells has not. In L6 myotubes treated for 24 h with insulin there was increased expression of the HK isoform, HKII, and increased glucose phosphorylation without a concomitant increase in glucose transport, indirectly suggesting that phosphorylation of glucose was a target of insulin action [Osawa, Printz, Whitesell and Granner (1995) Diabetes 44, 1426-1432]. In the present work the same treatment led to a 2-fold rise in G6P, suggesting that transport and/or HK were important targets of insulin action. We used a method to identify the site of rate control involving the specificity of phosphorylation towards 2-deoxy-[1-14C]glucose and D-[2-3H]glucose. Glucose transport does not greatly discriminate between these two tracers while HK shows increased specificity for glucose. Specificity of the glucose phosphorylation of the cells increased with addition of insulin and when extracellular glucose was raised. Specificity was reduced at low glucose concentrations or when the inhibitor of transport, cytochalasin B, was added. We conclude that transport and HK share nearly equal control over glucose phosphorylation in these cells. A computer program was used to test models for compatibility with the different types of experiments. The predicted intracellular glucose and transport rates associated with phosphorylation activity were lower than their measured values for the whole cell. In the most likely model, 15+/-4% of the glucose transporters serve a proportionate volume of the cytoplasm. Insulin activation of glucose phosphorylation might then result from stimulation of these transporters together with HK recruitment or relief from inhibition by G6P.

All Diels-Alder reactions between 1,3-butadiene and cyclopentadiene or 2H-phosphole have been examined at the MP4SDQJ6-3 lG*//HF/6-31G* level. There is remarkable similarity between the two systems. The thermodynamic product is the bicyclo[4.2.0]nonadiene while the kinetic product is the norbornene product. There is a slight kinetic preference for the endo addition and for the butadiene to be in the s-trans conformation. Except for the case where butadiene is the diene component and addition is endo, the reactions are concerted and synchronous. In these other two cases, the reaction is stepwise with a diradical intermediate.Key words: phosphole, Diels-Alder reaction, topological electron density analysis.RCsumC : On a CtudiC toutes les rkactions de Diels-Alder entre le buta-1,3-dikne et le cyclopentadikne ou le 2H-phosphole au niveau MP4SDQJ6-3 lG*NHF/6-3 lG*. I1 existe une similarit6 remarquable entre les deux systkmes. Le produit thermodynamique est le bicyclo[4.2.0]nonadikne alors que le produit cinCtique est le produit norbornkne. I1 y a une 1Cgkre prCf6rence cinCtique en faveur de I'addition etldo et pour que le butadikne soit dans la conformation s-trans. A l'exception du cas ou le butadikne agit comme butadikne et que I'addition est endo, toutes les rCactions sont concertCes et synchrones. Dans ces deux autres cas, la rCaction se fait par Ctape avec un intermkdiaire diradicalaire.Mots clPs : phosphole, rCaction de Diels-Alder, analyse topologique de la densit6 Clectronique.[Traduit par la rCdaction]

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The first steps of glucose metabolism are carried out by members of the families of GLUTs (glucose transporters) and HKs (hexokinases). Previous experiments using the inhibitor of glucose transport, CB (cytochalasin B), revealed that compartmentalization of GLUTs and HKs is a major factor in the control of glucose uptake in L6 myotubes [Whitesell, Ardehali, Printz, Beechem, Knobel, Piston, Granner, Van Der Meer, Perriott and May (2003) Biochem. J. 370, 47-56]. In the present paper, we evaluate compartmentalization of GLUTs and HKs in a hepatoma cell line, H4IIE, which is characterized by excess GLUT activity, HKI in a particulate and a cytosolic fraction, and insignificant G6Pase (glucose-6-phosphatase) activity. The measured activity of glucose transport exceeded the rate of phosphorylation approx. 30-fold. Treatment with 25 microM CB (K(i) approximately 3 microM in H4IIE cells) paradoxically increased the excess of GLUTs over phosphorylation (GLUTs are inhibited 80%, while phosphorylation is inhibited 98%). The global relationships of the data could be reconciled most simply by a two-compartment model. In this model, phosphorylation of glucose is carried out by a subset of HK molecules supplied by a subset of GLUTs that are more sensitive to CB than the other GLUTs. The agent, DCC (dicyclohexylcarbodi-imide) caused HKI to translocate from the particulate compartment to the cytosolic compartment and potently inhibited glucose phosphorylation. The particulate compartment may represent the mitochondria, to which the more CB-sensitive GLUTs may control the transport of glucose.