Activation of the sympathetic nervous system inhibits insulin-stimulated glucose uptake. However, the underlying mechanisms are incompletely understood. Therefore, we studied the effects of catecholamines on insulin-stimulated glucose uptake and insulin-stimulated translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. We found that epinephrine (1 microM) nearly halved insulin-stimulated 2-deoxyglucose uptake. The beta-adrenoceptor antagonist propranolol (0.3 microM) completely antagonized the inhibitory effect of epinephrine on insulin-stimulated glucose uptake, whereas the alpha-adrenoceptor antagonist phentolamine (10 microM) had no effect. When norepinephrine was used instead of epinephrine, the results were identical. None of the individual selective beta-adrenoceptor antagonists (1 microM, beta(1): metoprolol, beta(2): ICI-118551, beta(3): SR-59230A) could counteract the inhibitory effect of epinephrine. Combination of ICI-118551 and SR-59230A, as well as combination of all three selective beta-adrenoceptor antagonists, abolished the effect of epinephrine on insulin-stimulated glucose uptake. After differential centrifugation, we measured the amount of GLUT1 and GLUT4 in the plasma membrane and in intracellular vesicles by means of Western blotting. Both epinephrine and norepinephrine reduced insulin-stimulated GLUT4 translocation to the plasma membrane. These results show that beta-adrenergic (but not alpha-adrenergic) stimulation inhibits insulin-induced glucose uptake in 3T3-L1 adipocytes, most likely via the beta(2)- and beta(3)-adrenoceptor by interfering with GLUT4 translocation from intracellular vesicles to the plasma membrane.

The mechanism via which diacylglycerol-sensitive protein kinase Cs (PKCs) stimulate glucose transport in insulin-sensitive tissues is poorly defined. Phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), are potent activators of conventional and novel PKCs. Addition of PMA increases the rate of glucose uptake in many different cell systems. We attempted to investigate the mechanism via which PMA stimulates glucose transport in 3T3-L1 adipocytes in more detail. We observed a good correlation between the rate of disappearance of PKCbetaII during prolonged PMA treatment and the increase in glucose uptake. Moreover, inhibition of PKCbetaII with a specific myristoylated PKCbetaC2-4 peptide inhibitor significantly increased the rate of glucose transport. Western blot analysis demonstrated that both PMA treatment and incubation with the myristoylated PKCbetaC2-4 pseudosubstrate resulted in more glucose transporter (GLUT)-1 but not GLUT-4 at the plasma membrane. To our knowledge, we are the first to demonstrate that inactivation of PKC, most likely PKCbetaII, elevates glucose uptake in 3T3-L1 adipocytes. The observation that PKCbetaII influences the rate of glucose uptake through manipulation of GLUT-1 expression levels at the plasma membrane might reveal a yet unidentified regulatory mechanism involved in glucose homeostasis.

Members of the PKC (protein kinase C) superfamily play key regulatory roles in glucose transport. How the different PKC isotypes are involved in the regulation of glucose transport is still poorly defined. PMA is a potent activator of conventional and novel PKCs and PMA increases the rate of glucose uptake in many different cell systems. In the present study, we show that PMA treatment increases glucose uptake in 3T3-L1 adipocytes by two mechanisms: a mitogen-activated protein kinase kinase-dependent increase in GLUT1 (glucose transporter 1) expression levels and a PKClambda-dependent translocation of GLUT1 towards the plasma membrane. Intriguingly, PKClambda co-immunoprecipitated with PKCbeta(II) and did not with PKCbeta(I). Previously, we have described that down-regulation of PKCbeta(II) protein levels or inhibiting PKCbeta(II) by means of the myristoylated PKCbetaC2-4 peptide inhibitor induced GLUT1 translocation towards the plasma membrane in 3T3-L1 adipocytes. Combined with the present findings, these results suggest that the liberation of PKClambda from PKCbeta(II) is an important factor in the regulation of GLUT1 distribution in 3T3-L1 adipocytes.

Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns of five classic galactosemia patients with POI were compared to the pattern obtained in two patients with a primary glycosylation disorder (phosphomannomutase-2-deficient congenital disorders of glycosylation, PMM2-CDG) and POI, and in five postmenopausal women as controls. We used FPLC chromatofocussing with measurement of FSH concentration per fraction, and discovered that there were no significant differences between galactosemia patients, PMM2-CDG patients and postmenopausal controls. Our results do not support that FSH dysfunction due to a less acidic isoform pattern because of hypoglycosylation is a key mechanism of POI in this disease.

Animal studies suggest that overactivity of the hexosamine pathway, resulting in increased UDP-hexosamines [UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc)] is an important mechanism by which hyperglycemia causes insulin resistance. This study was performed to test this hypothesis in patients with type 2 diabetes mellitus (DM). Eight obese patients with uncontrolled DM type 2 and severe insulin resistance were treated with iv insulin for 28 +/- 6 d aimed at euglycemia. Before and after iv insulin treatment, insulin sensitivity was measured using a hyperinsulinemic euglycemic clamp, and a muscle biopsy was taken for measurement of UDP-GlcNAc, UDP-GalNAc, UDP-glucose, and UDP-galactose levels. Also, isoelectric focusing patterns of serum transferrin and the urinary excretion of glycosaminoglycans as measures of final products of the hexosamine pathway were examined. After euglycemia, insulin resistance improved, as demonstrated by an increase in the glucose infusion rate during the clamp from 12.7 +/- 5.6 to 22.4 +/- 8.8 micro mol/kg.min (P < 0.0005) and a decrease in insulin requirement from 1.7 +/- 0.9 to 1.1 +/- 0.6 U/kg.d (P < 0.005), whereas metabolic control improved. Surprisingly, both UDP-GlcNAc, from 8.81 +/- 1.21 to 12.31 +/- 2.52 nmol/g tissue (P < 0.005), and UDP-GalNAc concentrations, from 4.49 +/- 0.85 to 5.89 +/- 1.55 nmol/g tissue (P < 0.05) increased. Isoelectric focusing patterns of serum transferrin and excretion of glycosaminoglycans were similar before and after euglycemia. In conclusion, after amelioration of hyperglycemia- induced insulin resistance, UDP-hexosamines increased in skeletal muscle of patients with type 2 DM. These results do not support the hypothesis that accumulation of products of the hexosamine pathway plays a major role in hyperglycemia-induced insulin resistance.