SHERMAN, W. M., J. E. FRIEDMAN, J.-P. GAO, M. J. REED, C. W. ELTON, and G. L. DOHM. Glycemia and exercise training alter glucose transport and GLUT4 in the Zucker rat. Med. Sci. Sports Exerc., Vol. 25, No. 3, pp. 341–348, 1993. The purpose of this study was to determine the effects of chronic hyperglycemia and/or exercise training on the muscle concentration of the insulin-responsive glucose transporter protein, GLUT4, and on maximally insulin-stimulated hindlimb muscle glucose transport. Five-wk-old lean and obese Zucker rats were randomly assigned to sham-operated control (CTL) or 90% pancreatectomized (PX) groups. Obese-PX animals were further randomized into sedentary or exercise trained groups (15-wk treadmill running for 2 h.d-1, 5 d.wk-1, 15% grade, at 15–18 m.min-1). Muscle GLUT4 protein content and maximally insulin-stimulated glucose transport were determined in gastrocnemius, plantaris, and soleus muscles. At 20 wk, lean-PX displayed mild fasting hyperglycemia but normal insulin levels. Obese-PX rats had insulin levels similar to lean-CTL rats but had severe hyperglycemia. Hyperglycemia in lean-PX was associated with a 28% decrease in maximal glucose transport and a 65% decrease in muscle GLUT4 (P < 0.05) compared with lean-CTL. In obese-PX, maximal glucose transport was not affected, but muscle GLUT4 was reduced by 62% (P < 0.05) compared to obese-CTL. Exercise training obese-PX reduced hyperglycemia, increased maximal glucose transport by 45%, and increased muscle GLUT4 by >2-fold (P < 0.05) compared with obese-CTL. Thus, hyperglycemia associated with PX may be an important factor in the reduction of muscle GLUT4 levels in lean and obese rats. The reduced GLUT4 was accompanied by reduced maximal glucose transport in lean but not obese rats. Exercise training reduced hyperglycemia, normalized glucose transport, and increased muscle GLUT4 in obese-PX. The beneficial effects of exercise training result in a reduction in hyperglycemia and chronic adaptations in skeletal muscle glucose transport capacity including increases in GLUT4.
There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of beta(2)-adrenergic receptors (beta(2)ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in beta-arrestin-dependent beta(2)AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nm isoproterenol for 5 min induced approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 mum isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)-beta-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-beta-Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent beta-arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced beta-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, beta-arrestin binding, and internalization of beta(2)ARs.
Phosphoinositide 3-kinase is a key signaling intermediate necessary for the metabolic actions of insulin. In this study, we assessed the effects of in vivo knockdown of the p85α subunit of phosphoinositide 3-kinase on insulin sensitivity, using an antisense oligonucleotide, in lean mice, diet-induced obese mice, and obese leptin-deficient Lep ob/ob mice. Mice were injected with either p85α-targeted antisense oligonucleotide or saline twice weekly for 4 weeks. Fasting levels of glycemia and insulinemia and insulin and glucose tolerance tests were used to determine insulin sensitivity. Western blot analysis and real-time polyacrylamide chain reaction were used to assess p85α protein and mRNA expression. In vivo administration of antisense oligonucleotide resulted in 50 and 60% knockdown of liver p85α protein and mRNA, respectively, in the lean, diet-induced obese and Lep ob/ob mice. This was associated with increased phosphoinositide 3-kinase activity and improved insulin sensitivity in diet-induced obese and Lep ob/ob mice. Thus, p85α could be an important therapeutic target to ameliorate insulin resistance.
Cerebrospinal fluid (CSF) was obtained from five patients by lumbar puncture and from two patients by Ommaya reservoir tap after the i.v. administration of the antitumor agent N-(phosphonacetyl)-L-aspartate (PALA). PALA was quantified enzymatically by inhibition of the target enzyme, aspartate carbamoyltransferase. After a 1-hr infusion of PALA, its CSF concentration steadily rose until the eighth hr, at which time it was 12 to 40% of concurrent plasma concentration. PALA concentration then declined more gradually in CSF than in plasma, and CSF concentrations exceeded plasma concentrations by 24 hr. PALA concentration X time product in CSF was 12 to 25% of that in plasma. PALA was infused i.v. for 30 to 60 min into eight patients undergoing surgical resection if intracerebral tumors. Its concentration in intracerebral tumor was greater than or comparable to concentration in temporalis muscle in four of six patients from whom muscle was obtained. The PALA concentration in edematous brain tissue was consistently lower than the concentration in tumor or muscle. In a patient undergoing occipital lobectomy, the PALA concentration in brain was inversely proportional to the distance from the tumor. PALA reached concentrations in intracerebral tumor that appeared to be similar to concentrations reported previously in s.c. tumors, although biopsy techniques and conditions differed.
Within the past five weeks I have had an opportunity of experimenting with tetanus antitoxin in the treatment of two cases of trismus nascentium. This disease of infancy has been almost uniformly fatal and, notwithstanding the fact that all kinds of treatment have been used, has thus far resisted every measure. On learning of the successful treatment of tetanus in the adult by the serum treatment, I determined to try the same remedy for the infantile disease. By reason of the extreme youth of the patient one has no guide as to the quantity of the serum to be used. As will be seen in the first case, knowing that the child would die under the ordinary plan of treatment, a large dose was used in the beginning, considering the weight of the child as compared with that of the adult, in which a full bottle is usually administered. In
Pretreatment of either intact wild type S49 lymphoma cells (WT) or the uncoupled variants, cyc-, H21a, or UNC with epinephrine results in the redistribution of 20-30% of the beta-adrenergic receptors into a light vesicle fraction in sucrose gradients. Since the variants are uncoupled with respect to hormonal stimulation of adenylate cyclase, it appears that productive interaction with Gs is not required for the sequestration of beta-adrenergic receptors. Characterization of the epinephrine-induced redistribution of the beta-adrenergic receptor has revealed the following: The EC50 for the redistribution in WT cells was between 100 and 200 nM. Pretreatment of WT cells with 50 nM epinephrine for 30 min induced only a slight redistribution of receptors in sucrose gradients but produced a significant desensitization of adenylate cyclase. The desensitization was characterized by an increase in the Kact of epinephrine stimulation of adenylate cyclase while the Vmax was unaltered. Pretreatment with 10 microM epinephrine resulted in a significant decrease in the Vmax (50%) of epinephrine stimulation of adenylate cyclase and a 3-fold increase in Kact in the heavy vesicles. The beta-receptors in the light vesicle fraction of WT were uncoupled from adenylate cyclase and displayed low affinity for epinephrine binding, comparable to the cyc-. The "desensitized" receptor in the light vesicle fractions of cyc- was capable of stimulating adenylate following reconstitution with cholate extracts of WT membranes containing Gs. The molecular weight of the photolabeled beta-receptor in the light vesicle fractions (65,000 +/- 2,000) was not significantly different from the Mr 65,000 polypeptide photolabeled in the heavy fractions. The Mr 55,000 beta-receptor polypeptide was not detected in the light vesicles. Our results suggest first that the redistribution of the beta-receptor into light vesicles may follow an earlier stage of desensitization, and second that the beta-receptor in light vesicles while sequestered from Gs is capable of activating adenylate cyclase.
Treatment with low physiological concentrations of epinephrine (5-50 nM) rapidly desensitizes β-adrenergic stimulation of cAMP formation in S49 wild-type (WT) lymphoma cells. Previous attempts to detect this early phase of desensitization in cell-free assays of adenylate cyclase (EC 4.6.1.1) after intact cell treatment were unsuccessful. We have now found that reducing the Mg2+ concentrations in the adenylate cyclase assays to <1.0 mm unmasked this rapid phase of desensitization of the WT cells, and that high Mg2+ concentrations (5-10 mm) largely obscured the desensitization. Submillimolar Mg2+ conditions also revealed a two- to threefold decrease in the affinity of epinephrine binding to the β-adrenergic receptor after desensitization with 20 nm epinephrine. Detection of 4β-phorbol 12-myristate 13-acetate (PMA) desensitization of the WT β-adrencrgic receptor was also dependent on low Mg2+ as measured either by the decrease in epinephrine stimulation of adenylate cyclase or by the reduction in the affinity of epinephrine binding. Unexpectedly, when cyc− cells were pretreated with 50 nm epinephrine, the β-adrenergic stimulation of reconstituted adenylate cyclase was not desensitized. The characteristics of the Mg2+ effect on epinephrine- and PMA-induced desensitizations suggest a similar mechanism of action with the most likely events being phosphorylations of the β-adrenergic receptors. Our data indicate that cAMP-dependent protein kinase (EC 2.7.1.37) may play a role in the desensitization caused by low epinephrine concentrations inasmuch as this phase of desensitization did not occur in the cyc−. For the PMA-induced desensitization, the phosphorylation may be mediated by protein kinase C (EC 2.7.1.37).—Clark, R. B.; Friedman, J.; Johnson, J. A.; Kunkel, M. W. β-Adrenergic receptor desensitization of wild-type but not cyc lymphoma cells unmasked by submillimolar Mg2+. FASEB J. 1: 289-297; 1987.
