Expression of human GLUT4 in mice results in increased insulin action.

Glucose metabolism was evaluated in transgenic mice expressing the human GLUT 4 glucose transporter. Fed GLUT 4 transgenic mice exhibited a 32% and 56% reduction in serum glucose and insulin and a 69% and 33% increase in non-esterified fatty acid and lactate levels, respectively. Transgenic mice exhibited a significant increase in whole-body glucose disposal during a euglycaemic-hyperinsulinaemic clamp. Insulin-stimulated glucose uptake in isolated soleus muscles and adipocytes was greater in transgenic compared to control mice due to increased basal glucose uptake. Transgenic mice displayed increased glycogen levels in liver and gastrocnemius muscle, and increased insulin-stimulated 14C-glycogen accumulation in isolated soleus muscle. We conclude that over-expression of the GLUT 4 glucose transporter in mice results in 1) an increase in whole-body glucose disposal and storage, and 2) an increase in both basal and insulin-stimulated glucose uptake and disposal in vitro. These changes resulted in the reduction of serum glucose and insulin levels. These results provide direct evidence that glucose transport (and GLUT 4 per se) plays a significant role in regulating whole-body glucose homeostasis. Additionally, these data support the idea that pharmacological strategies directed at increasing the expression of GLUT 4 protein may have beneficial (hypoglycaemic) effects in the diabetic state.

Insulin action on whole body glucose utilization and on muscle glucose transporter translocation in mice.

Murine models of insulin resistance and diabetes are versatile and have been used to investigate genetic and metabolic disorders. However, the principal assays to assess insulin action, i.e., the euglycemic-hyperinsulinemic clamp and subcellular distribution of glucose transporters, have not been implemented in this species. Here we describe procedures which allow these methods to be adapted to mice. When normal C57bl/6j mice were infused with graded doses of insulin (1, 3, 10 or 30 mU/kg/min) during a euglycemic-hyerinsulinemic clamp, the glucose infusion rate necessary to maintain euglycemia increased in a dose-dependent manner (7.4 +/- 1.7, 13.1 +/- 3.6, 24.1 +/- 2.3 or 34.8 +/- 7.5 mg/kg/min), respectively. Hindlimb muscles were isolated and samples of 2-3 g were subjected to subcellular fractionation finalizing on 25%, 30% and 35% sucrose gradients. Fraction F25 (plasma membranes) was enriched in alpha 2 Na+/K(+)-ATPase and GLUT1 glucose transporters, whereas fraction F35 (intracellular membranes) was enriched in Ca(2+)-ATPase and GLUT4 glucose transporters. Following insulin treatment, GLUT4 increased in F25 and decreased in F35. Insulin treatment had no effect on GLUT1 in F25. However, unlike in rat skeletal muscle, GLUT1 was detectable in F35 and its content decreased in this fraction following insulin treatment. The results demonstrate that whole-body glucose utilization can be assessed in mice using euglycemic-hyperinsulinemic clamps and demonstrate how subcellular fractionation procedures can be applied to murine muscle. Murine muscle GLUT4 translocates from an intracellular storage site to the plasma membrane in response to insulin.

Amylin or CGRP (8-37) fragments reverse amylin-induced inhibition of 14C-glycogen accumulation.

The peptides amylin and calcitonin-gene related peptide (CGRP) have been shown to have similar effects on glycogen metabolism in vivo and in vitro. However, it is not clear whether they act via separate receptors. Peptide fragments based on the amino acid sequence of amylin or CGRP were evaluated for their ability to inhibit the action of the peptides in vitro. Insulin-stimulated glycogen turnover, as measured by 14C-glycogen accumulation, was inhibited about 70% by amylin (10nM) and 85% by CGRP (10nM). In the absence of exogenous peptide, peptide fragments based on the 8-37 and 10-37 amino acid sequences of rat amylin (10 uM) had no affect on 14C-glycogen accumulation. In the presence of amylin (10nM), the 8-37 and 10-37 fragments blocked amylin-induced inhibition of 14C-glycogen accumulation 100% and 11.4%, respectively. The 8-37 and 10-37 amylin fragments blocked CGRP inhibition of 14C-glycogen accumulation by 23.2% or 28.6%, respectively. The CGRP 8-37 fragment was equally effective as the amylin 8-37 reversing the effects of amylin than at reversing the effects of CGRP. These results demonstrate that amylin (8-37) completely antagonizes the effects of amylin with limited ability to block CGRP. Removing the eighth and ninth amino acids reduced the effectiveness of the inhibitor by about 90%.

Amylin activates glycogen phosphorylase and inactivates glycogen synthase via a cAMP-independent mechanism.

Although the novel pancreatic peptide amylin has been shown to induce insulin resistance and decrease glucose uptake, the mechanism of amylin's actions is unknown. The following study evaluated the effect of amylin on glycogen metabolism in isolated soleus muscles in the presence and absence of insulin (200 microU/ml). Total glycogen, glycogen phosphorylase and glycogen synthases activities, and cAMP levels were measured. Total glycogen levels were significantly decreased by amylin (100 nM) in fed or fasted muscles under conditions of insulin stimulation. Amylin (100 nM) activated glycogen phosphorylase by as much as 100% and decreased glycogen synthase activity by over 60%, depending on the metabolic state of the muscles. These effects where comparable to those of the beta adrenergic agonist isoproterenol. A lower concentration of amylin (1 nM) did not significantly affect glycogen levels, glycogen phosphorylase, or glycogen synthase activity. Cyclic AMP levels were increased two-fold by isoproterenol but were unaffected by amylin. In conclusion, amylin induces glycogenolysis by decreasing glycogen synthesis and increasing breakdown. The effect of amylin on enzyme activity is consistent with a phosphorylation-dependent mechanism. It is likely that these events are mediated via a cAMP independent protein kinase.

Effects of amylin on glucose metabolism and glycogenolysis in vivo and in vitro.

The effects of amylin on glucose metabolism and glycogenolysis were examined in vivo and in vitro. Eighteen-hour-fasted rats were infused with 5 nmol.kg-1.min-1 amylin and [3-3H]glucose for 120 min. Blood glucose levels increased an average of 45% during the infusion. Glucose turnover measurements indicated that the overall rate of glucose appearance (Ra) did not change, but the metabolic clearance rate of glucose was decreased by 42%. Samples of liver, gastrocnemius, and soleus muscles were freeze-clamped at the end of the infusion period and analyzed for glycogen and glucose 6-phosphate levels. Glycogen levels were decreased in all tissue samples, whereas glucose 6-phosphate was elevated in gastrocnemius and soleus muscles. Isolated soleus muscles were incubated in vitro with 200 microU/ml of insulin and 1, 10, or 100 nM amylin. Amylin treatment had no effect on 3-O-methyl-D-glucose transport; however, 2-deoxy-D-glucose uptake was inhibited by 33 or 48% at 10 or 100 nM amylin, respectively. Glycogen levels were also decreased after treatment with 10 and 100 nM amylin. Glucose 6-phosphate levels were not affected by amylin treatment in the presence of insulin but were increased nearly twofold in its absence. The data suggest that amylin stimulates glycogenolysis and inhibits glucose uptake both in vivo and in vitro and that the inhibition of glucose uptake is due to inhibition of glucose phosphorylation (i.e., hexokinase).

Innovative concepts in public-private coordination of benefits for retirees.

Benefits--in the broadest and most literal sense of the word--can be coordinated between Medicare and employer group health plans. What that may entail, however, ranges from (1) researching complementary plans' effect on Medicare utilization and costs; to (2) incorporating those findings into rate setting for the Medicare capitation payment that would be administered by the EGHPs; to (3) some potential cost-containing redesign of complementary plans that would be acceptable to employees/retirees and their unions and would involve improvements in information provided to consumers; to (4) some consideration given to allowing savings achieved through capitation to become part of a trust that would prefund the EGHP retiree plan.

Evidence for distinct systemic extravasation effects of platelet activating factor, leukotrienes B4, C4, D4 and histamine in the guinea pig.

The relative potencies of platelet-activating factor (PAF), leukotrienes B4 (LTB4), C4 (LTC4), D4 (LTD4) and histamine to induce hemoconcentration (HC) were evaluated in the guinea pig. The maximal hematocrit increase (MHI) from PAF, LTD4 and histamine occurred 5-7 min after i.v. injection, whereas the MHI of LTC4 occurred 13-15 min after injection. LTB4 (2.97-5.95 nmol kg-1) did not produce HC. The magnitude of PAF-induced MHI was 2-fold that of LTC4 or LTD4, regardless of the dose of leukotrienes used. The doses (nmol kg-1) needed to produce 30% HC were: 0.14-PAF, 0.71-LTD4 and 3.37-LTC4 and 2,400 histamine. The HC effects of LTD4 were markedly reduced by prior administration of FPL-55712. However, neither LTC4 or LTD4 HC effects were significantly reduced by prior i.v. injection of CV-3988 (3.4 mg kg-1), a competitive receptor antagonist to PAF which is 98% effective in abolishing HC response to 0.14 nmol kg-1 PAF. Diphenhydramine abolished histamine-induced hemoconcentration but was without effect on PAF or LTD4. These results suggest that the responses of PAF, leukotrienes and histamine differ in their potency and may involve separate vascular recognition sites related to acute increases in vascular permeability.

Impact of Medicare on the use of medical services by disabled beneficiaries, 1972-1974.

The extension of Medicare coverage in 1973 to disabled persons receiving cash benefits under the Social Security Act provided an opportunity to examine the impact of health insurance coverage on utilization and expenses for Part B services. Data on medical services used both before and after coverage, collected through the Current Medicare Survey, were analyzed. Results indicate that access to care (as measured by the number of persons using services) increased slightly, while the rate of use did not. The large increase in the number of persons eligible for Medicare reflected the large increase in the number of cash beneficiaries. Significant increases also were found in the amount charged for medical services. The absence of large increases in access and service use may be attributed, in part, to the already existing source of third party payment available to disabled cash beneficiaries in 1972, before Medicare coverage.