Tissue- and time-dependent effects of endothelin-1 on insulin-stimulated glucose uptake.

Hyperendothelinaemia is associated with various insulin-resistant states, e.g., diabetes, obesity and heart failure, but whether endothelin-1 (ET-1) has a direct effect on insulin-mediated glucose uptake is unclear because the interpretation of in vivo metabolic studies is complicated by ET-1 effects on muscle blood flow and insulin secretion. This study investigated the effects of ET-1 (1-10 nM) on basal and insulin-stimulated 2-deoxy-D-[3H]glucose (2-DOG) uptake in cultured L6 myoblasts and 3T3-adipocytes. RT-PCR analysis showed that both cell types express ET(A) but not ET(B) receptors. ET-1 had no effect on basal (non-insulin-mediated) glucose transport, but there was evidence of a tissue- and time-dependent inhibitory effect of ET-1 on insulin-stimulated glucose uptake. Specifically, ET-1 10 nM had a transient (0.5 h) inhibitory effect on glucose uptake in 3T3 cells (C(I-150) [dose of insulin required to increase glucose uptake by 50%, relative to control 100%] increased from 89 +/- 14 nM to 270 +/- 12 nM at 30 mins, P < 0.05) but no effect on insulin sensitivity in L6 myoblasts (C(I-150) was 56 +/- 14 nM [control], 43 +/- 14 [30 mins] and 26 +/- 16 [2 h]). In conclusion, the inhibitory effect of ET-1 on insulin-stimulated glucose uptake is transient and occurs in 3T3-L1 adipocytes but not skeletal muscle-derived cells, perhaps reflecting tissue differences in ET(A)-receptor signaling. It is therefore unlikely that chronic hyperendothelinaemia has a direct insulin-antagonist effect contributing to peripheral (ie muscle/fat) insulin resistance in vivo.

Possible interactions between angiotensin II and insulin: effects on glucose and lipid metabolism in vivo and in vitro.

Angiotensin II (ANGII) increases insulin sensitivity in diabetic and non-diabetic subjects, even at subpressor doses, and because there is 'crosstalk' between ANGII and insulin-signaling pathways the underlying mechanism may not be due solely to changes in regional blood flow. A series of experimental studies was undertaken to evaluate the effects of ANGII on glucose and lipid metabolism in vivo and in vitro. Groups of fructose-fed, insulin-resistant Sprague-Dawley (SD) rats were pre-treated with 0.3 mg/kg per day of the AT(1)-receptor antagonist L-158 809 (n=16), or vehicle (n=16), by oral gavage. This was prior to an oral glucose tolerance test (day 5) and measurement of the effects of ANGII infusion (20 ng/kg per min i.v. for 3 h) on whole-body insulin sensitivity using the insulin suppression test (day 7). The effect of ANGII infusion on total triglyceride secretion rate (TGSR) was evaluated in normal SD rats pretreated for 7 days with L-158 809 (n=12) or vehicle (n=12). AT(1)- and AT(2)- receptor mRNA expression and [(3)H]2-deoxyglucose uptake were assessed in cultured L6 myoblasts. Short-term treatment with L-158 809 had no effect on glucose tolerance or fasting triglyceride levels in fructose-fed rats. ANGII infusion had no effect on insulin sensitivity in fructose-fed rats pretreated with vehicle (steady-state plasma glucose (SSPG) values 8.1+/-1.6 vs 8. 4+/-0.4 mmol/l), but pretreatment with L-158 809 resulted in ANGII having a modest insulin antagonist effect in this insulin-resistant model (SSPG values 9.6+/-0.3 vs 7.1+/-0.6, P<0.03). ANGII infusion had no significant effect on TGSR (e.g. 24.6+/-1.4 vs 28.4+/-0.9 mg/100 g per h in vehicle-treated animals). RT-PCR analysis showed that L6 cells express both AT(1)- and AT(2)-receptor mRNA. Incubation with ANGII (10(-9) and 10(-8) M) had no significant effect on the dose-response curve for insulin-stimulated [(3)H]2-deoxyglucose uptake. For example, C(I200) values (dose of insulin required to increase glucose uptake by 200%) were 4.5 x 10(-9) M (control) vs 3.9 x 10(-9) M and 6.2 x 10(-9) M, whereas the positive control (glucagon-like peptide-1) increased insulin sensitivity. Thus, ANGII infusion may have a modest insulin antagonist effect on glucose disposal in insulin-resistant fructose-fed rats pretreated with an AT(1)-blocker, but ANGII has no effect on TGSR or in vitro glucose uptake in L6 myoblasts. These findings are relevant to recent clinical discussions about the metabolic effects of ANGII and renin-angiotensin system blockade.

Effects of tumour necrosis factor-alpha and inhibition of protein kinase C on glucose uptake in L6 myoblasts.

Clinical and experimental studies have implicated high circulating levels of the cytokine tumour necrosis factor-alpha (TNF-alpha) in the pathogenesis of insulin resistance, not only in obesity and diabetes, but also in clinical conditions associated with cachexia and sepsis. TNF-alpha impairs insulin-mediated glucose uptake in adipocytes, but because of lipolytic effects the interpretation of clinical studies and the extent to which TNF-alpha affects muscle insulin sensitivity are unclear. In addition, protein kinase C (PKC) has recently been implicated in the mechanism of TNF-alpha-induced insulin resistance. The present study investigated the effects of TNF-alpha and a PKC inhibitor (RO-318220) on basal and insulin-stimulated 2-[(3)H]deoxyglucose uptake in cultured L6 myoblasts. Reverse transcriptase-PCR analysis confirmed that L6 myoblasts express TNF-alpha receptors I and II (p60 and p80). Dose-response curves for glucose uptake were fitted to a quadratic function to derive C(I-150) values (concentration of insulin required to increase glucose uptake by 50%). Incubation with TNF-alpha at 1 or 10 ng/ml for 24 h had no significant effect on basal glucose uptake, insulin sensitivity or maximal insulin responsiveness. C(I-150) values (means+/-S.E.M.) were as follows: basal, 91.2+/-13 nM; 1 ng/ml TNF-alpha, 102+/-12 nM; and basal, 70.8+/-13 nM; 10 ng/ml TNF-alpha, 43.7+/-40 nM. PKC inhibition markedly attenuated glucose uptake, but there was no difference in insulin sensitivity with RO-318220 alone compared with RO-318220+TNF-alpha. In conclusion, although increased TNF-alpha expression and plasma concentrations have been implicated in the pathogenesis of insulin resistance in various clinical states, there is no evidence that TNF-alpha impairs insulin-stimulated glucose uptake in a skeletal-muscle-derived cell line.