High levels of dietary fat may contribute to both insulin resistance and obesity in humans but evidence is limited. The euglycemic clamp technique combined with tracer administration was used to study insulin action in vivo in liver and individual peripheral tissues after fat feeding. Basal and nutrient-stimulated metabolic rate was assessed by open-circuit respirometry. Adult male rats were pair-fed isocaloric diets high in either carbohydrate (69% of calories; HiCHO) or fat (59% of calories; HiFAT) for 24 +/- 1 days. Feeding of the HiFAT diet resulted in a greater than 50% reduction in net whole-body glucose utilization at midphysiological insulin levels (90-100 mU/l) due to both reduced glucose disposal and, to a lesser extent, failure to suppress liver glucose output. Major suppressive effects of the HiFAT diet on glucose uptake were found in oxidative skeletal muscles (29-61%) and in brown adipose tissue (BAT; 78-90%), the latter accounting for over 20% of the whole-body effect. There was no difference in basal metabolic rate but thermogenesis in response to glucose ingestion was higher in the HiCHO group. In contrast to their reduced BAT weight, the HiFAT group accumulated more white adipose tissue, consistent with reduced energy expenditure. HiFAT feeding also resulted in major decreases in basal and insulin-stimulated conversion of glucose to lipid in liver (26-60%) and brown adipose tissue (88-90%) with relatively less effect in white adipose (0-43%). We conclude that high-fat feeding results in insulin resistance due mainly to effects in oxidative skeletal muscle and BAT.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined peripheral insulin action in conscious rats chronically fed high fat (60% calories as fat) or high carbohydrate (lab chow) diets using the euglycaemic clamp plus 3H-2-deoxyglucose technique. A response parameter of individual tissue glucose metabolic rate (the glucose metabolic index, based on tissue deoxyglucose phosphorylation) was used to assess diet effects in eight skeletal muscle types, heart, lung and white and brown adipose tissue. Comparing high fat with high carbohydrate fed rats, basal glucose metabolism was only mildly reduced in skeletal muscle (only diaphragm was significant, p less than 0.05), but was more substantially reduced in other tissues (e.g. white adipose tissue 61% and heart 33%). No evidence of basal hyperinsulinaemia was found. In contrast, widespread insulin resistance was found during the hyperinsulinaemic clamp (150 mU/l) in high fat fed animals; mean whole body net glucose utilization was 34% lower (p less than 0.01), and the glucose metabolic index was lower in skeletal muscle (14 to 56%, p less than 0.05 in 6 out of 8 muscles), white adipose (27%, p less than 0.05) and brown adipose tissue (76%, p less than 0.01). The glucose metabolic index was also lower at maximal insulin levels in muscle and fat, suggesting the major effect of a high fat diet was a loss of insulin responsiveness. White adipose tissue differed from muscle in that incremental responses (maximal insulin minus basal) were not reduced by high fat feeding. The heart showed an effect opposite to other tissues, with an increase in insulin-stimulated glucose metabolism in high fat versus chow fed rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Numerous structurally diverse ligands were developed to target the human histamine H 3 receptor (hH 3 R), a presynaptic G i /G ocoupled auto-and heteroreceptor. Proxyfan was identified to be functionally selective, with different efficacies toward G i /G odependent hH 3 R signaling pathways. However, the underlying molecular mechanism of functional selectivity of proxyfan is still unclear. In the current study, we investigated the role of different G␣ i/o proteins in hH 3 R signaling, using a baculovirus/Sf9 cell expression system. We tested the hypothesis that ligandspecific coupling differences to defined G i /G o -heterotrimers are responsible for functional selectivity of proxyfan at hH 3 R. In Sf9 membranes, full-length hH 3 R (445 amino acids) was expressed in combination with an excess of different mammalian G proteins (G␣ i1 , G␣ i2 , G␣ i3 , or G␣ o1 and ␤ 1 ␥ 2 dimers, respectively). In addition, we constructed the fusion proteins hH 3 R-G␣ i2 and hH 3 R-G␣ o1 to ensure clearly defined receptor/G protein stoichiometries. Steady-state GTPase experiments were performed to directly measure the impact of each G protein on hH 3 R signal transduction. The hH 3 R coupled similarly to all G proteins. We also observed similar ligand-independent or constitutive activity. Proxyfan and various other imidazole-containing ligands, including full agonists, partial agonists, and inverse agonists, showed very similar pharmacological profiles not influenced by the type of G protein coexpressed. Selected ligands, examined in membranes expressing the fusion proteins hH 3 R-G␣ i2 and hH 3 R-G␣ o1 (plus ␤ 1 ␥ 2 dimers), yielded very similar results. Collectively, our data indicate that hH 3 R couples similarly to different G␣ i/o -subunits and that ligand-specific active receptor conformations, resulting in G protein-coupling preferences, do not exist for proxyfan or other imidazole compounds investigated.

The influence of blood flow (BF) and basal neuromotor tone on in vivo insulin-stimulated glucose uptake (Rg') in muscle was examined using the euglycemic clamp plus deoxyglucose/glucose tracer (insulin action) and labeled microsphere (BF) techniques. Anesthesia was used to produce perturbations in BF and/or activity compared with conscious rats. An index of muscle glycolytic flux (Gf) was estimated from Rg' in excess of glycogen synthesis. Gf and glycogen synthesis were significantly increased in soleus and red gastrocnemius (RG) during insulin infusion (150 mU/l) in conscious rats. Rg' was related to muscle BF in conscious rats (r = 0.92). In anesthetized rats, Rg' and BF were reduced in soleus, RG, red quadriceps, and plantaris (e.g., soleus 69%, P less than 0.001, and 80%, P less than 0.001, respectively). However, it is unlikely that Rg' and BF are causally related because fractional extraction of glucose by muscle was low (approximately 9%); fractional extraction increased during anesthesia (17%, P less than 0.01); BF but not Rg' was reduced by anesthesia in extensor digitorum longus, white gastrocnemius, and white quadriceps; and reduced Rg' during anesthesia in soleus and RG was mainly due to reduced GF. In conclusion, BF is not a major contributor to the heterogeneity in insulin-stimulated glucose uptake among individual muscles under basal conditions. These data suggest that neuromotor tone may indirectly influence the magnitude of muscle glucose uptake during insulin elevation via a substrate switching effect. This may give rise to a significant increase in glycolytic flux of glucose in those muscles that exhibit elevated activity even at rest (e.g., soleus).(ABSTRACT TRUNCATED AT 250 WORDS)

Physical training causes hypoinsulinemia and enhanced insulin sensitivity. Insulin sensitivity is also enhanced by a single bout of exercise. However, changes in beta-cell responsiveness with acute exercise are ill defined. To clarify these relationships intravenous glucose tolerance tests were performed in 1) physically trained rats (3 different levels), 48 h after the last bout of exercise; 2) untrained rats, 0.5, 4, and 24 h after a single bout of exercise; or 3) sedentary control rats. The total area under the glucose-stimulated insulin response curve (GSIR) was negatively correlated with total distance run during training (r = -0.45, P less than 0.05), whereas glucose tolerance was improved (P less than 0.005 vs. controls) with intensive training. GSIR was suppressed by 38% (P less than 0.01 vs. controls) 0.5 h after a single bout of exercise. This effect persisted for 4 h but was not present after 24 h. These results indicate that a single bout of exercise induces suppression of GSIR which lasts less than 24 h. In contrast, physical training induces prolonged suppression (for at least 48 h) of GSIR, proportional to the intensity of training, and improved glucose tolerance.

The relative time dependency of enhancement of glucose metabolism by insulin in individual tissues has not been examined previously in vivo. Using the glucose clamp technique in the rat combined with radiolabeled 2-deoxyglucose (3H or 14C-2DG) administration, we have estimated a glucose metabolic index (Rg') in major insulin-sensitive tissues at two different times (between 40-55 min and 80-95 min) after onset of hyperinsulinemia (insulin levels of approximately 130 mU/L). At the completion of the clamp (125 min), eight different skeletal muscles and three different adipose tissue beds were rapidly removed for estimation of Rg', based on the accumulation of intracellular tracer 2DG-6-phosphate (2DGP). Insulin-stimulated Rg' was 60-70% higher at 80-95 min than at 40-55 min after elevation of insulin levels in all three adipose tissue beds (P less than 0.01). In contrast, insulin-stimulated whole body glucose utilization and Rg' in all skeletal muscles were not significantly different at these two times. The constancy of insulin action in skeletal muscles during the 2-h euglycemic clamp, as determined in these studies, does not support the concept that the glucose clamp represents an integral of many rapidly changing individual tissue responses. In conclusion, while the amount of insulin-stimulated glucose uptake in adipose tissue is low compared with muscle, the fraction increases with duration of insulin elevation. This effect may have significant implications in hyperinsulinemic states.

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