Insulin-and glyburide-stimulated changes in cytosolic free calcium concentrations (QCa2+Ii) were studied in gluteal adipocytes obtained from six obese women (139±3% ideal body wt) and six healthy, normal weight age-and sex-matched controls. Biopsies were performed after an overnight fast and twice (at 3 and 6 h) during an insulin infusion (40 mU/m2 per min) (euglycemic clamp). In adipocytes obtained from normal subjects before insulin infusion, insulin (10 ng/ml) increased [Ca2+ji from 146±26 nM to 391±66 nM. Similar increases were evoked by 2 tiM glyburide (329±41 nM). After 3 h of insulin infusion, basal ICa2+ii rose to 234±21 nM, but the responses to insulin and glyburide were completely abolished. In vitro insulin-stimulated 2-deoxyglucose uptake was reduced by insulin and glucose infusion (25% stimulation before infusion, 5.4% at 3 h, and 0.85% at 6 h of infusion). Rat adipocytes were preincubated with 1-10 mM glucose and 10 ng/ml insulin for 24 h. Measurements of 2-deoxyglucose uptake demonstrated insulin resistance in these cells.Under these experimental conditions, increased levels of [Ca2+Ii that were no longer responsive to insulin were demonstrated. Verapamil in the preincubation medium prevented the development of insulin resistance.

In an attempt to elucidate the cellular mechanism(s) of the insulin resistance associated with impaired glucose tolerance (IGT) and non-insulin-dependent diabetes (NIDDM), insulin-sensitive glucose transport was studied employing isolated adipocytes obtained from these subjects using the nonmetabolized glucose analogue, 3-O-methyl glucose. In the subjects with IGT, basal and maximal rates of 3-O-methyl glucose uptake were normal while the responses at submaximal insulin concentrations were decreased, i.e., the dose-response curves were shifted rightward, indicative of decreased insulin sensitivity. In contrast, the dose-response curves for insulin-stimulated 3-O-methyl glucose uptake in adipocytes obtained from subjects with NIDDM were right-shifted and, in addition, there was a marked decrease in both the basal and maximally stimulated rates of glucose transport (i.e., decreased insulin responsiveness). Thus, the adipocytes from subjects with IGT show decreased insulin sensitivity consistent with decreased insulin binding, whereas the adipocytes from subjects with NIDDM exhibit both decreased insulin sensitivity and decreased insulin responsiveness consistent with a combined receptor and postreceptor defect in cellular insulin action. In the groups as a whole, the magnitude of the rightward shift in the dose-response curve for insulin-stimulated glucose transport correlated with the reduction in adipocyte insulin binding (r = 0.47, P < 0.02). In these subjects, the level of fasting hyperglycemia was correlated (P < 0.01) to the magnitude of the decrease in maximal glucose transport and a highly significant correlation was found between the maximal insulinstimulated rate of glucose transport and the maximal in vivo rate of insulin-stimulated glucose disposal (r = 0.49, P < 0.01). Therefore, we conclude that the insulin resistance in subjects with IGT is due solely to a decrease in insulin binding, whereas subjects with NIDDM exhibit both decreased insulin binding and decreased maximal rates of insulin-stimulated adipocyte glucose transport due to a postreceptor defect at this site in the insulin action pathway. If adipocytes are reflective of changes in glucose transport in other insulin target tissues, then these findings suggest that the cellular lesion responsible for the postreceptor defect in insulin action, previously demonstrated in vivo in subjects with NIDDM, resides in part at the level of the glucose transport system.

The insulin resistance of type II diabetes mellitus is due to both receptor and postreceptor defects of in vivo insulin action, with the postreceptor defect being the predominant abnormality. Diminished glucose transport has been found in adipocytes from patients with type II diabetes, suggesting that decreased cellular glucose transport activity may be responsible in part for the in vivo postreceptor defect observed in these patients. Recent studies have shown that the in vivo postreceptor defect initially present in patients with Type II diabetes is significantly reversed by insulin therapy. For these reasons, we speculated that the defect in adipocyte glucose transport might also be corrected with exogenous insulin therapy. Therefore, we measured adipocyte 3-O-methylglucose transport in cells from five type II diabetic subjects before and after a 2-week period of intensive insulin treatment. Glycemic control was significantly improved by this regimen. The mean (+/- SE) fasting serum glucose level fell from 292 +/- 24 to 135 +/- 29 mg/100 ml (P less than 0.005), and the mean integrated glucose area under a 7-h meal tolerance test curve decreased from 171,212 +/- 20,403 to 72,408 +/- 9,292 mg/min . dl. The mean 3-O-methylglucose transport activity increased after treatment at all insulin concentrations studied, including basal (before, 0.18 +/- 0.05; after, 0.45 +/- 0.09 pmol/2 X 10(5) cells . 10 sec; P less than 0.005) and maximally effective (25 ng/ml) insulin concentrations (before, 0.50 +/- 0.14; after, 1.32 +/- 0.30 pmol/2 X 10(5) cells . 10 sec; P less than 0.025), although the mean maximal glucose transport activity was still 25% decreased compared to normal values, indicating that a residual in vitro postreceptor defect remained. These results corresponded well with the degree of reversal (75%) of the in vivo postreceptor defect, as assessed by the euglycemic glucose clamp technique. These studies demonstrated that the decrease in adipocyte glucose transport activity in type II diabetes is practically reversible by intensive insulin therapy. This closely corresponds to the reversal by insulin therapy of the postreceptor defect expressed in vivo and provides further evidence that a cellular cause of the postreceptor defect in type II diabetes is a decrease in glucose transport system activity in the major insulin target tissues.

The alpha-subunit gene of the glycoprotein hormones is normally expressed in pituitary thyrotropes and gonadotropes and in placental cells. Thus, this gene must contain elements that mediate expression and hormonal responses in different cell types. The localization of DNA regions important for expression and regulation of the alpha-subunit gene in thyrotrope cells has not previously been reported. In these studies luciferase expression constructs containing 1700 basepairs of 5' flanking DNA derived from the mouse alpha-subunit gene were introduced by electroporation into freshly dispersed cells from TSH-producing mouse pituitary tumors (TtT 97). This promoter functioned with greater efficiency in thyrotropes than in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Primer extension confirmed that transcription from the alpha-subunit constructs initiated at the same site as the endogenous gene. Studies using 5' truncations showed a progressive loss of alpha-subunit promoter activity in thyrotropes between -480 and -120, with regions upstream of -254 contributing substantially to expression in thyrotrope cells. Thyroid hormone inhibited alpha-subunit promoter activity in a dose-dependent fashion, although in vivo treatment of tumors with thyroid hormone before transfection was necessary to achieve maximal inhibition. Thyroid hormone inhibition of alpha-subunit promoter activity also occurred in GH4 cells, but no effect was observed in L-cells. Studies using 5' truncations localized a region responsible for thyroid hormone inhibition between -62 and +43, encompassing the TATA sequence and the transcriptional initiation site. When this region was compared to the thyroid hormone inhibitory regions of the alpha-subunit genes from other species and the mouse TSH beta-subunit gene, a 6-basepair motif, 5' (G/A)GTG(G/A)G 3', emerged as a possible consensus sequence for a thyroid hormone inhibitory element.

We have studied the effects of dexamethasone and prednisolone in vitro and in vivo on insulin binding, deoxyglucose uptake and glucose oxidation in rat adipocytes. In the studies in vivo, rats were treated for 22 h with dexamethasone (30 micrograms/kg) or prednisolone (200 micrograms/kg). Following sacrifice, adipocytes were prepared and the results demonstrated that cells from prednisolone treated rats showed a 17% increase in insulin binding and increased rates of basal and insulin stimulated deoxyglucose uptake and glucose oxidation. Conversely, dexamethasone administration resulted in a 22% decrease in insulin binding, and decreased rates of deoxyglucose uptake and glucose oxidation by the cells. Thus, prednisolone and dexamethasone had opposite effects in vivo. In contrast to the opposite effects of the two glucocorticoids in vivo, dexamethasone and prednisolone (each at a concentration of 1 mumol/l) had similar effects on adipocytes in vitro. Incubation of adipocytes with the steroids did not alter insulin binding, while both agents led to a comparable decrease in the rates of basal and insulin stimulated deoxyglucose uptake and glucose oxidation. Thus, dexamethasone and prednisolone have opposite effects on adipocyte glucose metabolism in vivo but have similar effects in vitro.