Gab-1-mediated IGF-1 signaling in IRS-1-deficient 3T3 fibroblasts

The insulin receptor substrate (IRS) family of proteins mediate a variety of intracellular signaling events by serving as signaling platforms downstream of several receptor tyrosine kinases including the insulin and insulin-like growth factor-1 (IGF-1) receptors. Recently, several new members of this family have been identified including IRS-3, IRS-4, and growth factor receptor-binding protein 2-associated binder-1 (Gab-1). 3T3 cell lines derived from IRS-1-deficient embryos exhibit a 70-80% reduction in IGF-1-stimulated S-phase entry and a parallel decrease in the induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase-2. Reconstitution of IRS-1 expression in IRS-1-deficient fibroblasts by retroviral mediated gene transduction is capable of restoring these defects. Overexpression of Gab-1 in IRS-1-deficient fibroblasts also results in the restoration of egr-1 induction to levels similar to those achieved by IRS-1 reconstitution and markedly increases IGF-1-stimulated S-phase progression. Gab-1 is capable of regulating these biological end points despite the absence of IGF-1 stimulated tyrosine phosphorylation. These data provide evidence that Gab-1 may serve as a unique signaling intermediate in insulin/IGF-1 signaling for induction of early gene expression and stimulation of mitogenesis without direct tyrosine phosphorylation.     

Synthesis of 1-deoxy-3-C-methyl-d-fructose and 1-deoxy-3-C-methyl-d-sorbose

Hydroxylation of trans-1,3,4-trideoxy-5,6-O-isopropylidene-3-C-methyl-d-glycero-hex-3-enulose with osmium tetraoxide gave a mixture of 1-deoxy-5,6-O-isopropylidene-3-C-methyl-d-arabino- and -d-xylo-hexulose that was partially resolved by acetonation to give 1-deoxy-2,3:4,5-di-O-isopropylidene-3-C-methyl-β-d-fructopyranose (4), 1-deoxy-3,4:5,6-di-O-isopropylidene-3-C-methyl-keto-d-fructose (5), and 1-deoxy-2,3:4,6-di-O-isopropylidene-3-C-methyl-α-d-sorbofuranose (6). Treatment of a mixture of 4 and 5 with sodium borohydride gave, after column chromatography, 4 and 1-deoxy-3,4:5,6-di-O-isopropylidene-3-C-methyl-d-manno- and -d-gluco-hexitol. Deuterated derivatives corresponding to 4–6 were obtained when isopropylidenation was carried out with acetone-d₆. Deacetonation of 4 and 5 yielded 1-deoxy-3-C-methyl-d-fructose, and 6 similarly afforded 1-deoxy-3-C-methyl-d-sorbose.     

Endothelin-1 induces lipolysis in 3T3-L1 adipocytes

Endothelin-1 (ET-1) affects glucose uptake in adipocytes and may play an important role in adipose physiology. One of the principal functions of adipose tissue is the provision of energy substrate through lipolysis. In the present study, we investigated the effects of ET-1 on lipolysis in 3T3-L1 adipocytes. When glycerol release in the culture medium was measured as an index of lipolysis, the results showed that ET-1 caused a significant increase that was time and dose dependent. With a concentration of 10 nM ET-1, stimulation of glycerol release plateaued after 4 h of exposure. This effect was inhibited by the ETA receptor antagonist BQ-610 (10 microM) but not by the ETB receptor antagonist BQ-788 (10 microM). To further explore the underlying mechanisms of ET-1 action, we examined the involvement of the cAMP-dependent protein kinase A-mediated, phospholipase A2 (PLA2)-mediated, protein kinase C (PKC)-mediated, phosphatidylinositol 3 (PI 3)-kinase-mediated, and the mitogen-activated protein kinase (MAPK)-mediated pathways. Inhibition of adenylyl cyclase activation by SQ-22536 (100 microM) did not block ET-1-induced lipolysis. Pretreatment of adipocytes with the PLA2 inhibitor dexamethasone (100 nM), the PKC inhibitor H-7 (6 microM), or the PI 3-kinase inhibitor wortmannin (100 nM) also had no effect. ET-1-induced lipolysis was blocked by inhibition of extracellular signal-regulated kinase (ERK) activation using PD-98059 (75 microM), whereas a p38 MAPK inhibitor (SB-203580; 20 microM) had no effect. Results of Western blot further demonstrated that ET-1 induced ERK phosphorylation. These data show that ET-1 induces lipolysis in 3T3-L1 adipocytes via a pathway that is different from the conventional cAMP-dependent pathway used by isoproterenol and that involves ERK activation.     

Endothelin-1 inhibits adipogenic differentiation of 3T3-L1 preadipocytes

The effect of endothelin (ET)-1 on the adipogenic differentiation of 3T3-L1 preadipocytes was examined. Cellular morphology and lipoprotein lipase activity were used as differentiation markers. ET-1 inhibited the hormone-induced adipogenic differentiation of 3T3-L1 preadipocytes morphologically and biochemically in a dose-dependent manner. These findings promote ET-1 as a potent inhibitor of adipogenic differentiation, playing an important role in cellular differentiation of preadipocytes and making it a significant regulator of lipid metabolism.     

Endothelin-1 inhibits resistin secretion in 3T3-L1 adipocytes

Resistin is an adipocyte-derived hormone whose role in the development of insulin resistance is controversial. Endothelin-1 (ET-1) is a 21 amino acid peptide demonstrated to possess vasoconstrictor, positive inotropic, mitogenic, and metabolic properties. In numerous disease states, including congestive heart failure, obesity, and diabetes, elevated levels of ET-1 have been reported and are thought to contribute to the pathology of the disease. A recent study demonstrated that ET-1 induces the expression and stimulates the secretion of the adipose tissue-derived hormone leptin. However, the effect of ET-1 on resistin secretion has not been determined. To characterize the effect of ET-1 on resistin secretion, 3T3-L1 fibroblasts were differentiated into adipocytes and allowed to mature for 14 days. Cells were incubated for 24h with ET-1 (1-100 nM), insulin (1-100 nM), insulin+ET-1 (100 nM I+E) or the appropriate vehicle or antagonist. At the end of the incubation period, resistin secretion was determined in the media by immunoblotting and densitometric analysis. ET-1 (1-100 nM) significantly decreased basal resistin secretion by 49% (1 nM), 43% (10nM), and 59% (100 nM). Insulin (1-100 nM) produced a concentration-dependent increase in resistin secretion from 3T3-L1 adipocytes (1 nM-42%, 10nM-55%, and 100 nM-86% vs. control). Insulin-stimulated resistin secretion (100 nM) was almost completely inhibited (94%) by ET-1 (100 nM). The effects of ET-1 on resistin protein secretion were inhibited by co-incubation with the ET(A) receptor antagonist BQ-610. In conclusion, our studies demonstrate that basal and hormonal stimulation of resistin secretion by insulin are inhibited by ET-1. Such findings demonstrate that resistin secretion is regulated in a similar manner to other adipose tissue factors, including leptin, in 3T3-L1 adipocytes. In addition, our findings suggest that vascular factors such as ET-1 may regulate whole body energy metabolism through adipocyte-derived hormones, including leptin and resistin.     

IGF-I regulates IRS-1 expression in 3T3-L1 adipocytes

IRS-1 (the insulin receptor substrate-1) is required for signaling by both insulin and IGF-I. Chronic treatment of 3T3-L1 adipocytes with insulin at all concentrations results in increased proteolysis of IRS-1. In contrast, treatment with low concentrations of IGF-I (EC50 = 625 pM) for 4 h caused an increase in IRS-1 to 170% of control. Actinomycin D and cycloheximide blocked the IGF-I effect, but not the insulin effect, suggesting that IGF-I stimulated the synthesis of IRS-1. Concentrations of IGF-I high enough to cause significant binding to the insulin receptor resulted in the down-regulation of IRS-1. Phosphatidylinositol 3'-kinase inhibitors blocked both the insulin and IGF-I effects. Chronic IGF-I treatment caused an increase in both acute insulin-stimulated dGlc uptake and acute IGF-I-stimulated dGlc uptake. Chronic insulin treatment caused a decrease in both acute insulin-stimulated dGlc uptake and acute IGF-I-stimulated dGlc uptake.     

Synthesis of 1-hydroxylated bile acids: Methyl 1α, 3α-dihydroxy 5β-cholan-24-oate

Methyl 1α, 3α-dihydroxy 5β-cholan-24-oate was synthesized to provide a model compound for the mass spectrometric identification of 1α-hydroxylated bile acids.