Differentiation dependent biphasic regulation of adipsin gene expression by insulin and insulin-like growth factor-1 in 3T3-F442A adipocytes

Adipsin is a serine protease with complement factor D activity that is synthesized by adipocytes and secreted into the blood stream. Expression of adipsin is deficient in models of genetic (ob/ob, db/db) and acquired (monosodium glutamate-lesioned) obesity, but the cellular mechanisms responsible for this deficiency are unknown. Because hyperinsulinemia is frequently associated with obesity, we evaluated the effects of this hormone and insulin-like growth factor 1 (IGF-1) on adipsin secretion and adipsin messenger RNA (mRNA) levels in 3T3-F442A adipocytes. In the present study, we report that in fully differentiated adipocytes (after 11 days post confluence), insulin exposure progressively decreases adipsin secretion by 40%, 67%, and 78% after 2, 4, and 6 days of treatment. The inhibition of adipsin secretion by insulin is the result of a corresponding decrease in adipsin mRNA and is specific since two other differentiation-dependent fat cell mRNAs encoding aP2 (a fatty acid binding protein) and glycerophosphate dehydrogenase (GPD), are unaffected. Insulin suppresses adipsin gene expression via high affinity insulin receptors, because physiological levels of insulin produce this effect, and dose-response curves for insulin stimulation of 2-deoxyglucose uptake and glucose utilization are similar to insulin's effect on adipsin. In contrast, insulin when present during days 1-8 post confluence (during differentiation) markedly increases adipsin secretion and adipsin mRNA levels. This stimulation is due to the ability of insulin to accelerate differentiation as evidenced by corresponding increases in aP2 and GPD mRNAs as well. Insulin and IGF-1 are equipotent in this effect, suggesting that both insulin and IGF-1 receptors can mediate this response. In summary, during the differentiation of 3T3-F442A adipocytes, insulin stimulates adipsin gene expression by accelerating differentiation. As the cells become mature adipocytes, they acquire some differentiation-dependent factor, which couples insulin receptor stimulation to inhibition of adipsin gene expression. This model should aid our search for the molecular links between insulin receptor stimulation and altered gene expression.     

Effects of hypoxia on the expression of proangiogenic factors in differentiated 3T3-F442A adipocytes

OBJECTIVE: Adipocyte hypertrophy combined with hyperplasia, observed during the growth of adipose tissue in obesity, might promote the occurrence of hypoxic areas within the tissue. The aim of the present study is to assess the influence of hypoxia on the expression and secretion of adipocyte-derived proangiogenic factors. DESIGN AND METHODS: Differentiated 3T3-F442A adipocytes were submitted either to ambient hypoxia (5% O(2)) or to chemically induced hypoxia by treatments with cobalt chloride or desferrioxamine. The activities of the matrix metalloproteinases 2 and 9 (MMP-2 and -9) were determined by gelatin zymography. The expression of vascular endothelial growth factor (VEGF), hypoxia inducible factor 1 alpha (HIF-1alpha), leptin, MMP-2 and -9 were studied by the use of Western blotting and RT-PCR analyses. RESULTS: Low oxygen pressure exposure and hypoxia mimics treatments were associated with increased glucose consumption and release of lactate in differentiated 3T3-F442A adipocytes. They also led to an upregulation of the expression of leptin, VEGF and MMPs. An enhanced accumulation of HIF-1alpha protein was observed in the hypoxic adipocyte nuclei. CONCLUSION: Hypoxia, in adipocytes, markedly enhances the expression of leptin, VEGF and MMPs and stimulates the HIF-1 pathway. The present data demonstrate that hypoxic adipocytes express more proangiogenic factors and suggest that hypoxia, if occurring in adipose tissue, might be a modulator of the angiogenic process.     

Antagonism by growth hormone of insulin-sensitive hexose transport in 3T3-F442A adipocytes

We have studied the effects of GH on basal and insulin-stimulated hexose transport by 3T3-F442A adipocytes in a hormonally defined serum-free medium. Adipocytes preincubated in defined medium exhibit a low level of hexose transport which is acutely (15 min) stimulated (greater than 5-fold) by insulin (EC50, 0.1-0.2 nM). GH has acute (15-45 min) insulin-mimetic (greater than 2-fold) and chronic (4-48 h) diabetogenic (50-80%) effects on basal and insulin-stimulated hexose transport. The insulin-mimetic effect of GH has a higher EC50 (2 nM) than its diabetogenic effect (EC50, 0.2 nM). Chronic GH exposure decreases the maximal responsiveness (50-80%) and the acute sensitivity (approximately 2-fold) of hexose transport to insulin. Insulin-stimulated transport is more (approximately 5-fold) sensitive to the diabetogenic effect of GH than is basal transport. Insulin binding and degradation were not altered by chronic exposure to GH. The diabetogenic effect of GH may occur at a postinsulin binding level.     

Growth hormone alters lipolysis and hormone-sensitive lipase activity in 3T3-F442A adipocytes

While growth hormone (GH) has long been known as a lipolytic hormone, it has been difficult to study the cellular mechanisms for this effect. Since cultured 3T3-F442A adipocytes have recently proven to be useful to study chronic effects of GH on adipocyte metabolism, we examined the effects of GH on lipolysis. In these cells, GH alone produced a dose-dependent increase in the release of glycerol after 24 to 48 hours. The maximum increase occurred with 10 ng/mL human GH. The effect of GH was similar in the presence and absence of dexamethasone. Under each condition, the stimulation of glycerol release was accompanied by a GH-induced increase in the activity of hormone-sensitive lipase (HSL), a key lipolytic enzyme. The increase in HSL required 24 hours with GH and lasted at least 48 hours. The increase in HSL activity by epinephrine, like glycerol release, was potentiated by GH. Although GH potently simulates the activity of the lipogenic enzyme glycerol phosphate dehydrogenase (GPD) in differentiating 3T3-F442A preadipocytes, GH had a negligible effect on GPD activity in the differentiated adipocytes with chronic or short-term incubation. However, in contrast to the chronic effect of GH, short-term (30-minute) incubation with GH inhibited epinephrine-stimulated glycerol release, a characteristic transient antilipolytic effect of GH. These studies indicate that chronic GH treatment is lipolytic in cultured 3T3-F442A adipocytes, and document that lipolytic responses to GH involve an increase in the activity of HSL.     

Effects of human growth hormone on insulin-stimulated glucose metabolism in 3T3-F442A adipocytes

GH is believed to play a role in promoting insulin resistance in patients with diabetes and with GH excess. The means by which GH produces insulin resistance may be through direct suppression of glucose metabolism in target cells (insulin-independent) or by interfering with the ability of insulin to stimulate glucose metabolism (insulin-dependent). In 3T3-F442A adipocytes, long term incubation (24-72 h) with GH directly inhibits glucose oxidation and lipid synthesis in the absence of insulin. To distinguish the insulin-independent effects of GH on glucose metabolism from the insulin-dependent effects of GH, we examined the effect of GH on insulin-stimulated lipid accumulation in cultured 3T3-F442A adipocytes. Cells were incubated for 48-72 h with GH and then treated with insulin. Insulin stimulated lipid accumulation in GH-pretreated and control cells. Compared to control, GH-treated cells had lower absolute levels of lipid accumulation in the absence of insulin and at each insulin concentration tested. Thus, GH directly suppresses basal lipid accumulation and lowers the response to insulin. In addition, a 10 times higher insulin concentration was required to reach maximum stimulation of lipid accumulation in GH-treated cells (50 ng/ml) than in control cells (5 ng/ml). When cells were exposed simultaneously to insulin and GH for 72 h, GH treatment inhibited the ability of insulin to stimulate lipid accumulation, and the degree of suppression by GH was related to the GH concentration present. These observations suggest that GH suppresses glucose metabolism not only in the absence but also in the presence of insulin. Since short term (4-h) incubation with GH increases glucose metabolism transiently in GH-deficient preparations, we also examined the influence of short term incubation with GH on insulin responses. Cells were incubated for 4 h with varying concentrations of insulin in the simultaneous presence or absence of GH. Insulin stimulated the conversion of glucose to lipid when tested alone or in the presence of GH. Short term exposure to GH alone also stimulated glucose metabolism. The stimulation of lipid accumulation at insulin concentrations less than 5 ng/ml was greater with GH, but responses were comparable above 5 ng/ml insulin. The ability of insulin to bind to its receptor was not affected by prior treatment with GH for either short or prolonged time periods.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of vesicular H+-ATPase in insulin-stimulated glucose transport in 3T3-F442A adipocytes

In secretory cells, osmotic swelling of secretory granules is proposed to be an intermediate step in exocytic fusion of the granules with the plasma membrane. For osmotic swelling of the granule, a H (+) gradient generated by vacuolar-type H (+) -ATPase (V-ATPase) may be a driving force for accumulation of K (+) via its exchange with H (+) , concurrent with accumulation of Cl (-) and H(2)O. Here, we investigated whether a similar chemiosmotic mechanism is involved in the insulin-stimulated recruitment of GLUT4 to the plasma membrane in 3T3-F442A adipocytes. Incubating cells in a hypo-osmotic medium significantly increased 2-deoxy glucose (2-DG) uptake and the plasma membrane GLUT4 content (possibly via induction of osmotic swelling of GLUT4-containing vesicles (G4V)) and also potentiated the insulin-stimulated 2-DG uptake. Promotion of the G4V membrane ionic permeability using nigericin, an electroneutral K (+) /H (+) exchange ionophore, increased 2-DG uptake and the plasma membrane GLUT4 content. However, co-treatment with nigericin and insulin did not show an additive effect. Bafilomycin A(1), a diagnostically specific inhibitor of V-ATPase, inhibited insulin- and nigericin-stimulated 2-DG uptake. Immunoadsorption plus immunoblotting demonstrated that GLUT4 and V-ATPase co-localize in the same intracellular membranes. Together, these results indicate that V-ATPases in the G4V membrane may play an important role in the insulin-stimulated exocytic fusion of G4V with the plasma membrane via its participation in osmotic swelling of the vesicle.     

Beta -Arrestin 1 down-regulation after insulin treatment is associated with supersensitization of beta 2 adrenergic receptor Galpha s signaling in 3T3-L1 adipocytes

beta-Arrestin 1 is required for internalization and mitogen-activated protein (MAP) kinase activation by the beta2 adrenergic receptor (beta2AR). Our previous studies have shown that chronic insulin treatment down-regulates cellular beta-arrestin 1 levels, leading to a marked impairment in G protein-coupled receptor and insulin-like growth factor-1 receptor-mediated MAP kinase and mitogenic signaling. In this study, we show that chronic insulin-treated, beta-arrestin 1depleted 3T3-L1 adipocytes display (i) increased isoproterenol-induced cAMP generation (53 +/- 38% at 1.5 min, 25 +/- 19% at 5 min, 63 +/- 14% at 30 min, and 59 +/- 2% at 60 min), a Galpha(s)-associated pathway; (ii) impaired isoproterenol-induced beta2AR internalization (reduced by 98 +/- 4%), which is required for MAP kinase signaling, a Galpha(i)-associated pathway; and (iii) increased beta-arrestin 1 phosphorylation at Ser-412. Taken together, these findings represent a hitherto unknown mechanism (degradation and phosphorylation of beta-arrestin, whereby the activation of the insulin receptor, belonging to the family of receptor tyrosine kinases, causes supersensitization of Galpha(s)-associated signaling and inhibition of Galpha(i)-associated signaling by the beta2AR, a prototypical G protein-coupled receptor.     

The beta3-adrenergic agonist CL316,243 inhibits insulin signaling but not glucose uptake in primary human adipocytes.

Insulin resistance and obesity are central components of the metabolic syndrome which has become the leading cause of cardiovascular morbidity and mortality worldwide. Direct interactions of the beta (3)-adrenoceptor system with adipocyte signaling and function in humans remain poorly understood. However, this might have important consequences for the regulation of energy homeostasis and insulin resistance in states of hyperinsulinemia and sympatho-adrenergic overactivity. We therefore investigated beta (3)-adrenoceptor-mediated effects on insulin signaling and glucose uptake in mammary adipocytes of healthy women that underwent breast reduction surgery. Glucose uptake was strongly induced by insulin stimulation. This was paralleled by robust induction of insulin receptor kinase activity, insulin receptor substrate-1-associated phosphatidylinositol-3 kinase activity, and protein kinase B phosphorylation. Treatment with the beta (3)-adrenoceptor-selective agonist CL316,243 alone, neither induced alterations in the early insulin signaling cascade nor changed the basal level of glucose uptake. By contrast, pretreatment with the beta (3)-adrenoceptor agonist inhibited the insulin-induced insulin receptor substrate-1-associated phosphatidylinositol-3 kinase activity by 50 % and protein kinase B phosphorylation by 40 % without affecting insulin receptor kinase activity upstream. However, on the functional level insulin-induced glucose uptake remained unchanged by beta (3)-adrenoceptor stimulation. Our data demonstrate an insulin receptor-independent negative influence of beta (3)-adrenoceptor stimulation on proximal insulin signaling. This inhibition is apparently dissociated from glucose uptake in human adipocytes.     

Growth hormone and insulin-like growth factors I and II produce distinct alterations in glucose metabolism in 3T3-F442A adipocytes.

In 3T3-F442A adipocytes, human growth hormone (hGH) stimulates glucose oxidation in 4 hr. A maximal increase is evident at hGH concentrations of 50-100 ng/ml and rarely exceeds 50% above control. The stimulation is transient; after 48 hr of incubation with GH, glucose oxidation is significantly suppressed to 35% below control values. In view of the concept that insulin-like growth factors (IGF) may mediate the effects of GH, we compared the effects of hGH (500 ng/ml) and several preparations of IGF on glucose metabolism in 3T3 adipocytes. After 4 hr of incubation, IGF-I from human plasma stimulated glucose oxidation in a dose-related manner, producing a 10-fold increase at 50 ng/ml. Methionyl-IGF-I produced by recombinant DNA techniques was 85-88% as effective as IGF-I. IGF-II stimulated glucose oxidation 3-fold at 50 ng/ml after 4 hr of incubation. In contrast to the suppression observed with hGH after 48 hr, all three of the IGF preparations stimulated glucose oxidation after 48 hr of incubation and were as effective as they were after 4 hr. When each of the IGF preparations was tested (at 5 ng/ml) in combination with hGH, both the stimulatory and suppressive effects of GH were superimposed on the stimulation by the IGFs. Thus, the stimulatory properties of IGF differed from those of GH in that the maximum extent to which IGF increased glucose oxidation, compared with hGH, was as much as 20-fold greater. Furthermore, all of the IGF preparations stimulated glucose oxidation after 48 hr under conditions in which hGH suppressed glucose metabolism. Thus, it is unlikely that extracellular IGFs mediate the effects of hGH on glucose metabolism in 3T3-F442A adipocytes.     

Detection of activated double-stranded RNA-dependent protein kinase in 3T3-F442A cells.

We have previously reported that cultured mouse 3T3-F442A cells exhibit a transient, double-stranded RNA (dsRNA)-dependent phosphorylation of the dsRNA-dependent eIF-2 alpha kinase (eIF-2 alpha, alpha-subunit of the eukaryotic initiation factor 2) (dsI). When dsI is activated by low levels of dsRNA, it is a potent inhibitor of protein synthesis. The transient expression of dsI is due to an autocrine effect of interferon at specific stages of growth and differentiation, and it may represent a mechanism for regulating cell growth and differentiation in 3T3-F442A cells. In this report, the purification of dsI from 3T3-F442A cell cultures by a two-step procedure is described. A specific immune serum to dsI was prepared by immunizing a rabbit with highly pure preparations. Immune precipitation studies demonstrate that the serum reacts with phosphorylated dsI both in vitro and in vivo and with de novo synthesized dsI after induction with interferon. We find that dsI of 3T3 cells can undergo phosphorylation in vitro without the addition of dsRNA and in vivo in the absence of viral infection. These results are consistent with a physiologic role for dsI in the growth and differentiation of these cells.     

Oestradiol and progesterone change beta3-adrenergic receptor affinity and density in brown adipocytes.

OBJECTIVE: To check if the oestradiol- and progesterone-driven reduction in noradrenaline responsiveness of brown adipocytes is due to a reduction in either the density or the affinity of beta3-adrenoceptors (beta3-AR). beta1/beta2-AR were also studied. DESIGN: Four groups of animals were considered. (i) control rats at thermoneutrality, (ii) cold-acclimated rats, to determine beta-AR under continuous sympathetic stimulation, which is known to decrease noradrenaline responsiveness, (iii) oestradiol- and (iv) progesterone-treated cold-acclimated rats to determine hormonal effects on beta-AR populations in thermogenically active brown adipocytes. METHODS: Oestradiol and progesterone were chronically elevated by means of s.c. Silastic implants. Densities and affinities of beta-AR populations were determined by binding studies using [3H]CGP-12177 as radioligand. RESULTS: Two populations of low and high binding affinities (K(d) 1.6 and 27.3 nmol/l) corresponding to beta3- and beta1/beta2-AR respectively were found at thermoneutrality. beta3-AR density was higher than that of beta1/beta2-AR (B(max) 419 and 143 fmol/mg protein respectively). Cold-acclimated rats showed a reduction of beta3-AR binding capacity (B(max) 308 fmol/mg protein). Oestradiol and progesterone reduced the density of beta3-AR to 167 and 185 fmol/mg protein respectively, while increasing their affinity for [3H]CGP-12177 (K(d) 9.5 and 4.0 nmol/l vs 16 nmol/l in cold-acclimated untreated rats). The density of beta1/beta2-AR was also reduced after oestradiol treatment (B(max) 51 fmol/mg protein). CONCLUSIONS: Both oestradiol and progesterone reduce the density of beta3-AR in brown adipose tissue (BAT) while increasing their affinity for [3H]CGP-12177. Oestradiol also reduces the density of beta1/beta2-AR whereas cold-acclimation reduces the density of beta3-AR.     

Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.

The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta 3AR, the C-terminal tail of the beta 2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.     

Alterations in insulin signalling pathway induced by prolonged insulin treatment of 3T3-L1 adipocytes

Summary Insulin-induced glucose transport stimulation, which results from the translocation of glucose transporter 4 (GLUT 4)-containing vesicles, is completely blocked after prolonged insulin treatment of 3T3-L1 adipocytes. Since GLUT 4 expression was reduced by only 30%, we looked at the insulin signalling pathway in this insulin-resistant model. Insulin-induced tyrosine phosphorylation of the major insulin receptor substrate IRS 1 was reduced by 50±7%, while its expression was decreased by 70±4%. When cells were treated with wortmannin (a PI3-kinase inhibitor) together with insulin, the expression of IRS 1 diminished to a much lower extent. Associated with the decrease in IRS 1 expression and phosphorylation, the activation by insulin of antiphosphotyrosine immunoprecipitable PI3-kinase activity and of p44^mapk and p42^mapk activities was altered. However, the expression of these proteins was normal and p44^mapk activity remained responsive to the tumour promoter TPA. Those results indicate that prolonged insulin treatment of 3T3-L1 adipocytes induces an insulin-resistant state with a reduced ability of insulin to stimulate the PI3-kinase and the MAP-kinases and a blockade of glucose transporter translocation.Abbreviations GLUT Glucose transporter - TPA tumour promoter - MAPK mitogen-activated protein kinase - IRS insulin receptor substrate - SH2 src homology 2 - GRB GRB: Growth factor Receptor bound protein - PVDF polyvinyliden difluoride - HDM/LDM high density/low density microsomes - MBP myelin basic protein - DMEM Dulbecco's modified Eagle's medium - PMSF phenylmethanesulphonyl fluoride - PI3-kinase phosphatidylinositol 3-kinase     

Growth hormone and adipose differentiation: growth hormone-induced antimitogenic state in 3T3-F442A preadipose cells.

An additional activity for pituitary growth hormone is described--i.e., the in vitro induction of an antimitogenic state in murine 3T3-F442A preadipocyte fibroblasts. We previously developed a serum-free, hormonally defined medium permissive for the adipose differentiation of 3T3-F442A cells. When 3T3-F442A fibroblasts were maintained in serum-free medium without insulin but with growth hormone (2 nM), typical adipose differentiation did not occur. However, we found that growth hormone induced a state of cellular refractoriness to the mitogenic stimulus of fetal bovine serum as assayed by de novo DNA synthesis. The mitogen refractory condition (i.e., the antimitogenic state) was time-dependent (half maximal at approximately 2.5 days) and growth hormone concentration-dependent (half maximal and maximal at approximately 0.05 and 2.0 nM, respectively). The antimitogenic state was specifically induced by growth hormone and was not mediated by insulin-like growth factor I or prolactin. The growth hormone-induced antimitogenic state was completely reversible. The antimitogenic state was not induced by growth hormone in 3T3-C2 cells, a sister clone of 3T3 cells that exhibits essentially no adipose conversion. The kinetics for growth hormone-dependent commitment to adipose differentiation and induction of the antimitogenic state were similar. We suggest a relationship of growth hormone-induced antimitogenic state and the growth hormone-induced adipose differentiation of 3T3-F442A cells.     

Latent insulin receptors and possible receptor precursors in 3T3-L1 adipocytes.

Cell surface and cryptic insulin receptors were solubilized from the particulate fraction of murine 3T3-L1 adipocytes with buffer containing 1% Triton X-100. Solubilized receptors were affinity crosslinked with 125I-labeled insulin and disuccinimidyl suberate and characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography after specific immunoprecipitation. Two insulin-binding polypeptides were identified: the more abundant protein had a Mr of 130,000, corresponding to the size of the hormone-binding subunit of insulin receptors on the surface of target cells; the second polypeptide exhibited a Mr of 200,000 and appears to be a component of the latent pool because it was unaffected when 3T3-L1 adipocytes were exposed to trypsin under conditions that result in a 95% reduction in cell surface insulin-binding activity and the loss of the Mr 130,000 polypeptide in crosslinking experiments. Unexpectedly, the population of Mr 200,000 molecules in intact cells was accessible for limited cleavage by chymotrypsin, yielding a Mr 195,000 insulin-binding polypeptide. When 3T3-L1 adipocytes received a 15-min pulse of [35S]methionine, the predominant immunoprecipitated polypeptide had a Mr of 180,000. During a 1.5-hr chase, radioactivity in the Mr 180,000 species rapidly declined while the latent Mr 200,000 polypeptide became intensely labeled. After a 5-hr chase period, broad protein bands with Mrs of 130,000 and 90,000 were visualized as the major immunoprecipitated radioactive polypeptides. Thus, the Mr 180,000 species may be a very early biosynthetic precursor that may be subsequently processed to a Mr 200,000 form and one or both of the smaller receptor subunits at the cell surface.     

小檗碱改善3T3-L1脂肪细胞胰岛素抵抗的分子机制

目的研究小檗碱对游离脂肪酸诱导的3T3-L1脂肪细胞胰岛素抵抗的作用，探讨小檗碱改善胰岛素抵抗的分子机制。方法以0．5mmol／L软脂酸诱导3T3-L1脂肪细胞产生胰岛素抵抗，予以小檗碱进行干预，同时以阿司匹林作为阳性对照，用葡萄糖氧化酶法检测培液中的葡萄糖消耗量，以2-脱氧-[^3H]-D-葡萄糖摄入法观察葡萄糖的转运率，用Western印迹检测IκB激酶B（IKKβ）、胰岛素受体底物1（IRS-1）、磷酸肌醇3激酶p85（PI-3K p85），葡萄糖转运子4（Glut4）的蛋白表达和IKKβ 181位丝氨酸（IKKβ Ser181）、IRS-1 307位丝氨酸（IRS-1 Ser307）的磷酸化。结果0．5mmol／L软脂酸作用24h使3T3-L1脂肪细胞葡萄糖消耗降低41％，胰岛素刺激的葡萄糖转运抑制67％，IKKβSer181和IRS-1 Ser307的磷酸化增加，IRS-1和PI-3K p85蛋白的表达减少；同时加入小檗碱或阿司匹林则可逆转上述效应。但软脂酸、小檗碱、阿司匹林对3T3-L1脂肪细胞IKKβ蛋白、Glut4蛋白的表达无明显影响。结论小檗碱可以明显改善游离脂肪酸诱导的胰岛素抵抗，其分子机制可能是通过抑制IKKβ Ser181磷酸化实现的。