Chi-Wei Liu

Green tea (−)-epigallocatechin gallate inhibits insulin stimulation of 3T3-L1 preadipocyte mitogenesis via the 67-kDa laminin receptor pathway

Insulin and (-)-epigallocatechin gallate (EGCG) have been reported to regulate fat cell mitogenesis and adipogenesis, respectively. This study investigated the pathways involved in EGCG modulation of insulin-stimulated mitogenesis in 3T3-L1 preadipocytes. EGCG inhibited insulin stimulation of preadipocyte proliferation in a dose- and time-dependent manner. EGCG also suppressed insulin-stimulated phosphorylation of the insulin receptor-beta, insulin receptor (IR) substrates 1 and 2 (IRS1 and IRS2), and mitogen-activated protein kinase pathway proteins, RAF1, MEK1/2, and ERK1/2, but not JNK. Furthermore, EGCG inhibited the association of IR with the IRS1 and IRS2 proteins, but not with the IRS4 protein. These data suggest that EGCG selectively affects particular types of IRS and MAPK family members. Generally, EGCG was more effective than epicatechin, epicatechin gallate, and epigallocatechin in modulating insulin-stimulated mitogenic signaling. We identified the EGCG receptor [also known as the 67-kDa laminin receptor (67LR)] in fat cells and found that its expression was sensitive to growth phase, tissue type, and differentiation state. Pretreatment of preadipocytes with 67LR antiserum prevented the effects of EGCG on insulin-stimulated phosphorylation of IRS2, RAF1, and ERK1/2 and insulin-stimulated preadipocyte proliferation (cell number and bromodeoxyuridine incorporation). Moreover, EGCG tended to increase insulin-stimulated associations between the 67LR and IR, IRS1, IRS2, and IRS4 proteins. These data suggest that EGCG mediates anti-insulin signaling in preadipocyte mitogenesis via the 67LR pathway.

Green tea epigallocatechin gallate inhibits insulin stimulation of adipocyte glucose uptake via the 67-kilodalton laminin receptor and AMP-activated protein kinase pathways.

Insulin and (-)-epigallocatechin gallate (EGCG) are reported to regulate obesity and fat accumulation, respectively. This study investigated the pathways involved in EGCG modulation of insulin-stimulated glucose uptake in 3T3-L1 and C3H10T1/2 adipocytes. EGCG inhibited insulin stimulation of adipocyte glucose uptake in a dose- and time-dependent manner. The concentration of EGCG that decreased insulin-stimulated glucose uptake by 50-60% was approximately 5-10 µM for a period of 2 h. At 10 µM, EGCG and gallic acid were more effective than (-)-epicatechin, (-)-epigallocatechin, and (-)-epicatechin 3-gallate. We identified the EGCG receptor [also known as the 67-kDa laminin receptor (67LR)] in fat cells and extended the findings for this study to clarify whether EGCG-induced changes in insulin-stimulated glucose uptake in adipocytes could be mediated through the 67LR. Pretreatment of adipocytes with a 67LR antibody, but not normal rabbit immunoglobulin, prevented the effects of EGCG on insulin-increased glucose uptake. This suggests that the 67LR mediates the effect of EGCG on insulin-stimulated glucose uptake in adipocytes. Moreover, pretreatment with an AMP-activated protein kinase (AMPK) inhibitor, such as compound C, but not with a glutathione (GSH) activator, such as N-acetyl-L-cysteine (NAC), blocked the antiinsulin effect of EGCG on adipocyte glucose uptake. These data suggest that EGCG exerts its anti-insulin action on adipocyte glucose uptake via the AMPK, but not the GSH, pathway. The results of this study possibly support that EGCG mediates fat content.

Octylphenol stimulates resistin gene expression in 3T3-L1 adipocytes via the estrogen receptor and extracellular signal-regulated kinase pathways

Resistin is known as an adipocyte-specific secretory hormone that can cause insulin resistance and decrease adipocyte differentiation. It can be regulated by sexual hormones. Whether environmental estrogens regulate the production of resistin is still not clear. Using 3T3-L1 adipocytes, we found that octylphenol upregulated resistin mRNA expression in dose- and time-dependent manners. The concentration of octylphenol that increased resistin mRNA levels by 50% was approximately 100 nM within 6 h of treatment. The basal half-life of resistin mRNA induced by actinomycin D was lengthened by octylphenol treatment, suggesting that octylphenol decreases the rate of resistin mRNA degradation. In addition, octylphenol stimulated resistin protein expression and release. The basal half-life of resistin protein induced by cycloheximide was lengthened by octylphenol treatment, suggesting that octylphenol decreases the rate of resistin protein degradation. While octylphenol was shown to increase activities of the estrogen receptor (ER) and MEK1, signaling was demonstrated to be blocked by pretreatment with either ICI-182780 (an ERalpha antagonist) or U-0126 (a MEK1 inhibitor), in which both inhibitors prevented octylphenol-stimulated phosphorylation of ERK. These results imply that ERalpha and ERK are necessary for the octylphenol stimulation of resistin mRNA expression. Moreover, U-0126 antagonized the octylphenol-increased resistin protein expression and release. These data suggest that the way octylphenol signaling increases resistin protein levels is similar to that by which it increases resistin mRNA levels; it is likely mediated through an ERK-dependent pathway. In vivo, octylphenol increased adipose resistin mRNA expression and serum resistin and glucose levels, supporting its in vitro effect.

Endothelin-1 stimulates suppressor of cytokine signaling-3 gene expression in adipocytes.

Endothelin (ET)-1 and suppressor of cytokine signaling (SOCS)-3 were respectively found to regulate energy metabolism and hormone signaling in fat cells. Although ET-1 can also regulate the expression of SOCS-3-stimulating hormones, it is still unknown whether ET-1 regulates SOCS-3 gene expression. This study investigated the pathways involved in ET-1s modulation of SOCS-3 gene expression in 3T3-L1 adipocytes. ET-1 upregulated SOCS-3 mRNA and protein expression in dose- and time-dependent manners. The concentration of ET-1 that increased SOCS-3 mRNA levels by 250-400% was ∼100nM with 2-4h of treatment. Treatment with actinomycin D prevented ET-1-stimulated SOCS-3 mRNA expression, suggesting that the effect of ET-1 requires new mRNA synthesis. Pretreatment with the ET type A receptor (ET(A)R) antagonist, BQ-610, but not the ET type B receptor (ET(B)R) antagonist, BQ-788, prevented the stimulatory effect of ET-1 on SOCS-3 gene expression. The specific inhibitors of either MEK1 (U-0126 and PD-98059), JAK (AG-490), JNK (SP-600125), or PI3K (LY-294002 and wortmannin) reduced ET-1-increased levels of SOCS-3 mRNA and respectively inhibited ET-1-stimulated activities of MEK1, JAK, JNK, and PI3K. These results imply that the ET(A)R, ERK, JAK, JNK, and PI3K are functionally necessary for ET-1s stimulation of SOCS-3 gene expression. Moreover, ET-1 was observed to upregulate expressions of SOCS-1, -2, -3, -4, -5, and -6 mRNAs, but not SOCS-7 or cytokine-inducible SH2-containing protein-1 mRNAs. This suggests that ET-1 selectively affects particular types of SOCS family members. Changes in SOCS gene expressions induced by ET-1 may help explain the mechanism by which ET-1 modulates hormone signaling of adipocytes.

Green tea (−)-epigallocatechin gallate suppresses IGF-I and IGF-II stimulation of 3T3-L1 adipocyte glucose uptake via the glucose transporter 4, but not glucose transporter 1 pathway

This study investigated the pathways involved in EGCG modulation of insulin-like growth factor (IGF)-stimulated glucose uptake in 3T3-L1 adipocytes. EGCG inhibited IGF-I and IGF-II stimulation of adipocyte glucose uptake with dose and time dependencies. EGCG at 20μM for 2h decreased IGF-I- and IGF-II-stimulated glucose uptake by 59% and 64%, respectively. Pretreatment of adipocytes with antibody against the EGCG receptor (also known as the 67-kDa laminin receptor; 67LR), prevented the effects of EGCG on IGF-increased glucose uptake, but pretreatment with normal rabbit immunoglobulin did not. This suggests that the 67LR mediates the anti-IGF effect of EGCG on adipocyte glucose uptake. Further analysis indicated EGCG, IGF-I, and IGF-II did not alter total levels of GLUT1 or GLUT4 protein. However, EGCG prevented the IGF-increased GLUT4 levels in the plasma membrane and blocked the IGF-decreased GLUT4 levels in low-density microsomes. Neither EGCG nor its combination with IGF altered GLUT1 protein levels in the plasma membrane and low-density microsomes. EGCG also suppressed the IGF-stimulated phosphorylation of IGF signaling molecules, PKCζ/λ, but not AKT and ERK1/2, proteins. This study suggests that EGCG suppresses IGF stimulation of 3T3-L1 adipocyte glucose uptake through inhibition of the GLUT4 translocation, but not through alterations of the GLUT1 pathway.

Endothelin-1 Stimulates Resistin Gene Expression

Resistin and endothelin (ET)-1 have been reported to inhibit adipogenesis and regulate adipocyte insulin resistance, respectively. Although both hormones interact with each other, the exact signaling pathway of ET-1 to act on resistin gene expression is still unknown. Using 3T3-L1 adipocytes, we investigated the signaling pathways involved in ET-1-stimulated resistin gene expression. The up-regulation of resistin mRNA expression by ET-1 depends on concentration and timing. The concentration of ET-1 that increased resistin mRNA levels by 100%-250% was approximately 100 nM for a range of 0.25-12 hours of treatment. Treatment with actinomycin D blocked ET-1-increased resistin mRNA levels, suggesting that the effect of ET-1 requires new mRNA synthesis. Treatment with an inhibitor of the ET type-A receptor, such as N-[1-Formyl-N-[N-[(hexahydro-1H-azepin-1-yl)carbonyl]-L-leucyl]-D-tryptophyl]-D-tryptophan (BQ610), but not with the ET type-B receptor antagonist N-[(cis-2,6-Dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl-1-(methoxycarbonyl)-D-tryptophyl-D-norleucine (BQ788), blocked ET-1, increased the levels of resistin mRNA, and phosphorylated levels of downstream signaling molecules, such as ERK1/2, c-Jun N-terminal kinases (JNKs), protein kinase B (AKT), and signal transducer and activator of transcription 3 (STAT3). Moreover, pretreatment of specific inhibitors of either ERK1/2 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene [U0126] and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one [PD98059], two inhibitors of MEK1), JNKs (SP600125), phosphatidylinositol 3-kinase/AKT (LY294002 and Wortmannin), or Janus kinase 2 (JAK2)/STAT3 ((E)-2-Cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide, AG490) prevented ET-1-increased levels of resistin mRNA and reduced the ET-1-stimulated phosphorylation of ERK1/2, JNKs, AKT, and STAT3, respectively. However, the p38 kinase antagonist 4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580) did not alter the effect of ET-1. These results imply that ET type-A receptor, ERK1/2, JNKs, AKT, and JAK2, but not ET type-B receptor or p38, are necessary for the ET-1 stimulation of resistin gene expression. In vivo observations that ET-1 increased resistin mRNA and protein levels in sc and epididymal adipose tissues support the in vitro findings.

Cycloheximide stimulates suppressor of cytokine signaling-3 gene expression in 3T3-L1 adipocytes via the extracellular signal-regulated kinase pathway

Suppressor of cytokine signaling (SOCS)-3 can act as a regulator of energy metabolism and cytokine signaling in fat cells. It is regulated by hormones and toxicological factors. However, the action of cycloheximide on expression of adipocyte SOCS-3 gene is unknown. Using 3T3-L1 adipocytes, we found that cycloheximide up-regulated SOCS-3 mRNA expression in dose- and time-dependent manners. Treatment with actinomycin D prevented cycloheximide-stimulated SOCS-3 mRNA expression, suggesting that the effect of cycloheximide requires new mRNA synthesis. While cycloheximide was shown to increase activities of MEK1 and JNK, signaling was demonstrated to be inhibited by pretreatment with either MEK1 inhibitors U0126 and PD98059, or with the JNK inhibitor SP600125. U0126 and PD98059, respectively, reduced cycloheximide-stimulated SOCS-3 mRNA expression, but SP600125 did not antagonize cycloheximide effect. Moreover, cycloheximide was observed to up-regulate expression of other SOCS family members, such as SOCS-1, -2, -4, -5, -6, -7, and cytokine-inducible SH2-containing protein (CIS)-1 mRNAs. Such effects varied with the dosage and duration of cycloheximide treatment. These results imply the functional MEK1/ERK-mediated pathway is necessary for the cycloheximide stimulation of SOCS-3 gene expression.

Chapter 88 – Effect of Green Tea (−)-Epigallocatechin Gallate on Adipocytes: Signaling Effects

Green tea catechins, especially (−)-epigallocatechin-3-gallate (EGCG), regulate fat cell functions. This chapter reviews the evidence for understanding EGCG signaling in modulating multiple processes of fat cells, such as growth, apoptosis, differentiation, metabolism, and endocrine activity. Based on a variety of laboratory data, EGCG signaling in regulating these processes may be related to certain pathways and molecules, including (1) the EGCG receptor in association with receptor tyrosine kinase (RTK) signaling molecules, (2) phosphorylation of RTK signaling elements, (3) production of reactive oxygen species, (4) activities and expression of cell cycle-controlling and differentiation-controlling factors, (5) activity and expression of apoptotic and metabolic enzymes, (6) activity of energy substrate transporters, and (7) expression and secretion of adipokines. These EGCG signaling pathways associated with fat cells may explain the in vivo actions of EGCG on adipose tissues and indicate its possible use in the prevention of obesity.

Effect of Green Tea (−)-Epigallocatechin Gallate on Adipocytes

Green tea catechins, especially (−)-epigallocatechin-3-gallate (EGCG), regulate fat cell functions. This chapter reviews the evidence for understanding EGCG signaling in modulating multiple processes of fat cells, such as growth, apoptosis, differentiation, metabolism, and endocrine activity. Based on a variety of laboratory data, EGCG signaling in regulating these processes may be related to certain pathways and molecules, including (1) the EGCG receptor in association with receptor tyrosine kinase (RTK) signaling molecules, (2) phosphorylation of RTK signaling elements, (3) production of reactive oxygen species, (4) activities and expression of cell cycle-controlling and differentiation-controlling factors, (5) activity and expression of apoptotic and metabolic enzymes, (6) activity of energy substrate transporters, and (7) expression and secretion of adipokines. These EGCG signaling pathways associated with fat cells may explain the in vivo actions of EGCG on adipose tissues and indicate its possible use in the prevention of obesity.

Effect of Green Tea (−)-Epigallocatechin Gallate on Adipocytes: Signaling Effects

Green tea catechins, especially (−)-epigallocatechin-3-gallate (EGCG), regulate fat cell functions. This chapter reviews the evidence for understanding EGCG signaling in modulating multiple processes of fat cells, such as growth, apoptosis, differentiation, metabolism, and endocrine activity. Based on a variety of laboratory data, EGCG signaling in regulating these processes may be related to certain pathways and molecules, including (1) the EGCG receptor in association with receptor tyrosine kinase (RTK) signaling molecules, (2) phosphorylation of RTK signaling elements, (3) production of reactive oxygen species, (4) activities and expression of cell cycle-controlling and differentiation-controlling factors, (5) activity and expression of apoptotic and metabolic enzymes, (6) activity of energy substrate transporters, and (7) expression and secretion of adipokines. These EGCG signaling pathways associated with fat cells may explain the in vivo actions of EGCG on adipose tissues and indicate its possible use in the prevention of obesity.