Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase C␣ (PKC␣). Here we show that HGA-induced PKC␣ activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKC␣ in HGA-treated L6 cells. A direct interaction of PKC␣ with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKC␣ co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKC␣ activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKC␣. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKC␣.

Hepatitis C virus-like particles (HCV-LPsHepatitis C virus (HCV) is the major etiology of non-A, non-B hepatitis that infects 170 million people worldwide. Approximately 70 to 80% of HCV patients develop chronic hepatitis, 20 to 30% of which progress to liver cirrhosis (52). At present, there is no vaccine available to prevent HCV infection, and current therapies are not optimal. The initial steps of HCV infection (binding and entry) that are critical for tissue tropism, and hence pathogenesis, are poorly understood. Studies to elucidate this process have been hampered by the lack of robust cell culture systems or convenient small animal models that can support HCV infection.HCV is an enveloped, positive-strand RNA virus that belongs to the Flaviviridae family. Based on the sequence heterogeneity of the genome, HCV is classified into six major genotypes and ϳ100 subtypes (52). The viral genome (ϳ9.6 kb) is translated into a single polyprotein of ϳ3,000 amino acids (aa). A combination of host and viral proteases are involved in polyprotein processing to give at least nine different proteins (for a review, see reference 4). The structural proteins of HCV are believed to comprise the core protein (ϳ21 kDa) and two envelope glycoproteins: E1 (ϳ31 kDa) and E2 (ϳ70 kDa). Like other enveloped viruses, E1 and E2 proteins most likely play a pivotal role in HCV life cycle: in the assembly of infectious particle and in the initiation of viral infection by binding to its cellular receptor(s). Since hepatocytes represent the primary site of HCV replication in vivo, the HCV genome has also been found in lymphoid cells. Infection of the lymphoid cells has been implicated in extrahepatic manifestations of HCV infection such as mixed cryoglobulinemia and B-lymphocyte proliferative disorders (2, 39, 42).Detail ultrastructural features of the HCV virion remain elusive since direct visualization of virus particles from infected serum and tissues has proven to be difficult. Previous studies have shown that HCV particles vary in size between 30 and 60 nm in diameter (24,38,43). In addition, HCV particles display significant heterogeneity in buoyant density on sucrose density gradient centrifugation, ranging from low (Ͻ1.07 g/ml) to high (1.25 g/ml) density (22,24,47,54). The heterogeneity of the particle density has been attributed to the variability in size (44), nonenveloped nucleocapsid particles (28,48), and an association with antibodies or ␤-lipoproteins (38, 47).To date, the cellular receptor(s) for HCV remains controversial. The observations that HCV can infect both hepatic and lymphoid cells suggest that HCV may use different cellular receptors to access different cell types. However, the absence of an in vitro system that supports HCV replication and particle assembly has hampered studies to elucidate the early steps of HCV infection, i.e., virus binding and entry. Association of HCV virions with ␤-lipoproteins in plasma has raised the possibility that HCV may use low-density lipoprotein receptor (LDL-R) for viral entry (1,...

We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.Hepatitis C virus (HCV) infection has become a major health problem affecting an estimated 170 million people worldwide. Persistent infection occurs in more than 70% of people infected with HCV, which may be complicated by cirrhosis and/or hepatocellular carcinoma (24, 33). Despite highly competitive and extensive research in this field, a highly effective treatment is not yet available. The mainstay of anti-HCV therapy, alpha interferon (IFN-␣) or pegylated IFN-␣, together with ribavirin leads, at best, to viral clearance in ca. 41 to 54% of patients infected with HCV genotype 1a (34,38). Since mechanisms of HCV infection remain unclear, characterization of these mechanisms is now a major issue for the development of new strategies for anti-HCV treatment and prevention.

Conditions perturbing the homeostasis of the endoplasmic reticulum (ER) cause accumulation of unfolded proteins and trigger ER stress. In PC Cl3 thyroid cells, thapsigargin and tunicamycin interfered with the folding of thyroglobulin, causing accumulation of this very large secretory glycoprotein in the ER. Consequently, mRNAs encoding BiP and XBP-1 were induced and spliced, respectively. In the absence of apoptosis, differentiation of PC Cl3 cells was inhibited. mRNA and protein levels of the thyroid-specific genes encoding thyroglobulin, thyroperoxidase and the sodium/iodide symporter and of the genes encoding the thyroid transcription factors TTF-1, TTF-2 and Pax-8 were dramatically downregulated. These effects were, at least in part, transcriptional. Moreover, they were selective and temporally distinct from the general and transient PERK-dependent translational inhibition. Thyroid dedifferentiation was accompanied by changes in the organization of the polarized epithelial monolayer. Downregulation of the mRNA encoding E-cadherin, and upregulation of the mRNAs encoding vimentin, α-smooth muscle actin, α(1)(I) collagen and SNAI1/SIP1, together with formation of actin stress fibers and loss of trans-epithelial resistance were found, confirming an epithelial-mesenchymal transition (EMT). The thyroid-specific and epithelial dedifferentiation by thapsigargin or tunicamycin were completely prevented by the PP2 inhibitor of Src-family kinases and by stable expression of a dominant-negative Src. Together, these data indicate that ER stress induces dedifferentiation and an EMT-like phenotype in thyroid cells through a Src-mediated signaling pathway.

We used in situ hybridization to evaluate thyroid transcription factor-1 (TTF-1) RNA expression in individual follicles and related this to thyroglobulin (Tg) synthesis in vivo, as estimated by immunohistochemical analysis. We studied the thyroids of Wistar rats treated with thyroxine (T4) or propylthiouracil (PTU), each of which modulates TSH levels, but affects follicular function and Tg accumulation in the follicular lumen very differently. We show that TTF-1 RNA levels in vivo correlate directly with an increase in the cytoplasmic accumulation of Tg within the cells of individual follicles. Because TTF-1 increases Tg gene expression, RNA levels, and protein synthesis in thyroid cell cultures and because there is no correlation with TSH-increased Tg degradation within the follicular lumen, the increased cytoplasmic accumulation of Tg in vivo is interpreted to reflect TTF-1-increased Tg synthesis. Increases in serum TSH levels in the PTU or T4 treated animals did not always correlate with increases in this measure of increased Tg synthesis; and TSH levels did not always correlate with changes in TTF-1 RNA levels that would be expected to accompany increased Tg synthesis. As one possibility, this suggested there might be a hitherto unrecognized suppressor of TTF-1 RNA levels and TSH-induced Tg synthesis in individual follicles. The immunohistochemical data suggested that this suppressor might be follicular Tg itself. Supporting this possibility, we show that physiological concentrations of highly purified 19S follicular Tg decrease TTF-1 RNA levels in rat FRTL-5 thyroid cells and inhibit the action of TSH to increase Tg synthesis. We therefore suggest that follicular Tg is a feedback autoregulator of thyroid function that can counterregulate TSH actions on thyroid function in vivo and in thyroid cells in culture. We suggest this phenomenon contributes to follicular heterogeneity in vivo.

During its initial folding in the endoplasmic reticulum (ER), newly synthesized thyroglobulin (Tg) is known to interact with calnexin and other ER molecular chaperones, but its interaction with calreticulin has not been examined previously. In the present study, we have investigated the interactions of endogenous Tg with calreticulin and with several other ER chaperones. We find that, in FRTL-5 and PC-Cl3 cells, calnexin and calreticulin interact with newly synthesized Tg in a carbohydrate-dependent manner, with largely overlapping kinetics that are concomitant with the maturation of Tg intrachain disulphide bonds, preceding Tg dimerization and exit from the ER. Calreticulin co-precipitates more newly synthesized Tg than does calnexin; however, using two different experimental approaches, calnexin and calreticulin were found in ternary complexes with Tg, making this the first endogenous protein reported in ternary complexes with calnexin and calreticulin in the ER of live cells. Depletion of Ca(2+) from the ER elicited by thapsigargin (a specific inhibitor of ER Ca(2+)-ATPases) results in retention of Tg in this organelle. Interestingly, thapsigargin treatment induces the premature exit of Tg from the calnexin/calreticulin cycle, while stabilizing and prolonging interactions of Tg with BiP (immunoglobulin heavy chain binding protein) and GRP94 (glucose-regulated protein 94), two chaperones whose binding is not carbohydrate-dependent. Our results suggest that calnexin and calreticulin, acting in ternary complexes with a large glycoprotein substrate such as Tg, might be engaged in the folding of distinct domains, and indicate that lumenal Ca(2+) strongly influences the folding of exportable glycoproteins, in part by regulating the balance of substrate binding to different molecular chaperone systems within the ER.

We present the first identification of transient folding intermediates of endogenous thyroglobulin (Tg; a large homodimeric secretory glycoprotein of thyrocytes), which include mixed disulfides with endogenous oxidoreductases servicing Tg folding needs. Formation of disulfide-linked Tg adducts with endoplasmic reticulum (ER) oxidoreductases begins cotranslationally. Inhibition of ER glucosidase activity blocked formation of a subgroup of Tg adducts containing ERp57 while causing increased Tg adduct formation with protein disulfide isomerase (PDI), delayed adduct resolution, perturbed oxidative folding of Tg monomers, impaired Tg dimerization, increased Tg association with BiP/GRP78 and GRP94, activation of the unfolded protein response, increased ER-associated degradation of a subpopulation of Tg, partial Tg escape from ER quality control with increased secretion of free monomers, and decreased overall Tg secretion. These data point towards mixed disulfides with the ERp57 oxidoreductase in conjunction with calreticulin/calnexin chaperones acting as normal early Tg folding intermediates that can be "substituted" by PDI adducts only at the expense of lower folding efficiency with resultant ER stress.Membrane and secretory proteins are cotranslationally translocated in the lumen of the endoplasmic reticulum (ER), where they acquire their three-dimensional structure (including the formation and isomerization of disulfide bonds), typically culminating in oligomeric assembly. This is a complex task, both facilitated and monitored by ER folding enzymes and molecular chaperones. Glycoproteins are an important subset of exportable proteins, and those bearing Asn-linked oligosaccharides fold preferentially with the aid of calreticulin (CRT) and calnexin (CNX), both of which possess a lectin-like binding site that prefers association with monoglucosylated oligosaccharide processing intermediates (4). CRT and CNX might directly influence protein folding (32), but an additional critical function of these proteins is to bring newly synthesized exportable glycoproteins in close proximity with ERp57 (47), an oxidoreductase that works in a complex with CRT/CNX and promotes proper disulfide bond formation (21,46,54).Another molecular chaperone is BiP (GRP78), which binds to unfolded polypeptides, helps to prevent protein aggregation through noncovalent associations regulated by its ATPase domain (9), and works cooperatively with protein disulfide isomerase (PDI) to promote oxidative protein folding (36). Indeed, recently the concept of two distinct chaperone-oxidoreductase complexes, one comprising CRT/CNX/ERp57 and the other including BiP/PDI (37), has emerged. This fits well with earlier proposals of a reticular-like matrix in the ER lumen in which different chaperone systems are organized (25,51). In this view, PDI plays a role in the BiP system analogous to that of ERp57 in the CRT/CNX system. However, while the absence of the CRT contribution to the ERp57 system can be functionally compensated for by the presence of CNX, the...

Follicular thyroglobulin (TG) decreases expression of the thyroid-restricted transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, thereby suppressing expression of the sodium iodide symporter, thyroid peroxidase, TG, and thyrotropin receptor genes (Suzuki, K., Lavaroni, S., Mori, A., Ohta, M., Saito, J., Pietrarelli, M., Singer, D. S., Kimura, S., Katoh, R., Kawaoi, A., and Kohn, L. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 95, 8251-8256). The ability of highly purified 27, 19, or 12 S follicular TG to suppress thyroid-restricted gene expression correlates with their ability to bind to FRTL-5 thyrocytes and is inhibited by a specific antibody to the thyroid apical membrane asialoglycoprotein receptor (ASGPR), which is related to the ASGPR of liver cells. Phosphorylating serine/threonine residues of TG, by autophosphorylation or protein kinase A, eliminates TG suppression and enhances transcript levels of the thyroid-restricted genes 2-fold in the absence of a change in TG binding to the ASGPR. Follicular TG suppression of thyroid-restricted genes is thus mediated by the ASPGR on the thyrocyte apical membrane and regulated by a signal system wherein phosphorylation of serine/threonine residues on the bound ligand is an important component. These data provide a hitherto unsuspected role for the ASGPR in transcriptional signaling, aside from its role in endocytosis. They establish a functional role for phosphorylated serine/threonine residues on the TG molecule.Thyrotropin (TSH), 1 in concert with insulin and insulin-like growth factor-1 (IGF-1), regulates thyroid function (1-3). TSH increases expression of the sodium iodide symporter (NIS), thyroglobulin (TG), and thyroid peroxidase (TPO) genes; this increases concentrative iodide uptake, TG synthesis, and thyroid hormone formation (1-4). NIS, TG, and TPO expression are controlled by thyroid-restricted transcription factors: thyroid transcription factor (TTF)-1, TTF-2, and Pax-8 (5-12). TTF-2 is regulated by insulin/IGF-1 (9, 10), TTF-1 and Pax-8 by TSH/cAMP (13-16).We have recently shown that TG accumulated in the follicular lumen acts as a feedback suppressor of hormonally-increased thyroid function (17-19). Thus, follicular TG selectively suppresses expression of TTF-1, TTF-2, and Pax-8 (17, 18), thereby altering expression of the TG, TPO, NIS, and TSHR genes, and counter regulating TSH-and insulin/IGF-1-induced changes in these genes (17-19). The follicular TG acts transcriptionally; its suppressive effect is not duplicated by thyroid hormones or iodide (17-19). The mechanism by which follicular TG can act as a transcriptional suppressor is unknown.TG is synthesized as a 12 S molecule (330 kDa), but forms a 19 S dimer and 27 S tetramer; all three exist in the follicular lumen (20, 21). It has been suggested that newly synthesized TG attaches to a specific binding protein related to the lectinlike asialoglycoprotein receptor (ASPGR) of the liver (22-26) 2 and that the thyroid ASPGR vectorially transports newly synthesized TG to the fol...