Adiponectin is a recently described adipokine that has been recognized as a key regulator of insulin sensitivity and tissue inflammation. It is produced by adipose tissue (white and brown) and circulates in the blood at very high concentrations. It has direct actions in liver, skeletal muscle and the vasculature, with prominent roles to improve hepatic insulin sensitivity, increase fuel oxidation [via up-regulation of adenosine monophosphateactivated protein kinase (AMPK) activity] and decrease vascular inflammation. Adiponectin exists in the circulation as varying molecular weight forms, produced by multimerization. Recent data indicate that the highmolecular weight (HMW) complexes have the predominant action in the liver. In contrast to other adipokines, adiponectin secretion and circulating levels are inversely proportional to body fat content. Levels are further reduced in subjects with diabetes and coronary artery disease. Adiponectin antagonizes many effects of tumour necrosis factor-a (TNF-a) and this, in turn, suppresses adiponectin production. Furthermore, adiponectin secretion from adipocytes is enhanced by thiazolidinediones (which also act to antagonize TNF-a effects). Thus, adiponectin may be the common mechanism by which TNF-a promotes, and the thiazolidinediones suppress, insulin resistance and inflammation. Two adiponectin receptors, termed AdipoR1 and AdipoR2, have been identified and these are ubiquitously expressed. AdipoR1 is most highly expressed in skeletal muscle and has a prominent action to activate AMPK, and hence promote lipid oxidation. AdipoR2 is most highly expressed in liver, where it enhances insulin sensitivity and reduces steatosis via activation of AMPK and increased peroxisome-proliferator-activated receptor a ligand activity. T-cadherin, which is expressed in endothelium and smooth muscle, has been identified as an adiponectin-binding protein with preference for HMW adiponectin multimers. Given the low levels of adiponectin in subjects with the metabolic syndrome, and the beneficial effect of the adipokine in animal studies, there is exciting potential for adiponectin replacement therapy in insulin resistance and related disorders.

Clathrin-coated pits and caveolae are two of the most recognizable features of the plasma membrane of mammalian cells. While our understanding of the machinery regulating and driving clathrin-coated pit-mediated endocytosis has progressed dramatically, including the elucidation of the structure of individual components and partial in vitro reconstitution, the role of caveolae as alternative endocytic carriers still remains elusive 50 years after their discovery. However, recent work has started to provide new insights into endocytosis by caveolae and into apparently related pathways involving lipid raft domains. These pathways, distinguished by their exquisite sensitivity to cholesterol-sequestering agents, can involve caveolae but also exist in cells devoid of caveolins and caveolae. This review examines the current evidence for the involvement of rafts and caveolae in endocytosis and the molecular players involved in their regulation.

Adiponectin is a secreted, multimeric protein with insulin-sensitizing, antiatherogenic, and antiinflammatory properties. Serum adiponectin consists of trimer, hexamer, and larger high-molecular-weight (HMW) multimers, and these HMW multimers appear to be the more bioactive forms. Multimer composition of adiponectin appears to be regulated; however, the molecular mechanisms involved are unknown. We hypothesize that regulation of adiponectin multimerization and secretion occurs via changes in posttranslational modifications (PTMs). Although a structural role for intertrimer disulfide bonds in the formation of hexamers and HMW multimers is established, the role of other PTMs is unknown. PTMs identified in murine and bovine adiponectin include hydroxylation of multiple conserved proline and lysine residues and glycosylation of hydroxylysines. By mass spectrometry, we confirmed the presence of these PTMs in human adiponectin and identified three additional hydroxylations on Pro71, Pro76, and Pro95. We also investigated the role of the five modified lysines in multimer formation and secretion of recombinant human adiponectin expressed in mammalian cell lines. Mutation of modified lysines in the collagenous domain prevented formation of HMW multimers, whereas a pharmacological inhibitor of prolyl- and lysyl-hydroxylases, 2,2'-dipyridyl, inhibited formation of hexamers and HMW multimers. Bacterially expressed human adiponectin displayed a complete lack of differentially modified isoforms and failed to form bona fide trimers and larger multimers. Finally, glucose-induced increases in HMW multimer production from human adipose explants correlated with changes in the two-dimensional electrophoresis profile of adiponectin isoforms. Collectively, these data suggest that adiponectin multimer composition is affected by changes in PTM in response to physiological factors.

Macrophage pre-treatment with bacterial lipopolysaccharide (LPS) boosts subsequent activation of the NLRP3 inflammasome, which controls caspase-1-dependent pro-inflammatory cytokine maturation. Previous work has attributed this phenomenon (known as LPS 'priming') to LPS-dependent induction of NLRP3 expression. Whilst this plays a role, here we demonstrate that rapid LPS priming of NLRP3 inflammasome activation can occur independently of NLRP3 induction, since the priming effect of LPS is still apparent at short pre-treatment times in which NLRP3 protein expression remains unchanged. Furthermore, rapid LPS priming is still evident in Nlrp3(-/-) primary macrophages with NLRP3 expression reconstituted using a constitutive promoter. Similarly, we found that LPS potentiates AIM2 inflammasome activation to submaximal doses of cytosolic DNA without concomitant upregulation of AIM2 protein expression. Our data suggest that, in addition to augmenting NLRP3 inflammasome activity via NLRP3 induction, LPS boosts caspase-1 activation by the NLRP3 and AIM2 inflammasomes by an acute mechanism that is independent of inflammasome sensor induction.

Simian virus 40 (SV40) is a nonenveloped virus that has been shown to pass from surface caveolae to the endoplasmic reticulum in an apparently novel infectious entry pathway. We now show that the initial entry step is blocked by brefeldin A and by incubation at 20°C. Subsequent to the entry step, the virus reaches a domain of the rough endoplasmic reticulum by an unknown pathway. This intracellular trafficking pathway is also brefeldin A sensitive. Infection is strongly inhibited by expression of GTP-restricted ADP-ribosylation factor 1 (Arf1) and Sar1 mutants and by microinjection of antibodies to ␤COP. In addition, we demonstrate a potent inhibition of SV40 infection by the dipeptide N-benzoyl-oxycarbonyl-Gly-Phe-amide, which also inhibits late events in cholera toxin action. Our results identify novel inhibitors of SV40 infection and show that SV40 requires COPI-and COPII-dependent transport steps for successful infection. INTRODUCTIONViruses have exploited endocytic pathways to overcome the barrier presented by the plasma membrane and enter animal cells. Although the entry of viruses via clathrin-coated pits has been extensively studied and is well understood (Marsh et al., 1984), entry of viruses by alternative pathways has been less well characterized. Simian virus 40 (SV40) is a nonenveloped virus that uses an apparently unique entry route to reach the nucleus of animal cells. SV40 binds to major histocompatibility complex (MHC) class I molecules on the cell surface (Breau et al., 1992) and then is observed in tight-fitting, nonclathrin-coated pits (Kartenbeck et al., 1989;Anderson et al., 1996;Stang et al., 1997;Norkin, 1999; Parton and Lindsay, 1999). These pits are enriched in caveolin-1 but are smaller than caveolae, raising the possibility that caveolin is recruited around the virus . Consistent with this model, the levels of caveolin-1 associated with virus-containing membranes were shown to increase with time . Subsequently, the virus is internalized in a process that is sensitive to cholesterol-perturbing agents . The role of caveolin in this process is suggested by the finding that viral infection is also inhibited by dominant negative mutants of caveolin (Roy et al., 1999). Viral entry apparently occurs without concomitant internalization of MHC class I (Anderson et al., 1998), in a process dependent on tyrosine kinase activity (Dangoria et al., 1996;Chen and Norkin, 1999;Norkin, 1999). Early electron microscopic studies suggested that the virus is then transported directly to the endoplasmic reticulum (ER) because no intermediate stations were identified (Kartenbeck et al., 1989). A direct pathway between the cell surface and ER would be a novel finding, although parallels can be made to the cholesterol oxidase-induced redistribution of caveolin-1 from caveolae to the lumen of the ER (Smart et al., 1994).The virus eventually reaches the nucleus where uncoating and replication occur. At some stage in the entry pathway the SV40 virions appear to translocate across a membrane into the cytosol and...

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These data demonstrate that the relationship between insulin resistance and adiponectin is similar in controls and patients with genotype 3 but not genotype 1 infection. The greater degree of insulin resistance in genotype 1 appears to be a genotype-specific effect.

These data show that AA and TZD have synergistic rather than simple additive effects on secretion of HMW adiponectin from human adipocytes and raise the possibility that differences in AA levels may contribute to the variability in adiponectin multimer profiles and efficacy of TZD in humans. Our results also provide a rationale for longitudinal clinical trials investigating the effects of AA supplementation with or without TZD on adiponectin and metabolic profiles.