Atsunori Fukuhara

Adipose Tissue Hypoxia in Obesity and Its Impact on Adipocytokine Dysregulation

Obesity is linked to a variety of metabolic disorders, such as insulin resistance and atherosclerosis. Dysregulated production of fat-derived secretory factors, adipocytokines, is partly responsible for obesity-linked metabolic disorders. However, the mechanistic role of obesity per se to adipocytokine dysregulation has not been fully elucidated. Here, we show that adipose tissue of obese mice is hypoxic and that local adipose tissue hypoxia dysregulates the production of adipocytokines. Tissue hypoxia was confirmed by an exogenous marker, pimonidazole, and by an elevated concentration of lactate, an endogenous marker. Moreover, local tissue hypoperfusion (measured by colored microspheres) was confirmed in adipose tissue of obese mice. Adiponectin mRNA expression was decreased, and mRNA of C/EBP homologous protein (CHOP), an endoplasmic reticulum (ER) stress–mediated protein, was significantly increased in adipose tissue of obese mice. In 3T3-L1 adipocytes, hypoxia dysregulated the expression of adipocytokines, such as adiponectin and plasminogen activator inhibitor type-1, and increased the mRNAs of ER stress marker genes, CHOP and GRP78 (glucose-regulated protein, 78 kD). Expression of CHOP attenuated adiponectin promoter activity, and RNA interference of CHOP partly reversed hypoxia-induced suppression of adiponectin mRNA expression in adipocytes. Hypoxia also increased instability of adiponectin mRNA. Our results suggest that hypoperfusion and hypoxia in adipose tissues underlie the dysregulated production of adipocytokines and metabolic syndrome in obesity.

Involvement of nectin in the localization of junctional adhesion molecule at tight junctions

Junctional adhesion molecule (JAM) is a Ca2+-independent immunoglobulin-like cell–cell adhesion molecule which localizes at tight junctions (TJs). Claudin is a key cell–cell adhesion molecule that forms TJ strands at TJs. JAM is associated with claudin through their cytoplasmic tail-binding protein, ZO-1. JAM is furthermore associated with Par-3, a cell polarity protein which forms a ternary complex with Par-6 and atypical protein kinase C. Nectin is another Ca2+-independent immunoglobulin-like cell–cell adhesion molecule which localizes at adherens junctions (AJs). Nectin is associated with E-cadherin through their respective cytoplasmic tail-binding proteins, afadin and catenins, and involved in the formation of AJs cooperatively with E-cadherin. We show here that nectin is furthermore involved in the localization of JAM at TJs. During the formation of the junctional complex consisting of AJs and TJs in Madin-Darby canine kidney (MDCK) cells, JAM was recruited to the nectin-based cell–cell adhesion sites. This recruitment of JAM was inhibited by nectin inhibitors, which inhibited the trans-interaction of nectin. Microbeads coated with the extracellular fragment of nectin, that interacted with cellular nectin, also recruited JAM to the bead–MDCK cell contact sites. Furthermore, when cadherin-deficient L fibroblasts stably expressing both exogenous JAM and nectin (nectin-JAM-L cells) were co-cultured with L fibroblasts expressing only nectin (nectin-L cells), JAM was concentrated at the cell–cell adhesion sites between nectin-JAM-L and nectin-L cells without the trans-interaction of JAM. Analyses of the localization and immunoprecipitation of JAM revealed that it was associated with nectin through afadin and ZO-1. These results suggest that nectin has a role in the localization of JAM at TJs in the process of the formation of the junctional complex in epithelial cells.

Antagonistic and agonistic effects of an extracellular fragment of nectin on formation of E-cadherin-based cell-cell adhesion

Background: Nectin is a Ca2+‐independent immunoglobulin‐like cell‐cell adhesion molecule at the E‐cadherin‐based cell‐cell adherens junctions (AJs), and comprises a family consisting of four members, nectin‐1, ‐2, ‐3, and ‐4. Nectin and E‐cadherin are associated with afadin and α‐catenin, actin filament (F‐actin)‐binding proteins connecting respective adhesion molecules to the actin cytoskeleton, but the role of nectin in the formation of the E‐cadherin‐based cell‐cell AJs has not yet been fully understood. To obtain evidence for this role of nectin, we attempted to develop an antagonist and/or agonist of nectin.

Role of nectin in organization of tight junctions in epithelial cells

Background In polarized epithelial cells, cell‐cell adhesion forms specialized membrane structures comprised of claudin‐based tight junctions (TJs) and of E‐cadherin‐based adherens junctions (AJs). These structures are aligned from the apical to the basal side of the lateral membrane, but the mechanism of this organization remains unknown. Nectin is a Ca2+ independent immunoglobulin‐like cell‐cell adhesion molecule which localizes at AJs. Nectin is associated with E‐cadherin through their respective cytoplasmic tail‐binding proteins, afadin and catenins, and involved in the formation of AJs in cooperation with E‐cadherin. We show here that nectin is also involved in the formation of TJs.

Involvement of Nectin-activated Cdc42 Small G Protein in Organization of Adherens and Tight Junctions in Madin-Darby Canine Kidney Cells

Nectins, Ca2+-independent immunoglobulin-like cell-cell adhesion molecules, trans-interact and form cell-cell adhesion, which increases the velocities of the formation of the E-cadherin-based adherens junctions (AJs) and the claudin-based tight junctions (TJs) in Madin-Darby canine kidney (MDCK) cells. The trans-interactions of nectins furthermore induce activation of Cdc42 and Rac small G proteins, but the roles of these small G proteins activated in this way remain unknown. We examined here the role and the mode of action of Cdc42 in the organization of AJs and TJs in MDCK cells. We first made the NWASP-Cdc42 and Rac interactive binding (CRIB) domain, an inhibitor of activated Cdc42, fused to the Ki-Ras CAAX motif (NWASP-CRIB-CAAX; where A is aliphatic amino acid), which was targeted to the cell-cell adhesion sites. We then found that overexpression of NWASP-CRIB-CAAX reduced the velocities of the formation of AJs and TJs. Conversely, overexpression of a constitutively active mutant of Cdc42 (V12Cdc42) increased their velocities, and the inhibitory effect of NWASP-CRIB-CAAX was suppressed by co-expression with V12Cdc42. The inhibitory effect of NWASP-CRIB-CAAX on the formation of AJs and TJs was suppressed by co-expression of nectin-1 of which trans-interaction activated endogenous Cdc42. Moreover, the formation of the claudin-based TJs required a greater amount of activated Cdc42 than that of the E-cadherin-based AJs. These results indicate that the Cdc42 activated by the trans-interactions of nectins is involved in the organization of AJs and TJs in different mechanisms in MDCK cells.

Effects of Statins on Adipose Tissue Inflammation Their Inhibitory Effect on MyD88-Independent IRF3/IFN-β Pathway in Macrophages

Objectives—Macrophage-mediated chronic inflammation of adipose tissue is causally linked to insulin resistance in obesity. The beneficial effects of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase inhibitors (statins) on glucose metabolism have been suggested, but the effects of these agents on adipose tissue inflammation are unclear. The aim of the present study is to define the effects of statins on adipose tissue inflammation and macrophages. Methods and Results—Pravastatin or pitavastatin treatment of obese mice attenuated an increase in mRNA expressions of proinflammatory genes, including MCP1 and IL6, in adipose tissue. The supernatant of TLR4-stimulated RAW264 macrophages strongly induced the expression of these genes in 3T3-L1 adipocytes, which was inhibited by pretreatment of macrophages with either statin. Statins inhibited TLR4-mediated activation of interferon (IFN) regulatory factor (IRF)3 by either lipopolysaccharide (LPS) or palmitic acid, resulting in suppression of IFN-&bgr; expression, but not that of NF-&kgr;B or JNK. Moreover, statins strongly downregulated TLR3-mediated gene expressions by poly(I:C), but not TLR2-stimulation by zymosan A. Neutralization of IFN-&bgr; attenuated proinflammatory activities of the macrophage supernatant. Conclusions—Statins partially attenuated the development of adipose tissue inflammation in obese mice, which might be associated with an inhibitory effect of statins on TLR4-triggered expression of IFN-&bgr; via MyD88-independent signaling pathway in macrophages.

Dysregulated glutathione metabolism links to impaired insulin action in adipocytes

Oxidative stress plays an important role in obesity-related metabolic diseases. Glutathione peroxidase (GPX) is an antioxidant enzyme downregulated in adipose tissue of obese mice. However, the role of GPX in adipocytes remains elusive. The objective of this study was to clarify the pathophysiological changes in GPX activity and glutathione metabolism and their roles in the pathogenesis of insulin resistance in adipocytes. To achieve this goal, we measured cellular GPX activity, glutathione (GSH) contents, GSH/GSSG ratio, and mRNA expression of gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme for de novo GSH synthesis, in adipose tissue of control and ob/ob mice and in 3T3-L1 adipocytes treated with insulin, H(2)O(2), free fatty acid (FFA), or TNFalpha. Furthermore, we investigated the effects of GPX inhibition with a specific GPX inhibitor or RNA interference against GPX, H(2)O(2), and reduced GSH on insulin signaling in 3T3-L1 adipocytes. ob/ob Mice showed not only a decrease in cellular activity of GPXs (GPX1, -4, and -7) but also an increase in gamma-GCS expression, resulting in increased GSH contents in adipose tissue. These alterations in glutathione metabolism were also observed during differentiation of 3T3-L1 cells and their exposure to insulin, FFA, or H(2)O(2). Inhibition of GPX activity, addition of GSH, and H(2)O(2) resulted in impaired insulin signaling in 3T3-L1 adipocytes. These results suggest that decreased GPX activity and increased gamma-GCS expression lead to overaccumulation of GSH, which might be involved in the pathogenesis of insulin resistance in obesity.

Requirement of the actin cytoskeleton for the association of nectins with other cell adhesion molecules at adherens and tight junctions in MDCK cells.

Nectins, Ca2+‐independent immunoglobulin‐like cell adhesion molecules (CAMs), first form cell‐cell adhesion where cadherins are recruited, forming adherens junctions (AJs) in epithelial cells and fibroblasts. In addition, nectins recruit claudins, occludin, and junctional adhesion molecules (JAMs) to the apical side of AJs, forming tight junctions (TJs) in epithelial cells. Nectins are associated with these CAMs through peripheral membrane proteins (PMPs), many of which are actin filament‐binding proteins. We examined here the roles of the actin cytoskeleton in the association of nectins with other CAMs in MDCK cells stably expressing exogenous nectin‐1. The nectin‐1‐based cell‐cell adhesion was formed and maintained irrespective of the presence and absence of the actin filament‐disrupting agents, such as cytochalasin D and latrunculin A. In the presence of these agents, only afadin remained at the nectin‐1‐based cell‐cell adhesion sites, whereas E‐cadherin and other PMPs at AJs, α‐catenin, β‐catenin, vinculin, α‐actinin, ADIP, and LMO7, were not concentrated there. The CAMs at TJs, claudin‐1, occludin and JAM‐1, or the PMPs at TJs, ZO‐1 and MAGI‐1, were not concentrated there, either. These results indicate that the actin cytoskeleton is required for the association of the nectin‐afadin unit with other CAMs and PMPs at AJs and TJs.

Cdc42 and Rac small G proteins activated by trans interactions of nectins are involved in activation of c-Jun N-terminal kinase, but not in association of nectins and cadherin to form adherens junctions, in fibroblasts

Background: Nectins are Ca2+‐independent immunoglobulin‐like cell‐cell adhesion molecules which associate with cadherins to form adherens junctions (AJs) in epithelial cells and fibroblasts. Nectin‐1 and ‐3 are members of the nectin family which most strongly trans‐interact, causing cell‐cell adhesion. The trans‐interaction between nectin‐1 and ‐3 induces the activation of both Cdc42 and Rac small G proteins in epithelial cells. We studied the roles of Cdc42 and Rac activated in this way in L fibroblasts stably expressing both nectin‐1 and E‐cadherin (nectin‐1‐EL cells).

Adenovirus-mediated gene transfer of adiponectin reduces the severity of collagen-induced arthritis in mice.

Adiponectin (APN) is a hormone released by adipose tissue with anti-inflammatory properties. The purpose of this study was to examine the therapeutic effects of systemic delivery of APN in murine arthritis model. Collagen-induced arthritis (CIA) was induced in male DBA1/J mice, and adenoviral vectors encoding human APN (Ad-APN) or beta-galactosidase (Ad-beta-gal) as control were injected either before or during arthritis progression. Systemic APN delivery at both time points significantly decreased clinical disease activity scores of CIA. In addition, APN treatment before arthritis progression significantly decreased histological scores of inflammation and cartilage damage, bone erosion, and mRNA levels of pro-inflammatory cytokines in the joints, without altering serum anti-collagen antibodies levels. Immunohistochemical staining showed significant inhibition of complement C1q and C3 deposition in the joints of Ad-APN infected CIA mice. These results provide novel evidence that systemic APN delivery prevents inflammation and joint destruction in murine arthritis model.