Shunbun Kita

Positive Feedback Regulation Between Adiponectin and T-Cadherin Impacts Adiponectin Levels in Tissue and Plasma of Male Mice

Adiponectin (Adipo), a multimeric adipocyte-secreted protein abundant in the circulation, is implicated in cardiovascular protective functions. Recent work documented that Adipo locally associates with responsive tissues through interactions with T-cadherin (Tcad), an atypical, glycosylphosphatidylinositol (GPI)-anchored cadherin cell surface glycoprotein. Mice deficient for Tcad lack tissue-associated Adipo, accumulate Adipo in the circulation, and mimic the Adipo knockout (KO) cardiovascular phenotype. In reverse, Tcad protein is visibly reduced from cardiac tissue in Adipo-KO mice, suggesting interdependent regulation of the 2 proteins. Here, we evaluate the effect of Adipo on Tcad protein expression. Adipo and Tcad proteins were colocalized in aorta, heart, and skeletal muscle. Adipo positively regulated levels of Tcad protein in vivo and in endothelial cell (EC) cultures. In Tcad-KO mice, binding of endogenous and exogenously administered Adipo to cardiovascular tissues was dramatically reduced. Consistently, knockdown of Tcad in cultured murine vascular ECs significantly diminished Adipo binding. In search for a possible mechanism, we found that enzymatic cleavage of Tcad with phosphatidylinositol-specific phospholipase C increases plasma Adipo while decreasing tissue-bound levels. Similarly, pretreatment of cultured ECs with serum containing Adipo attenuated phosphatidylinositol-specific phospholipase C-mediated Tcad cleavage. In vivo administration of adenovirus producing Adipo suppressed plasma levels of GPI phospholipase D, the endogenous cleavage enzyme for GPI-anchored proteins. In conclusion, our data show that both circulating and tissue-bound Adipo levels are dependent on Tcad and, in reverse, regulate tissue Tcad levels through a positive feedback loop that operates by suppressing phospholipase-mediated Tcad release from the cell surface.

Adiponectin association with T-cadherin protects against neointima proliferation and atherosclerosis

Adiponectin, an adipocyte‐derived protein abundant in the circulation, is thought to be protective against atherosclerosis. However, it is not fully understood how the association of adiponectin with vascular cells and its antiatherogenic effect are connected. In this study, T‐cadherin was essential for accumulation of adiponectin in the neointima and atherosclerotic plaque lesions, and the adiponectin–T‐cadherin association protected against vascular injury. In the apolipoprotein E‐knockout (ApoE‐KO) mice, adiponectin and T‐cadherin colocalized on en‐dothelial cells and synthetic smooth muscle cells in the aortic intima. Notably, aortic adiponectin protein disappeared in T‐cadherin/ApoE double‐knockout (Tcad/ApoE‐DKO) mice with significant elevation of blood adiponectin concentration. Furthermore, in Tcad/ApoE‐DKO mice, carotid artery ligation resulted in a significant increase of neointimal thickness compared with ApoE‐KO mice. Finally, on a high‐cholesterol diet, Tcad/ApoE‐DKO mice increased atherosclerotic plaque formation, despite a 5‐fold increase in plasma adiponectin level compared with that in ApoE‐KO mice. In vitro, knockdown of T‐cadherin from human aortic smooth muscle cells (HASMCs) with synthetic phenotype significantly reduced adiponectin accumulation on HASMCs and negated the inhibitory effect of adiponectin on proinflammatory change. Collective evidence showed that adiponectin accumulates in the vasculature via T‐cadherin, and the adiponectin–T‐cadherin association plays a protective role against neointimal and atheroscle‐rotic plaque formations. —Fujishima, Y., Maeda, N., Matsuda, K., Masuda, S., Mori, T., Fukuda, S., Sekimoto, R., Yamaoka, M., Obata, Y., Kita, S., Nishizawa, H., Funahashi, T., Ranscht, B., Shimomura, I. Adiponectin association with T‐cadherin protects against neointima proliferation and atherosclerosis. FASEB J. 31, 1571–1583 (2017) www.fasebj.org

Competitive binding of Musclin to natriuretic peptide receptor 3 with atrial natriuretic peptide.

Musclin is a novel skeletal muscle-derived secretory factor that was isolated by our group. Musclin contains a region homologous to natriuretic peptides (NPs). This study investigated the interaction between musclin and NP receptors (NPRs). Musclin specifically bound to NPR3, but not to NPR1 or NPR2. Musclin and atrial natriuretic peptide (ANP) competed for binding to NPR3. We conducted binding assays using various synthetic musclin peptides and mutant musclin proteins. The first NP-homologous region in musclin ((88)LDRL(91)) and the second homologous region ((117)MDRI(120)) were responsible cooperatively for high-affinity binding to NPR3. The first NP-homologous region was more importantly associated with binding to NPR3, than the second homologous region. The competitive nature of musclin with ANP for the natriuretic clearance receptor NPR3 was also confirmed in vivo. We conclude that musclin binds to NPR3 competitively with ANP and may affect ANP concentrations in a local or systemic manner.

Increased Dynamics of Tricarboxylic Acid Cycle and Glutamate Synthesis in Obese Adipose Tissue: In vivo Metabolic Turnover Analysis

Obesity is closely associated with various metabolic disorders. However, little is known about abnormalities in the metabolic change of obese adipose tissue. Here we use static metabolic analysis and in vivo metabolic turnover analysis to assess metabolic dynamics in obese mice. The static metabolic analyses showed that glutamate and constitutive metabolites of the TCA cycle were increased in the white adipose tissue (WAT) of ob/ob and diet-induced obesity mice but not in the liver or skeletal muscle of these obese mice. Moreover, in vivo metabolic turnover analyses demonstrated that these glucose-derived metabolites were dynamically and specifically produced in obese WAT compared with lean WAT. Glutamate rise in obese WAT was associated with down-regulation of glutamate aspartate transporter (GLAST), a major glutamate transporter for adipocytes, and low uptake of glutamate into adipose tissue. In adipocytes, glutamate treatment reduced adiponectin secretion and insulin-mediated glucose uptake and phosphorylation of Akt. These data suggest that a high intra-adipocyte glutamate level potentially relates to adipocyte dysfunction in obesity. This study provides novel insights into metabolic dysfunction in obesity through comprehensive application of in vivo metabolic turnover analysis in two obese animal models.

The unique prodomain of T-cadherin plays a key role in adiponectin binding with the essential extracellular cadherin repeats 1 and 2

Adiponectin, an adipocyte-derived circulating protein, accumulates in the heart, vascular endothelium, and skeletal muscles through an interaction with T-cadherin (T-cad), a unique glycosylphosphatidylinositol-anchored cadherin. Recent studies have suggested that this interaction is essential for adiponectin-mediated cardiovascular protection. However, the precise protein-protein interaction between adiponectin and T-cad remains poorly characterized. Using ELISA-based and surface plasmon analyses, we report here that T-cad fused with IgG Fc as a fusion tag by replacing its glycosylphosphatidylinositol-anchor specifically bound both hexameric and larger multimeric adiponectin with a dissociation constant of ∼1.0 nm and without any contribution from other cellular or serum factors. The extracellular T-cad repeats 1 and 2 were critical for the observed adiponectin binding, which is required for classical cadherin-mediated cell-to-cell adhesion. Moreover, the 130-kDa prodomain-bearing T-cad, uniquely expressed on the cell surface among members of the cadherin family and predominantly increased by adiponectin, contributed significantly to adiponectin binding. Inhibition of prodomain-processing by a prohormone convertase inhibitor increased 130-kDa T-cad levels and also enhanced adiponectin binding to endothelial cells both by more preferential cell-surface localization and by higher adiponectin-binding affinity of 130-kDa T-cad relative to 100-kDa T-cad. The preferential cell-surface localization of 130-kDa T-cad relative to 100-kDa T-cad was also observed in normal mice aorta in vivo. In conclusion, our study shows that a unique key feature of the T-cad prodomain is its involvement in binding of the T-cad repeats 1 and 2 to adiponectin and also demonstrates that adiponectin positively regulates T-cad abundance.

Adiponectin/T-cadherin system enhances exosome biogenesis and decreases cellular ceramides by exosomal release

Adiponectin, an adipocyte-derived circulating protein, accumulates in vasculature, heart, and skeletal muscles through interaction with a unique glycosylphosphatidylinositol-anchored cadherin, T-cadherin. Recent studies have demonstrated that such accumulation is essential for adiponectin-mediated cardiovascular protection. Here, we demonstrate that the adiponectin/T-cadherin system enhances exosome biogenesis and secretion, leading to the decrease of cellular ceramides. Adiponectin accumulated inside multivesicular bodies, the site of exosome generation, in cultured cells and in vivo aorta, and also in exosomes in conditioned media and in blood, together with T-cadherin. The systemic level of exosomes in blood was significantly affected by adiponectin or T-cadherin in vivo. Adiponectin increased exosome biogenesis from the cells, dependently on T-cadherin, but not on AdipoR1 or AdipoR2. Such enhancement of exosome release accompanied the reduction of cellular ceramides through ceramide efflux in exosomes. Consistently, the ceramide reduction by adiponectin was found in aortas of WT mice treated with angiotensin II, but not in T-cadherin-knockout mice. Our findings provide insights into adiponectin/T-cadherin-mediated organ protection through exosome biogenesis and secretion.

Identification of Mouse Mesenteric and Subcutaneous in vitro Adipogenic Cells

Fat accumulation and the dysfunction of visceral white adipose tissue (WAT), but not subcutaneous WAT, cause abnormalities in whole body metabolic homeostasis. However, no current drugs specifically target visceral WAT. The primary reason for this is that a practical in vitro culture system for mesenteric adipocytes has not been established. To resolve this issue, we sought to identify in vitro adipogenic cells in mesenteric and subcutaneous WATs. First, we examined the expression pattern of surface antigens in stromal-vascular fraction (SVF) cells from mouse mesenteric and subcutaneous WATs, and found the expression of 30 stem cell-related surface antigens. Then, to evaluate the adipogenic ability of each fraction, we performed in vitro screening, and identified five candidate markers for mesenteric adipogenic cells and one candidate marker for subcutaneous adipogenic cells. To investigate whether in vitro adipogenic ability accurately reflects the conditions in vivo, we performed transplantation experiments, and identified CD9− CD201+ Sca-1− cells and CD90+ cells as mesenteric and subcutaneous in vitro adipogenic cells, respectively. Furthermore, mature adipocytes derived from mesenteric and subcutaneous adipogenic cells maintained each characteristic phenotype in vitro. Thus, our study should contribute to the development of a useful culture system for visceral adipocytes.

Hypoxanthine Secretion from Human Adipose Tissue and its Increase in Hypoxia

The production of uric acid in murine white adipose tissue (mWAT), and that such production was augmented in obese mice, was recently reported. However, little is known about the secretion of metabolites associated with purine catabolism in human WAT (hWAT). The present study analyzed this in hWAT.

Multiple Gouty Tophi with Bone Erosion and Destruction: A Report of an Early-onset Case in an Obese Patient

A 27 year-old severely obese man (BMI, 35.1) had hyperuricemia and multiple gouty tophi with bone erosion and destruction, resulting in gait disturbance for 6 years after the early onset of gout at 21 years of age. His hyperuricemia was associated with hyperinsulinemia in obesity and a genetic variant of the ABCG2 gene. In addition, multiple gouty tophi with bone erosion and destruction might have been caused by hypoadiponectinemia and the elevation of the patient’s pro-inflammatory cytokine (IL-1β) level with the accumulation of visceral fat. In this case, bone and Ga-67 scintigraphy were useful for detecting the location and magnitude of gouty tophi.