Adipogenesis is a primary energy valancing response in physiological status and critical in embryo development. One of the essential factors for initiation and maintaining of adipogenesis is the composition of extracellular matrix. Previously, we confirmed the effects of diphlorethohydroxycarmalol (DPHC), an extract of Ishige okamurae, on the antiobesity effects and ECM stability in adipose tissue. In vitro model for adipogenesis study, 3T3-L1, a precursor cell type of adipocyte, and the adipose-tissue derived stem cell (ADSC) can be used. Usually the induction period for adipocyte is shorter in 3T3-L1 than in ADSCs. However, so far, the difference of the expression patterns of ECM components in 3T3-L1 and ADSCs, and the effects of DPHC are not much known. We induced differentiation of 3T3-L1 and ADSCs into adipocyte with or without DPHC (0, 0.4, 2, 10, 50 μg/mL) and confirmed the adipogenesis with adipogenic markers (PPAR-γ, LDL). After then, the levels of collagen type 1 alpha 1 (Col1a1), collagen type 3 alpha 1 (Col3a1), collagen type 4 (Col4), collagen type 6 (Col6), Elastin (Eln) and microfibrillar associated protein 5 (Mfap5) were analyzed with real-time RT-PCR. During early adipogenesis of ADSC, the expression levels of Col1, Col3, Col6, and Mfap5 mRNA were decreased but Col4 and Eln mRNA were increased. In the matured adipocyte, the expression levels of Col1, Col3, Col4, Mfap5 mRNA were decreased but not Eln. In the case of early differentiation of 3T3-L1, the expression levels of Col1, Col3, Eln mRNA were decreased but the expression levels of Col6 and Mfap5 were increased. In matured adipocyte of 3T3-L1, the expression levels of Col1, Col3, Eln, Mfap5 mRNA were increase but the expression level of Col6 mRNA was decreased. The expression levels of Col4, Eln mRNA were suppressed by 50 mg/mL DPHC treatment during early adipogenic period of ADSC. On the other hand in 3T3-L1, the expression levels of Col3 and Col6 mRNA were not changed by the DPHC treatment during early induction period. In the matured adipocytes derived from ADSC, Col1 mRNA levels was not decreased by the treatment of 50 mg/mL DPHC. Col4 mRNA levels was not increased by DPHC treatment. In the case of matured adipocytes derived from 3T3-L1, DPHC suppressed the increase of Col1, Col3, Col6 mRNA expression and the expression of Col4 and Eln mRNA was decreased. In summary, these data show that expression levels of each ECM component types are dramatically changed with some common patterns in two cell types, and the treatment of DPHC can modify the expression patterns of some ECM components in each cell types. It is suggested that one of the reason of antiadipogenic effect of DPHC may be the ECM modification.

Diphlorethohydroxycarmalol (DPHC) is a known to modulate the expression of extracellular matrix (ECM) components in 3T3-L1. However, the possible role of DPHC in integument stability during obesity induction is not clear yet. We evaluated the effects of DPHC on collagen or elastic fiber quantity in integument during obesity induction with high-fat diet. The dorsal back integument sections were stained with hematoxylin–eosin, Masson trichrome, and Verhoff-Van Gieson. The intensities of collagen fibers and elastin fibers were analyzed with ImageJ. The number of fibroblasts was counted at ×1,000 fields. The number of fibroblast was increased by obesity induction, but DPHC suppressed it in a concentrationdependent manner both in lean and obese mice. On the other hand, the intensities of collagen fibers were increased by DPHC treatment in obese mice groups but not in lean mice groups. The intensities of collagen fibers of obese mice were lower than that of the lean mice in 0% group. However, the number became similar between lean and obese mice by the treatment of DPHC. The intensity of elastic fibers was increased in the lean mice with the concentration of DPHC. In the obese mice group, there were increasing patterns but only significant at 10% DPHC group. The intensity of elastic fibers of obese mice was higher than lean mice in 0%, 1%, and 10% groups. Histologically epithelial cells and follicle cells which were diffused nuclear staining forms were increased by DPHC treatment. The results suggest that the activity of integument cells during obesity induction can be modulated by DPHC.

Adipogenesis is critical in development and homeostasis of energy metabolism. However, in these days, the obesity has become prevalent and became a cause of medial complication. Various applications have been suggested to prevent or decrease accumulaiton of energy in fat cells. However, those have little usefulness and have various side effects. Diphlorethohydroxy-carmalol (DPHC) is a phlorotannin compound, with various biological activities in vitro and in vivo. In here, we studied that DPHC could modify the accumulation of fat on integument. The size of adipocytes and thickness of the subcutanous fat tissue was analyzed after treatment of cosmetics contained 0, 0.01, 0.1, 1, or 10 % DPHC using NIS Element D 4.10.00 software (Nikon). The viability and proliferation of cell was analyzed after 0, 0.4, 2, 10, or 50μg/ml of DPHC treatment using MTT (3-[4,5-dimethylthiazo-2-yl]-2,5-diphenyl tetrazolium bromide) assay (R&D system, Cat # 48090-025-k) and measurment of doubling time. Accumulation of lipids in differentiating preadipocytes was analyzed with spectrophotometer after Oil Red-O staining. The size of adipocyte and thickness in skin was decreased in DPHC treated mice. The metabolic activity and doubling of 3T3-L1 were suppressed by DPHC in concentration dependent manner. DPHC also inhibit accumulation of lipids in the adipocyte. The expression of the marker genes for adipocyte differentiation coincided with cytochemical results. Base on them, it is suggested that DPHC has antiobesity effects in integument through suppress accumulation of lipids and suppress the proliferation and differentiation both of adipose stem cells and precursor cells.

Although, one of the etiologies of localized lipodystrophy of the subcutaneous connective tissue (cellulite) is the histological alternation of adipose tissue, the characteristics of expression of the components of extracellular matrix (ECM) components during adipogenesis are not uncovered. In this study, the effects of caffeine and Ishige okamurae originated diphlorethohydroxycarmalol (DPHC) on the expression of extracellualr fibers was analyzed with quantitative RT-PCR during differentiation induction of mouse subcutaneous adipose derived stem cells (msADSC) into adipocyte. The expression levels of Col1a, Col3a1, and Col61a were decreased by the adipogenci induction in a time-dependent manners. However, Col2a mRNA and Col4a1 mRNA expressions were oposit to them. Caffeine and DPHC stimulated the changes of the expression of these collagens. Eln mRNA expression was increased by induction. DPHC stimulated the expression of it. Mfap5 mRNA expression was deceased in both adipogenic cell and matured adipocytes. Caffeine suppressed the expression of Mfap5 but the effect of DPHC was different by the concentration. The expression of bioglycan, decorin, and lumican were also modified by caffeine and DPHC in a concentration-dependent manner. Based on this study, we revealed firstly the effects of caffeine and DPHC on the expression of collagens, elastin, and glycoproteins during adipogenesis of msADSCs. Those results suggest that DPHC may have antiadipogenic effect and has more positive effets on normal adipose tissue generation and work as suppressor the abnormality of ECM structure. Such results indicate that DPHC can be applied in keeping the stability of the ECM of adipogenic tissues.

Brown algae is variety of biological compounds, including xanthophyll, pigments, fucoidans, phycocolloids, and phlorotannins. Several studies concerning these types of compounds have pointed out the variety of biological benefits associated with the algae, including antioxidant, anticoagulant, antihypertension, antibacterial, and antitumor activities. Diphlorethohydroxy- carmalol (DPHC) is a phlorotannin compound isolated from the brown algae Ishige okamurae, with various biological activities in vitro and in vivo. Numerous studies have shown that antioxidant assist inhibition of accumulation of fat. So we studied that effect of DPHC isolated from Ishige okamurae modified the accumulation of fat on preadipocyte, 3T3-L1 cells in vitro. First, the viability of cell was analyzed after 0.4, 2, 10, 50 μg/ml of DPHC treatment using MTT (3-[4,5-dimethylthiazo-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Second, proliferation of cell was analyzed after 0.4, 2, 10, 50 μg/ml of DPHC treatment through measure doubling time. 3T3-L1 cell differentiation into adipocyte was analyzed after induction in the induction medium containing DPHC. The metabolic activity was suppressed by DPHC in concentration dependent manner. Doubling of 3T3-L1 was delayed by the treatment of DPHC in concentration dependent manner. DPHC also inhibit accumulation of triglyceride in the adipocyte. The expression of the marker genes for adipocyte differentiation coincided with cytochemical results. Base on them, it is suggested that DPHC suppress proliferation of adipose precursor cell and differentiation into adipocytes.