Myoung-Sool Do

Berberine reduces the expression of adipogenic enzymes and inflammatory molecules of 3T3-L1 adipocyte.

Berberine (BBR), an isoquinoline alkaloid, has a wide range of pharmacological effects, yet its exact mechanism is unknown. In order to understand the anti-adipogenic effect of BBR, we studied the change of expression of several adipogenic enzymes of 3T3-L1 cells by BBR treatment. First, we measured the change of leptin and glycerol in the medium of 3T3-L1 cells treated with 1 µM, 5 µM and 10 µM concentrations of BBR. We also measured the changes of adipogenic and lipolytic factors of 3T3-L1. In 3T3-L1 cells, both leptin and adipogenic factors (SREBP-1c, C/EBP-α, PPAR-γ, fatty acid synthase, acetyl-CoA carboxylase, acyl-CoA synthase and lipoprotein lipase) were reduced by BBR treatment. Glycerol secretion was increased, whereas expression of lipolytic enzymes (hormone-sensitive lipase and perilipin) mRNA was slightly decreased. Next, we measured the change of inflammation markers of 3T3-L1 cells by BBR treatment. This resulted in the down-regulation of mRNA level of inflammation markers such as TNF-α, IL-6, C- reactive protein and haptoglobin. Taken together, our data shows that BBR has both anti-adipogenic and anti-inflammatory effects on 3T3-L1 adipocytes, and the anti-adipogenic effect seems to be due to the down-regulation of adipogenic enzymes and transcription factors.

B cell activation factor (BAFF) is a novel adipokine that links obesity and inflammation

B cell activation factor (BAFF) is a novel member of the TNF ligand superfamily, mainly produced by myeloid cells. BAFF has been shown to participate in B-cell survival and B- and T-cell maturation. BAFF expression in adipocytes has been recently demonstrated. In the current study, we verified that BAFF expression is increased during adipocyte differentiation. BAFF expression was augmented by TNF-α treatment and was decreased by rosiglitazone treatment. BAFF secretion in lean and in ob/ob mice sera were compared and smaller amount of BAFF was secreted in ob/ob mice. mRNA and protein expression were different between epididymal and visceral adipose tissue. BAFF expression was also increased in ob/ob mouse adipose tissue. We sought to identify known BAFF receptors (BAFF-R, BCMA, and TACI) in adipocytes, and determined that all three were present and upregulated during adipocyte differentiation. However, the expression of TACI was distinct from that of BAFF-R and BCMA under TNF-α and BAFF ligand treatment. BAFF-R and BCMA expression levels were upregulated under pro-inflammatory conditions, but TACI was reduced. Conversely, BAFF-R and BCMA expression levels were downregulated by rosiglitazone treatment, but TACI was increased. Taken together, our results suggest that BAFF may be a new adipokine, representing a link between obesity and inflammation.

The probiotic Lactobacillus rhamnosus BFE5264 and Lactobacillus plantarum NR74 promote cholesterol efflux and suppress inflammation in THP‐1 cells

BACKGROUND The balance between the rate of cholesterol uptake/accumulation and the rate of cholesterol efflux is reflected in the amount of lipid accumulation in macrophages. Based upon the fact that liver X receptors (LXRs) play a role in cholesterol efflux, we studied the effects of probiotics on cholesterol efflux and anti-inflammatory action in macrophages. We confirmed changes in LXR expression by treatment of LXR-transfected CHO-K1 cells with lactic acid bacteria (LAB), and co-cultured THP-1 cells with LAB to investigate changes in cholesterol efflux and inflammation. RESULTS The experiment with CHO-K1 cells showed upregulation of LXR-β by LAB. Treatment of THP-1 cells with LAB promoted LXR expression in THP-1, which eventually led to significant upregulation of ABCA1 and ABCG1 expression. The treatment with live LAB also significantly promoted cholesterol efflux. LAB suppressed expression of interleukin (IL)-1β and tumor necrosis factor (TNF)-α, which resulted from activation of LXR. CONCLUSION Our study shows that Lactobacillus rhamnosus BFE5264 and Lactobacillus plantarum NR74 activated LXR and induced cholesterol efflux by promoting expression of ABCA1 and ABCG1. Both strains also suppressed proinflammatory cytokines including IL-1β and TNF-α. This study could account for the observation that LAB may block foam cell formation by cholesterol efflux and immune modulation.

The Korean Mistletoe (Viscum album coloratum) Extract Has an Antiobesity Effect and Protects against Hepatic Steatosis in Mice with High-Fat Diet-Induced Obesity.

This study investigates the inhibitory effects of Korean mistletoe extract (KME) on adipogenic factors in 3T3-L1 cells and obesity and nonalcoholic fatty liver disease (NAFLD) in mice fed a high-fat diet. Male C57Bl/6 mice fed a high-fat diet were treated with KME (3 g/kg/day) for 15 weeks for the antiobesity and NAFLD experiments. Body weight and daily food intake were measured regularly during the experimental period. The epididymal pad was measured and liver histology was observed. The effects of KME on thermogenesis and endurance capacity were measured. The effects of KME on adipogenic factors were examined in 3T3-L1 cells. Body and epididymal fat pad weights were reduced in KME-treated mice, and histological examination showed an amelioration of fatty liver in KME-treated mice, without an effect on food consumption. KME potently induces mitochondrial activity by activating thermogenesis and improving endurance capacity. KME also inhibited adipogenic factors in vitro. These results demonstrate the inhibitory effects of KME on obesity and NAFLD in mice fed a high-fat diet. The effects appear to be mediated through an enhanced mitochondrial activity. Therefore, KME may be an effective therapeutic candidate for treating obesity and fatty liver caused by a high-fat diet.

Anti-obesity and antioxidative effects of purple sweet potato extract in 3T3-L1 adipocytes in vitro.

The purpose of the current study was to determine the anti-obesity and anti-inflammatory effects of an extract of purple sweet potatoes (PSPs) on 3T3-L1 adipocytes. For this purpose, differentiated 3T3-L1 adipocytes were treated with a PSP extract at concentrations of 1,000, 2,000, and 3,000 μg/mL for 24 hours. Then, we measured the changes in the sizes of the adipocytes, the secretion of leptin, and the mRNA/protein expression of lipogenic, inflammatory, and lipolytic factors after the treatment with the PSP extract. The PSP extract diminished leptin secretion, indicating that growth of fat droplets was suppressed. The extract also suppressed the expression of mRNAs of lipogenic and inflammatory factors and promoted lipolytic action. The antioxidative activity of the PSP extract was also measured using three different in vitro methods: 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity, ferric reducing ability potential assay, and chelating activity of transition metal ions. Taken together, our study shows that PSP extract has antilipogenic, anti-inflammatory, and lipolytic effects on adipocytes and has radical scavenging and reducing activity.

Reduction in cholesterol absorption in Caco-2 cells through the down-regulation of Niemann-Pick C1-like 1 by the putative probiotic strains Lactobacillus rhamnosus BFE5264 and Lactobacillus plantarum NR74 from fermented foods.

Hypercholesterolaemia is a major risk factor related to atherosclerosis, and it may be influenced by our diet. This study addresses the impact of Lactobacillus rhamnosus BFE5264 (isolated from Maasai fermented milk) and Lactobacillus plantarum NR74 (from Korean kimchi) on the control of cholesterol absorption through down-regulation of Niemann-Pick C1-like 1 (NPC1L1) expression. Caco-2 enterocytes were treated with the live, heat-killed (HK) bacteria, bacterial cell wall extracts and metabolites; mRNA level and protein expression were measured. Caco-2 cells showed lower NPC1L1 expression in the presence of the live test strains than the control, elucidating down-regulation of cholesterol uptake, and were compared well with the positive control, L. rhamnosus GG. This effect was also observed with HK bacteria and cell wall fractions but not with their metabolites. The potential of some Lactobacillus strains associated with traditional fermented foods to suppress cholesterol uptake and promote its efflux in enterocytes has been suggested from these data.

Lactobacillus rhamnosus BFE 5264 and Lactobacillus plantarum NR74 Promote Cholesterol Excretion Through the Up-Regulation of ABCG5/8 in Caco-2 Cells.

The effect of two putative probiotic strains, Lactobacillus rhamnosus BFE5264 and Lactobacillus plantarum NR74, on the control of cholesterol efflux in enterocytes was assessed by focusing on the promotion of ATP-binding cassette sub-family G members 5 and 8 (ABCG5 and ABCG8). Differentiated Caco-2 enterocytes were treated with live bacteria, heat-killed bacteria, a bacterial cell wall fraction, and metabolites and were subjected to cholesterol uptake assay, mRNA analysis, and protein analyses. Following LXR-transfection by incubation with CHO-K1 cells in DNA-lipofectin added media, the luciferase assay was conducted for LXR analysis. Treatment of Caco-2 cells with L. rhamnosus BFE5264 (isolated from traditional fermented Maasai milk) and L. plantarum NR74 (isolated from Korean kimchi) resulted in the up-regulation of LXR, concomitantly with the elevated expression of ABCG5 and ABCG8. This was associated with the promotion of cholesterol efflux at significantly higher levels compared to the positive control strain L. rhamnosus GG (LGG). The experiment with CHO-K1 cells confirmed up-regulation of LXR-beta by the test strains, and treatment with the live L. rhamnosus BFE5264 and L. plantarum NR74 strains significantly increased cholesterol efflux. Heat-killed cells and cell wall fractions of both LAB strains induced the upregulation of ABCG5/8 through LXR activation. By contrast, LAB metabolites did not show any effect on ABCG5/8 and LXR expression. Data from this study suggest that LAB strains, such as L. rhamnosus BFE5264 and L. plantarum NR74, may promote cholesterol efflux in enterocytes, and thus potentially contribute to the prevention of hypercholesterolemia and atherosclerosis.

Expression of eotaxin in 3T3-L1 adipocytes and the effects of weight loss in high-fat diet induced obese mice

Eotaxin is an important inflammatory chemokine in eosinophil chemotaxis and activation and, thus, is implicated in asthma. Recently, obesity was associated with an increased prevalence of asthma, but the relationship between obesity and eotaxin expression has only been partially understood in obese mice and human studies. Therefore, we studied the expression patterns of eotaxin in 3T3-L1 preadipocytes/adipocytes to determine whether eotaxin levels are influenced by body weight gain and/or reduction in diet-induced obese mice. First, we investigated eotaxin expression during differentiation in 3T3-L1 adipocytes. Then, we treated 3T3-L1 preadipocytes/adipocytes with tumor necrosis factor-alpha (TNF-α), eotaxin, interleukin (IL)-4, IL-5, or leptin. To examine the effects of weight loss in high-fat diet induced obese mice, we fed C57BL/6 mice a high-fat diet or a normal diet for 26 weeks. Then, half of the high-fat diet group were fed a normal diet until 30 weeks to reduce weight. Epididymal adipose tissue, visceral adipose tissue, serum, and bronchoalveolar fluid of mice were examined for eotaxin expression. The results showed that eotaxin expression levels increased with adipocyte differentiation and that more eotaxin was expressed when the cells were stimulated with TNF-α, eotaxin, IL-4, IL-5, or leptin. An in vivo study showed that eotaxin levels were reduced in visceral adipose tissues when high-fat diet fed mice underwent weight loss. Taken together, these results indicate a close relationship between eotaxin expression and obesity as well as weight loss, thus, they indirectly show a relation to asthma.

Modification of PDGFα receptor expression or function alters the metastatic phenotype of 3LL cells

Functional PDGFα receptors are selectively expressed on highly lung-metastasizing clones of the 3LL Lewis lung carcinoma, but not on low-mestastatic clones. The highly metastatic clones are also growth induced in vitro by PDGF and lung conditioned medium. To investigate whether modification of PDGFα receptor expression or function can affect metastatic capability, we transfected cells of a low-metastatic 3LL clone with a full length PDGFα receptor gene and cells of a highly-metastatic clone with a truncated kinase domain PDGFα receptor gene. Introduction of the full length PDGFα receptor conferred upon low-metastatic cells the ability to grow in vitro in the presence of PDGF-AA and to colonize the lung in experimental and spontaneous metastases assays. Conversely, introduction of a truncated version of the PDGFα receptor into highly metastatic cells reduced their metastatic load to control levels. Accordingly, their responses to PDGF-AA, including growth stimulation and receptor autophosphorylation, were reduced. These results demonstrate that PDGFα receptor expression and function can control the capacity of tumor cells to generate metastases in the lung. The response of this receptor to lung-derived PDGF-like factors may define a paracrine mode of metastatic cell growth in the target organ.

BAFF knockout improves systemic inflammation via regulating adipose tissue distribution in high-fat diet-induced obesity

Obesity is recognized as a chronic low-grade inflammatory state due to adipose tissue expansion being accompanied by an increase in the production of proinflammatory adipokines. Our group is the first to report that B-cell-activating factor (BAFF) is produced from adipocytes and functions as a proinflammatory adipokine. Here, we investigated how loss of BAFF influenced diet-induced obesity in mice by challenging BAFF−/− mice with a high-fat diet for 10 weeks. The results demonstrated that weight gain in BAFF−/− mice was >30% than in control mice, with a specific increase in the fat mass of the subcutaneous region rather than the abdominal region. Expression of lipogenic genes was examined by quantitative real-time PCR, and increased lipogenesis was observed in the subcutaneous adipose tissue (SAT), whereas lipogenesis in the epididymal adipose tissue (EAT) was reduced. A significant decrease in EAT mass resulted in the downregulation of inflammatory gene expression in EAT, and more importantly, overall levels of inflammatory cytokines in the circulation were reduced in obese BAFF−/− mice. We also observed that the macrophages recruited in the enlarged SAT were predominantly M2 macrophages. 3T3-L1 adipocytes were cultured with adipose tissue conditioned media (ATCM), demonstrating that EAT ATCM from BAFF−/− mice contains antilipogenic and anti-inflammatory properties. Taken together, BAFF−/− improved systemic inflammation by redistributing adipose tissue into subcutaneous regions. Understanding the mechanisms by which BAFF regulates obesity in a tissue-specific manner would provide therapeutic opportunities to target obesity-related chronic diseases.