A convenient and time-saving method for primary culture of mature white adipocytes from mice / 南方医科大学学报

OBJECTIVE@#To establish a simple, low-cost and time-saving method for primary culture of mature white adipocytes from mice.@*METHODS@#Mature white adipocytes were isolated from the epididymis and perirenal area of mice for primary culture using a modified mature adipocyte culture method or the ceiling culture method. The morphology of the cultured mature adipocytes was observed using Oil Red O staining, and the cell viability was assessed with CCK8 method. The expression of PPARγ protein in the cells was detected with Western blotting, and the mRNA expressions of CD36, FAS, CPT1A and FABP4 were detected using RT-qPCR.@*RESULTS@#Oil Red O staining showed a good and uniform morphology of the adipocytes in primary culture using the modified culture method, while the cells cultured using the ceiling culture method exhibited obvious morphological changes. CCK8 assay showed no significant difference in cell viability between freshly isolated mature white adipocytes and the cells obtained with the modified culture method. Western blotting showed that the freshly isolated adipocytes and the cells cultured for 72 h did not differ significantly in the expression levels of PPARγ protein (P=0.759), which was significantly lowered in response to treatment with GW9662 (P < 0.001). GW9662 treatment of the cells upregulated mRNA expressions of CD36 (P < 0.001) and CPT1A (P=0.003) and down-regulated those of FAS (P=0.001) and FABP4 (P < 0.001).@*CONCLUSION@#We established a convenient and time-saving method for primary culture mature white adipocytes from mice to facilitate further functional studies of mature adipocytes.

Papel de la célula grasa en el riesgo cardiovascular / Role of the fat cell in cardiovascular risk

Resumen La obesidad es una enfermedad crónica, no transmisible, que recientemente ha tenido una connotación especial debido al aumento de su prevalencia en países en vía de desarrollo. Este incremento está relacionado con un aumento en la aparición de enfermedades metabólicas y el riesgo cardiovascular. Si bien la prevalencia de obesidad está aumentando en todos los países del mundo, existen diferencias regionales tanto en la prevalencia como en las tendencias de la obesidad. Por consiguiente, comprender los impulsores de estas diferencias regionales podría ayudar a proporcionar orientación para las estrategias de intervención más prometedoras. A pesar de considerarse una eventualidad simple en una proporción de lo que se ingiere y lo que se gasta, existen muchos factores que regulan esta enfermedad. No es sencillo encontrar medidas terapéuticas para la obesidad, pues sus causas son múltiples. En forma reciente, ha despertado un especial interés la caracterización funcional de los adipocitos, específicamente de los adipocitos beige, dado que su función está íntimamente relacionada con las circunstancias externas del ambiente y tienen una flexibilidad que les permite producir energía y mejorar muchos de los parámetros metabólicos en los individuos. En este manuscrito se hará énfasis en las características de las células adiposas y su influencia en el riesgo cardiovascular.

Abstract Obesity is a chronic non-transmissible disease that has recently had a special connotation due to the increase of its prevalence in developing countries. The increase in obesity is related to an expansion in the appearance of metabolic diseases and cardiovascular risk. Although the prevalence of obesity is increasing in all countries of the world, there are regional differences in both the prevalence and trends of obesity. Therefore, understanding the circumstances of these regional differences could help provide guidance for the most promising intervention strategies. Despite being considered a simple outcome in a proportion of what is ingested and what is spent, there are many factors that regulate this disease. It is not easy to find therapeutic measures for obesity, because their causes are multiple. Recently, the functional characterization of adipocytes, especially Beige adipocytes, has been of particular interest since their function is intimately related to the external circumstances of the environment and they have a flexibility that allows them to produce energy and improve many of the metabolic parameters in individuals. In the present manuscript we will focus on the characteristics of fat cells and their influence on cardiovascular risk.

Mitochondrial Dysfunction in Adipocytes as a Primary Cause of Adipose Tissue Inflammation

Adipose tissue inflammation is considered a major contributing factor in the development of obesity-associated insulin resistance and cardiovascular diseases. However, the cause of adipose tissue inflammation is presently unclear. The role of mitochondria in white adipocytes has long been neglected because of their low abundance. However, recent evidence suggests that mitochondria are essential for maintaining metabolic homeostasis in white adipocytes. In a series of recent studies, we found that mitochondrial function in white adipocytes is essential to the synthesis of adiponectin, which is the most abundant adipokine synthesized from adipocytes, with many favorable effects on metabolism, including improvement of insulin sensitivity and reduction of atherosclerotic processes and systemic inflammation. From these results, we propose a new hypothesis that mitochondrial dysfunction in adipocytes is a primary cause of adipose tissue inflammation and compared this hypothesis with a prevailing concept that “adipose tissue hypoxia” may underlie adipose tissue dysfunction in obesity. Recent studies have emphasized the role of the mitochondrial quality control mechanism in maintaining mitochondrial function. Future studies are warranted to test whether an inadequate mitochondrial quality control mechanism is responsible for mitochondrial dysfunction in adipocytes and adipose tissue inflammation.

Complications of the surgical excision of encapsulated versus nonencapsulated lipomas: A retrospective analysis

BACKGROUND: Lipomas are common benign soft tissue tumors composed of mature white adipocytes, with histological features including a well-circumscribed and lobular mass covered with a thin fibrous capsule. However, lipomas that are poorly demarcated from the surrounding fat are often encountered during surgery despite a postoperative histological diagnosis. We investigated the complications associated with different types of lipomas. METHODS: This retrospective study included 119 patients who underwent lipoma excision and computed tomography (CT) imaging at our clinic between January 2011 and August 2018. We classified the lipomas as encapsulated or nonencapsulated according to the histology, CT findings, and clinical criteria. Nonencapsulated lipomas were defined as relatively heterogeneous without a distinct capsule, whereas encapsulated lipomas were homogeneous with a distinct capsule. The analyzed complications included delayed wound healing, which can cause prominent scarring, hematoma or seroma, and recurrence. RESULTS: Encapsulated and nonencapsulated lipomas were diagnosed in 89 (74.8%) and 30 (25.2%) patients, respectively. Encapsulated lipomas occurred most commonly on the head, whereas nonencapsulated lipomas occurred most commonly on the neck and trunk (P=0.000, P=0.002, and P=0.031, respectively). The Fisher exact test showed a significantly higher incidence of delayed wound healing for nonencapsulated than encapsulated lipomas (P=0.014). CONCLUSIONS: Preoperative classification of lipomas using CT imaging is important for predicting the incidence of postoperative complications. Direct excision is adequate for removing encapsulated lipomas. However, nonencapsulated lipomas might require alternative methods, such as ultrasonic liposuction, to prevent postoperative complications. Our results will help reduce the incidence of scarring by providing guidance on surgical methods.

Brown Adipocyte and Splenocyte Co-Culture Maintains Regulatory T Cell Subset in Intermittent Hypobaric Conditions

BACKGROUND: Brown adipocytes have thermogenic characteristics in neonates and elicit anti-inflammatory responses. We postulated that thermogenic brown adipocytes produce distinctive intercellular effects in a hypobaric state. The purpose of this study is to analyze the correlation between brown adipocyte and regulatory T cell (T(reg)) expression under intermittent hypobaric conditions. METHODS: Brown and white adipocytes were harvested from the interscapular and flank areas of C57BL6 mice, respectively. Adipocytes were cultured with syngeneic splenocytes after isolation and differentiation. Intermittent hypobaric conditions were generated using cyclic negative pressure application for 48 h in both groups of adipocytes. Expression levels of T(regs) (CD4 + CD25 + Foxp3 + T cells), cytokines [tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10), and the programmed death-ligand 1 (PD-L1)] co-inhibitory ligand were examined. RESULTS: Splenocytes, cultured with brown and white adipocytes, exhibited comparable T(reg) expression in a normobaric state. Under hypobaric conditions, brown adipocytes maintained a subset of T(regs). However, a decrease in T(regs) was found in the white adipocyte group. TNF-α levels increased in both groups under hypobaric conditions. In the brown adipocyte group, anti-inflammatory IL-10 expression increased significantly; meanwhile, IL-10 expression decreased in the white adipocyte group. PD-L1 levels increased more significantly in brown adipocytes than in white adipocytes under hypobaric conditions. CONCLUSION: Both brown and white adipocytes support T(reg) expression when they are cultured with splenocytes. Of note, brown adipocytes maintained T(reg) expression in intermittent hypobaric conditions. Anti-inflammatory cytokines and co-inhibitory ligands mediate the immunomodulatory effects of brown adipocytes under altered atmospheric conditions. Brown adipocytes showed the feasibility as a source of adjustment in physical stresses.

Alteration in plasma chemokine profile in a high-fat diet-induced obesity mouse model / 한국영양학회지

PURPOSE: Obesity is associated with a dysregulation of metabolic balance and is regarded as a low grade chronic inflammation. Western-style diet and physical inactivity are leading causes of obesity. This study examined the profiles of forty plasma cytokines and chemokines at the same time in the early stages of high-fat diet-induced obesity using a mouse model. METHODS: A total of 30 male CD1 mice, 12 ~ 14 weeks of age, were enrolled. The mice were fed a high-fat diet for 6 weeks to induce obesity. The plasma glucose and triglyceride concentrations were measured using a hexokinase colorimetric assay kit and a serum triglyceride determination kit, respectively. The relative levels of multiple cytokines and chemokines in the plasma were determined using a mouse cytokine array kit. RESULTS: The mice exhibited significant weight gain after 6 weeks of a high-fat diet. The genital fat depot was enlarged along with an increase in the number and the mean size of white adipocytes as early as 4 weeks after a high-fat diet. In addition, the plasma glucose and triglyceride levels increased significantly after 4 weeks of a high-fat diet. Cytokine array analysis revealed a remarkable increase in the expression of both CXCL12 and CXCL13, whereas the proinflammatory cytokines remained low after 4 weeks of a high-fat diet. CONCLUSION: A significant increase in plasma levels of CXCL12 and CXCL13 was observed after 4 weeks of a high-fat diet, which might induce the migration of B lymphocytes, T lymphocytes, and monocytes from the blood to expanding adipose tissue or fat associated lymphoid clusters, playing a key role in adipose tissue remodeling and local immunity during the early stages of high-fat diet-induced obesity.

The Effects of High Fat Diet and Resveratrol on Mitochondrial Activity of Brown Adipocytes

BACKGROUND: Resveratrol (RSV) is a polyphenolic phytoalexin that has many effects on metabolic diseases such as diabetes and obesity. Given the importance of brown adipose tissue (BAT) for energy expenditure, we investigated the effects of RSV on brown adipocytes. METHODS: For the in vitro study, interscapular BAT was isolated from 7-week-old male Sprague Dawley rats. For the in vivo study, 7-week-old male Otsuka Long Evans Tokushima Fatty (OLETF) rats were divided into four groups and treated for 27 weeks with: standard diet (SD); SD+RSV (10 mg/kg body weight, daily); high fat diet (HFD); HFD+RSV. RSV was provided via oral gavage once daily during the in vivo experiments. RESULTS: RSV treatment of primary cultured brown preadipocytes promoted mitochondrial activity, along with over-expression of estrogen receptor α (ER-α). In OLETF rats, both HFD and RSV treatment increased the weight of BAT and the differentiation of BAT. However, only RSV increased the mitochondrial activity and ER-α expression of BAT in the HFD-fed group. Finally, RSV improved the insulin sensitivity of OLETF rats by increasing the mitochondrial activity of BAT, despite having no effects on white adipocytes and muscles in either diet group. CONCLUSION: RSV could improve insulin resistance, which might be associated with mitochondrial activity of brown adipocyte. Further studies evaluating the activity of RSV for both the differentiation and mitochondrial activity of BAT could be helpful in investigating the effects of RSV on metabolic parameters.

BAY 41-2272 activates host defence against local and disseminated Candida albicans infections

In our previous study, we have found that 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), a guanylate cyclase agonist, activates human monocytes and the THP-1 cell line to produce the superoxide anion, increasing in vitro microbicidal activity, suggesting that this drug can be used to modulate immune functioning in primary immunodeficiency patients. In the present work, we investigated the potential of the in vivo administration of BAY 41-2272 for the treatment of Candida albicans and Staphylococcus aureus infections introduced via intraperitoneal and subcutaneous inoculation. We found that intraperitoneal treatment with BAY 41-2272 markedly increased macrophage-dependent cell influx to the peritoneum in addition to macrophage functions, such as spreading, zymosan particle phagocytosis and nitric oxide and phorbol myristate acetate-stimulated hydrogen peroxide production. Treatment with BAY 41-2272 was highly effective in reducing the death rate due to intraperitoneal inoculation of C. albicans, but not S. aureus. However, we found that in vitro stimulation of peritoneal macrophages with BAY 41-2272 markedly increased microbicidal activities against both pathogens. Our results show that the prevention of death by the treatment of C. albicans-infected mice with BAY 41-2272 might occur primarily by the modulation of the host immune response through macrophage activation. .

Leptin: A Multifunctional Role as an Immunomodulator in Mycobacterial Lung Disease

Leptin is a 16 kDa protein which consists of 167 amino acids. Leptin is considered as one of the adipokines, secreted by white adipocytes, and is the product of the obese (ob) gene. Recently, leptin is recognized as the immuno-stimulator which belongs to the same class of long chain helical cytokines such as interleukin (IL)-6. Leptin is related to the immune responses evoked by Mycobacterium tuberculosis infection. Thus, studies of association between immunomolecules including leptin and tuberculosis may contribute to provide an essential solution regulating adverse immune responses in several mycobacterial diseases. Leptin has a multifunctional role in the secretion of acute-phase cytokines including IL-1beta and tumor-necrosis factor-alpha (TNF-alpha), and links to T helper 1 (Th1) immune response. Moreover, the binding of leptin to leptin receptor (LepR) is important in that this binding involves janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. In addition, the activation of LepR mediates extra-cellular signal-regulated kinase (ERK) and phosphoinositide 3 kinase (PI3K) pathways. Furthermore, many studies suggest that leptin may play a critical role in respiratory diseases including chronic obstructive pulmonary disease (COPD) and asthma as well as tuberculosis. These findings indicate that leptin is one of the important regulators for immune responses in respiratory diseases. We herein discuss the multifunctional role of leptin in mycobacterial lung disease, especially focusing on the related pathway to immune responses.

Comparing the osteogenic potential of canine mesenchymal stem cells derived from adipose tissues, bone marrow, umbilical cord blood, and Wharton's jelly for treating bone defects

Alternative sources of mesenchymal stem cells (MSCs) for replacing bone marrow (BM) have been extensively investigated in the field of bone tissue engineering. The purpose of this study was to compare the osteogenic potential of canine MSCs derived from adipose tissue (AT), BM, umbilical cord blood (UCB), and Wharton's jelly (WJ) using in vitro culture techniques and in vivo orthotopic implantation assays. After canine MSCs were isolated from various tissues, the proliferation and osteogenic potential along with vascular endothelial growth factor (VEGF) production were measured and compared in vitro. For the in vivo assay, MSCs derived from each type of tissue were mixed with beta-tricalcium phosphate and implanted into segmental bone defects in dogs. Among the different types of MSCs, AT-MSCs had a higher proliferation potential and BM-MSCs produced the most VEGF. AT-MSCs and UCB-MSCs showed greater in vitro osteogenic potential compared to the other cells. Radiographic and histological analyses showed that all tested MSCs had similar osteogenic capacities, and the level of new bone formation was much higher with implants containing MSCs than cell-free implants. These results indicate that AT-MSCs, UCB-MSCs, and WJ-MSCs can potentially be used in place of BM-MSCs for clinical bone engineering procedures.

[Comparison of leptin blood levels and correlation of leptin with LH and FSH in PCOS patients and normal individuals]

There are high concentration of LH in Polycystic Ovary syndrome [PCOS] patients. These patients are usually obese and have increased leptin levels in their blood. It is not clear whether obesity or increased leptin levels cause changes in the hypothalamus- pituitary axis. This study evaluated the blood leptin, LH and FSH levels in PCOS group and compared them with a control group. The study included 27 PCOS patients and 27 normal individuals as control. They were matched for age [18-40 year]. A fasting blood sample was taken and then freezed at -80 degrees Celsius. This sample was used for measurement of leptin, FSH, LH and insulin levels. There was a significant difference between leptin, insulin and glucose levels in the two groups [P<0.05] Leptin levels were higher in group with BMI>25 as compared to group with BMI< 25 [P<0.05]. The mean LH level in PCOS group was significantly higher than the control group. It is possible that in PCOS patients, high levels of LH could be related to an increase in leptin levels along with insulin resistance

Cell-cell communication between mouse mammary epithelial cells and 3T3-L1 preadipocytes: effect on triglyceride accumulation and cell proliferation

Interaction between parenchyma and stroma is essential for organogenesis, morphogenesis, and differentiation. Mammary gland has being the chosen model for developmental biologist because the most striking changes in morphology and function take place after birth. We have demonstrated a regulation of triglyceride accumulation by protein factors synthesized by normal mouse mammary gland epithelial cells (NMMG), acting on a cell line, 3T3-L1, long used as a model for adipogenesis. In this paper, we demonstrate that this inhibitory effect seems to be shared by other cells of epithelial origin but not by other cell types. We found a regulation of cell proliferation when NMMG cells are cultured in the presence of conditioned media from Swiss 3T3 or 3T3-L1 cells. We found a possible point of regulation for the mammary factor on a key enzyme of the lipid metabolic pathway, the glycerol-3-phosphate dehydrogenase. The inhibitory factor seems to have an effect on this enzyme's activity and reduces it. The results presented herein contribute to the understanding of cell-cell communication in a model of a normal mammary gland.