Culture on Tissue‐Specific Coatings Derived from α‐Amylase‐Digested Decellularized Adipose Tissue Enhances the Proliferation and Adipogenic Differentiation of Human Adipose‐Derived Stromal Cells

While extracellular matrix (ECM)‐derived coatings have the potential to direct the response of cell populations in culture, there is a need to investigate the effects of ECM sourcing and processing on substrate bioactivity. To develop improved cell culture models for studying adipogenesis, the current study examines the proliferation and adipogenic differentiation of human adipose‐derived stem/stromal cells (ASCs) on a range of ECM‐derived coatings. Human decellularized adipose tissue (DAT) and commercially available bovine tendon collagen (COL) are digested with α‐amylase or pepsin to prepare the coatings. Physical characterization demonstrates that α‐amylase digestion generates softer, thicker, and more stable coatings, with a fibrous tissue‐like ultrastructure that is lost in the pepsin‐digested thin films. ASCs cultured on the α‐amylase‐digested ECM have a more spindle‐shaped morphology, and proliferation is significantly enhanced on the α‐amylase‐digested DAT coatings. Further, the α‐amylase‐digested DAT provides a more pro‐adipogenic microenvironment, based on higher levels of adipogenic gene expression, glycerol‐3‐phosphate dehydrogenase (GPDH) enzyme activity, and perilipin staining. Overall, this study supports α‐amylase digestion as a new approach for generating bioactive ECM‐derived coatings, and demonstrates tissue‐specific bioactivity using adipose‐derived ECM to enhance ASC proliferation and adipogenic differentiation.     

mRNA Levels of the Insulin‐Signaling Molecule SORBS1 in the Adipose Depots of Nondiabetic Women

Objectives: The SORBS1 gene has been shown to be an important adaptor protein in the insulin‐signaling pathway in many molecular and cellular biology studies. However, its roles in humans either in health or disease are rarely explored. In this report, we measured the SORBS1 mRNA levels in human adipose tissues. Research Methods and Procedures: Adipose tissues of both the abdominal subcutaneous and omental depots were obtained from 62 nondiabetic women. The relative SORBS1 mRNA levels were quantified using real‐time polymerase chain reaction. Results: The relative SORBS1 mRNA levels from these two depots significantly correlated with each other (γ = 0.85, p = 0.0000). The relative SORBS1 mRNA levels in the omental depots were lower than those in the subcutaneous depots (p = 0.053 by two‐tailed test, p = 0.026 by one‐tailed paired Student's t test). The mean SORBS1 expression level in the omental depots was ～70% that in the subcutaneous depots. Moreover, the relative SORBS1 mRNA levels in the omental depots were significantly related to BMI using either correlation analysis (γ = ?0.41, p = 0.0008) or multivariate linear regression analysis (β = ?0.20 ± 0.09, p = 0.031) with adjustment for age, plasma glucose, serum insulin, triglyceride, and total cholesterol levels. Discussion: Our preliminary results indicate the depot‐specific differential expression of SORBS1 in relation to BMI. Further investigation of the functional significance of this phenomenon in human obesity is warranted.     

人钙周期蛋白结合蛋白（hCacyBP）基因的克隆与表达

筛选cDNA文库得到了人的钙周期蛋白结合蛋白基因 ,将此基因的全编码区克隆到原核表达载体pET2 8上 ,诱导目的蛋白质表达以后将重组蛋白质用亲和层析的方法进行纯化 ,得到了纯度很好的重组的目的蛋白质 ,以此作为抗原免疫动物 ,得到抗钙周期蛋白结合蛋白的特异多克隆抗体。Western印迹的结果表明 ,该基因在小鼠多种组织中广泛表达 ;免疫组化的结果表明 ,BT32 5细胞诱导分化后钙周期蛋白结合蛋白分布有变化 ,由分布于胞质中转向分布于胞核和核周胞质     

Leptin Responses to Weight Loss in Postmenopausal Women: Relationship to Sex‐Hormone Binding Globulin and Visceral Obesity

Objective: Leptin concentrations increase with obesity and tend to decrease with weight loss. However, there is large variation in the response of serum leptin levels to decreases in body weight. This study examines which endocrine and body composition factors are related to changes in leptin concentrations following weight loss in obese, postmenopausal women. Research Methods and Procedures: Body composition (DXA), visceral obesity (computed tomography), leptin, cortisol, insulin, and sex hormone‐binding globulin (SHBG) concentrations were measured in 54 obese (body mass index [BMI] = 32.0 ± 4.5 kg/m²; mean ± SD), women (60 ± 6 years) before and after a 6‐month hypocaloric diet (250 to 350 kcal/day deficit). Results: Body weight decreased by 5.8 ± 3.4 kg (7.1%) and leptin levels decreased by 6.6 ± 11.9 ng/mL (14.5%) after the 6‐month treatment. Insulin levels decreased 10% (p < 0.05), but mean SHBG and cortisol levels did not change significantly. Relative changes in leptin with weight loss correlated positively with relative changes in body weight (r = 0.50, p < 0.0001), fat mass (r = 0.38, p < 0.01), subcutaneous fat area (r = 0.52, p < 0.0001), and with baseline values of SHBG (r = 0.38, p < 0.01) and baseline intra‐abdominal fat area (r = ?0.27, p < 0.06). Stepwise multiple regression analysis showed that baseline SHBG levels (r² = 0.24, p < 0.01), relative changes in body weight (cumulative r² = 0.40, p < 0.05), and baseline intra‐abdominal fat area (cumulative r² = 0.48, p < 0.05) were the only independent predictors of the relative change in leptin, accounting for 48% of the variance. Discussion: These results suggest that obese, postmenopausal women with a lower initial SHBG and more visceral obesity have a greater decrease in leptin with weight loss, independent of the amount of weight lost.     

Treatment of Morbid Obesity in Inner‐City Women

Objective: To explore the use of the very‐low‐calorie formula diet (VLCD) in the indigent population of Newark, NJ, with the goal of achieving 10% weight loss within a relatively short period of 10 weeks. Research Methods and Procedures: We accepted 131 morbidly obese indigent women into our study program. The study was limited to women only and the average starting weight was 292.3 ± 5.9 lbs (± SE; 50.3 ± 0.9 body mass index [kg/m²]). We used three treatment paradigms: total cost‐free program for 10 weeks; cost‐free, but compliance requirements; and a weekly charge of $25. The results obtained were compared with two control populations: women enrolled during the same recruitment period in a comparable suburban VLCD program and a historical control population of suburban women treated from 1985 through 1995. Results: In group A (total cost‐free), 79% of patients completed the 10‐week program, but only 18% of patients achieved the goal of 10% weight loss. In group B when attendance and weight loss requirements were imposed, the dropout rate accelerated such that only 37% of patients completed the 10‐week course, and 16% of the women were successful with their weight loss. In group C, imposing $25/wk financial outlay also accelerated dropouts but had little effect on weight loss success, which was 10% of the starting group. By comparison, the suburban patients and the historical control group exhibited 67% and 76% attendance rates after 10 weeks, and 33% and 55% success rates, respectively, in achieving the weight loss goal. Discussion: We conclude that inner‐city patients exhibit great interest in weight loss when financial barriers are removed. Successful weight loss was achieved in 10% to 18% of patients using the VLCD approach, approximately one‐half of that obtained in affluent suburban women. Imposing financial or compliance restrictions to the inner‐city patients served only to enhance dropouts.     

Treatment of Morbid Obesity in Low‐income Adolescents: Effects of Parental Self‐monitoring

Objective: This study examined the extent to which consistency of self‐monitoring by participants and their parents was related to weight control over an initial period of 3 months within the context of a treatment program for morbidly obese low‐income minority adolescents. Research Methods and Procedures: Eighty‐three obese adolescents (mean age, 13.0 years; 51% boys; 92% African American; mean BMI, 43.0 kg/m²; mean BMI z‐score, 6.0) and at least one parent participated in a long‐term treatment program that included a very‐low‐fat dietary focus, weekly group cognitive‐behavior therapy, monthly nutrition education classes, a 12‐week physical therapy class, and medical monitoring. Results: Participants who self‐monitored on the majority of days compared with those who did not self‐monitor at all or who self‐monitored infrequently attended more sessions and generally lost more weight over the first 3 months. Although parents signed behavioral contracts committing to self‐monitor their own eating and exercising over the first month, only 12% did so. Nonetheless, participants whose parents self‐monitored were much more likely to self‐monitor consistently and lose weight during the first 3 months. Discussion: These results indicate that self‐monitoring is a cornerstone of successful weight control even for morbidly obese low‐income minority adolescents; targeting consistency of self‐monitoring among these high‐risk weight controllers and their parents should be just as important as it is for more affluent and less overweight adolescents.     

Transgenic expression of n‐3 fatty acid desaturase (fat‐1) in C57/BL6 mice: Effects on glucose homeostasis and body weight

The fat‐1 gene, derived from Caenorhabditis elegans, encodes for a fatty acid n‐3 desaturase. In order to study the potential metabolic benefits of n‐3 fatty acids, independent of dietary fatty acids, we developed seven lines of fat‐1 transgenic mice (C57/BL6) controlled by the regulatory sequences of the adipocyte protein‐2 (aP2) gene for adipocyte‐specific expression (AP‐lines). We were unable to obtain homozygous fat‐1 transgenic offspring from the two highest expressing lines, suggesting that excessive expression of this enzyme may be lethal during gestation. Serum fatty acid analysis of fat‐1 transgenic mice (AP‐3) fed a high n‐6 unsaturated fat (HUSF) diet had an n‐6/n‐3 fatty acid ratio reduced by 23% (P < 0.025) and the n‐3 fatty acid eicosapentaenoic acid (EPA) concentration increased by 61% (P < 0.020). Docosahexaenoic acid (DHA) was increased by 19% (P < 0.015) in white adipose tissue. Male AP‐3‐fat‐1 line of mice had improved glucose tolerance and reduced body weight with no change in insulin sensitivity when challenged with a high‐carbohydrate (HC) diet. In contrast, the female AP‐3 mice had reduced glucose tolerance and no change in insulin sensitivity or body weight. These findings indicate that male transgenic fat‐1 mice have improved glucose tolerance likely due to increased insulin secretion while female fat‐1 mice have reduced glucose tolerance compared to wild‐type mice. Finally the inability of fat‐1 transgenic mice to generate homozygous offspring suggests that prolonged exposure to increased concentrations of n‐3 fatty acids may be detrimental to reproduction. J. Cell. Biochem. 107: 809–817, 2009. © 2009 Wiley‐Liss, Inc.