Enlarged adipocytes are associated with insulin resistance and are an independent predictor of type 2 diabetes. To understand the molecular link between these diseases and adipocyte hypertrophy, we developed a technique to separate human adipocytes from an adipose tissue sample into populations of small cells (mean 57.6+/-3.54 microm) and large cells (mean 100.1+/-3.94 microm). Microarray analysis of the cell populations separated from adipose tissue from three subjects identified 14 genes, of which five immune-related, with more than fourfold higher expression in large cells than small cells. Two of these genes were serum amyloid A (SAA) and transmembrane 4 L six family member 1 (TM4SF1). Real-time RT-PCR analysis of SAA and TM4SF1 expression in adipocytes from seven subjects revealed 19-fold and 22-fold higher expression in the large cells, respectively, and a correlation between adipocyte size and both SAA and TM4SF1 expression. The results were verified using immunohistochemistry. In comparison with 17 other human tissues and cell types by microarray, large adipocytes displayed by far the highest SAA and TM4SF1 expression. Thus, we have identified genes with markedly higher expression in large, compared with small, human adipocytes. These genes may link hypertrophic obesity to insulin resistance/type 2 diabetes.

Research Methods and Procedures:Using 52 human adipose tissue expression profiles (HU95), 10 putative reference genes with the lowest variation in expression levels were selected for further studies. Expression stability of these 10 novel and 5 previously established reference genes was evaluated by real-time reverse transcriptase-polymerase chain reaction analysis. For this purpose, 44 adipose tissue biopsies from 27 subjects were chosen to include a wide range of parameters such as sex, age, BMI, depot origin, biopsy procedure, and effects of nutrition. Results: LRP10 was identified as the gene with the least variation in expression levels. The frequently used reference genes RPLP0, 18S rRNA, PPIA, ACTB, and GAPD were ranked as 4, 6, 7, 8, and 10, respectively. Discussion: Our results suggest that LRP10 is a better choice as reference for expression studies of human adipose tissue compared with the most frequently used reference genes.

Fluorescence recovery after photobleaching (FRAP) is a versatile tool for determining diffusion and interaction/binding properties in biological and material sciences. An understanding of the mechanisms controlling the diffusion requires a deep understanding of structure-interaction-diffusion relationships. In cell biology, for instance, this applies to the movement of proteins and lipids in the plasma membrane, cytoplasm and nucleus. In industrial applications related to pharmaceutics, foods, textiles, hygiene products and cosmetics, the diffusion of solutes and solvent molecules contributes strongly to the properties and functionality of the final product. All these systems are heterogeneous, and accurate quantification of the mass transport processes at the local level is therefore essential to the understanding of the properties of soft (bio)materials. FRAP is a commonly used fluorescence microscopy-based technique to determine local molecular transport at the micrometer scale. A brief high-intensity laser pulse is locally applied to the sample, causing substantial photobleaching of the fluorescent molecules within the illuminated area. This causes a local concentration gradient of fluorescent molecules, leading to diffusional influx of intact fluorophores from the local surroundings into the bleached area. Quantitative information on the molecular transport can be extracted from the time evolution of the fluorescence recovery in the bleached area using a suitable model. A multitude of FRAP models has been developed over the years, each based on specific assumptions. This makes it challenging for the non-specialist to decide which model is best suited for a particular application. Furthermore, there are many subtleties in performing accurate FRAP experiments. For these reasons, this review aims to provide an extensive tutorial covering the essential theoretical and practical aspects so as to enable accurate quantitative FRAP experiments for molecular transport measurements in soft (bio)materials.

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Confocal or multi-photon laser scanning microscopes are convenient tools to perform FRAP diffusion measurements. Despite its popularity, accurate FRAP remains often challenging since current methods are either limited to relatively large bleach regions or can be complicated for non-specialists. In order to bring reliable quantitative FRAP measurements to the broad community of laser scanning microscopy users, here we have revised FRAP theory and present a new pixelbased FRAP method relying on the photobleaching of rectangular regions of any size and aspect ratio. The method allows for fast and straightforward quantitative diffusion measurements due to a closed-form expression for the recovery process utilising all available spatial and temporal data. After a detailed validation, its versatility is demonstrated by diffusion studies in heterogeneous biopolymer mixtures. 2010 Optical Society of America