Towards high resolution analysis of metabolic flux in cells and tissues.

Metabolism extracts chemical energy from nutrients, uses this energy to form building blocks for biosynthesis, and interconverts between various small molecules that coordinate the activities of cellular pathways. The metabolic state of a cell is increasingly recognized to determine the phenotype of not only metabolically active cell types such as liver, muscle, and adipose, but also other specialized cell types such as neurons and immune cells. This review focuses on methods to quantify intracellular reaction flux as a measure of cellular metabolic activity, with emphasis on studies involving cells of mammalian tissue. Two key areas are highlighted for future development, single cell metabolomics and noninvasive imaging, which could enable spatiotemporally resolved analysis and thereby overcome issues of heterogeneity, a distinctive feature of tissue metabolism.

Adipogenesis of adipose-derived stem cells may be regulated via the cytoskeleton at physiological oxygen levels in vitro.

INTRODUCTION: Obesity, which is excessive expansion of white adipose tissue, is a major risk factor for several serious health issues, including diabetes, cardiovascular disease and cancer. Efforts to combat obesity and related diseases require understanding the basic biology of adipogenesis. However, in vitro studies do not result in lipid composition and morphology that are typically seen in vivo, likely because the in vitro conditions are not truly representative of in vivo adipose tissue formation. In vitro, low oxygen tension and cytoskeletal tension have been shown to independently regulate adipogenesis, but in vivo, these two factors simultaneously influence differentiation. METHODS: The purpose of our study was to examine the influence of physiological oxygen tension on cytoskeletal tension-mediated adipogenesis. Adipose-derived stem cells (ASCs) were differentiated under both ambient (20%) and physiological (5%) oxygen conditions and treated with cytoskeletal inhibitors, cytochalasin D or blebbistatin. Adipogenesis was assessed on the basis of gene expression and adipocyte metabolic function. RESULTS: Adipose tissue metabolic markers (glycerol-3-phosphate dehydrogenase (GPDH) and triglycerides) were significantly down-regulated by physiological oxygen levels. Reducing cytoskeletal tension through the use of chemical inhibitors, either cytochalasin D or blebbistatin, resulted in an up-regulation of adipogenic gene expression (peroxisome proliferator-activated receptor γ (PPARγ), lipoprotein lipase (LPL) and fatty acid binding protein 4 (FABP4)) and metabolic markers, regardless of oxygen levels. Cytochalasin D and blebbistatin treatment altered cytoskeletal organization and associated tension via different mechanisms; however, both conditions had similar effects on adipogenesis, suggesting that physiological oxygen-mediated regulation of adipogenesis in ASCs is modulated, in part, by cytoskeletal tension. CONCLUSIONS: These results demonstrated that interactions between the cytoskeleton and oxygen tension influence adipogenic differentiation of ASCs.

Adipocyte induction of preadipocyte differentiation in a gradient chamber.

Adipose tissue expansion involves enlargement of mature adipocytes and the formation of new adipocytes through the differentiation of locally resident preadipocytes. Factors released by the enlarged adipocytes are potential cues that induce the differentiation of the preadipocytes. Currently, there are limited options to investigate these cues in isolation from confounding systemic influences. A gradient generating microfluidic channel-based cell culture system was designed to enable solution patterning, while supporting long-term culture and differentiation of preadipocytes. Solution patterning was confirmed by selectively staining a fraction of uniformly seeded preadipocytes. An adipogenic cocktail gradient was used to induce the differentiation of a fraction of uniformly seeded preadipocytes and establish a spatially defined coculture of adipocytes and preadipocytes. Varying the adipogenic cocktail gradient generated cocultures of preadipocytes and adipocytes with different compositions. Transient application of the cocktail gradient, followed by basal medium treatment showed a biphasic induction of differentiation. The two phases of differentiation correlated with a spatial gradient in adipocyte size. Our results provide in vitro data supporting the size-dependent release of preadipocyte differentiation factors by enlarged adipocytes. Prospectively, the coculture system developed in this study could facilitate controlled, yet physiologically meaningful studies on paracrine interactions between adipocytes and preadipocytes during adipose tissue development.