Imaging of adipose tissue.

Adipose tissue is an endocrine organ that specializes in lipid metabolism and is distributed throughout the body in distinct white adipose tissue (WAT) and brown adipose tissue (BAT) depots. These tissues have opposing roles in lipid metabolism with WAT storing excessive caloric intake in the form of lipid, and BAT burning lipid through nonshivering thermogenesis. As accumulation of lipid in mature adipocytes of WAT leads to obesity and increased risk of comorbidity (Pi-Sunyer et al., 1998), detailed understanding of the mechanisms of BAT activation and WAT accumulation could produce therapeutic strategies for combatting metabolic pathologies. As morphological changes accompany alterations in adipose function, imaging of adipose tissue is one of the most important tools for understanding how adipose tissue mass fluctuates in response to various physiological contexts. Therefore, this chapter details several methods of processing and imaging adipose tissue, including bright-field colorimetric imaging of paraffin-sectioned adipose tissue with a detailed protocol for automated adipocyte size analysis; fluorescent imaging of paraffin and frozen-sectioned adipose tissue; and confocal fluorescent microscopy of whole mounted adipose tissue. We have also provided many example images showing results produced using each protocol, as well as commentary on the strengths and limitations of each approach.

Neuropeptide Y impairs insulin-stimulated translocation of glucose transporter 4 in 3T3-L1 adipocytes through the Y1 receptor.

Neuropeptide Y (NPY) is expressed in adipose tissue and is involved in adipocyte metabolism. Although NPY impacts on glucose utilization in vivo, the underlying cellular mechanism is yet to be fully elucidated. In this study we investigated the effect of NPY on the insulin-stimulated translocation of glucose transporter 4 (GLUT4) from intracellular stores to the cell surface in vitro. Using cellular fractionation and immunofluorescence we analyzed the cellular localization and content of GLUT4 in 3T3-L1 adipocytes. Additionally we investigated the effect of NPY on insulin action in adipocyte cultures by assessing the phosphorylation of Akt and [(3)H]-deoxyglucose uptake. Our data suggest that in 3T3-L1 adipocytes NPY inhibits insulin-stimulated glucose uptake in a GLUT4-dependent manner. The insulin induced translocation of GLUT4 was attenuated by the Y1 receptor agonist [Phe(7),Pro(34)] pNPY, demonstrating an essential role of the Y1 receptor in GLUT4 translocation. Additionally, we observed an NPY dose-dependent impairment of Akt phosphorylation. This study provides evidence that NPY impairs the insulin sensitivity of adipocytes and suggests that the Y1 receptor could be a potential therapeutic target for type 2 diabetes.

Receptors for NPY and PACAP differ in expression and activity during adipogenesis in the murine 3T3-L1 fibroblast cell line.

BACKGROUND AND PURPOSE: Neuropeptides are involved in the regulation of food intake in the central nervous system, but they might also act on peripheral fat tissue via neuropeptide receptors. EXPERIMENTAL APPROACH: We investigated the receptor expression and activity of pituitary adenylate cyclase-activating polypeptide (PACAP) and of neuropeptide Y at the mRNA and protein levels in the 3T3-L1 fibroblast line during differentiation into adipocytes. Intracellular calcium concentration was measured by calcium imaging. KEY RESULTS: The PACAP receptors PAC(1) and VPAC(2) as well as the neuropeptide Y(1) receptor were expressed at the mRNA level in fibroblasts, pre-adipocytes and adipocytes. The mRNA profile of the PAC(1) receptor isoforms showed the HOP sequence, whereas the HIP-isoform was present in subconfluent 3T3-L1 fibroblasts only. At the protein level, the mature 3T3-L1 adipocytes produced the PAC(1) and Y(1) receptors; only the PAC(1) receptor showed carbohydrate residues. Both neuropeptides induced an increase of intracellular calcium in mature adipocytes, which was absent in the precursor cells. These changes in calcium were mediated by Y(1) and PAC(1) receptors as demonstrated by the effects of specific receptor agonists and antagonists. CONCLUSIONS AND IMPLICATIONS: As the PAC(1)-HOP receptor variant seems to be responsible for PACAP-mediated calcium influx in many cell types, the HOP sequence might also mediate the increase in intracellular calcium in adipocytes. Because a high intracellular calcium level is associated with lipogenesis, peptidergic innervation of adipose tissue might be involved in stress-induced obesity.