OBJECTIVETo determine the cellular architecture of the inflammatory infiltrate in adipose tissue from obese mice, and identify the source of inflammatory cytokines in adipose tissue at a single cell level.DESIGN AND METHODSAdipose tissue from diet-induced obese mice was digested by collagenase treatment and fractionated by density centrifugation to obtain an adipocyte floating layer and a pellet of stromal vascular cells. The cellular architecture of the adipocyte-macrophage interaction in both intact white adipose tissue and the separated density gradient floating layer fraction was analyzed by confocal immunohistochemistry. Cytokine expression was detected by semi-quantitative real time PCR and immunohistochemical analysis.RESULTSThree dimensional image analysis of white adipose tissue and the separated ‘adipocyte’ floating layer revealed lipid-engorged macrophages, macrophages in contact with lipid droplets and sheath-like assemblies of macrophages surrounding adipocytes. The macrophages immunostained for TNFα and to a lesser extent for the immunoregulatory cytokine IL-10. TNFα staining was associated only with macrophages indicating that macrophages and not adipocytes are the source of TNFα expression in the adipocyte floating layer.CONCLUSIONMacrophages form assemblies that tightly adhere to and cover adipocytes and lipid droplets. TNFα found in low density adipocyte preparations is due to contamination with macrophages.
Tubby-like protein-1 (Tulp1) is a photoreceptor-specific protein involved in the transport of specific proteins from the inner segment (IS) to the outer segment (OS) in photoreceptor cells. Mutations in the human TULP1 gene cause an early onset form of retinitis pigmentosa. Our previous work has shown an association between Tulp1 and the microtubule-associated protein, MAP1B. An allele of Mtap1a, which encodes the MAP1A protein, significantly delays photoreceptor degeneration in Tulp1 mutant mice. MAP1 proteins are important in stabilizing microtubules in neuronal cells, but their role in photoreceptors remains obscure. To investigate the relationship between Tulp1 and MAP1 proteins, we performed western blots, immunoprecipitations (IP), immunohistochemistry and proximity ligand assays (PLA) in wild-type and tulp1-/- mouse retinas. Our IP experiments provide evidence that Tulp1 and MAP1B interact while PLA experiments localize their interaction to the outer nuclear layer and IS of photoreceptors. Although MAP1A and MAP1B protein levels are not affected in the tulp1-/- retina, they are no longer localized to the OS of photoreceptors. This may be the cause for disorganized OSs in tulp1-/- mice, and indicate that their transport to the OS is Tulp1-dependent.
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