Adipocyte hypertrophy, inflammation and fibrosis characterize subcutaneous adipose tissue of healthy, non-obese subjects predisposed to type 2 diabetes.

BACKGROUND: The adipose tissue is important for development of insulin resistance and type 2 diabetes and adipose tissue dysfunction has been proposed as an underlying cause. In the present study we investigated presence of adipocyte hypertrophy, and gene expression pattern of adipose tissue dysfunction in the subcutaneous adipose tissue of healthy, non-obese subjects predisposed to type 2 diabetes compared to matched control subjects with no known genetic predisposition for type 2 diabetes. METHOD: Seventeen healthy and non-obese subjects with known genetic predisposition for type 2 diabetes (first-degree relatives, FDRs) and 17 control subjects were recruited. The groups were matched for gender and BMI and had similar age. Glucose tolerance was determined by an oral glucose tolerance test and insulin sensitivity was calculated using HOMA-index. Blood samples were collected and subcutaneous abdominal adipose tissue biopsies obtained for gene expression analysis and adipocyte cell size measurement. RESULTS: Our findings show that, in spite of similar age, BMI and percent body fat, FDRs displayed adipocyte hypertrophy, as well as higher waist/hip ratio, fasting insulin levels, HOMA-IR and serum triglycerides. Adipocyte hypertrophy in the FDR group, but not among controls, was associated with measures of impaired insulin sensitivity. The adipocyte hypertrophy was accompanied by increased inflammation and Wnt-signal activation. In addition, signs of tissue remodeling and fibrosis were observed indicating presence of early alterations associated with adipose tissue dysfunction in the FDRs. CONCLUSION: Genetic predisposition for type 2 diabetes is associated with impaired insulin sensitivity, adipocyte hypertrophy and other markers of adipose tissue dysfunction. A dysregulated subcutaneous adipose tissue may be a major susceptibility factor for later development of type 2 diabetes.

Characterization of integrin and anchorage dependence in mammary epithelial cells following c-erbB2-induced epithelial-mesenchymal transition.

Signalling from the proto-oncogene c-erbB2 in mammary epithelial cells has earlier been shown to result in epithelial-mesenchymal transition (EMT) giving rise to fibroblast-like cells, and acquisition of anchorage-independent growth (AIG) usually determined by growth capacity in soft agar. In this study, we have analysed AIG associated with c-erbB2-induced EMT in a human mammary epithelial cell line. Intriguingly, cells capable of growth in soft agar were shown to be dependent on the function of beta(1) integrin extracellular matrix receptors for growth in collagen. We therefore tested the hypothesis that apparent AIG was due to deposition of extracellular matrix in the agar. Although the fibroblastic cells had strongly upregulated expression of the fibronectin receptor subunit integrin alpha(5) andabundant fibronectin fibrils, these properties did not have a positive correlation with AIG. Furthermore, antibody blocking of integrin alpha(5) and beta(1) failed to inhibit AIG. These results indicate that the anchorage-independent cells are not dependent on connection to extracellular matrix, but instead may be subject to a growth-inhibitory effect from the collagen in the absence of integrin signalling. This notion was supported by the finding that integrin blocking of the fibroblastic cells in fibrin was without effect on proliferation.

c-erbB2-induced epithelial-mesenchymal transition in mammary epithelial cells is suppressed by cell-cell contact and initiated prior to E-cadherin downregulation.

Prolonged signalling from the growth factor receptor subunit and proto-oncogene c-erbB2 has been shown to cause epithelial-mesenchymal transition (EMT) in mammary epithelial cells. Using a system where c-erbB2 homodimer signalling can be induced in human mammary epithelial cells, we characterised the properties of c-erbB2-induced EMT. The cells resulting from this transdifferentiation showed a pronounced and stable fibroblastic phenotype with spindle-like morphology, homogeneous high expression of vimentin, N-cadherin, and integrin alpha5 as well as loss of E-cadherin and desmoplakin. However, the rate at which EMT occurred was very slow compared to other reported systems, as complete conversion was not seen until after 12-15 weeks of c-erbB2 signalling. This time delay was however not due to the presence of long-lived intermediate cell types as measured by expression of combinations of markers. By studying morphological time-courses of individual colonies of epithelial cells subjected to c-erbB2 signalling, we could conclude that apart from EMT, c-erbB2 could also cause transition to very large cells retaining cell-cell contact but with little or no proliferative capacity. EMT preferentially occurred in small colonies, suggesting that extensive cell-cell contact inhibits EMT. When testing this conclusion by exposing cells to c-erbB2 signalling at different cell densities, we concluded that only cells kept at low density would undergo EMT. High cell density also prevented the proliferative decrease associated with prolonged c-erbB2 signalling. Immunofluorescence microscopy revealed that disruption of cell-cell contact was preceded by partial relocalisation of beta-catenin to the cytoplasm whereas downregulation of E-cadherin appeared to occur after initiation of cell scattering.

PKB mediates c-erbB2-induced epithelial beta1 integrin conformational inactivation through Rho-independent F-actin rearrangements.

Signalling from the growth factor receptor subunit and proto-oncogene c-erbB2 has been shown to inhibit the adhesive function of the collagen receptor integrin alpha(2)beta(1) in human mammary epithelial cells. This anti-adhesive effect is mediated by the MAP ERK kinase 1/2 (MEK1/2) and protein kinase B (PKB) pathways. Here, we show that both pathways mediate suppression of matrix adhesion by causing the extracellular domain of the beta(1) integrin subunit to adopt an inactive conformation. The conformational switch was also dependent on rapid and extensive actin depolymerisation. While neither activation nor inhibition of the Rho GTPase affected this rearrangement, Rho was found to be activated by c-erbB2 and to be necessary for conformation-dependent integrin inactivation and, apparently by a different mechanism, a delayed re-formation of stress fibers which did not restore integrin function. Interestingly, the initial actin depolymerisation as well as its effects on integrin function was shown to be mediated by PKB. These results demonstrate how oncogenic growth factor signalling inhibits matrix adhesion by multiple pathways converging on integrin conformation and how Rho signalling can profoundly influence integrin activation in a cytoskeleton-independent manner.