Long-acting insulin analog detemir displays reduced effects on adipocyte differentiation of human subcutaneous and visceral adipose stem cells

Background and aims: Since treatment with insulin detemir results in a lower weight gain compared to human insulin, we investigated whether detemir is associated with lower ability to promote adipogenesis and/or lipogenesis in human adipose stem cells (ASC). Methods and results: Human ASC isolated from both the subcutaneous and visceral adipose tissues were differentiated for 30 days in the presence of human insulin or insulin detemir. Nile Red and Oil-Red-O staining were used to quantify the rate of ASC conversion to adipocytes and lipid accumulation, respectively. mRNA expression levels of early genes, including Fos and Cebpb, as well as of lipogenic and adipogenic genes, were measured at various phases of differentiation by qRT-PCR. Activation of insulin signaling was assessed by immunoblotting. ASC isolated from subcutaneous and visceral adipose tissue were less differentiated when exposed to insulin detemir compared to human insulin, showing lower rates of adipocyte conversion, reduced triglyceride accumulation, and impaired expression of late-phase adipocyte marker genes, such as Pparg2, Slc2a4, Adipoq, and Cidec. However, no differences in activation of insulin receptor, Akt and Erk and induction of the early genes Fos and Cebpb were observed between insulin detemir and human insulin. Conclusion: Insulin detemir displays reduced induction of the Pparg2 adipocyte master gene and diminished effects on adipocyte differentiation and lipogenesis in human subcutaneous and visceral ASC, in spite of normal activation of proximal insulin signaling reactions. These characteristics of insulin detemir may be of potential relevance to its weight-sparing effects observed in the clinical setting

Exendin-4 protects pancreatic beta cells from palmitate-induced apoptosis by interfering with GPR40 and the MKK4/7 stress kinase signalling pathway

Aims/hypothesis: The mechanisms of the protective effects of exendin-4 on NEFA-induced beta cell apoptosis were investigated. Methods: The effects of exendin-4 and palmitate were evaluated in human and murine islets, rat insulin-secreting INS-1E cells and murine glucagon-secreting alpha-TC1-6 cells. mRNA and protein expression/phosphorylation were measured by real-time RT-PCR and immunoblotting or immunofluorescence, respectively. Small interfering (si)RNAs for Ib1 and Gpr40 were used. Cell apoptosis was quantified by two independent assays. Insulin release was assessed with an insulin ELISA. Results: Exposure of human and murine primary islets and INS-1E cells, but not alpha-TC1-6 cells, to exendin-4 inhibited phosphorylation of the stress kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and prevented apoptosis in response to palmitate. Exendin-4 increased the protein content of islet-brain 1 (IB1), an endogenous JNK blocker; however, siRNA-mediated reduction of IB1 did not impair the ability of exendin-4 to inhibit JNK and prevent apoptosis. Exendin-4 reduced G-protein-coupled receptor 40 (GPR40) expression and inhibited palmitate-induced phosphorylation of mitogen-activated kinase kinase (MKK)4 and MKK7. The effects of exendin-4 were abrogated in the presence of the protein kinase A (PKA) inhibitors, H89 and KT5720. Knockdown of GPR40, as well as use of a specific GPR40 antagonist, resulted in diminished palmitate-induced JNK and p38 MAPK phosphorylation and apoptosis. Furthermore, inhibition of JNK and p38 MAPK activity prevented palmitate-induced apoptosis. Conclusions/interpretation: Exendin-4 counteracts the proapoptotic effects of palmitate in beta cells by reducing GPR40 expression and inhibiting MKK7- and MKK4-dependent phosphorylation of the stress kinases, JNK and p38 MAPK, in a PKA-dependent manner. © 2013 Springer-Verlag Berlin Heidelberg

Differences in Gene Expression and Cytokine Release Profiles Highlight the Heterogeneity of Distinct Subsets of Adipose Tissue-Derived Stem Cells in the Subcutaneous and Visceral Adipose Tissue in Humans

<div><p>Differences in the inherent properties of adipose tissue-derived stem cells (ASC) may contribute to the biological specificity of the subcutaneous (Sc) and visceral (V) adipose tissue depots. In this study, three distinct subpopulations of ASC, i.e. ASC_SVF, ASC_Bottom, and ASC_Ceiling, were isolated from Sc and V fat biopsies of non-obese subjects, and their gene expression and functional characteristics were investigated. Genome-wide mRNA expression profiles of ASC_SVF, ASC_Bottom and ASC_Ceiling from Sc fat were significantly different as compared to their homologous subsets of V-ASCs. Furthermore, ASC_SVF, ASC_Ceiling and ASC_Bottom from the same fat depot were also distinct from each other. In this respect, both principal component analysis and hierarchical clusters analysis showed that ASC_Ceiling and ASC_SVF shared a similar pattern of closely related genes, which was highly different when compared to that of ASC_Bottom. However, larger variations in gene expression were found in inter-depot than in intra-depot comparisons. The analysis of connectivity of genes differently expressed in each ASC subset demonstrated that, although there was some overlap, there was also a clear distinction between each Sc-ASC and their corresponding V-ASC subsets, and among ASC_SVF, ASC_Bottom, and ASC_Ceiling of Sc or V fat depots in regard to networks associated with regulation of cell cycle, cell organization and development, inflammation and metabolic responses. Finally, the release of several cytokines and growth factors in the ASC cultured medium also showed both inter- and intra-depot differences. Thus, ASC_Ceiling and ASC_Bottom can be identified as two genetically and functionally heterogeneous ASC populations in addition to the ASC_SVF, with ASC_Bottom showing the highest degree of unmatched gene expression. On the other hand, inter-depot seem to prevail over intra-depot differences in the ASC gene expression assets and network functions, contributing to the high degree of specificity of Sc and V adipose tissue in humans.</p> </div

Quantitative analysis of specific genes previously identified in the microarray analysis and found to be differentially expressed in the distinct ASC subsets.

<p>mRNA levels were analyzed by <i>q</i>RT-PCR, as described under Methods. Open bars, Sc-ASC; filled bars, V-ASC. All data represent mean ± SE from 9 independent adipose tissue donors. *<i>p</i><0.05 vs. V-ASC; #<i>p</i><0.05 vs. other ASC populations from the same adipose tissue depot (ANOVA test followed by Fisher’s post-hoc test). SVF, stromo-vascular; B, bottom; C, ceiling.</p

Significant top list of canonical pathways from IPA in Sc and V ASCs.

<p>Significant top list of canonical pathways from IPA in Sc and V ASCs.</p

Venn diagrams summarizing the number of differentially expressed genes in Sc and V ASC populations.

<p><i>A</i>. Genes found to be differentially expressed by comparing Sc-ASC and V-ASC subsets (inter-depot analysis). <i>B</i>. Genes found to be differentially expressed by comparing ASC_SVF, ASC_Bottom, and ASC_Ceiling from the Sc adipose tissue (Sc intra-depot analysis). <i>C</i>. Genes found to be differentially expressed by comparing ASC_SVF, ASC_Bottom, and ASC_Ceiling from the V adipose tissue (V intra-depot analysis). In each panel, figures for conjoint (and non-conjoint) differentially expressed genes are also indicated.</p

Isolation and characterization of human ASC.

<p><i>A</i>. Morphology of human ASC populations, as detected under bright field. <i>B</i>. Expression of specific markers measured by <i>q</i>RT-PCR. Cells were harvested at passage 0, and RNA was analyzed for markers of adipose-derived stem cells (CD105, CD44, CD49d), human leukocytes and macrophages (CD45, CD11b), and mature endothelial cells (CD31).+or – signs indicate the relative levels of marker expression. Results are from 5 independent adipose tissue donors.</p

Release of cytokines from Sc-ASC and V-ASC populations.

<p>Culture medium from Sc-ASC (open bars) and V-ASC (filled bars) subsets (10⁶ cells) was collected after a 16-h period, and levels of specific cytokines were determined using the multiplex technique, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057892#s2" target="_blank">Materials and Methods</a>. Data represent the mean ± SE of results from 9 independent adipose tissue donors. *<i>p</i><0.05 vs. V; #<i>p</i><0.05 vs. other ASC subsets from the same adipose tissue depot (ANOVA test followed by Fisher’s post-hoc test).</p

Significant top list of associated network functions from IPA in Sc and V ASCs.

<p>Significant top list of associated network functions from IPA in Sc and V ASCs.</p