Sirt3 Pharmacologically Promotes Insulin Sensitivity through PI3/AKT/mTOR and Their Downstream Pathway in Adipocytes.

Sirtuin-3 (Sirt3) is a major mitochondrial deacetylase enzyme that regulates multiple metabolic pathways, and its expression is decreased in diabetes type 1 and type 2 diabetes. This study aimed to elucidate Sirt3's molecular mechanism in regulating insulin sensitivity in adipocytes that can contribute to the effort of targeting Sirt3 for the treatment of obesity and type 2 diabetes. We found that the Sirt3 activator honokiol (HNK) induced adipogenesis compared to the control, in contrast to Sirt3 inhibitor, 3-TYP. Accordingly, HNK increased expression of adipocyte gene markers, gene-involved lipolysis and glucose transport (GLUT4), while 3-TYP reduced expression of those genes. Interestingly, 3-TYP caused an increase in gene expression of adipocyte-specific cytokines including IL6, resistin, and TNF-α. However, changes in adipocyte-specific cytokines in HNK treated cells were not significant. In addition, HNK stimulated insulin pathway by promoting insulin receptor beta (IRß) and PI3K/AKT/mTOR pathways, resulting in an increase in phosphorylation of the forkhead family FoxO1/FoxO3a/FoxO4 and glycogen synthase kinase-3 (GSK-3ß), opposing 3-TYP. In line with these findings, HNK increased free fatty acid and glucose uptake, contrary to 3-TYP. In conclusion, Sirt3 activator-HNK induced adipogenesis and lipolysis reduced adipocytes specific cytokines. Intriguingly, HNK activated insulin signaling pathway and increased free fatty acid as well as glucose uptake and transport, in sharp contrast to 3-TYP. These results indicate that, via insulin signaling regulation, Sirt3 activation by HNK improves insulin resistance, while Sirt3 inhibition by 3-TYP might precipitate insulin resistance.

Absence of myocyte regeneration by mobilization of bone marrow stem cells after myocardial infarction.

The study aimed to test the potential for bone marrow stem cells (BMSC) mobilized by granulocyte macrophage colony stimulating factor (GM-CSF) to promote neovascularization and cardiomyocytes regeneration in a rat model of myocardial infarction. The myocardial infracted rats were randomly assigned to receive GM-CSF injection as GM-CSF group or received saline injection as control group. Evaluation of CD34+ stem cells was performed by flow cytometry. Cardiac functions were monitored using a multiple channel recorder via cardiac catheterization. Immunobiological staining including factor VIII and Ki67, and phosphotungstic acid-hematoxylin (PTAH) staining, were performed to assess angiogenesis and myogenesis and calculated myocardial infarction size. The CD34+ stem cells in blood and bone marrow of GM-CSF group increased significantlyon day 7 and day 14 comparing with control group, and declined on day 28. Immunobiological staining showed neovasculature formation and more Ki67 expression in the infracted regions of theGM-CSF group.Ki67 and PTAH double staining showed Ki67 positive signals were overlap with lymphocytes, fibroblasts and endothelial cells but not myocytes. No significant decrease of infracted size occurred in the GM-CSF group. These results suggested BMSC could be mobilized effectively by GM-CSF after myocardial infarction, which could only promote neovascularization without myogenesis.

[Effects of mobilization of bone marrow stem cells on regenerative potential of cardiac tissues after myocardial infarction].

OBJECTIVE: To test the potential of bone marrow stem cells mobilized by granulocyte macrophage colony stimulating factor (GM-CSF) to promote regeneration of myocyte and neovascularization in a rat model of myocardial infarction. METHODS: A myocardial infarction model was created by ligation of the left anterior descending coronary artery in adult Sprague-Dawley rats. Then the infracted rats were randomly assigned to receive GM-CSF injection subcutaneously 50 microg x kg(-1) x day(-1) as experimental group or receive saline injection as control group. Enumeration of CD(34)(+) stem cells in peripheral blood and bone marrow was performed by flow cytometric analysis. Histologic and immunohistological staining such as Factor VIII, Ki67 and PTAH staining (phosphotungstic acid hematoxylin) was performed to assess angiogenesis and myogenesis, calculate myocardial infarction size in the heart. RESULTS: The CD(34)(+) stem cells in the blood and bone marrow increased from Day 3, peaked between Day 7 (0.350% +/- 0.026%, 2.250% +/- 0.140%) and Day 14 (0.260% +/- 0.022%, 2.060% +/- 0.110%) in experimental group compared with control group (0.170% +/- 0.015%, 1.240% +/- 0.064%) (P < 0.01), declined on Day 28 and no difference versus control group (P > 0.05). Immunohistological staining showed apparent neovasculature formation and much more Ki67 expression in the infracted regions in experimental group (313 +/- 10, 275 +/- 5) compared with control group (264 +/- 10, 207 +/- 10) (P < 0.05). The Ki67 positive cells in the experimental hearts were significantly positively correlated with CD(34)(+) stem cells in the blood and bone marrow (r = 0.961, P = 0.019, r = 0.975, P = 0.005). Ki67 and PTAH double staining showed that the Ki67 positive nucleoli overlay the lymphocytes, fibroblasts and endothelial cells but not myocytes. No significant decrease of infracted size occurred in experimental group (P > 0.05). CONCLUSIONS: Bone marrow and peripheral blood stem cells could be mobilized effectively by GM-CSF after myocardial infarction which could only promote neovascularization without myogenesis.

Is it possible to obtain "true endothelial progenitor cells" by in vitro culture of bone marrow mononuclear cells?

In vitro-cultured bone marrow cells have been shown to contain some low-density lipoprotein (LDL) uptake-positive cells. Although a small portion of LDL uptake-positive cells had expression for endothelial markers, all of them demonstrated a phagocytosis function similar to monocyte/macrophages and expression of the panleukocyte surface marker CD45 and monocyte marker CD14. These LDL uptake-positive cells did not show significant proliferative capacity and died out gradually in long-term culture. In contrast, the bone marrow-derived LDL uptake-negative cells showed strong proliferation and expression of typical mesenchymal surface markers CD29 and CD44. Although cultured under endothelial promoting conditions, these mesenchymal stem cells (MSCs) did not show any sign of differentiation toward endothelial cells. In conclusion, adult bone marrow-derived LDL uptake-positive cells that have been reported so far actually are monocytes/macrophages that can express some endothelial markers but are not "true endothelial progenitor cells" (EPCs). MSCs, which are the only cell type that shows strong proliferation during long-term adherent culture for bone marrow cells, do not differentiate toward the endothelial lineage when grown under endothelial promoting conditions.

Transplantation of mesenchymal stem cells from human bone marrow improves damaged heart function in rats.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (MSCs) are of great therapeutic potential after myocardial ischemic injury. However, little is known about the biological characteristics of MSCs in patients with coronary artery disease and their effects on infracted myocardium. The present study evaluated the biological characteristics of MSCs from patients with coronary artery disease and their effects after being transplanted into infarcted myocardium using a rat model. METHODS: Sternal bone marrow aspirates were taken at the time of coronary artery bypass graft surgery. Mononuclear cells isolated from bone marrow were cultured based on plastic adherence. The morphology and growth characteristics of MSCs were observed in primary and successive passages. A myocardial infarction model was created in 27 adult rats. Two weeks later, animals were randomized into two groups: culture medium (group I, n=13) or MSCs (2x10(6)) from early passages labeled with BrdU (group II, n=14) were injected into the infarcted myocardium. Echocardiography, histological examination, and reverse transcription-polymerase chain reaction (RT-PCR) were performed four weeks after cell transplantation. RESULTS: Flow cytometry analyses demonstrated that adherent spindle cells from bone marrow are mesenchymal stem cells (positive for CD29 and CD44, but negative for CD34 and CD45). Growth curves showed that MSCs have great proliferative capability especially at early passages. MSCs implantation in the infarcted border zone improved left ventricular function significantly in group II compared with group I. However, despite improved left ventricular function, we did not observe significant regeneration of cardiac myocytes. Immunohistochemistry revealed only the expression of desmin in the engrafted MSCs, a marker of premature myocyte. Moreover, the improved left ventricular function in this study seemed to be secondary to the beneficial reverse remodeling induced by the increase of collagen in infarcted zone, the decrease in the adjacent myocardium, and the increase of neovascularization (capillary density: 192+/-7.8/mm2 in group II vs. 165+/-5.9/mm2 in group I, P<0.05). Reverse transcription-polymerase chain reaction (RT-PCR) results showed the expression levels of collagen I, collagen III, SDF-1 (stromal cell-derived factor-1), and VEGF (vascular endothelia growth factor) in the infarcted border zone were significantly higher in the MSCs treated group. CONCLUSIONS: The MSCs from patients with coronary artery disease have a typical phenotype with highly proliferative potential and the engrafted MSCs may regulate extracellular collagens and cytokines to prevent the ventricular scar from pathologic thinning and attenuate the contractile dysfunction of the infarcted heart.

Adult endothelial progenitor cells from human peripheral blood maintain monocyte/macrophage function throughout in vitro culture.

Mononuclear cells (MNCs) isolated from peripheral blood by density gradient centrifugation were plated on human fibronectin-coated culture plates and cultured in EGM-2 medium. Attached spindle-shaped cells, reported as endothelial progenitor cells (EPCs) by some investigators, had elongated from adherent round cells, but had not proliferated from a small number of cells as supposed previously. The growth curve of the primary EPCs showed that the cells had little proliferative capacity. Flow cytometry analysis showed that the cells could express some of the endothelial lineage markers, while they could also express CD14, which is considered a marker of monocyte/macrophage lineages throughout culture. In endothelial function assays, the cells demonstrated a lower level of expression of eNOS than mature endothelial cells in the reverse transcription-polymerase chain reaction and did not show an ability to develop tube-like structures in angiogenesis assay in vitro. In this study, we identified the monocytoid function of EPCs by the combined Dil-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) and Indian ink uptake tests. All the cells were double positive for Dil-Ac-LDL and Indian ink uptake at days 4, 14 and 28 of culture, which means the EPCs maintained monocytoid function throughout the culture. Therefore, although adult EPCs from peripheral MNCs have some endothelial lineage properties, they maintain typical monocytic function and have little proliferative capacity.