Mechanisms of inhibition of lipolysis by insulin, vanadate and peroxovanadate in rat adipocytes

Vanadate and peroxovanadate (pV), potent inhibitors of tyrosine phosphatases, mimic several of the metabolic actions of insulin. Here we compare the mechanisms for the anti-lipolytic action of insulin, vanadate and pV in rat adipocytes. Vanadate (5 mM) and pV (0.01 mM) inhibited lipolysis induced by 0.01-1 microM isoprenaline, vanadate being more and pV less efficient than insulin (1 nM). A loss of anti-lipolytic effect of pV was observed by increasing the concentration of isoprenaline and/or pV. pV induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 to a greater extent than insulin, whereas vanadate affected these components little if at all. In addition, only a higher concentration (0.1 mM) of pV induced the tyrosine phosphorylation of p85, the 85 kDa regulatory subunit of phosphoinositide 3-kinase (PI-3K). Vanadate activated PI-3K-independent (in the presence of 10 nM isoprenaline) and PI-3K-dependent (in the presence of 100 nM isoprenaline) anti-lipolytic pathways, both of which were found to be independent of phosphodiesterase type 3B (PDE3B). pV (0.01 mM), like insulin, activated PI-3K- and PDE3B-dependent pathways. However, the anti-lipolytic pathway of 0.1 mM pV did not seem to require insulin receptor substrate-1-associated PI-3K and was found to be partly independent of PDE3B. Vanadate and pV (only at 0.01 mM), like insulin, decreased the isoprenaline-induced activation of cAMP-dependent protein kinase. Overall, these results underline the complexity and the diversity in the mechanisms that regulate lipolysis

Degerman E. Mechanisms of inhibition of lipolysis by insulin, vanadate and peroxovanadate in rat adipocytes

Vanadate and peroxovanadate (pV), potent inhibitors of tyrosine phosphatases, mimic several of the metabolic actions of insulin. Here we compare the mechanisms for the anti-lipolytic action of insulin, vanadate and pV in rat adipocytes. Vanadate (5 mM) and pV (0.01 mM) inhibited lipolysis induced by 0.01-1 µM isoprenaline, vanadate being more and pV less efficient than insulin (1 nM). A loss of anti-lipolytic effect of pV was observed by increasing the concentration of isoprenaline and\or pV. pV induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 to a greater extent than insulin, whereas vanadate affected these components little if at all. In addition, only a higher concentration (0.1 mM) of pV induced the tyrosine phosphorylation of p85, the 85 kDa regulatory subunit of phosphoinositide 3-kinase (PI-3K). Vanadate activated PI-3K-independent (in the presence of 10 nM isoprenaline

Insulin-induced translocation of protein kinase B to the plasma membrane in rat adipocytes

Protein kinase B (PKB) has previously been shown to be activated in response to insulin and growth factor stimulation. The activation mechanism has been suggested to involve translocation of PKB to membranes, where it is phosphorylated and activated. Insulin-induced translocation of PKB has not been demonstrated in a physiological target cell. Therefore we have used the primary rat adipocyte to investigate insulin-induced translocation of PKB. In the presence of 1 nM insulin translocation of PKB was detected within 30 seconds and was blocked by wortmannin, a selective phosphatidylinositol 3-kinase inhibitor. This translocation was potentiated by the tyrosine phosphatase inhibitor vanadate. Subcellular localization studies revealed that PKB translocated to the plasma membrane

Green, Black and Rooibos Tea Inhibit Prostaglandin E2 Formation in Human Monocytes by Inhibiting Expression of Enzymes in the Prostaglandin E2 Pathway

The formation of prostaglandin E2 (PGE2) is associated with adverse inflammatory effects. However, long-term treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) comes with risk of severe side effects. Therefore, alternative ways to inhibit PGE2 are warranted. We have investigated the effects of tea extracts and the polyphenols epigallocatechin gallate (EGCG) and quercetin on PGE2 formation, determined by immunoassay, and protein expression, determined by immunoblotting, of cytosolic phospholipase A2 (cPLA2), cyclooxygenase 2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) in human monocytes. Green and black tea extracts, and with a lower potency, Rooibos tea extract, inhibited lipopolysaccharide (LPS) and calcium ionophore-induced PGE2 formation. In addition, all tea extracts inhibited the LPS-induced expression of mPGES-1, and the green and black tea extracts also inhibited, to a lesser extent, COX-2 expression. The tea extracts only marginally reduced cPLA2 expression and had no effect on COX-1 expression. EGCG, present in green and black tea, and quercetin, present in all three teas, also inhibited PGE2 formation and expression of mPGES-1, COX-2 and cPLA2. Cell-based and cell-free assays were also performed to evaluate direct effects on the enzymatic activity of COX and PGE synthases. Mainly, the cell-free assay demonstrated partial inhibition by the tea extracts and polyphenols. However, the inhibition required higher doses compared to the effects demonstrated on protein expression. In conclusion, green and black tea, and to a lesser extent Rooibos tea, are potent inhibitors of PGE2 formation in human monocytes, and mediate their effects by inhibiting the expression of the enzymes responsible for PGE2 formation, especially mPGES-1Ingick som manuskript i avhandlingen med titeln Cancer and Inflammation: Role of Macrophages and Monocytes</p

Conditioned media from macrophages of M1, but not M2 phenotype, inhibit the proliferation of the colon cancer cell lines HT-29 and CACO-2

Solid tumors are infiltrated by stroma cells including macrophages and these cells can affect tumor growth, metastasis and angiogenesis. We have investigated the effects of conditioned media (CM) from different macrophages on the proliferation of the colon cancer cell lines HT-29 and CACO-2. CM from THP-1 macrophages and monocyte-derived human macrophages of the M1 phenotype, but not the M2 phenotype, inhibited proliferation of the tumor cells in a dose-dependent manner. Lipopolysaccaharide and interferon γ was used for differentiation of macrophages towards the M1 phenotype and CM were generated both during differentiation (M1DIFF) and after differentiation (M1). M1 and M1DIFF CM as well as THP-1 macrophage CM resulted in cell cycle arrest in HT-29 cells with a decrease of cells in S phase and an increase in G2/M phase. Treatment of HT-29 cells with M1DIFF, but not M1 or THP-1 macrophage CM, resulted in apoptosis of about 20% of the tumor cells and this was accompanied by lack of recovery of cell growth after removal of CM and subsequent culture in fresh media. A protein array was used to identify cytokines released from M1 and M2 macrophages. Among the cytokines released by M1 macrophages, tumor necrosis factor α and CXCL9 were tested by direct addition to HT-29 cells, but neither affected proliferation. Our results indicate that M1 macrophages inhibit colon cancer cell growth and have the potential of contributing to reducing tumor growth in vivo

Conditioned media from human macrophages of M1 phenotype attenuate the cytotoxic effect of 5‑fluorouracil on the HT‑29 colon cancer cell line

Resistance of tumor cells to chemotherapy, such as 5‑fluorouracil (5‑FU), is an obstacle for successful treatment of cancer. As a follow‑up of a previous study we have investigated the effect of conditioned media (CM) from macrophages of M1 or M2 phenotypes on 5‑FU cytotoxicity on the colon cancer cell lines HT‑29 and CACO‑2. HT‑29 cells, but not CACO‑2 cells, having been treated with a combination of M1 CM and 5‑FU recovered their cell growth to a much larger extent compared to cells having been treated with 5‑FU alone when further cultured for 7 days in fresh media. M1 CM treatment of HT‑29, but not CACO‑2 cells, induced cell cycle arrest in the G0/G1 and G2/M phases. 5‑FU treatment induced accumulation of cells in S‑phase in both HT‑29 and CACO‑2 cells. This accumulation of cells in S‑phase was attenuated by combined M1 CM and 5‑FU treatment in HT‑29 cells, but not in CACO‑2 cells. The mRNA expression of cell cycle regulatory proteins and 5‑FU metabolic enzymes were analyzed in an attempt to find possible mechanisms for the M1 CM induced attenuation of 5‑FU cytotoxicity in HT‑29. Thymidylate synthetase (TS) and thymidine phosphorylase (TP) were found to be substantially downregulated and upregulated, respectively, in HT‑29 cells treated with M1 CM, making them unlikely as mediators of reduced 5‑FU cytotoxicity. Among cell cycle regulating proteins, p21 was induced in HT‑29 cells, but not in CACO‑2 cells, in response to M1 CM treatment. However, small interfering RNA (siRNA) knockdown of p21 had no effect on the M1 CM induced cell cycle arrest seen in HT‑29 and neither did it change the growth recovery after combined treatment of HT‑29 cells with M1 CM and 5‑FU. In conclusion, treatment of HT‑29 cells with M1 CM reduces the cytotoxic effect of 5‑FU and this is mediated by a M1 CM induced cell cycle arrest in the G0/G1 and G2/M phases. So far, we lack an explanation why this action is absent in the CACO‑2 cells. The current findings may be important for optimization of chemotherapy in colon cancer