Angiogenesis is a fundamental process to normal and abnormal tissue growth and repair, which consists of recruiting endothelial cells toward an angiogenic stimulus. The cells subsequently proliferate and differentiate to form endothelial tubes and capillary-like structures. Little is known about the metabolic adaptation of endothelial cells through such a transformation. We studied the metabolic changes of endothelial cell activation by growth factors using human umbilical vein endothelial cells (HUVECs), [1,2-(13)C(2)]-glucose and mass isotopomer distribution analysis. The metabolism of [1,2-(13)C(2)]-glucose by HUVEC allows us to trace many of the main glucose metabolic pathways, including glycogen synthesis, the pentose cycle and the glycolytic pathways. So we established that these pathways were crucial to endothelial cell proliferation under vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) stimulation. A specific VEGF receptor-2 inhibitor demonstrated the importance of glycogen metabolism and pentose cycle pathway. Furthermore, we showed that glycogen was depleted in a low glucose medium, but conserved under hypoxic conditions. Finally, we demonstrated that direct inhibition of key enzymes to glycogen metabolism and pentose phosphate pathways reduced HUVEC viability and migration. In this regard, inhibitors of these pathways have been shown to be effective antitumoral agents. To sum up, our data suggest that the inhibition of metabolic pathways offers a novel and powerful therapeutic approach, which simultaneously inhibits tumor cell proliferation and tumor-induced angiogenesis.

Cell cycle regulation is dependent on multiple cellular and molecular events. Cell proliferation requires metabolic sources for the duplication of DNA and cell size. However, nucleotide reservoirs are not sufficient to support cell duplication and, therefore, biosynthetic pathways should be upregulated during cell cycle. Here, we reveal that glucose-6-phosphate dehydrogenase (G6PDH) and transketolase (TKT), the 2 key enzymes of oxidative and nonoxidative branches of the pentose phosphate pathway (PPP), respectively, which is necessary for nucleotide synthesis, are enhanced during cell cycle progression of the human colon cancer cell line HT29. These enhanced enzyme activities coincide with an increased ratio of pentose monophosphate to hexose monophosphate pool during late G1 and S phase, suggesting a potential role for pentose phosphates in proliferating signaling. Isotopomeric analysis distribution of nucleotide ribose synthesized from 1,2-13 C 2 -glucose confirms the activation of the PPP during late G1 and S phase and reveals specific upregulation of the oxidative branch. Our data sustain the idea of a critical oxidative and nonoxidative balance in cancer cells, which is consistent with a late G1 metabolic check point. The distinctive modulation of these enzymes during cell cycle progression may represent a new strategy to inhibit proliferation in anticancer treatments. ' 2009 UICC

In type 2 diabetes, beta-cell dysfunction is thought to be due to several causes, one being the formation of toxic protein aggregates called islet amyloid, formed by accumulations of misfolded human islet amyloid polypeptide (hIAPP). The process of hIAPP misfolding and aggregation is one of the factors that may activate the unfolded protein response (UPR), perturbing endoplasmic reticulum (ER) homeostasis. Molecular chaperones have been described to be important in regulating ER response to ER stress. In the present work, we evaluate the role of chaperones in a stressed cellular model of hIAPP overexpression. A rat pancreatic beta-cell line expressing hIAPP exposed to thapsigargin or treated with high glucose and palmitic acid, both of which are known ER stress inducers, showed an increase in ER stress genes when compared to INS1E cells expressing rat IAPP or INS1E control cells. Treatment with molecular chaperone glucose-regulated protein 78 kDa (GRP78, also known as BiP) or protein disulfite isomerase (PDI), and chemical chaperones taurine-conjugated ursodeoxycholic acid (TUDCA) or 4-phenylbutyrate (PBA), alleviated ER stress and increased insulin secretion in hIAPP-expressing cells. Our results suggest that the overexpression of hIAPP induces a stronger response of ER stress markers. Moreover, endogenous and chemical chaperones are able to ameliorate induced ER stress and increase insulin secretion, suggesting that improving chaperone capacity can play an important role in improving beta-cell function in type 2 diabetes.

Human islet amyloid polypeptide (hIAPP) aggregation is associated with β-cell dysfunction and death in type 2 diabetes (T2D). we aimed to determine whether treatment with chemical chaperone 4-phenylbutyrate (PBA) ameliorates hIAPP-induced β-cell dysfunction and islet amyloid formation. Oral administration of PBA in hIAPP transgenic (hIAPP Tg) mice expressing hIAPP in pancreatic β cells counteracted impaired glucose homeostasis and restored glucose-stimulated insulin secretion. Moreover, PBA treatment almost completely prevented the transcriptomic alterations observed in hIAPP Tg islets, including the induction of genes related to inflammation. PBA also increased β-cell viability and improved insulin secretion in hIAPP Tg islets cultured under glucolipotoxic conditions. Strikingly, PBA not only prevented but even reversed islet amyloid deposition, pointing to a direct effect of PBA on hIAPP. This was supported by calculations uncovering potential binding sites of PBA to monomeric, dimeric, and pentameric fibrillar structures, and by assays showing inhibition of hIAPP fibril formation by PBA. Collectively, these results uncover a novel beneficial effect of PBA on glucose homeostasis by restoring β-cell function and preventing amyloid formation in mice expressing hIAPP in β cells, highlighting the therapeutic potential of PBA for the treatment of T2D.-Montane, J., de Pablo, S., Castaño, C., Rodríguez-Comas, J., Cadavez, L., Obach, M., Visa, M., Alcarraz-Vizán, G., Sanchez-Martinez, M., Nonell-Canals, A., Parrizas, M., Servitja, J.-M., Novials, A. Amyloid-induced β-cell dysfunction and islet inflammation are ameliorated by 4-phenylbutyrate (PBA) treatment.

Human islet amyloid polypeptide (hIAPP), or amylin, has the tendency to aggregate into insoluble amyloid fibrils, a typical feature of islets from type 2 diabetes individuals. Thus, we investigated comparatively the impact of hIAPP on key pathways involved in pancreatic beta survival. INS1E-hIAPP cells present a hyperactivation of MTORC1 and an inhibition of autophagy signaling, those cells showing an increase in cell size. Resveratrol, a MTORC1 inhibitor, can reverse TSC2 degradation that occurs in INS1E-hIAPP cells and diminished MTORC1 hyperactivation with concomitant autophagy stimulation. At the same time, a blockade in mitophagy was found in INS1E-hIAPP cells, as compared with control or INS1E-rIAPP cells. Consistently, human amylin overexpression generates a basal induction of nitrotyrosine levels and polyubiquitinated aggregates. Failure of the protein degradation machinery finally results in an accumulation of damaged and fissioned mitochondria, ROS production, and increased susceptibility to endoplasmic reticulum (ER)-stress-induced apoptosis. Overall, hIAPP overexpression in INS1E cells induced MTORC1 activation and mitophagy inhibition, favoring a pro-fission scenario of damaged mitochondria, these cells turn out to be more susceptible to the ER-stress-induced apoptosis and malfunction.

No abstract

Diet plays a decisive role in promoting or preventing colon cancer. However, the specific effects of some nutrients remain unclear. The capacity of fruit and vegetables to prevent cancer has been associated with their fiber and antioxidant composition. We investigated whether consumption of a lyophilized red grape pomace containing proanthocyanidin-rich dietary fiber (grape antioxidant dietary fiber, GADF) by female C57BL/6J mice would affect the serum metabolic profile or colon mucosa gene expression using NMR techniques and DNA microarray, respectively. The mice were randomly assigned to 2 groups that for 2 wk consumed a standard rodent diet and were gavaged with 100 mg/kg body weight GADF suspended in water or an equivalent volume of plain tap water (10 mL/kg body weight). The amount of fiber supplemented was calculated to equal the current recommended daily levels of fiber consumption for humans. The inclusion of dietary GADF induced alterations in the expression of tumor suppressor genes and proto-oncogenes as well as the modulation of genes from pathways, including lipid biosynthesis, energy metabolism, cell cycle, and apoptosis. Overexpression of enzymes pertaining to the xenobiotic detoxifying system and endogenous antioxidant cell defenses was also observed. In summary, the genetic and metabolic profiles induced by GADF were consistent with the preventive effects of fiber and polyphenols. On the basis of these observations, we propose that GADF may contribute to reducing the risk of colon cancer.

Cell differentiation is an orderly process that begins with modifications in gene expression. This process is regulated by the acetylation state of histones. Removal of the acetyl groups of histones by specific enzymes (histone deacetylases, HDAC) usually downregulates expression of genes that can cause cells to differentiate, and pharmacological inhibitors of these enzymes have been shown to induce differentiation in several colon cancer cell lines. Butyrate at high (mM) concentration is both a precursor for acetyl-CoA and a known HDAC inhibitor that induces cell differentiation in colon cells. The dual role of butyrate raises the question whether its effects on HT29 cell differentiation are due to butyrate metabolism or to its HDAC inhibitor activity. To distinguish between these two possibilities, we used a tracer-based metabolomics approach to compare the metabolic changes induced by two different types of HDAC inhibitors (butyrate and the non-metabolic agent trichostatin A) and those induced by other acetyl-CoA precursors that do not inhibit HDAC (caprylic and capric acids). [1,2-13C2]-d-glucose was used as a tracer and its redistribution among metabolic intermediates was measured to estimate the contribution of glycolysis, the pentose phosphate pathway and the Krebs cycle to the metabolic profile of HT29 cells under the different treatments. The results demonstrate that both HDAC inhibitors (trichostatin A and butyrate) induce a common metabolic profile that is associated with histone deacetylase inhibition and differentiation of HT29 cells whereas the metabolic effects of acetyl-CoA precursors are different from those of butyrate. The experimental findings support the concept of crosstalk between metabolic and cell signalling events, and provide an experimental approach for the rational design of new combined therapies that exploit the potential synergism between metabolic adaptation and cell differentiation processes through modification of HDAC activity.