Biomedical subjects

Nica M Borradaile

Publications and source records attributed to Nica M Borradaile.

8 recordsLinked to original sources

PubMed2006

Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death.

Cell dysfunction and death induced by lipid accumulation in nonadipose tissues, or lipotoxicity, may contribute to the pathogenesis of obesity and type 2 diabetes. However, the mechanisms leading to lipotoxic cell death are poorly understood. We recently reported that, in Chinese hamster ovary (CHO) cells and in H9c2 cardiomyoblasts, lipid overload induced by incubation with 500 muM palmitate leads to intracellular accumulation of reactive oxygen species, which subsequently induce endoplasmic reticulum (ER) stress and cell death. Here, we show that palmitate also impairs ER function through a more direct mechanism. Palmitate was rapidly incorporated into saturated phospholipid and triglyceride species in microsomal membranes of CHO cells. The resulting membrane remodeling was associated with dramatic dilatation of the ER and redistribution of protein-folding chaperones to the cytosol within 5 h, indicating compromised ER membrane integrity. Increasing beta-oxidation, through the activation of AMP-activated protein kinase, decreased palmitate incorporation into microsomes, decreased the escape of chaperones to the cytosol, and decreased subsequent caspase activation and cell death. Thus, palmitate rapidly increases the saturated lipid content of the ER, leading to compromised ER morphology and integrity, suggesting that impairment of the structure and function of this organelle is involved in the cellular response to fatty acid overload.

Animals↗

PubMed2005

A critical role for eukaryotic elongation factor 1A-1 in lipotoxic cell death.

The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide- and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease.

Animals↗

PubMed2005

Lipotoxicity in the heart.

Cardiomyopathy is associated with both rare genetic metabolic abnormalities and highly prevalent diseases characterized by elevated serum triglycerides and nonesterified fatty acids, such as obesity and type 2 diabetes. In these disorders, an imbalance between fatty acid uptake and utilization leads to the inappropriate accumulation of free fatty acids and neutral lipids within cardiomyocytes. Through the process of lipotoxicity, this lipid overload causes cellular dysfunction, cell death, and eventual organ dysfunction. This review focuses on lipotoxicity in the heart, with an emphasis on the contribution of this process to the pathogenesis of cardiomyopathy associated with obesity, diabetes, and the metabolic syndrome. The magnitude of the current worldwide epidemic of obesity and type 2 diabetes suggests that understanding the pathogenesis of cardiac complications associated with these diseases will contribute substantially to improvements in health care.

Animals↗

PubMed2005

Inhibition of microsomal triglyceride transfer protein expression and apolipoprotein B100 secretion by the citrus flavonoid naringenin and by insulin involves activation of the mitogen-activated protein kinase pathway in hepatocytes.

Microsomal triglyceride transfer protein (MTP) is necessary for hepatocyte assembly and secretion of apolipoprotein (apo)B100-containing lipoproteins. The citrus flavonoid naringenin, like insulin, decreased MTP expression in HepG2 cells, resulting in inhibition of apoB100 secretion; however, the mechanism for naringenin is independent of insulin receptor substrate-1/2. Recently, it was reported that insulin decreased MTP expression in HepG2 cells via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) (MAPK(erk)) pathway. We hypothesized that naringenin acts via a similar mechanism. Inhibition of MAPK kinase (MEK) 1/2 in HepG2 cells significantly attenuated the naringenin- and insulin-induced reduction in MTP expression. Both naringenin and insulin increased ERK1/2 phosphorylation, which was completely inhibited by MEK1/2 inhibition and enhanced by inhibition of MAPK(p38), a negative regulator of MAPK(erk) activity. Inhibition of MEK1/2 significantly attenuated both the naringenin- and insulin-induced decrease in apoB100 secretion demonstrating a direct link between MAPK(erk) activation and apoB100 secretion. Furthermore, both compounds increased MAPK(p38) activation, and therefore inhibition of MAPK(p38) amplified thenaringenin- and insulin-induced decrease in apoB100 secretion. We conclude that MAPK(erk) signaling in hepatocytes is critical for inhibition of apoB100 secretion by naringenin and insulin. Therefore, naringenin may prove useful for activating insulin-signaling pathways important for regulation of hepatocyte lipid homeostasis.

Animals↗

PubMed2003

Hepatocyte apoB-containing lipoprotein secretion is decreased by the grapefruit flavonoid, naringenin, via inhibition of MTP-mediated microsomal triglyceride accumulation.

Naringenin, the principal flavonoid in grapefruit, reduces plasma lipids in vivo and inhibits apoB secretion, cholesterol esterification, and MTP activity in HepG2 human hepatoma cells. Although naringenin inhibits ACAT, we recently demonstrated that CE availability in the microsomal lumen does not regulate apoB secretion in HepG2 cells. We therefore hypothesized that inhibition of TG accumulation in the ER lumen, secondary to MTP inhibition, is the primary mechanism whereby naringenin blocks lipidation and subsequent secretion of apoB. Multicompartmental modeling of pulse-chase studies was used to compare cellular apoB kinetics in the presence of either naringenin or the specific MTP inhibitor, BMS-197636. At concentrations that reduced apoB secretion by 50%, both compounds selectively enhanced degradation via a kinetically defined, rapid, proteasomal pathway to the same extent. Subcellular fractionation experiments revealed that naringenin and BMS-197636 reduced accumulation of newly synthesized TG in the microsomal lumen by 48% and 54%, respectively. Newly synthesized CE accumulation in the lumen was reduced by 80% and 33% with naringenin and BMS-197636, respectively, demonstrating for the first time that MTP is involved in CE accumulation in the microsomal lumen. Reduced TG availability at this initial site of lipoprotein assembly was associated with significant reductions in the secretion of apoB-containing lipoproteins. Both naringenin and BMS-197636 were most effective in reducing secretion of IDL and LDL, but also inhibited secretion of apoB-containing HDL-sized particles. Furthermore, in McA-RH7777-derived cell lines, naringenin reduced secretion of hapoB72 and hapoB100, which require significant assembly with lipid to be secreted, but did not reduce secretion of hapoB17, hapoB23, and hapoB48, which require only minimal lipidation. Taken together, our results indicate that naringenin inhibits the lipidation and subsequent secretion of apoB-containing lipoproteins primarily by limiting the accumulation of TG in the ER lumen, secondary to MTP inhibition.

Apolipoprotein B-100↗

PubMed2003

Inhibition of net HepG2 cell apolipoprotein B secretion by the citrus flavonoid naringenin involves activation of phosphatidylinositol 3-kinase, independent of insulin receptor substrate-1 phosphorylation.

The flavonoid naringenin improves hyperlipidemia and hyperglycemia in streptozotocin-treated rats. In HepG2 human hepatoma cells, naringenin inhibits apolipoprotein B (apoB) secretion primarily by inhibiting microsomal triglyceride transfer protein and enhances LDL receptor (LDLr)-mediated apoB-containing lipoprotein uptake. Phosphatidylinositol 3-kinase (PI3K) activation by insulin increases sterol regulatory element-binding protein (SREBP)-1 and LDLr expression and inhibits apoB secretion in hepatocytes. Thus, we determined whether naringenin activates this pathway. Insulin and naringenin induced PI3K-dependent increases in cytosolic and nuclear SREBP-1 and LDLr expression. Similar PI3K-mediated increases in SREBP-1 were observed in McA-RH7777 rat hepatoma cells, which express predominantly SREBP-1c. Reductions in HepG2 cell media apoB with naringenin were partially attenuated by wortmannin, whereas the effect of insulin was completely blocked. Both treatments reduced apoB100 secretion in wild-type and LDLr(-/-) mouse hepatocytes to the same extent. Insulin and naringenin increased HepG2 cell PI3K activity and decreased insulin receptor substrate (IRS)-2 levels. In sharp contrast to insulin, naringenin did not induce tyrosine phosphorylation of IRS-1. We conclude that naringenin increases LDLr expression in HepG2 cells via PI3K-mediated upregulation of SREBP-1, independent of IRS-1 phosphorylation. Although this pathway may not regulate apoB secretion in primary hepatocytes, PI3K activation by this novel mechanism may explain the insulin-like effects of naringenin in vivo.

Animals↗

PubMed2002

Soya phytoestrogens, genistein and daidzein, decrease apolipoprotein B secretion from HepG2 cells through multiple mechanisms.

Diets containing the soya-derived phytoestrogens, genistein and daidzein, decrease plasma cholesterol in humans and experimental animals. The mechanisms responsible for the hypocholesterolaemic effects of these isoflavones are unknown. The present study was conducted to determine if genistein and daidzein regulate hepatocyte cholesterol metabolism and apolipoprotein (apo) B secretion in cultured human hepatoma (HepG2) cells. ApoB secretion was decreased dose-dependently by up to 63% and 71% by genistein and daidzein (100 microM; P<0.0001) respectively. In contrast, no effect on apoAI secretion was observed. Cellular cholesterol synthesis was inhibited 41% by genistein (100 microM; P<0.005) and 18% by daidzein (100 microM; P<0.05), which was associated with significant increases in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA. Cellular cholesterol esterification was decreased 56% by genistein (100 microM; P<0.04) and 29% by daidzein (100 microM; P<0.04); however, mRNA levels for acyl-CoA:cholesterol acyltransferase (ACAT) 1 and ACAT2 were unaffected. At 100 microM, both isoflavones equally inhibited the activities of both forms of ACAT in cells transfected with either ACAT1 or ACAT2. Genistein (100 microM) and daidzein (100 microM) significantly decreased the activity of microsomal triacylglycerol transfer protein (MTP) by 30% and 24% respectively, and significantly decreased MTP mRNA levels by 35% and 55%. Both isoflavones increased low-density lipoprotein (LDL)-receptor mRNA levels by 3- to 6-fold (100 microM; P<0.03) and significantly increased the binding, uptake and degradation of (125)I-labelled LDL, suggesting that enhanced reuptake of newly secreted apoB-containing lipoproteins contributed to the net decrease in apoB secretion. These results indicate that genistein and daidzein inhibit hepatocyte apoB secretion through several mechanisms, including inhibition of cholesterol synthesis and esterification, inhibition of MTP activity and expression and increased expression of the LDL-receptor.

Apolipoproteins B↗

PubMed2002

Inhibition of hepatocyte apoB secretion by naringenin: enhanced rapid intracellular degradation independent of reduced microsomal cholesteryl esters.

The grapefruit flavonoid, naringenin, is hypocholesterolemic in vivo, and inhibits basal apolipoprotein B (apoB) secretion and the expression and activities of both ACAT and microsomal triglyceride transfer protein (MTP) in human hepatoma cells (HepG2). In this report, we examined the effects of naringenin on apoB kinetics in oleate-stimulated HepG2 cells and determined the contribution of microsomal lumen cholesteryl ester (CE) availability to apoB secretion. Pulse-chase studies of apoB secretion and intracellular degradation were analyzed by multicompartmental modeling. The model for apoB metabolism in HepG2 cells includes an intracellular compartment from which apoB can be either secreted or degraded by both rapid and slow pathways. In the presence of 0.1 mM oleic acid, naringenin (200 micro M) reduced the secretion of newly synthesized apoB by 52%, due to a 56% reduction in the rate constant for secretion. Intracellular degradation was significantly increased due to a selective increase in rapid degradation, while slow degradation was unaffected. Incubation with either N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) or lactacystin showed that degradation via the rapid pathway was largely proteasomal. Although these changes in apoB metabolism were accompanied by significant reductions in CE synthesis and mass, subcellular fractionation experiments comparing naringenin to specific ACAT and HMG-CoA reductase inhibitors revealed that reduced accumulation of newly synthesized CE in the microsomal lumen is not consistently associated with reduced apoB secretion. However, naringenin, unlike the ACAT and HMG-CoA reductase inhibitors, significantly reduced lumenal TG accumulation. We conclude that naringenin inhibits apoB secretion in oleate-stimulated HepG2 cells and selectively increases intracellular degradation via a largely proteasomal, rapid kinetic pathway. Although naringenin inhibits ACAT, CE availability in the endoplasmic reticulum (ER) lumen does not appear to regulate apoB secretion in HepG2 cells. Rather, inhibition of TG accumulation in the ER lumen via inhibition of MTP is the primary mechanism blocking apoB secretion.

Acetylcysteine↗