Toxicity induced by orellanine from the mushroom Cortinarius orellanus in primary renal tubular proximal epithelial cells (RPTEC): Novel mechanisms of action.

The toxicity of Orellanine (OR), a significant factor in mushroom poisoning, has severe effects on the kidneys, particularly the proximal tubules. This study investigated the acute toxicity of OR from the Cortinarius orellanus mushroom in human Primary Renal Tubular Proximal Epithelial Cells (RPTEC). Additionally, the half maximal inhibitory concentration (IC50) of OR in MCF-7 cells was established. RPTEC were subjected to a 6.25 µg/ml dose of orellanine for 24 h, while Control cells were exposed to 0.05% DMSO (vehicle). The RT2 Profiler™ PCR Array Human Nephrotoxicity was utilized to identify genes that were upregulated or downregulated. Western blotting confirmed the protein product of some significantly regulated genes compared to control cells. The IC50 of OR was found to be 319.2 µg/ml. The mechanism of OR toxicity involved several pathways including apoptosis, metal ion binding, cell proliferation, tissue remodeling, xenobiotic metabolism, transporters, extracellular matrix molecules, and cytoskeleton pathways. Other genes from non-specific pathways were also identified. These findings enhance our understanding of OR nephrotoxicity and pave the way for future research into potential treatments or antidotes for natural mushroom poisoning.

The influence of adverse drug effects on health-related quality of life in chronic obstructive pulmonary disease patients.

OBJECTIVES: Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Concerns have been raised about the influence of adverse drug effects on patient's health-related quality of life (HRQoL) in COPD patients. This study aimed to evaluate the impact of COPD treatment-related adverse effects on HRQoL in COPD patients. METHODS: In a cross-sectional study, COPD patients aged 40 years or older were identified and interviewed during their hospital visits. The EuroQol 5 Dimension 5 Level (EQ-5D-5L) questionnaire was used for evaluating HRQoL. Potential treatment adverse effects were evaluated as experienced by participants during the last 2 weeks preceding the interview. The intensity of adverse effects was reported in the following categories: never, mild, moderate and severe. Multivariable linear regression model was performed to evaluate the influence of adverse drug effects on utility scores as an indicator of HRQoL. KEY FINDINGS: A total of 203 patients diagnosed with COPD were recruited in the current study. The mean utility score of the study sample was 0.68 (SD = 0.36). Moderate-severe constipation, moderate-severe confusion, mild urinary hesitation, moderate-severe urinary hesitation, moderate-severe dry eyes and moderate-severe drowsiness were significant predictors/determinants for the average utility scores (coefficients were -0.099, -0.191, -0.111, -0.157 and -0.144, respectively). In addition, having higher COPD Assessment Test scores and severe disease was negatively associated with average utility scores (coefficients were -0.287 and -0.124, respectively). CONCLUSIONS: Higher intensity of COPD treatment-related adverse effects has a negative influence on HRQoL in COPD patients. Anticholinergic drug effects are of concern in COPD adults' population.

Reduced Hepatic Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Level in Obesity.

Impairment of insulin clearance is being increasingly recognized as a critical step in the development of insulin resistance and metabolic disease. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes insulin clearance. Null deletion or liver-specific inactivation of Ceacam1 in mice causes a defect in insulin clearance, insulin resistance, steatohepatitis, and visceral obesity. Immunohistological analysis revealed reduction of hepatic CEACAM1 in obese subjects with fatty liver disease. Thus, we aimed to determine whether this occurs at the hepatocyte level in response to systemic extrahepatic factors and whether this holds across species. Northern and Western blot analyses demonstrate that CEACAM1 mRNA and protein levels are reduced in liver tissues of obese individuals compared to their lean age-matched counterparts. Furthermore, Western analysis reveals a comparable reduction of CEACAM1 protein in primary hepatocytes derived from the same obese subjects. Similar to humans, Ceacam1 mRNA level, assessed by quantitative RT-PCR analysis, is significantly reduced in the livers of obese Zucker (fa/fa, ZDF) and Koletsky (f/f) rats relative to their age-matched lean counterparts. These studies demonstrate that the reduction of hepatic CEACAM1 in obesity occurs at the level of hepatocytes and identify the reduction of hepatic CEACAM1 as a common denominator of obesity across multiple species.

Loss of Hepatic CEACAM1: A Unifying Mechanism Linking Insulin Resistance to Obesity and Non-Alcoholic Fatty Liver Disease.

The pathogenesis of human non-alcoholic fatty liver disease (NAFLD) remains unclear, in particular in the context of its relationship to insulin resistance and visceral obesity. Work on the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in mice has resolved some of the related questions. CEACAM1 promotes insulin clearance by enhancing the rate of uptake of the insulin-receptor complex. It also mediates a negative acute effect of insulin on fatty acid synthase activity. This positions CEACAM1 to coordinate the regulation of insulin and lipid metabolism. Fed a regular chow diet, global null mutation of Ceacam1 manifest hyperinsulinemia, insulin resistance, obesity, and steatohepatitis. They also develop spontaneous chicken-wire fibrosis, characteristic of non-alcoholic steatohepatitis. Reduction of hepatic CEACAM1 expression plays a significant role in the pathogenesis of diet-induced metabolic abnormalities, as bolstered by the protective effect of hepatic CEACAM1 gain-of-function against the metabolic response to dietary fat. Together, this emphasizes that loss of hepatic CEACAM1 links NAFLD to insulin resistance and obesity.

Role for hepatic CEACAM1 in regulating fatty acid metabolism along the adipocyte-hepatocyte axis.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates insulin sensitivity by promoting hepatic insulin clearance and mediating suppression of fatty acid synthase activity. Feeding C57BL/6J male mice with a high-fat (HF) diet for 3-4 weeks triggered a >60% decrease in hepatic CEACAM1 levels to subsequently impair insulin clearance and cause systemic insulin resistance and hepatic steatosis. This study aimed at investigating whether lipolysis drives reduction in hepatic CEACAM1 and whether this constitutes a key mechanism leading to diet-induced metabolic abnormalities. Blocking lipolysis with a daily intraperitoneal injection of nicotinic acid in the last two days of a 30-day HF feeding regimen demonstrated that white adipose tissue (WAT)-derived fatty acids repressed hepatic CEACAM1-dependent regulation of insulin and lipid metabolism in 3-month-old male C57BL/6J mice. Adenoviral-mediated CEACAM1 redelivery countered the adverse metabolic effect of the HF diet on insulin resistance, hepatic steatosis, visceral obesity, and energy expenditure. It also reversed the effect of HF diet on inflammation and fibrosis in WAT and liver. This assigns a causative role for lipolysis-driven decrease in hepatic CEACAM1 level and its regulation of insulin and lipid metabolism in sustaining systemic insulin resistance, hepatic steatosis, and other abnormalities associated with excessive energy supply.

PPARα (Peroxisome Proliferator-activated Receptor α) Activation Reduces Hepatic CEACAM1 Protein Expression to Regulate Fatty Acid Oxidation during Fasting-refeeding Transition.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed at high levels in the hepatocyte, consistent with its role in promoting insulin clearance in liver. CEACAM1 also mediates a negative acute effect of insulin on fatty acid synthase activity. Western blot analysis reveals lower hepatic CEACAM1 expression during fasting. Treating of rat hepatoma FAO cells with Wy14,643, an agonist of peroxisome proliferator-activated receptor α (PPARα), rapidly reduces Ceacam1 mRNA and CEACAM1 protein levels within 1 and 2 h, respectively. Luciferase reporter assay shows a decrease in the promoter activity of both rat and mouse genes by Pparα activation, and 5'-deletion and block substitution analyses reveal that the Pparα response element between nucleotides -557 and -543 is required for regulation of the mouse promoter activity. Chromatin immunoprecipitation analysis demonstrates binding of liganded Pparα toCeacam1promoter in liver lysates ofPparα(+/+), but notPparα(-/-)mice fed a Wy14,643-supplemented chow diet. Consequently, Wy14,643 feeding reduces hepatic Ceacam1 mRNA and CEACAM1 protein levels, thus decreasing insulin clearance to compensate for compromised insulin secretion and maintain glucose homeostasis and insulin sensitivity in wild-type mice. Together, the data show that the low hepatic CEACAM1 expression at fasting is mediated by Pparα-dependent mechanisms. Changes in CEACAM1 expression contribute to the coordination of fatty acid oxidation and insulin action in the fasting-refeeding transition.

High-fat diet amplifies renal renin angiotensin system expression, blood pressure elevation, and renal dysfunction caused by Ceacam1 null deletion.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAMl), a substrate of the insulin receptor tyrosine kinase, regulates insulin action by promoting insulin clearance. Global null mutation of Ceacam1 gene (Cc1(-/-)) results in features of the metabolic syndrome, including insulin resistance, hyperinsulinemia, visceral adiposity, elevated blood pressure, and albuminuria. It also causes activation of the renal renin-angiotensin system (RAS). In the current study, we tested the hypothesis that high-fat diet enhances the expression of RAS components. Three-month-old wild-type (Cc1(+/+)) and Cc1(-/-) mice were fed either a regular or a high-fat diet for 8 wk. At baseline under regular feeding conditions, Cc1(-/-) mice exhibited higher blood pressure, urine albumin-to-creatinine ratio (UACR), and renal expression of angiotensinogen, renin/prorenin, angiotensin-converting enzyme, (pro)renin receptor, angiotensin subtype AT1 receptor, angiotensin II, and elevated PI3K phosphorylation, as detected by p85α (Tyr(508)) immunostaining, inflammatory response, and the expression of collagen I and collagen III. In Cc1(+/+) mice, high-fat diet increased blood pressure, UACR, the expression of angiotensin-converting enzyme and angiotensin II, PI3K phosphorylation, inflammatory response, and the expression of collagen I and collagen III. In Cc1(-/-) mice, high-fat intake further amplified these parameters. Immunohistochemical staining showed increased p-PI3K p85α (Tyr(508)) expression in renal glomeruli, proximal, distal, and collecting tubules of Cc1(-/-) mice fed a high-fat diet. Together, this demonstrates that high-fat diet amplifies the permissive effect of Ceacam1 deletion on renal expression of all RAS components, PI3K phosphorylation, inflammation, and fibrosis.

Hepatic CEACAM1 Over-Expression Protects Against Diet-Induced Fibrosis and Inflammation in White Adipose Tissue.

CEACAM1 promotes insulin extraction, an event that occurs mainly in liver. Phenocopying global Ceacam1 null mice (Cc1(-/-) ), C57/BL6J mice fed a high-fat (HF) diet exhibited reduced hepatic CEACAM1 levels and impaired insulin clearance, followed by hyperinsulinemia, insulin resistance, and visceral obesity. Conversely, forced liver-specific expression of CEACAM1 protected insulin sensitivity and energy expenditure, and limited gain in total fat mass by HF diet in L-CC1 mice. Because CEACAM1 protein is barely detectable in white adipose tissue (WAT), we herein investigated whether hepatic CEACAM1-dependent insulin clearance pathways regulate adipose tissue biology in response to dietary fat. While HF diet caused a similar body weight gain in L-CC1, this effect was delayed and less intense relative to wild-type (WT) mice. Histological examination revealed less expansion of adipocytes in L-CC1 than WT by HF intake. Immunofluorescence analysis demonstrated a more limited recruitment of crown-like structures, and qRT-PCR analysis showed no significant rise in TNFα mRNA levels in response to HF intake in L-CC1 than WT mice. Unlike WT, HF diet did not activate TGF-ß in WAT of L-CC1 mice, as assessed by Western analysis of Smad2/3 phosphorylation. Consistently, HF diet caused relatively less collagen deposition in L-CC1 than WT mice, as shown by Trichrome staining. Coupled with reduced lipid redistribution from liver to visceral fat, lower inflammation and fibrosis could contribute to protected energy expenditure against HF diet in L-CC1 mice. The data underscore the important role of hepatic insulin clearance in the regulation of adipose tissue inflammation and fibrosis.

Forced Hepatic Overexpression of CEACAM1 Curtails Diet-Induced Insulin Resistance.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates insulin sensitivity by promoting hepatic insulin clearance. Liver-specific inactivation or global null-mutation of Ceacam1 impairs hepatic insulin extraction to cause chronic hyperinsulinemia, resulting in insulin resistance and visceral obesity. In this study we investigated whether diet-induced insulin resistance implicates changes in hepatic CEACAM1. We report that feeding C57/BL6J mice a high-fat diet reduced hepatic CEACAM1 levels by >50% beginning at 21 days, causing hyperinsulinemia, insulin resistance, and elevation in hepatic triacylglycerol content. Conversely, liver-specific inducible CEACAM1 expression prevented hyperinsulinemia and markedly limited insulin resistance and hepatic lipid accumulation that were induced by prolonged high-fat intake. This was partly mediated by increased hepatic ß-fatty acid oxidation and energy expenditure. The data demonstrate that the high-fat diet reduced hepatic CEACAM1 expression and that overexpressing CEACAM1 in liver curtailed diet-induced metabolic abnormalities by protecting hepatic insulin clearance.

Development of nonalcoholic steatohepatitis in insulin-resistant liver-specific S503A carcinoembryonic antigen-related cell adhesion molecule 1 mutant mice.

BACKGROUND & AIMS: Liver-specific inactivation of carcinoembryonic antigen-related cell adhesion molecule 1 causes hyperinsulinemia and insulin resistance, which result from impaired insulin clearance, in liver-specific S503A carcinoembryonic antigen-related cell adhesion molecule 1 mutant mice (L-SACC1). These mice also develop steatosis. Because hepatic fat accumulation precedes hepatitis, lipid peroxidation, and apoptosis in the pathogenesis of nonalcoholic steatohepatitis (NASH), we investigated whether a high-fat diet, by causing inflammation, is sufficient to induce hepatitis and other features of NASH in L-SACC1 mice. METHODS: L-SACC1 and wild-type mice were placed on a high-fat diet for 3 months, then several biochemical and histologic analyses were performed to investigate the NASH phenotype. RESULTS: A high-fat diet caused hepatic macrosteatosis and hepatitis, characterized by increased hepatic tumor necrosis factor alpha levels and activation of the NF-kappaB pathway in L-SACC1 but not in wild-type mice. The high-fat diet also induced necrosis and apoptosis in the livers of the L-SACC1 mice. Insulin resistance in L-SACC1 fed a high-fat diet increased the hepatic procollagen protein level, suggesting a role in the development of fibrosis. CONCLUSIONS: A high-fat diet induces key features of human NASH in insulin-resistant L-SACC1 mice, validating this model as a tool to study the molecular mechanisms of NASH.

Aerobic interval training versus continuous moderate exercise as a treatment for the metabolic syndrome: a pilot study.

BACKGROUND: Individuals with the metabolic syndrome are 3 times more likely to die of heart disease than healthy counterparts. Exercise training reduces several of the symptoms of the syndrome, but the exercise intensity that yields the maximal beneficial adaptations is in dispute. We compared moderate and high exercise intensity with regard to variables associated with cardiovascular function and prognosis in patients with the metabolic syndrome. METHODS AND RESULTS: Thirty-two metabolic syndrome patients (age, 52.3+/-3.7 years; maximal oxygen uptake [o(2)max], 34 mL x kg(-1) x min(-1)) were randomized to equal volumes of either moderate continuous moderate exercise (CME; 70% of highest measured heart rate [Hfmax]) or aerobic interval training (AIT; 90% of Hfmax) 3 times a week for 16 weeks or to a control group. o(2)max increased more after AIT than CME (35% versus 16%; P<0.01) and was associated with removal of more risk factors that constitute the metabolic syndrome (number of factors: AIT, 5.9 before versus 4.0 after; P<0.01; CME, 5.7 before versus 5.0 after; group difference, P<0.05). AIT was superior to CME in enhancing endothelial function (9% versus 5%; P<0.001), insulin signaling in fat and skeletal muscle, skeletal muscle biogenesis, and excitation-contraction coupling and in reducing blood glucose and lipogenesis in adipose tissue. The 2 exercise programs were equally effective at lowering mean arterial blood pressure and reducing body weight (-2.3 and -3.6 kg in AIT and CME, respectively) and fat. CONCLUSIONS: Exercise intensity was an important factor for improving aerobic capacity and reversing the risk factors of the metabolic syndrome. These findings may have important implications for exercise training in rehabilitation programs and future studies.

Insulin acutely decreases hepatic fatty acid synthase activity.

Insulin is viewed as a positive regulator of fatty acid synthesis by increasing fatty acid synthase (FAS) mRNA transcription. We uncover a new mechanism by which insulin acutely reduces hepatic FAS activity by inducing phosphorylation of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and its interaction with FAS. Ceacam1 null mice (Cc1(-/-)) show loss of insulin's ability to acutely decrease hepatic FAS activity. Moreover, adenoviral delivery of wild-type, but not the phosphorylation-defective Ceacam1 mutant, restores the acute effect of insulin on FAS activity in Cc1(-/-) primary hepatocytes. Failure of insulin to acutely reduce hepatic FAS activity in hyperinsulinemic mice, including L-SACC1 transgenics with liver inactivation of CEACAM1, and Ob/Ob obese mice, suggests that the acute effect of insulin on FAS activity depends on the prior insulinemic state. We propose that this mechanism acts to reduce hepatic lipogenesis incurred by insulin pulses during refeeding.

CEACAM1 modulates epidermal growth factor receptor--mediated cell proliferation.

Phosphorylation of the cell adhesion protein CEACAM1 increases insulin sensitivity and decreases insulin-dependent mitogenesis in vivo. Here we show that CEACAM1 is a substrate of the EGFR and that upon being phosphorylated, CEACAM1 reduces EGFR-mediated growth of transfected Cos-7 and MCF-7 cells in response to EGF. Using transgenic mice overexpressing a phosphorylation-defective CEACAM1 mutant in liver (L-SACC1), we show that the effect of CEACAM1 on EGF-dependent cell proliferation is mediated by its ability to bind to and sequester Shc, thus uncoupling EGFR signaling from the ras/MAPK pathway. In L-SACC1 mice, we also show that impaired CEACAM1 phosphorylation leads to ligand-independent increase of EGFR-mediated cell proliferation. This appears to be secondary to visceral obesity and the metabolic syndrome, with increased levels of output of free fatty acids and heparin-binding EGF-like growth factor from the adipose tissue of the mice. Thus, L-SACC1 mice provide a model for the mechanistic link between increased cell proliferation in states of impaired metabolism and visceral obesity.

Interaction between altered insulin and lipid metabolism in CEACAM1-inactive transgenic mice.

Inactivation of CEACAM1 in L-SACC1 mice by a dominant-negative transgene in liver impairs insulin clearance and increases serum free fatty acid (FFA) levels, resulting in insulin resistance. The contribution of elevated FFAs in the pathogenesis of insulin resistance is herein investigated. Treatment of L-SACC1 female mice with carnitine restored plasma FFA content. Concomitantly, it normalized insulin levels without directly regulating receptor-mediated insulin internalization and prevented glucose tolerance in these mice. Similarly, treatment with nicotinic acid, a lipolysis inhibitor, restored insulin-stimulated receptor uptake in L-SACC1 mice. Taken together, these data suggest that chronic elevation in plasma FFAs levels contributes to the regulation of insulin metabolism and action in L-SACC1 mice.