Microsomal Protein Levels Research Articles

Dietary nicotinic acid promotes the growth and tryptophan metabolome of Chinese mitten crab (Eriocheir sinensis) through ACMSD and NADs

This paper did research on the effect of dietary nicotinic acid (NA) on lipid metabolism and tryptophan metabolome of Chinese mitten crab (Eriocheir sinensis) to promote its growth. Three diets to administer to E. sinensis (37.25 ± 0.5 g), respectively, for 10 weeks were designed to contain 0% NA (control), 0.02% NA, and 0.10% NA. According to the results, when feeding crabs with 0.02% NA diet, the specific growth rate (SGR) increased markedly while the feed conversion ratio (FCR) was the opposite compared with 0% NA group. The content of total cholesterol (T-CHO) and low-density lipoprotein cholesterol (LDL) in hemolymph decreased significantly when feeding crabs with 0.02% NA diet while the content of high-density lipoprotein cholesterol (HDL) was the opposite. In addition, the relative expression level of microsomal triglyceride transfer protein (mttp) and kynureninase (KFase) decreased significantly in 0.10% NA group while the relative expression level of sterol regulatory element-binding protein 1 (srebp-1) and nicotinamide adenine dinucleotide synthetase (NADs) increased significantly in 0.02% NA group. Meanwhile, the protein expression of 2-amino-3-carboxymuconic semialdehyde decarboxylase (ACMSD) in 0.02% NA group had a very significant increase compared with the other two groups. The result of tryptophan metabolome in hepatopancreas indicates that 0.02% NA in the diet could inhibit the downstream metabolic pathway of tryptophan. According to the results of this research, 0.02% NA in the diet can improve the growth and tryptophan metabolome through ACMSD and NADs, and lipid metabolism is also improved in this group, while 0.10% NA in the diet has no effect on crabs in growth performance.

Predicting the systemic exposure and lung concentration of nafamostat using physiologically-based pharmacokinetic modeling.

Nafamostat has been actively studied for its neuroprotective activity and effect on various indications, such as coronavirus disease 2019 (COVID-19). Nafamostat has low water solubility at a specific pH and is rapidly metabolized in the blood. Therefore, it is administered only intravenously, and its distribution is not well known. The main purposes of this study are to predict and evaluate the pharmacokinetic (PK) profiles of nafamostat in a virtual healthy population under various dosing regimens. The most important parameters were assessed using a physiologically based pharmacokinetic (PBPK) approach and global sensitivity analysis with the Sobol sensitivity analysis. A PBPK model was constructed using the SimCYP® simulator. Data regarding the in vitro metabolism and clinical studies were extracted from the literature to assess the predicted results. The model was verified using the arithmetic mean maximum concentration (Cmax), the area under the curve from 0 to the last time point (AUC0-t), and AUC from 0 to infinity (AUC0-∞) ratio (predicted/observed), which were included in the 2-fold range. The simulation results suggested that the 2 dosing regimens for the treatment of COVID-19 used in the case reports could maintain the proposed effective concentration for inhibiting severe acute respiratory syndrome coronavirus 2 entry into the plasma and lung tissue. Global sensitivity analysis indicated that hematocrit, plasma half-life, and microsomal protein levels significantly influenced the systematic exposure prediction of nafamostat. Therefore, the PBPK modeling approach is valuable in predicting the PK profile and designing an appropriate dosage regimen.

Tebuconazole Fungicide Induces Lipid Accumulation and Oxidative Stress in HepG2 Cells.

Tebuconazole (TEB), a triazole fungicide, is frequently applied to agriculture for the increase of food production. Although TEB causes liver toxicity, its effects on cellular lipid accumulation are rarely investigated. Therefore, this study aimed to study the effects of TEB on lipid metabolism and accumulation in HepG2 cells. HepG2 cells were exposed to 0–320 µM TEB for 1–24 h. TEB (20–80 µM, 24 h)-treated cells showed lipid accumulation. Further, TEB (20–80 µM, 1–12 h) increased the nuclear translocation of peroxisome proliferator-activated receptors and the expression of lipid uptake and oxidation-related markers such as cluster of differentiation 36, fatty acid transport protein (FATP) 2, FATP5, and carnitine palmitoyltransferase 1. Oxidative stress levels in TEB-treated cells (20–80 µM, 24 h) were higher, compared to those in the control. TEB (20–80 µM, 24 h) also induced the loss of mitochondrial membrane potential and lower levels of microsomal triglyceride transfer protein in the cells. Thus, TEB can induce lipid accumulation by altering the expression of lipid-metabolizing molecules and can therefore impair lipid metabolism. Our data suggest that human exposure to TEB may be a risk factor for non-alcoholic fatty liver disease.

Berberine ameliorates nonalcoholic fatty liver disease by decreasing the liver lipid content via reversing the abnormal expression of MTTP and LDLR

The global incidence of nonalcoholic fatty liver disease (NAFLD) is increasing. The present study explored the effect and mechanism of berberine (BBR) on NAFLD in rats. Thirty-five Sprague-Dawley rats were randomly divided into the control and NAFLD groups, which were fed a normal diet or high-fat diet, respectively, for 8 weeks. Hematoxylin and eosin staining was performed on liver tissues and establishment of the NAFLD model was confirmed by microscopy. NAFLD rats were subsequently randomly subdivided and treated with saline or BBR for 8 weeks. The liver wet weight of rats in each group was measured, the liver tissue structure was observed by microscopy, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), fasting blood glucose (FBG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels were detected using a semi-automatic biochemical detector. Reverse transcription-quantitative PCR and western blotting were performed to determine the mRNA and protein expression levels of microsomal triglyceride transfer protein (MTTP), apolipoprotein B and low-density lipoprotein receptor (LDLR). Compared with the control group, the liver wet weight of the NAFLD rats was higher; the liver showed obvious fatty degeneration and liver TG levels increased significantly, as did serum levels of ALT, AST, TC, TG, FBG, HDL and LDL, while expression of MTTP and LDLR significantly decreased. Compared with the saline-treated NAFLD rats, the BBR-treated rats had reduced liver wet weight, improved liver steatosis and a significant decrease in liver TG levels, while ALT, AST, TC, TG, and LDL serum levels significantly decreased and MTTP levels were significantly upregulated. In conclusion, BBR treatment ameliorated the fatty liver induced by a high-fat diet in rats. Furthermore, BBR reversed the abnormal expression of MTTP and LDLR in rats with high-fat diet induced-NAFLD. The present findings suggest that fatty liver could be improved by BBR administration, via reversing the abnormal expression of MTTP and LDLR and inhibiting lipid synthesis.

Lipid overload impairs hepatic VLDL secretion via oxidative stress-mediated PKCδ-HNF4α-MTP pathway in large yellow croaker (Larimichthys crocea)

Lipid overload-induced hepatic steatosis is a major public health problem worldwide. However, the potential molecular mechanism is not completely understood. Herein, we found that high-fat diet (HFD) or oleic acid (OA) treatment induced oxidative stress which prevented the entry of hepatocyte nuclear factor 4 alpha (HNF4α) into the nucleus by activating protein kinase C delta (PKCδ) in vivo and in vitro in large yellow croaker (Larimichthys crocea). This reduced the level of microsomal triglyceride transfer protein (MTP) transcription, resulting in the impaired secretion of very-low-density lipoprotein (VLDL) and the abnormal accumulation of triglyceride (TG) in hepatocytes. Meanwhile, the detrimental effects induced by lipid overload could be partly alleviated by pretreating hepatocytes with Go6983 (PKCδ inhibitor) or N-acetylcysteine (NAC, reactive oxygen species (ROS) scavenger). In conclusion, for the first time, we revealed that lipid overload impaired hepatic VLDL secretion via oxidative stress-mediated PKCδ-HNF4α-MTP pathway in fish. This study may provide critical insights into potential intervention strategies against lipid overload-induced hepatic steatosis of fish and human beings.

The Diagnostic Value of Combined Detection of Kidney Injury Molecule-1 and Urine Microsomal Protein in Early Stage Nephropathy of Gestational Hypertension

To investigate the diagnostic value of urine kidney injury molecule-1 and urine microsomal protein combined test in early stage nephropathy of gestational hypertension. 72 patients with gestational hypertension treated at the hospital from December 2018 to December 2019 were randomly selected as subjects and 72 normal pregnant women who gave birth at the hospital during the same period were randomly selected as controls. Urinary microsomal protein levels were measured by immune enhanced turbidimetric assay, while urinary kidney injury molecule-1 levels were measured by enzyme linked immunosorbent assay. Blood urea nitrogen, high-sensitivity C-reactive protein levels, and specificity and sensitivity of urine kidney injury molecule-1 and urine microsomal serum proteins in the two groups and their subgroups were analyzed comparatively. The levels of urinary kidney injury molecule-1 and urinary microsomal serum protein were higher in the study group than in the control group. Among the three subgroups of the study group, urinary kidney injury molecule-1 and urinary microsomal serum protein levels were significantly increased in the mid and late pregnancy groups compared to the early pregnancy group. In the subgroup with impaired renal function, the urinary kidney injury molecule-1 and urinary microalbumin levels were significantly higher than those in the subgroup with normal renal function and the difference was statistically significant (p<0.05). In terms of blood urea nitrogen and high-sensitivity C-reactive protein levels, blood urea nitrogen and high-sensitivity C-reactive protein levels in the study group were higher than those in the healthy control group and estimated glomerular filtration rate was lower than those in the healthy control group. Urinary kidney injury molecule-1 level and urinary microsomal protein in the study group showed positive correlation with blood urea nitrogen and high-sensitivity C-reactive protein (both p<0.05), and negative correlation with Estimated glomerular filtration rate. In terms of sensitivity and specificity, the results showed that the specificity and sensitivity of the combined test were significantly higher than that of the individual test and the difference was statistically significant (p<0.05). The detection of serum kidney injury molecule-1 and microsomal protein and other indicators to determine the degree of early renal impairment in pregnancy induced hypertension is beneficial for early clinical treatment and is of great clinical significance for pregnant women and pregnancy outcomes.

Lomitapide treatment in a female with homozygous familial hypercholesterolaemia: a case report.

BackgroundHomozygous familial hypercholesterolaemia (FH) is an autosomal-dominant inherited disease presenting with highly elevated low-density lipoprotein cholesterol (LDL-C) levels. Untreated, the patient can develop atherosclerosis and cardiovascular disease already in adolescence. Treatment with statins and ezetimibe is usually not sufficient and LDL apheresis is often required. Lomitapide, an inhibitor of the microsomal triglyceride transfer protein, reduces LDL-C and triglyceride levels and can be used alone or in combination with other therapies in homozygous FH. However, experience with this agent is still limited.Case summaryWe present a young female who was diagnosed with homozygous FH at 6 years of age. She shows a complete lack of normal LDL receptor activity and no cholesterol-lowering effect from statins. The patient was treated with LDL apheresis from 7 years of age. When LDL apheresis treatment extended to twice a week, she began to experience adverse effects, including catheter-related complications, infections, and hospital admissions. When lomitapide treatment was initiated, the frequency of apheresis reduced, the LDL-C levels improved and she has not had any further hospital admissions since. Initially, she suffered from gastrointestinal disturbances. However, after 3 years of treatment with lomitapide 20 mg/day, the patient has not experienced any adverse effects.DiscussionIn this female with homozygous FH adding lomitapide treatment to LDL apheresis has contributed to improved LDL-C levels, a reduction in LDL apheresis sessions and enhanced quality of life. No adverse effects have been reported. These findings suggest that lomitapide can be a drug of choice in patients with homozygous FH.

MiR-130b is a potent stimulator of hepatic very-low-density lipoprotein assembly and secretion via marked induction of microsomal triglyceride transfer protein

miR-130b is a microRNA whose expression is particularly elevated within adipose tissue and in the circulation in diabetic states. Hepatic miR-130b expression has been linked to hepatocellular carcinoma and changes in lipid metabolism. Here, we investigated the role of miR-130b in hepatic lipid homeostasis and lipoprotein export. We observed that overexpression of miR-130b-3p or -5p in HepG2 cells markedly enhanced the secretion of very-low-density lipoprotein (VLDL) particles, enhanced the secretion of [3H]glycerol metabolically labeled triglyceride (TG), and significantly increased the number or the average size of lipid droplets (LDs), respectively. Overexpression of miR-130b also altered the expression of key genes involved in lipid metabolism and in particular markedly increased both mRNA and protein expression levels of microsomal triglyceride transfer protein (MTP). Conversely, the miR-130b inhibitor decreased mRNA levels of MTP and fatty acid synthase (FAS) in HepG2 cells. However, dual-luciferase reporter assays indicated that MTP is not a direct target of miR-130b-3p. miR-130b overexpression did not alter de novo synthesized TG or the stability and secretion of apolipoprotein B 100. Interestingly, knockdown of phosphatase and tensin homolog (PTEN) blocked the upregulation of MTP mRNA induced by miR-130b. Finally, miR-130b-induced stimulation of VLDL secretion was also observed in a second hepatocyte cell culture model, immortalized human hepatocytes, confirming the effects observed in HepG2 cells. Overall, these data suggest a potential role for miR-130b in promoting hepatic VLDL assembly and secretion mediated by marked stimulation of MTP expression and TG mobilization. Thus miR-130b overexpression corrects the defect in VLDL production in HepG2 cells.

Increased fat synthesis and limited apolipoprotein B cause lipid accumulation in the liver of broiler chickens exposed to chronic heat stress.

Chronic heat stress can enhance fat synthesis in broilers, and excessive triglyceride (TG) synthesized by the liver needs to be transported to extrahepatic tissues by very low density lipoprotein (VLDL) otherwise will accumulate in the liver, which may even result in hepatic steatosis. To investigate the molecular mechanisms by which chronic heat stress enhances fat synthesis and results in lipid accumulation in the liver of chickens, 144 broilers (Arbor Acres, 28-day-old) were randomly allocated to the normal control (NC, 22°C), heat stress (HS, consistent 32°C), or pair-fed (PF, 22°C) groups for a 14-D trial. The 7 D of heat exposure significantly increased the respiratory rate, relative weight of abdominal fat, the levels of glucose, TG, corticosterone, insulin, and VLDL in plasma, as well as the levels of TG, total cholesterol, acyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) in the liver, and mRNA expression levels of carbohydrate response element-binding protein (ChREBP), ACC, FAS, and microsomal triglyceride transfer protein (MTTP) in comparison with the other 2 groups. After 14 D of heat exposure, the relative weights of abdominal fat and liver and levels of TG and FAS in the liver were significantly higher in the HS group than in the other 2 groups, and there were no significant differences in the respiratory rate, plasma corticosterone concentration, apolipoprotein B (ApoB) level in the liver, and mRNA expression levels of key genes of fat synthesis among the 3 groups. In conclusion, chronic heat exposure activated LXRα pathway and enhanced fat synthesis in the liver after 7 D of heat exposure. After 14 D of heat exposure, heat-stressed broilers exhibited an adaptation to the high temperature in parameters of stress and fat synthesis gene expression levels. Moreover, chronic heat stress resulted in lipid accumulation in the liver of broilers, which is probably because the limited ApoB was not enough to transport the excessive TG synthesized by the liver in chronic heat-stressed broilers.

Short-term regulation of the hepatic activities of cytochrome P450 and glutathione S-transferase by nose-only cigarette smoke exposure in mice

The present study aimed to determine the effects of cigarette smoke on the regulation of hepatic cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes in male BALB/c mice exposed to nose-only cigarette smoke for 4 days. There were no significant increases in serum liver injury markers (alanine aminotransferase and aspartate aminotransferase) or oxidative stress (total antioxidant capacity, malondialdehyde, and glutathione disulfide/reduced glutathione) following cigarette smoke exposure, but malondialdehyde was elevated in the bronchoalveolar lavage fluid of smoke-exposed mice. Additionally, the hepatic microsomal protein levels of Cyp1a and Cyp2b, and the activities of ethoxyresorufin O-deethylase, pentoxyresorufin O-depenylase, and chlorzoxazone 6-hydrxylase, were elevated in smoke-exposed mice. Interestingly, the hepatic activities of GST toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and ethacrynic acid, but not cumene hydroperoxide were enhanced by cigarette smoke exposure, which was consistent with the increased expression levels of mu- and pi-class GSTs, but not alpha-class GSTs, observed in immunoblot analyses. These findings indicate that the short-term inhalation of cigarette smoke induces drug-metabolizing enzymes such as CYP1A, CYP2B, and mu/pi-class GSTs in the absence of hepatic injury and oxidative stress. Furthermore, smoking may alter hepatic drug metabolism, as well as the disposition and toxicity of xenobiotics, including some therapeutic drugs and cigarette smoke constituents.

A diet-induced Sprague–Dawley rat model of nonalcoholic steatohepatitis-related cirrhosis

Certain modified diets containing saturated fatty acids, cholesterol or fructose lead to the development of nonalcoholic steatohepatitis (NASH)-related fibrosis in rodents; however, progression to cirrhosis is rare. Experimental liver cirrhosis models have relied on genetic manipulation or administration of hepatotoxins. This study aimed to establish a reliable dietary model of NASH-related cirrhosis in a relatively short period. Male Sprague–Dawley rats (9 weeks of age) were randomly assigned to normal, high-fat (HF), or two types (1.25% or 2.5% cholesterol) of high-fat and high-cholesterol (HFC) diets for 18 weeks. All HFC diets contained 2% cholic acid by weight. Histopathological analysis revealed that the HFC diets induced obvious hepatic steatosis, inflammation with hepatocyte ballooning and advanced fibrosis (stage 3–4) in all 12 rats at 27 weeks of age. In contrast, all five rats given the HF diet developed mild steatosis and inflammation without fibrosis. The amount of cholesterol in the liver and hepatocellular mitochondrial and microsomal fractions was significantly higher in rats fed the HFC diets than the normal or HF diets. The HFC diets significantly suppressed mRNA levels of microsomal triglyceride transfer protein, adenosine triphosphate binding cassette transporter G5, bile acid CoA: amino acid N-acyltransferase and bile salt export pump, as well as the enzymatic activity of carnitine palmitoyltransferase in the liver. In conclusion, the HFC diets induced liver cirrhosis in conjunction with hepatic features of NASH in Sprague–Dawley rats within 18 weeks, and altered gene expression and enzyme activity to accumulate lipid and bile acid in the liver.

In Vitro effect of DDE exposure on the regulation of lipid metabolism and secretion in McA-RH7777 hepatocytes: A potential role in dyslipidemia which may increase the risk of type 2 diabetes mellitus

Organochlorine compounds (OC), such as the legacy insecticides, were widespread environmental contaminants. OC including dichlorodiphenyldichloroethylene (DDE), a metabolite of the insecticide DDT, have an epidemiological association with type 2 diabetes mellitus (T2D) and may play a role in risk factors that contribute to T2D such as dyslipidemia. The liver, a potential target for DDE, plays a role in dyslipidemia. The in vitro effect of DDE on hepatocyte lipid metabolism and secretion was investigated using McArdle-RH7777 (McA) rodent hepatoma liver cells. When stimulated by the free fatty acid oleic acid (OA), DDE increased the secretion of apolipoprotein B (ApoB) suggesting a role for DDE in increasing lipid secretion. Intracellular protein levels of microsomal triglyceride transfer protein (MTP) were increased while sortilin-1 (Sort-1) levels were decreased suggesting a role for DDE in increasing lipid transport and decreasing lipid degradation. Neutral lipids such as intracellular triglycerides (TG) were decreased suggesting that DDE may alter lipid accumulation in liver cells. DDE may play a role in dyslipidemia by affecting mechanisms that regulate lipid metabolism and secretion. These in vitro results on biochemical markers of liver cell dyslipidemia support the concept that DDE exposure may play a role in the dyslipidemia frequently observed in T2D.

Hepatic expression of cytochrome P450 in Zucker diabetic fatty rats

In this study, the hepatic expression of cytochrome P450 (CYP) enzymes, including CYP1A1/2, 2B1, 2C11, 2E1, 3A1/2, and 4A, was investigated in 5-week-old (insulinresistant state) and 11-week-old (diabetic) Zucker diabetic fatty (ZDF) rats. Serum glucose and glycated hemoglobin levels were increased in 11-week-old ZDF rats, but not in 5-weekold ZDF rats. Hyperinsulinemia was observed in both age groups. The microsomal protein, total CYP, CYP reductase, CYP1A1/2, and CYP3A1 levels did not differ between 5- and 11-week-old ZDF rats and their respective control rats, while CYP4A was up-regulated in both groups. Hepatic levels of cytochrome b5, CYP2B1, CYP2C11, CYP2E1, and CYP3A2 were decreased in 5-week-old ZDF rats, but not in 11-week-old ZDF rats. Similarly, pentoxyresorufin O-depentylase, testosterone 2α- and 16α-hydroxylase, chlorzoxazone 6- hydroxylase, and midazolam 1′- and 4-hydroxylase activities were decreased only in 5-weekold ZDF rats. Based on these results, the 5-week-old ZDF rats exhibited down-regulation of the major CYP enzymes. These results suggest that hepatic expression of CYP enzymes may be dysregulated during development in ZDF rats. With the exception of CYP2B1 and CYP4A, the hepatic levels and activities of CYP were comparable between 11-week-old ZDF and control rats, suggesting that xenobiotic metabolism is normally regulated in the early diabetic state.

Differential effect of fructose on fat metabolism and clock gene expression in hepatocytes vs. myotubes

In the liver, fructose bypasses the main rate-limiting step of glycolysis at the level of phosphofructokinase, allowing it to act as an unregulated substrate for de novo lipogenesis. It has been reported that consumption of large amounts of fructose increases de novo lipogenesis in the liver. However, the effect of fructose on ectopic deposition of muscle fat has been under dispute. Our aim was to study the effect of fructose on levels of genes and proteins involved in fatty acid oxidation and synthesis in hepatocytes vs. muscle cells. In addition, as fat accumulation leads to disruption of daily rhythms, we tested the effect of fructose treatment on clock gene expression. AML-12 hepatocytes and C2C12 myotubes were treated with fructose or glucose for 2 consecutive 24-h cycles and harvested every 6h. In contrast to glucose, fructose disrupted clock gene rhythms in hepatocytes, but in myotubes, it led to more robust rhythms. Fructose led to low levels of phosphorylated AMP-activated protein kinase (pAMPK) and high levels of LIPIN1 in hepatocytes compared with glucose. In contrast, fructose led to high pAMPK and low LIPIN1 and microsomal triacylglycerol transfer protein (MTTP) levels in myotubes compared with glucose. Analysis of fat content revealed that fructose led to less fat accumulation in myotubes compared to hepatocytes. In summary, fructose shifts metabolism towards fatty acid synthesis and clock disruption in hepatocytes, but not in myotubes.

Effects of β-hydroxybutyricacid on the synthesis and assembly of very low-density lipoprotein in bovine hepatocytes invitro.

β-Hydroxybutyricacid (BHBA) is an important metabolite that involved in the development of ketosis and fatty liver in dairy cows. Dairy cows with fatty liver displayed high blood concentration of BHBA and very low-density lipoprotein (VLDL) assembly. The effects of BHBA on VLDL synthesis and assembly in hepatocytes of cows were unclear. In this study, bovine hepatocytes were cultured and treated with different concentrations of BHBA. We found that BHBA treatment upregulated the mRNA and protein levels of apolipoprotein B100 (ApoB 100), apolipoprotein E (ApoE) and microsomal triglyceride transfer protein (MTTP) and showed in a firstly increased and then decreased trend. Meanwhile, the mRNA and protein levels of LDLR showed in a reverse trend. Consequently, VLDL content was significantly increased in medium-dose BHBA treatment group, while decreased in high-dose group. These results indicate that the effects of BHBA on the VLDL synthesis showed in a dose-dependent manner that low levels of BHBA increase VLDL synthesis and high levels of BHBA decrease VLDL synthesis.

Evaluating toxic endpoints of exposure to a commercial PCB mixture: an &lt;em&gt;in vivo&lt;/em&gt; laboratory study

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that produce a wide range of toxic effects. To determine sensitive endpoints in various organ systems, the effects of Aroclor 1260 on immune, endocrine, and hepatic systems were evaluated in a dose-response study. Nine-week old male rats were treated with Aroclor 1260 by oral gavage at dosages ranging from 0.025 to 156 mg/kg/day for 10 consecutive days and killed two days after the last treatment. Eight days prior to sacrifice, rats were injected i.v. with sheep red blood cells (SRBC) for determination of humoral immunity. No observable adverse effect level (NOAEL) and lowest observable adverse effect level (LOAEL) were determined for liver, thymus and genital organ weights, body weight, serum luteinizing hormone (LH), testosterone, thyroxine and thyroid-stimulating hormone (TSH) concentrations, hepatic microsomal testosterone hydroxylase activities, and hepatic microsomal cytochrome P450 (CYP) 1A1, CYP1A2, CYP2B1 and CYP2B2 protein levels. Treatment with Aroclor 1260, at all dosages, had no effect on testis, seminal vesicle or ventral prostate weights, on thymus weight or on serum LH or testosterone levels. Among the endpoints altered by Aroclor 1260, the most sensitive, with a LOAEL of 1.25 mg/kg/day, were increased testosterone 16β-hydroxylase activity and androstenedione formation. The LOAEL for increased liver weight, testosterone 16α-hydroxylase activity and CYP2B1 protein content was 3.13 mg/kg/day, while the LOAEL for decreased serum thyroxine levels and anti-SRBC IgM titer was 6.25 mg/kg/day. Less sensitive responses, as reflected by larger LOAEL values, included CYP1A enzyme induction and decreased body weight. In summary, comparison of NOAEL and LOAEL values indicated that hepatic CYP2B-mediated activities were a more sensitive response to Aroclor 1260 exposure in male rats than immune or endocrine endpoints.

A Calibrated Human PBPK Model for Benzene Inhalation with Urinary Bladder and Bone Marrow Compartments

A physiologically-based pharmacokinetic (PBPK) model of benzene inhalation based on a recent mouse model was adapted to include bone marrow (target organ) and urinary bladder compartments. Empirical data on human liver microsomal protein levels and linked CYP2E1 activities were incorporated into the model, and metabolite-specific conversion rate parameters were estimated by fitting to human biomonitoring data and adjusting for background levels of urinary metabolites. Human studies of benzene levels in blood and breath, and phenol levels in urine were used to validate the rate of human conversion of benzene to benzene oxide, and urinary benzene metabolites from Chinese benzene worker populations provided model validation for rates of human conversion of benzene to muconic acid (MA) and phenylmercapturic acid (PMA), phenol (PH), catechol (CA), hydroquinone (HQ), and benzenetriol (BT). The calibrated human model reveals that while liver microsomal protein and CYP2E1 activities are lower on average in humans compared to mice, the mouse also shows far lower rates of benzene conversion to MA and PMA, and far higher conversion of benzene to BO/PH, and of BO/PH to CA, HQ, and BT. The model also differed substantially from existing human PBPK models with respect to several metabolic rate parameters of importance to interpreting benzene metabolism and health risks in human populations associated with bone marrow doses. The model provides a new methodological paradigm focused on integrating linked human liver metabolism data and calibration using biomonitoring data, thus allowing for model uncertainty analysis and more rigorous validation.

Hypomorphic sialidase expression decreases serum cholesterol by downregulation of VLDL production in mice

Lipoprotein metabolism is an important contributing factor in the development and progression of atherosclerosis. Plasma lipoproteins and their receptors are heavily glycosylated and sialylated, and levels of sialic acids modulate their biological functions. Sialylation is controlled by the activities of sialyltranferases and sialidases. To address the impact of sialidase (neu1) activity on lipoprotein metabolism, we have generated a mouse model with a hypomorphic neu1 allele (B6.SM) that displays reduced sialidase expression and sialidase activity. The objectives of this study are to determine the impact of sialidase on the rate of hepatic lipoprotein secretion and lipoprotein uptake. Our results indicate that hepatic levels of cholesterol and triglycerides are significantly higher in B6.SM mice compared with C57Bl/6 mice; however, VLDL-triglyceride production rate is lower. In addition, B6.SM mice show significantly lower levels of hepatic microsomal triglyceride transfer protein (MTP) and active sterol-regulatory element binding protein (SREBP)-2 but higher levels of diglyceride acyltransferase (DGAT)2; these are all indicative of increased hepatic lipid storage. Rescue of sialidase activity in hypomorphic sialidase mice using helper-dependent adenovirus resulted in increased VLDL production and an increase in MTP levels. Furthermore, hypomorphic sialidase expression results in stabilization of hepatic LDL receptor (LDLR) protein expression, which enhances LDL uptake. These findings provide novel evidence for a central role of sialidase in the cross talk between the uptake and production of lipoproteins.

Lysophosphatidylcholine Acyltransferase 3 Knockdown-mediated Liver Lysophosphatidylcholine Accumulation Promotes Very Low Density Lipoprotein Production by Enhancing Microsomal Triglyceride Transfer Protein Expression

After de novo biosynthesis phospholipids undergo extensive remodeling by the Lands' cycle. Enzymes involved in phospholipid biosynthesis have been studied extensively but not those involved in reacylation of lysophosphopholipids. One key enzyme in the Lands' cycle is fatty acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which utilizes lysophosphatidylcholine (LysoPC) and fatty acyl-CoA to produce various phosphatidylcholine (PC) species. Four isoforms of LPCAT have been identified. In this study we found that LPCAT3 is the major hepatic isoform, and its knockdown significantly reduces hepatic LPCAT activity. Moreover, we report that hepatic LPCAT3 knockdown increases certain species of LysoPCs and decreases certain species of PC. A surprising observation was that LPCAT3 knockdown significantly reduces hepatic triglycerides. Despite this, these mice had higher plasma triglyceride and apoB levels. Lipoprotein production studies indicated that reductions in LPCAT3 enhanced assembly and secretion of triglyceride-rich apoB-containing lipoproteins. Furthermore, these mice had higher microsomal triglyceride transfer protein (MTP) mRNA and protein levels. Mechanistic studies in hepatoma cells revealed that LysoPC enhances secretion of apoB but not apoA-I in a concentration-dependent manner. Moreover, LysoPC increased MTP mRNA, protein, and activity. In short, these results indicate that hepatic LPCAT3 modulates VLDL production by regulating LysoPC levels and MTP expression.

Increased Intestinal Lipid Absorption Caused by Ire1β Deficiency Contributes to Hyperlipidemia and Atherosclerosis in Apolipoprotein E–Deficient Mice

High fasting serum lipid levels are significant risk factors for atherosclerosis. However, the contributions of postprandial excursions in serum lipoproteins to atherogenesis are less well-characterized. This study aims to delineate whether changes in intestinal lipid absorption associated with loss of inositol-requiring enzyme 1β (Ire1β) would affect the development of hyperlipidemia and atherosclerosis in Apoe(-/-) mice. We used Ire1β-deficient mice to assess the contribution of intestinal lipid absorption to atherosclerosis. Here, we show that Ire1b(-/-)/Apoe(-/-) mice contain higher levels of intestinal microsomal triglyceride transfer protein, absorb more lipids, exhibit hyperlipidemia, and have higher levels of atherosclerotic plaques compared with Apoe(-/-) mice when fed chow and western diets. These studies indicate that Ire1β regulates intestinal lipid absorption and that increased intestinal lipoprotein production contributes to atherosclerosis.