A mouse model of human familial hypercholesterolemia: markedly elevated low density lipoprotein cholesterol levels and severe atherosclerosis on a low-fat chow diet.

Mutations in the low density lipoprotein (LDL) receptor gene cause familial hypercholesterolemia, a human disease characterized by premature atherosclerosis and markedly elevated plasma levels of LDL cholesterol and apolipoprotein (apo) B100. In contrast, mice deficient for the LDL receptor (Ldlr-/-) have only mildly elevated LDL cholesterol levels and little atherosclerosis. This difference results from extensive editing of the hepatic apoB mRNA in the mouse, which limits apoB100 synthesis in favor of apoB48 synthesis. We have generated Ldlr-/- mice that cannot edit the apoB mRNA and therefore synthesize exclusively apoB100. These mice had markedly elevated LDL cholesterol and apoB100 levels and developed extensive atherosclerosis on a chow diet. This authentic model of human familial hypercholesterolemia will provide a new tool for studying atherosclerosis.

Insulin increases expression of apobec-1, the catalytic subunit of the apolipoprotein B mRNA editing complex in rat hepatocytes.

We have previously shown that chronic insulin treatment of rat hepatocytes increases the fraction of edited apolipoprotein B (apoB) mRNA from approximately 50% to as much as 90%. We have now examined the effect of insulin on apobec-1 mRNA abundance and demonstrate that increased editing of apoB mRNA following insulin treatment is accompanied by elevated apobec-1 mRNA levels in primary rat hepatocytes. Time-course measurements of the effects of insulin on apoB mRNA editing and apobec-1 mRNA abundance showed that both were elevated almost maximally within 48 hours and sustained for at least 5 days of insulin treatment.

Autofluorescence in indomethacin-induced gastric mucosal lesions in rats.

Autofluorescence observations enable scientists to sensitively identify various lesions. Non-steroidal anti-inflammatory drugs such as aspirin and indomethacin are well known to induce gastric mucosal injuries. Our purpose was to clarify whether the observation of mucosal autofluorescence could help us to recognize indomethacin-induced gastric lesion formation. Gastric mucosal fluorescence intensity and gastric lesion scores were time-sequentially measured after indomethacin treatment in rats. To identify the localization of autofluorescent substances, stomach cryosections were observed with an epifluorescence microscope. Fluorescent substances from damaged tissue were also analyzed by high-performance liquid chromatography. In addition, to elucidate whether oxidative stress directly generates fluorescent substances from heme, we investigated the reaction between hydrogen peroxide and hemoglobin in a cell-free system. Treatment with indomethacin induced gastric lesions by tissue peroxidation, with mucosal fluorescence intensity increasing time-dependently. The fluorescence products were mesoporphyrin and protoporphyrin, and they were localized in disrupted mucosal tissue. In the cell-free system, porphyrins were directly generated by hydrogen peroxide from hemoglobin. These findings indicate that indomethacin treatment increased the intensity of porphyrin fluorescence. Gastric mucosal lesion formation can be sensitively detected with fluorescence observations.

Escherichia coli sepsis increases hepatic apolipoprotein B secretion by inhibiting degradation.

Sepsis leads to hypertriglyceridemia in both humans and animals. Previously, we reported that plasma very low density lipoprotein apolipoprotein (apo) B and hepatic production of apoB increased during Escherichia coli sepsis. The present experiments were undertaken to determine whether the altered hepatic secretion of apoB was associated with an increase in synthesis or a decrease in degradation rate. Sepsis was induced in male, Lewis rats (225-275 g) by intravenous injection of 3.8 x 10(8) live E. coli colonies/100 g body. Twenty-four hours later rats were sacrificed, and primary hepatocytes were prepared and incubated overnight with 35S-methionine. Hepatocytes from E. coli-treated rats secreted twice as much apoB-48 and total apoB than the hepatocytes from control rats. Escherichia coil sepsis increased cellular triglyceride mass by 86%, which was due to a stimulation in triglyceride synthesis from newly synthesized fatty acids, measured by 3H2O incorporation into triglycerides. The apoB synthesis rate, apoB mRNA levels, and apoB mRNA editing were not altered during E. coil sepsis. The pulse-chase experiments showed that the rate of apoB degradation decreased in E. coli-treated rats. These findings demonstrate that the secretion of apoB is regulated posttranslationally during E. coli sepsis by decreasing the degradation of newly synthesized apoB, which contributes to the development of hypertriglyceridemia.

Aminoacylase pellets.

Aminoacylase was immobilized on the mycelium pellets of Aspergillus ochraceus by using albumin and glutaraldehyde. No difference in the optimum pH was observed between native aminoacylase and aminoacylase pellets. The aminoacylase pellets were stable in pH 4-8 but they were unstable in alkaline conditions. The aminoacylase pellets were more stable against heavy metal ions and inhibitors than native aminoacylase. However, the degree of the activation of aminoacylase with cobalt ion decreased with the immobilization. It was suggested that most of aminoacylase was covalently coupled to the mycelium with glutaraldehyde.

Kupffer cell-independent acute hepatocellular oxidative stress and decreased bile formation in post-cold-ischemic rat liver.

The purpose of this study was to examine distribution and time history of oxidative stress during the hyperacute period of reperfusion in the liver grafts undergoing cold ischemia and to investigate roles of Kupffer cells as a potential oxidant source. Rat livers were harvested at 4 degrees C in University of Wisconsin solution and followed by reperfusion with Krebs-Henseleit buffer under monitoring bile excretion. To investigate oxidative changes, laser-confocal microfluorography was performed in reperfused livers preloaded with dichlorodihydrofluorescein diacetate succinimidyl ester, a fluorescence precursor sensing intracellular hydroperoxide generation. Livers undergoing the 16-hour cold storage displayed an impaired recovery of bile acid-dependent bile output concurrent with a marked increase in hydroperoxide generation in hepatocytes, which occurred as early as 5 minutes after the onset of reperfusion, whereas the status of lobular perfusion was well maintained. Pretreatment with liposome-encapsulated dichloromethylene diphosphonate, a Kupffer cell-depleting reagent, did neither alter the reperfusion-induced periportal oxidative changes nor improve the recovery of bile output in the graft. On the other hand, EPCK, a hepatotropic antioxidant composed of vitamin E phosphate ester bound to vitamin C, not only diminished the oxidative changes but also improved the reduction of bile acid-dependent bile output. Furthermore, the reagent was capable of inhibiting H(2)O(2)-induced oxidative stress in cultured hepatocytes. These results suggest that hepatocytes constitute a major site of the oxidative insult triggered through Kupffer cell-independent mechanisms and serve as an important cellular component to be protected by antioxidant therapeutics.