Newly synthesized apolipoprotein B (apoB) undergoes rapid degradation in a pre-Golgi compartment in HepG2 cells. A major site of this early degradation seems to be on the cytosolic side of the endoplasmic reticulum (ER) membrane and is sensitive to N-acetylleucinyl-leucinyl-norleucinal (ALLN), which can inhibit neutral cysteine proteases and/or proteasome activity. Oleate (OA) treatment, which facilitates translocation of nascent apoB across the ER membrane, also reduces early degradation. In the present studies, we have used brefeldin A (BFA), which inhibits vesicular transport from the ER to the Golgi, to demonstrate that apoB can also be degraded by an ER luminal proteolytic activity that is distinct from the ALLN-sensitive proteases. Thus, when BFA-treated HepG2 cells were cotreated with ALLN, which protects apoB but does not facilitate its translocation into the ER lumen, degradation of newly synthesized apoB was significantly reduced compared with cells incubated with BFA alone. However, apoB degradation was rapid and complete when OA was added to media containing either BFA or ALLN/BFA. These results suggested that OA, by increasing translocation of nascent apoB into the ER lumen, exposed apoB to an ALLN-resistant proteolytic pathway. When we incubated HepG2 cells with dithiothreitol (DTT)/OA/BFA or DTT/OA/ALLN/BFA, degradation of apoB was inhibited. Furthermore, addition of DTT resulted in the accumulation of a 70-kDa amino-terminal fragment of apoB. Both full-length and amino-terminal apoB were degraded if DTT was removed from the incubation media; both were secreted if only BFA was removed. Thus, even after apoB is translocated into the ER lumen (thereby avoiding the initial proteolytic pathway), it can potentially be degraded by a lumenal proteolytic process that is ALLN-resistant but DTT-sensitive. The present results, together with previous studies, suggest that at least two distinct steps may be involved in the posttranslational degradation of apoB: 1) the first occurs while apoB is partially translocated and is ALLNsensitive; and 2) the second occurs in the ER lumen and is DTT-sensitive. Finally, our results support the hypothesis that degradation of partially translocated apoB generates a 70-kDa amino-terminal fragment that is mainly degraded in the ER lumen by a DTT-sensitive pathway. ApoB1 secretion from cultured liver cells is regulated mainly at the posttranslational level. Thus, apoB mRNA levels are relatively stable under many conditions, whereas secretion of apoB-containing lipoproteins is altered (1-5). The impact of this posttranslational regulation is demonstrated by the observations that only a small to moderate proportion of the newly synthesized apoB is eventually secreted from primary rat hepatocytes (6, 7), McArdle cells (8), and HepG2 cells (9). A major portion of newly synthesized apoB undergoes rapid intracellular degradation in a pre-Golgi or ER compartment (10 -12) in HepG2 cells as well as in apoB cDNA-transfected Chinese hamster ovary cells (13). ApoB can be protected from e...

A magnetic resonance spectroscopy (MRS) procedure for in vivo measurement of lipid levels in mouse liver is described and validated. The method uses respiratory-gated, localized spectroscopy to collect proton spectra from voxels within the mouse liver. Bayesian probability theory analysis of these spectra allows the relative intensities of the lipid and water resonances within the liver to be accurately measured. All spectral data were corrected for measured spin-spin relaxation. A total of 48 mice were used in this study, including wild-type mice and two different transgenic mouse strains. Different groups of these mice were fed high-fat or low-fat diets or liquid diets with and without the addition of alcohol. Proton spectra were collected at baseline and, subsequently, Alcoholic (1, 2) and nonalcoholic fatty liver (NAFL) (3-7) are highly prevalent in human populations and may develop into steatohepatitis and in some cases into cirrhosis requiring liver transplantation. Animal models of both conditions have been developed. Mouse models are particularly useful because genetic manipulations are highly developed in inbred mice. However, longitudinal studies require the killing of animals because no noninvasive method for quantifying liver fat is available. Here, we describe a noninvasive, nondestructive method for quantifying the liver fat contents of the mouse using a NAFL mouse model. The overwhelming majority of NAFL cases are associated with obesity, dyslipidemia, hypertension, insulin-resistant type 2 diabetes mellitus, and atherosclerotic cardiovascular disease (8)(9)(10)(11)(12). This constellation defines the metabolic syndrome (13,14). One naturally occurring cause of fatty liver is familial hypobetalipoproteinemia (FHBL). FHBL is defined by less than fifth percentile plasma levels of LDL-cholesterol and/or total apolipoprotein B (apoB), segregating in families as an autosomal dominant trait (15-17). The mean liver triglyceride content in apoBimpaired FHBL subjects is 3-to 5-fold greater than that of controls (18,19).In an attempt to understand the cellular/molecular bases of hypobetalipoproteinemia, several recombinant mice mimicking human FHBL have been produced (19)(20)(21)(22)(23)(24)(25). The resulting mice closely resemble their human counterparts with respect to the fatty liver phenotype. Thus, these mice could serve as good models of one genetic form of fatty liver for studies of the progression of fatty liver and on the effects of metabolic, hormonal, and therapeutic perturbations over time.One current limitation of such studies is the absence of an accurate, quantifiable, noninvasive method for repeat assessments of liver fat in animals. In humans, the gold standard for quantifying liver fat is the liver biopsy, which is invasive and, at best, semiquantitative. The analogous procedure in animals is killing of the animal and direct analysis of liver fat by standard chemical methods after extraction. Obviously, direct sampling of livers for fat analysis is not ideal for longitudinal, fol...

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