NAFLD: Mechanisms, Treatments, and Biomarkers.

Nonalcoholic fatty liver disease (NAFLD), recently renamed metabolic-associated fatty liver disease (MAFLD), is one of the most common causes of liver diseases worldwide. NAFLD is growing in parallel with the obesity epidemic. No pharmacological treatment is available to treat NAFLD, specifically. The reason might be that NAFLD is a multi-factorial disease with an incomplete understanding of the mechanisms involved, an absence of accurate and inexpensive imaging tools, and lack of adequate non-invasive biomarkers. NAFLD consists of the accumulation of excess lipids in the liver, causing lipotoxicity that might progress to metabolic-associated steatohepatitis (NASH), liver fibrosis, and hepatocellular carcinoma. The mechanisms for the pathogenesis of NAFLD, current interventions in the management of the disease, and the role of sirtuins as potential targets for treatment are discussed here. In addition, the current diagnostic tools, and the role of non-coding RNAs as emerging diagnostic biomarkers are summarized. The availability of non-invasive biomarkers, and accurate and inexpensive non-invasive diagnosis tools are crucial in the detection of the early signs in the progression of NAFLD. This will expedite clinical trials and the validation of the emerging therapeutic treatments.

Non-Alcoholic Fatty Pancreas Disease (NAFPD): A Silent Spectator or the Fifth Component of Metabolic Syndrome? A Literature Review.

BACKGROUND AND OBJECTIVE: Fat accumulation in the pancreas has remained a relatively unknown disease since it was initially described in 1926. However, it has gained increasing attention in the past two decades with the emergence of the obesity epidemic. Pancreatic steatosis is a general term used for fat accumulation in the pancreas. It is further classified into fatty replacement, fatty infiltration, lipomatous pseudo-hypertrophy, non-alcoholic fatty pancreas disease (NAFPD) and non-alcoholic fatty steatopancreatitis (NASP). NAFPD is defined as obesity-associated accumulation of fat in the pancreas without significant alcohol consumption. Data on the prevalence of NAFPD are limited due to a lack of standardized screening tests. METHODS: MEDLINE/PubMed was searched to find relevant studies and abstracts on pancreatic steatosis. RESULTS: Pancreatic fat can be quantified by various imaging techniques including ultrasonography, computed tomography, magnetic resonance imaging and magnetic resonance spectroscopy. The pathophysiology of NAFPD has not been completely understood. Chronic exposure of ß-cells to hyperglycemia and higher levels of free fatty acids results in increased intracellular triglyceride accumulation, which ultimately causes reduced insulin secretion, insulin resistance, cell apoptosis and subsequent fatty replacement. This vicious cycle likely is a determining factor in the development of diabetes mellitus and metabolic syndrome. There is no approved pharmacologic therapy for NAFPD. Caloric restriction might have a role in normalization of ß-cell function by reducing pancreatic fat content. Troglitazone (blend of telmisartan and sitagliptin) has demonstrated effectiveness in animal models but is still in experimental stages. CONCLUSION: The cause and effect relationship between the metabolic syndrome and NAFPD has not yet been established. Further studies are required to study the effect of NAFPD on glucose hemostasis.

Regulation of mitochondrial trifunctional protein modulates nonalcoholic fatty liver disease in mice.

Mitochondrial trifunctional protein (MTP) plays a critical role in the oxidation of long-chain fatty acids. We previously reported that aging mice (>9 months old) heterozygous for an MTP defect (MTP+/-) develop nonalcoholic fatty liver disease (NAFLD). We tested whether a high-fat diet (HFD) accelerates NAFLD in young MTP+/-mice, and whether overexpression of the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase sirtuin 3 (SIRT3) deacetylates MTP and improves mitochondrial function and NAFLD. Three-month-old WT and MTP+/- mice were fed HFD (60% cal fat) for 16 weeks and livers were assessed for fatty acid oxidation (FAO) and NAFLD. Compared with WT, MTP+/- mice displayed reduced hepatic SIRT3 levels and reduced FAO, with increased hepatic steatosis and the inflammatory marker CD68. Hepatic overexpression of SIRT3 in HFD-fed MTP+/- mice increased hepatic MTP protein levels at the posttranscriptional level. Immunoprecipitation of MTP from liver mitochondria followed by Western blot with acetyl-lysine antibody showed higher acetylation of MTP in MTP+/- compared with WT mice. Overexpression of SIRT3 in MTP+/- mice significantly reduced the acetylation of MTP compared with ß-galactosidase controls, increased mitochondrial FAO, and reduced hepatic steatosis, CD68, and serum ALT levels. Taken together, our data indicate that deacetylation of MTP by SIRT3 improves mitochondrial function and rescues NAFLD in MTP+/- mice.

Sirtuins and nonalcoholic fatty liver disease.

Mammalian sirtuins are seven members belonging to the silent information regulator 2 family, a group of Class III histone/protein deacetylases. Sirtuins (SIRT 1-7) have different subcellular localization and function and they regulate cellular protein function through various posttranslational modifications. SIRT1 and 3, the most studied sirtuins, use the product of cellular metabolism nicotinamide adenine dinucleotide as a cofactor to post-translationally deacetylate cellular proteins and consequently link the metabolic status of the cell to protein function. Sirtuins have been shown to play a key role in the development and rescue of various metabolic diseases including non-alcoholic fatty liver disease (NAFLD). NAFLD is currently the most chronic liver disease due mainly to high-calorie consumption and lower physical activity. No pharmacological approach is available to treat NAFLD, the current recommended treatment are lifestyle modification such as weight loss through calorie restriction and exercise. Recent studies have shown downregulation of sirtuins in human as well as animal models of NAFLD indicating an important role of sirtuins in the dynamic pathophysiology of NAFLD. In this review, we highlight the recent knowledge on sirtuins, their role in NAFLD and their unique potential role as novel therapeutic target for NAFLD treatment.

Pathogenesis and Prevention of Hepatic Steatosis.

Hepatic steatosis is defined as intrahepatic fat of at least 5% of liver weight. Simple accumulation of triacylglycerols in the liver could be hepatoprotective; however, prolonged hepatic lipid storage may lead to liver metabolic dysfunction, inflammation, and advanced forms of nonalcoholic fatty liver disease. Nonalcoholic hepatic steatosis is associated with obesity, type 2 diabetes, and dyslipidemia. Several mechanisms are involved in the accumulation of intrahepatic fat, including increased flux of fatty acids to the liver, increased de novo lipogenesis, and/or reduced clearance through ß-oxidation or very-low-density lipoprotein secretion. This article summarizes the mechanisms involved in the accumulation of triacylglycerols in the liver, the clinical implications, and the prevention of hepatic steatosis, with a focus on the role of mitochondrial function and lifestyle modifications.

Role of mitochondria in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) affects about 30% of the general population in the United States and includes a spectrum of disease that includes simple steatosis, non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Significant insight has been gained into our understanding of the pathogenesis of NALFD; however the key metabolic aberrations underlying lipid accumulation in hepatocytes and the progression of NAFLD remain to be elucidated. Accumulating and emerging evidence indicate that hepatic mitochondria play a critical role in the development and pathogenesis of steatosis and NAFLD. Here, we review studies that document a link between the pathogenesis of NAFLD and hepatic mitochondrial dysfunction with particular focus on new insights into the role of impaired fatty acid oxidation, the transcription factor peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and sirtuins in development and progression of NAFLD.

Role of mitochondria in alcoholic liver disease.

Alcohol abuse is the leading cause of liver related morbidity and mortality. Chronic or binge alcohol drinking causes hepatic steatosis which can develop to steatohepatitis, cirrhosis and ultimately hepatocellular carcinoma. The pathogenesis of alcoholic liver disease (ALD) is poorly characterized, however several recent studies point to a major role of mitochondria in this process. Mitochondria play a crucial role in cellular energy metabolism and in reactive species formation. Alcohol treatment causes mitochondrial DNA damage, lipid accumulation and oxidative stress. Studies in both animal models and in humans showed that alcohol administration causes changes in the mitochondrial morphology and function suggesting a role of these changes in the pathogenesis of ALD. We review recent findings on mechanisms by which alcohol negatively impacts mitochondrial biogenesis and function and we will discuss the specific intracellular pathways affected by alcohol consumption. Interestingly, recent findings indicate that a large number of mitochondrial proteins are acetylated and that mitochondrial proteins acetylation and sirtuins are modulated by alcohol. Understanding the mechanisms behind alcohol mediated impaired mitochondrial biogenesis and function may help identify potential therapeutic targets for treating ALD in humans.

CD36 deletion reduces VLDL secretion, modulates liver prostaglandins, and exacerbates hepatic steatosis in ob/ob mice.

Recent findings described the role of CD36-mediated signaling in regulating cellular calcium and the release of various bioactive molecules, including the prostaglandins, neurotransmitters, cholecystokinin, and secretin. Here we document the role of CD36 in the secretion of hepatic VLDL. CD36 deletion resulted in 60% suppression of VLDL output in vivo, and VLDL secretion was reduced in vitro using incubated liver slices. The effect of CD36 deletion was mediated by enhancing formation of hepatic prostaglandins D2, F2, and E2. Treatment of CD36-deficient slices with inhibitors of cyclooxygenases reversed the reduction in triglyceride secretion. We also examined the effect of CD36 deletion on the obesity-associated spontaneous steatosis of the ob/ob mouse that is driven by enhanced de novo lipogenesis. Homozygous ob/ob mice lacking CD36 (ob-CD36⁻/⁻) were generated and studied for hepatic triglyceride accumulation and VLDL secretion. Livers of ob/ob mice were steatotic as expected and had 5-fold more CD36 on Kupffer cells and hepatocytes. CD36 deletion exacerbated the steatosis by impairing hepatic triglyceride and apoB secretion through increasing prostaglandin levels. These findings suggest an unappreciated role of CD36 in regulating VLDL secretion, which might have relevance to some forms of fatty liver. They provide insight into the association reported in humans between CD36 protein expression and serum levels of apoB and VLDL particle number.

CD36-dependent signaling mediates fatty acid-induced gut release of secretin and cholecystokinin.

Genetic variants in the fatty acid (FA) translocase FAT/CD36 associate with abnormal postprandial lipids and influence risk for the metabolic syndrome. CD36 is abundant on apical enterocyte membranes in the proximal small intestine, where it facilitates FA uptake and FA-initiated signaling. We explored whether CD36 signaling influences FA-mediated secretion of cholecystokinin (CCK) and secretin, peptides released by enteroendocrine cells (EECs) in the duodenum/jejunum, which regulate events important for fat digestion and homeostasis. CD36 was immunodetected on apical membranes of secretin- and CCK-positive EECs and colocalized with cytosolic granules. Intragastric lipid administration to CD36 mice released less secretin (-60%) and CCK (-50%) compared with wild-type mice. Likewise, diminished secretin and CCK responses to FA were observed with CD36 intestinal segments in vitro, arguing against influence of alterations in fat absorption. Signaling mechanisms underlying peptide release were examined in STC-1 cells stably expressing human CD36 or a signaling-impaired mutant (CD36K/A). FA stimulation of cells expressing CD36 (vs. vector or CD36K/A) released more secretin (3.5- to 4-fold) and CCK (2- to 3-fold), generated more cAMP (2- to 2.5-fold), and enhanced protein kinase A activation. Protein kinase A inhibition (H-89) blunted secretin (80%) but not CCK release, which was reduced (50%) by blocking of calmodulin kinase II (KN-62). Coculture of STC-1 cells with Caco-2 cells stably expressing CD36 did not alter secretin or CCK release, consistent with a minimal effect of adjacent enterocytes. In summary, CD36 is a major mediator of FA-induced release of CCK and secretin. These peptides contribute to the role of CD36 in fat absorption and to its pleiotropic metabolic effects.

Luminal lipid regulates CD36 levels and downstream signaling to stimulate chylomicron synthesis.

The membrane glycoprotein CD36 binds nanomolar concentrations of long chain fatty acids (LCFA) and is highly expressed on the luminal surface of enterocytes. CD36 deficiency reduces chylomicron production through unknown mechanisms. In this report, we provide novel insights into some of the underlying mechanisms. Our in vivo data demonstrate that CD36 gene deletion in mice does not affect LCFA uptake and subsequent esterification into triglycerides by the intestinal mucosa exposed to the micellar LCFA concentrations prevailing in the intestine. In rodents, the CD36 protein disappears early from the luminal side of intestinal villi during the postprandial period, but only when the diet contains lipids. This drop is significant 1 h after a lipid supply and associates with ubiquitination of CD36. Using CHO cells expressing CD36, it is shown that the digestion products LCFA and diglycerides trigger CD36 ubiquitination. In vivo treatment with the proteasome inhibitor MG132 prevents the lipid-mediated degradation of CD36. In vivo and ex vivo, CD36 is shown to be required for lipid activation of ERK1/2, which associates with an increase of the key chylomicron synthesis proteins, apolipoprotein B48 and microsomal triglyceride transfer protein. Therefore, intestinal CD36, possibly through ERK1/2-mediated signaling, is involved in the adaptation of enterocyte metabolism to the postprandial lipid challenge by promoting the production of large triglyceride-rich lipoproteins that are rapidly cleared in the blood. This suggests that CD36 may be a therapeutic target for reducing the postprandial hypertriglyceridemia and associated cardiovascular risks.

Genetic ablation of calcium-independent phospholipase A2gamma prevents obesity and insulin resistance during high fat feeding by mitochondrial uncoupling and increased adipocyte fatty acid oxidation.

Phospholipases are critical enzyme mediators participating in many aspects of cellular function through modulating the generation of lipid 2nd messengers, membrane physical properties, and cellular bioenergetics. Here, we demonstrate that mice null for calcium-independent phospholipase A(2)γ (iPLA(2)γ(-/-)) are completely resistant to high fat diet-induced weight gain, adipocyte hypertrophy, hyperinsulinemia, and insulin resistance, which occur in iPLA(2)γ(+/+) mice after high fat feeding. Notably, iPLA(2)γ(-/-) mice were lean, demonstrated abdominal lipodystrophy, and remained insulin-sensitive despite having a marked impairment in glucose-stimulated insulin secretion after high fat feeding. Respirometry of adipocyte explants from iPLA(2)γ(-/-) mice identified increased rates of oxidation of multiple different substrates in comparison with adipocyte explants from wild-type littermates. Shotgun lipidomics of adipose tissue from wild-type mice demonstrated the anticipated 2-fold increase in triglyceride content after high fat feeding. In sharp contrast, the adipocyte triglyceride content was identical in iPLA(2)γ(-/-) mice fed either a standard diet or a high fat diet. Respirometry of skeletal muscle mitochondria from iPLA(2)γ(-/-) mice demonstrated marked decreases in state 3 respiration using multiple substrates whose metabolism was uncoupled from ATP production. Shotgun lipidomics of skeletal muscle revealed a decreased content of cardiolipin with an altered molecular species composition thereby identifying the mechanism underlying mitochondrial uncoupling in the iPLA(2)γ(-/-) mouse. Collectively, these results identify iPLA(2)γ as an obligatory upstream enzyme that is necessary for efficient electron transport chain coupling and energy production through its participation in the alterations of cellular bioenergetics that promote the development of the metabolic syndrome.

Enhanced hepatic apoA-I secretion and peripheral efflux of cholesterol and phospholipid in CD36 null mice.

CD36 facilitates oxidized low density lipoprotein uptake and is implicated in development of atherosclerotic lesions. CD36 also binds unmodified high and very low density lipoproteins (HDL, VLDL) but its role in the metabolism of these particles is unclear. Several polymorphisms in the CD36 gene were recently shown to associate with serum HDL cholesterol. To gain insight into potential mechanisms for these associations we examined HDL metabolism in CD36 null (CD36(-/-)) mice. Feeding CD36(-/-) mice a high cholesterol diet significantly increased serum HDL, cholesterol and phospholipids, as compared to wild type mice. HDL apolipoproteins apoA-I and apoA-IV were increased and shifted to higher density HDL fractions suggesting altered particle maturation. Clearance of dual-labeled HDL was unchanged in CD36(-/-) mice and cholesterol uptake from HDL or LDL by isolated CD36(-/-) hepatocytes was unaltered. However, CD36(-/-) hepatocytes had higher cholesterol and phospholipid efflux rates. In addition, expression and secretion of apoA-I and apoA-IV were increased reflecting enhanced PXR. Similar to hepatocytes, cholesterol and phospholipid efflux were enhanced in CD36(-/-) macrophages without changes in protein levels of ABCA1, ABCG1 or SR-B1. However, biotinylation assays showed increased surface ABCA1 localization in CD36(-/-) cells. In conclusion, CD36 influences reverse cholesterol transport and hepatic ApoA-I production. Both pathways are enhanced in CD36 deficiency, increasing HDL concentrations, which suggests the potential benefit of CD36 inhibition.

CD36 is important for fatty acid and cholesterol uptake by the proximal but not distal intestine.

CD36, a membrane protein that facilitates fatty acid uptake, is highly expressed in the intestine on the luminal surface of enterocytes. Cd36 null (Cd36(-/-)) mice exhibit impaired chylomicron secretion but no overall lipid absorption defect. Because chylomicron production is most efficient proximally we examined whether CD36 function is important for proximal lipid absorption. CD36 levels followed a steep decreasing gradient along three equal-length, proximal to distal intestinal segments (S1-S3). Enterocytes isolated from the small intestines of Cd36(-/-) mice, when compared with wild type counterparts, exhibited reduced uptake of fatty acid (50%) and cholesterol (60%) in S1. The high affinity fatty acid uptake component was missing in Cd36(-/-) cells. Fatty acid incorporation into triglyceride and triglyceride secretion were also reduced in Cd36(-/-) S1 enterocytes. In vivo, proximal absorption was monitored using mass spectrometry from oleic acid enrichment of S1 lipids, 90 min (active absorption) and 5 h (steady state) after intragastric olive oil (70% triolein). Oleate enrichment was 50% reduced at 90 min in Cd36(-/-) tissue consistent with defective uptake whereas no differences were measured at 5 h. In Cd36(-/-) S1, mRNA for L-fabp, Dgat1, and apoA-IV was reduced. Protein levels for FATP4, SR-BI, and NPC1L1 were similar, whereas those for apoB48 and apoA-IV were significantly lower. A large increase in NPC1L1 was observed in Cd36(-/-) S2 and S3. The findings support the role of CD36 in proximal absorption of dietary fatty acid and cholesterol for optimal chylomicron formation, whereas CD36-independent mechanisms predominate in distal segments.

CD36 is important for chylomicron formation and secretion and may mediate cholesterol uptake in the proximal intestine.

BACKGROUND & AIMS: Studies are aimed to determine the role of CD36 in intestinal lipid absorption. METHODS: Knock-out (KO) and wild-type (WT) lymph fistula mice were used to study fatty acids (FA) and cholesterol uptake, and chylomicron formation and secretion. Uptake of FA and cholesterol was studied by using sucrose polybehenate and fecal dual isotope methods, respectively. RESULTS: The CD36 KO exhibited significant accumulation of dietary cholesterol in the intestinal lumen at the end of 6-hour lipid infusion and significant reduction of dietary cholesterol transport into the lymph. Fecal dual isotope studies, however, did not show any significant difference in cholesterol uptake, suggesting that given sufficient time, the KO intestine could compensate for the reduced cholesterol uptake observed in the acute lymph fistula studies. Recovery of dietary FA in the intestinal lumen was comparable between WT and KO, consistent with the sucrose polybehenate study. However, the KO mice accumulated more, albeit not significantly, dietary triacylglycerols in the intestine, followed by a significant reduction in lymphatic transport. The ratio of intestinal dietary triacylglycerols to FA was not higher in WT than KO, arguing against impaired lipid esterification. It is rather a deficiency in the formation and secretion of chylomicrons, as supported by the significantly less apolipoprotein B-48 and the smaller, albeit not significantly, lipoprotein particles secreted into the lymph of the KO. CONCLUSIONS: CD36 may play an important role in chylomicron formation and secretion and may also facilitate cholesterol uptake in the proximal intestine.

CD36 deficiency impairs intestinal lipid secretion and clearance of chylomicrons from the blood.

CD36 mediates the transfer of fatty acids (FAs) across the plasma membranes of muscle and adipose cells, thus playing an important role in regulating peripheral FA metabolism in vivo. In the proximal intestine, CD36 is localized in abundant quantities on the apical surface of epithelial cells, a pattern similar to that of other proteins implicated in the uptake of dietary FAs. To define the role of CD36 in the intestine, we examined FA utilization and lipoprotein secretion by WT and CD36-null mice in response to acute and chronic fat feeding. CD36-null mice given a fat bolus by gavage or fed a high-fat diet accumulated neutral lipid in the proximal intestine, which indicated abnormal lipid processing. Using a model in which mice were equipped with lymph fistulae, we obtained evidence of defective lipoprotein secretion by directly measuring lipid output. The secretion defect appeared to reflect an impaired ability of CD36-null enterocytes to efficiently synthesize triacylglycerols from dietary FAs in the endoplasmic reticulum. In the plasma of intact mice, the reduced intestinal lipid secretion was masked by slow clearance of intestine-derived lipoproteins. The impaired clearance occurred despite normal lipoprotein lipase activity and likely reflected feedback inhibition of the lipase by FAs due to their defective removal from the plasma. We conclude that CD36 is important for both secretion and clearance of intestinal lipoproteins. CD36 deficiency results in hypertriglyceridemia both in the postprandial and fasting states and in humans may constitute a risk factor for diet-induced type 2 diabetes and cardiovascular disease.

Hepatic secretion of small lipoprotein particles in apobec-1-/- mice is regulated by the LDL receptor.

Recent studies have examined the role of the LDL receptor (LDLR) in regulating murine hepatic lipoprotein production and apolipoprotein B (apoB) secretion, with divergent conclusions from in vivo versus in vitro approaches. We have re-examined this question, both in vivo and in vitro, using apobec-1-/- mice to model the pattern of human hepatic apoB-100 secretion. Hepatic triglyceride production in vivo (using Triton WR-1339) was unchanged in wild-type (WT) C57BL/6, apobec-1-/-, ldlr-/-, and [apobec-1-/-, ldlr-/-] mice, while apoB-100 production (using [35S]methionine incorporation) was increased > 2-fold in [apobec-1-/-, ldlr-/-] mice. Although > 90% of newly synthesized apoB floated within the d < 1.006 fraction of serum from all genotypes, fast-performance liquid chromatography separation revealed that nascent triglyceride-rich particles from [apobec-1-/-, ldlr-/-] mice, but not WT, apobec-1-/-, or ldlr-/- mice, distributed into smaller (intermediate and LDL-sized) particles. Studies in isolated hepatocytes from these different genotypes confirmed secretion of smaller particles exclusively from [apobec-1-/-, ldlr-/-] mice, and pulse-chase analysis demonstrated increased secretion of apoB-100 with virtual elimination of posttranslational degradation. These results directly support the suggestion that the LDLR regulates hepatic apoB-100 production and modulates secretion of small, triglyceride-rich particles, both in vivo and in vitro.

Intestinal lipoprotein assembly in apobec-1-/- mice reveals subtle alterations in triglyceride secretion coupled with a shift to larger lipoproteins.

Mammalian enterocytes express apolipoprotein (apo)B-48, which is produced after posttranscriptional RNA editing of the nuclear apoB-100 transcript by the catalytic deaminase apobec-1. Earlier studies in apobec-1-/- mice revealed an apoB-100-only lipoprotein profile but no gross defects in triglyceride absorption. However, subtle defects may have been obscured by the mixed genetic background. In addition, the intrinsic susceptibility to proteolytic degradation of intestinal apoB-100 and apoB-48 has been questioned. Accordingly, we examined triglyceride absorption, intestinal apoB expression, and lipoprotein secretion in apobec-1-/- mice backcrossed into a C57BL/6 background. Inbred apobec-1-/- mice absorb triglyceride normally, yet secrete triglyceride-rich lipoproteins more slowly than wild-type congenic controls. There was comparable induction of apoB synthesis in response to fat feeding in both genotypes, but apoB-100 was preferentially retained and more extensively degraded than apoB-48. By contrast, synthesis, secretion, and content of apo A-IV were indistinguishable in apobec-1-/- and wild-type mice with 100% recovery, suggesting no degradation of this apoprotein in either genotype. Newly secreted lipoproteins from isolated enterocytes of wild-type mice revealed apoB-48 in both high-density lipoproteins and very low-density lipoproteins. By contrast, apobec-1-/- mice secreted apoB-100-containing particles that were almost exclusively in the low and very low-density lipoproteins range with no apoB-100-containing high-density lipoproteins. These studies establish the existence of preferential degradation of intestinal apoB-100 and subtle defects in triglyceride secretion in apobec-1-/- mice, coupled with a shift to the production of larger particles, findings that suggest an important divergence in intestinal lipoprotein assembly pathways with the different isoforms of apoB.

Apolipoprotein[a] secretion from hepatoma cells is regulated in a size-dependent manner by alterations in disulfide bond formation.

Apolipoprotein[a] (apo[a]) is a large disulfide linked glycoprotein synthesized by hepatocytes. We have examined the role of disulfide bond formation in the processing of apo[a] using human and rat hepatoma cells expressing apo[a] isoforms containing varying numbers of kringle 4 (K4) domains, following treatment with DTT. Hepatoma cells expressing 6- or 9-K4 isoforms revealed approximately 90% inhibition of apo[a] secretion following DTT treatment, although larger isoforms containing 13- or 17-K4 domains demonstrated continued secretion (up to 30% of control values), suggesting that a fraction of the larger isoforms is at least partially DTT resistant. Wash-out experiments demonstrated that these effects were completely reversible for all isoforms studied, with no enhanced degradation associated with prolonged intracellular retention. DTT treatment was associated with enhanced binding of apo[a] with the endoplasmic reticulum-associated chaperone proteins calnexin, calreticulin, and BiP, which was reversible upon DTT removal. The chemical chaperone 6-aminohexanoic acid, previously demonstrated by others to rescue defective apo[a] secretion associated with alterations in glycosylation, failed to alter the secretion of apo[a] following DTT treatment. The demonstration that DTT modulates apo[a] secretion in a manner influenced by both the type and number of K4 repeats extends understanding of the mechanisms that regulate its exit from the endoplasmic reticulum.

Thyroid hormone regulates hepatic triglyceride mobilization and apolipoprotein B messenger ribonucleic Acid editing in a murine model of congenital hypothyroidism.

Thyroid hormone modulates the expression of numerous genes that in turn regulate lipoprotein metabolism in vivo. We have examined the thyroid hormone-dependent regulation of apolipoprotein B (apoB) RNA editing in a strain of congenitally hypothyroid mice (Pax8(-/-)) that lacks thyroid follicular cells. Neonatal Pax8(-/-) mice demonstrate an approximately 10-fold increase in hepatic triglyceride content associated with a decrease in hepatic apoB RNA editing. Thyroid hormone administration resulted in hepatic triglyceride mobilization in conjunction with an increase in hepatic, but not intestinal, apoB RNA editing and without changing total apoB RNA abundance. ApoB RNA editing is mediated by a multicomponent enzyme complex whose catalytic core contains two proteins, apobec-1 and apobec-1 complementation factor (ACF). Hepatic ACF mRNA and protein abundance decreased in Pax8(-/-) mice, with restoration after thyroid hormone administration, whereas apobec-1 mRNA and protein abundance were unchanged. Immunohistochemical analysis revealed increased staining intensity of ACF within hepatocyte nuclei of treated mice, findings confirmed by Western analysis of isolated nuclei. In vitro RNA editing assays demonstrated that supplementation with recombinant ACF alone restored enzymatic activity of S100 extracts from hypothyroid, Pax8(-/-) mice. These data demonstrate that thyroid hormone modulates murine hepatic lipoprotein metabolism in association with tissue-specific effects on apoB RNA editing mediated through alterations in ACF gene expression.

Disruption of hedgehog signaling reveals a novel role in intestinal morphogenesis and intestinal-specific lipid metabolism in mice.

BACKGROUND & AIMS: The hedgehog (hh) signaling pathway has been shown to play crucial roles in the development of embryonic gut. However, its role in intestinal development and function beyond the embryonic stage is still undefined. METHODS: Expression of hh and its receptor, Patched, were examined by Western blot and X-gal staining. An anti-hh monoclonal antibody was administered into developing embryos or postnatal mice and histologic analyses were performed. Effects on lipid metabolism were examined by Oil Red O and Sudan III stainings, messenger RNA (mRNA) analysis, and electron microscopy. Serum apolipoprotein IV level, a marker for lipid absorption, was quantified by Western blot. RESULTS: Mice receiving anti-hh monoclonal antibody in utero or after birth exhibited progressive runting and died before weaning. Histology revealed hyperproliferation of intestinal crypt epithelial cells and disorganization of the villi with prominent vacuolation and accumulation of neutral lipid. Fecal fat microscopy revealed numerous large fat droplets. Intestinal mRNA abundance of 2 candidate genes involved in lipid transport, mtp and apob, was unchanged, although serum levels of apolipoprotein A-IV were reduced. CONCLUSIONS: Abnormal villus structure, lipid-filled enterocytes, and fatty stools in anti-hh monoclonal antibody-treated mice indicate a novel role for hh signaling in intestinal morphogenesis and lipid transport in postnatal mice.