Faculty Bibliography

Hyperlipidemia is a risk factor for various cardiovascular and metabolic disorders. Overproduction of lipoproteins, a process that is dependent on microsomal triglyceride transfer protein (MTP), can contribute to hyperlipidemia. We show that microRNA-30c (miR-30c) interacts with the 3' untranslated region of MTP mRNA and induces its degradation, leading to reductions in MTP activity and in apolipoprotein B (APOB) secretion. miR-30c also reduces lipid synthesis independently of MTP. Hepatic overexpression of miR-30c reduced hyperlipidemia in Western diet-fed mice by decreasing lipid synthesis and the secretion of triglyceride-rich ApoB-containing lipoproteins and decreased atherosclerosis in Apoe(-/-) mice. Furthermore, inhibition of hepatic miR-30c by anti-miR-30c increased hyperlipidemia and atherosclerosis. Therefore, miR-30c coordinately reduces lipid biosynthesis and lipoprotein secretion, thereby regulating hepatic and plasma lipid concentrations. Raising miR-30c levels might be useful in treating hyperlipidemias and associated disorders.

Endothelial lipase (EL) is a major negative regulator of plasma HDL levels in mice, rabbits, and most probably, humans. Although this regulatory function is critically dependent on EL's hydrolysis of HDL phospholipids, as yet there is no phospholipase assay specific for EL in plasma. We developed such an assay for the mouse enzyme using a commercially available phospholipid-like fluorescent substrate in combination with an EL neutralizing antibody. The specificity of the assay was established using EL knockout mice and its utility demonstrated by detection of an increase in plasma EL phospholipase activity following exposure of wild-type mice to lipopolysaccharide. The assay revealed that murine pre-heparin plasma does not contain measurable EL activity, indicating that the hydrolysis of HDL phospholipids by EL in vivo likely occurs on the cell surface.

PURPOSE OF REVIEW/OBJECTIVE:To summarize the new knowledge about the regulation of dietary lipid absorption by circadian locomotor output cycles kaput (Clock) and Nocturnin. RECENT FINDINGS/RESULTS:Recent findings have shown that Clock and Nocturnin, proteins involved in circadian regulation, play an important role in the regulation of dietary lipid absorption. Clock deficiency increases, whereas Nocturnin deficiency decreases lipid absorption. Clock plays a role in turning off the genes involved in lipid absorption at the onset of the day. Molecular studies revealed that Clock binds to the promoter of small heterodimer partner to enhance its transcription. When levels are high, small heterodimer partner interacts with the transcription factors associated with the promoter of microsomal triglyceride transfer protein to repress transcription. Reduced microsomal triglyceride transfer protein levels are correlated with low intestinal lipid absorption and plasma lipid levels. In contrast, Nocturnin assists in lipid absorption by regulating their partitioning in different intracellular compartments. SUMMARY/CONCLUSIONS:Clock and Nocturnin regulate lipid absorption involving different mechanisms. It is likely that other clock genes also modulate lipid absorption and plasma lipid levels.

RATIONALE: 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. OBJECTIVE: This study aims to delineate whether changes in intestinal lipid absorption associated with loss of inositol-requiring enzyme 1beta (Ire1beta) would affect the development of hyperlipidemia and atherosclerosis in Apoe(-/-) mice. METHODS AND RESULTS: We used Ire1beta-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. CONCLUSIONS: These studies indicate that Ire1beta regulates intestinal lipid absorption and that increased intestinal lipoprotein production contributes to atherosclerosis.

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.

Our knowledge of how the body absorbs triacylglycerols (TAG) from the diet and how this process is regulated has increased at a rapid rate in recent years. Dietary TAG are hydrolyzed in the intestinal lumen to free fatty acids (FFA) and monoacylglycerols (MAG), which are taken up by enterocytes from their apical side, transported to the endoplasmic reticulum (ER) and resynthesized into TAG. TAG are assembled into chylomicrons (CM) in the ER, transported to the Golgi via pre-chylomicron transport vesicles and secreted towards the basolateral side. In this review, we mainly focus on the roles of key proteins involved in uptake and intracellular transport of fatty acids, their conversion to TAG and packaging into CM. We will also discuss intracellular transport and secretion of CM. Moreover, we will bring to light few factors that regulate gut triglyceride production. Furthermore, we briefly summarize pathways involved in cholesterol absorption. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.

BACKGROUND:Most physiologic processes exhibit diurnal fluctuations controlled by the circadian regulation of sleep-wake behavior and feeding cycles. In addition, many cell types express endogenous circadian rhythms that affect cell-specific processes. Independent reports support the hypothesis that thrombopoietin (TPO) is under circadian control. OBJECTIVES/OBJECTIVE:The current study tested the hypothesis that CLOCK, a circadian transcription factor, may regulate Thpo, the gene encoding TPO. METHODS:Circadian gene expression patterns were analyzed in mice and in human cell lines, Small interfering RNA was used to knock down CLOCK expression in cell lines, and gene expression was also examined in Clock(Δ19/Δ19) mutant mice. RESULTS:It was found that there was a diurnal rhythm in the expression of Thpoin vivo in mice, and that this was associated with concomitant rhythms of protein abundance. Thpo was rhythmically expressed in human cell lines, consistent with the gene being directly or indirectly regulated by the circadian clock. Silencing of CLOCK in the Huh7 human hepatoma cell line led to a significant reduction in the rhythmicity of Thpo expression. The expression of Mpl in murine marrow also displayed diurnal rhythmicity in vivo. In Clock(Δ19/Δ19) mutant mice, Thpo and Mpl expression was disrupted and there was an increase in the number of mature megakaryocytes, but no change in the ploidy distribution within the megakaryocyte population. CONCLUSIONS:These findings establish that Clock regulates Thpo and Mpl expression in vivo, and demonstrate an important link between the body's circadian timing mechanisms and megakaryopoiesis.

BACKGROUND:Efficient metabolic function in mammals depends on the circadian clock, which drives temporal regulation of metabolic processes. Nocturnin is a clock-regulated deadenylase that controls its target mRNA expression posttranscriptionally through poly(A) tail removal. Mice lacking nocturnin (Noc(-/-) mice) are resistant to diet-induced obesity and hepatic steatosis yet are not hyperactive or hypophagic. RESULTS:Here we show that nocturnin is expressed rhythmically in the small intestine and is induced by olive oil gavage and that the Noc(-/-) mice have reduced chylomicron transit into the plasma following the ingestion of dietary lipids. Genes involved in triglyceride synthesis and storage and chylomicron formation have altered expression, and large cytoplasmic lipid droplets accumulate in the apical domains of the Noc(-/-) enterocytes. The physiological significance of this deficit in absorption is clear because maintenance of Noc(-/-) mice on diets that challenge the chylomicron synthesis pathway result in significant reductions in body weight, whereas diets that bypass this pathway do not. CONCLUSIONS:Therefore, we propose that nocturnin plays an important role in the trafficking of dietary lipid in the intestinal enterocytes by optimizing efficient absorption of lipids.

Coordinated regulation of fat storage and utilization is essential for energy homeostasis, and its disruption is associated with metabolic syndrome and atherosclerosis in humans. Across species, Krüppel-like transcription factors (KLFs) have been identified as key components of adipogenesis. In humans, KLF14 acts as a master transregulator of adipose gene expression in type 2 diabetes and cis-acting expression quantitative trait locus associated with high-density lipoprotein cholesterol. Herein we report that, in Caenorhabditis elegans, mutants in klf-3 accumulate large fat droplets rich in neutral lipids in the intestine; this lipid accumulation is associated with an increase in triglyceride levels. The klf-3 mutants show normal pharyngeal pumping; however, they are sterile or semisterile. We explored important genetic interactions of klf-3 with the genes encoding enzymes involved in fatty acid (FA) β-oxidation in mitochondria or peroxisomes and FA synthesis in the cytosol, namely acyl-CoA synthetase (acs-1 and acs-2), acyl-CoA oxidase (F08A8.1 and F08A8.2), and stearoyl-CoA desaturase (fat-7). We show that mutations or RNA interference in these genes increases fat deposits in the intestine of acs-1, acs-2, F08A8.1, and F08A8 animals. We further show that acs-1 and F08A8.1 influence larval development and fertility, respectively. Thus, KLF3 may regulate FA utilization in the intestine and reproductive tissue. We demonstrate that depletion of F08A8.1 activity, but not of acs-1, acs-2, F08A8.2, or fat-7 activity, enhances the fat phenotype of the klf-3 mutant. Taken together, these results suggest that klf-3 regulates lipid metabolism, along with acs-1, acs-2, F08A8.1, and F08A8.2, by promoting FA β-oxidation and, in parallel, may contribute to normal reproductive behavior and fecundity in C. elegans.

Apolipoprotein (apo) A-IV overexpression enhances chylomicron (CM) assembly and secretion in newborn swine intestinal epithelial cells by producing larger particles (Lu S, Yao Y, Cheng X, Mitchell S, Leng S, Meng S, Gallagher JW, Shelness GS, Morris GS, Mahan J, Frase S, Mansbach CM, Weinberg RB, Black DD. J Biol Chem 281: 3473-3483, 2006). To determine the impact of apo A-IV on microsomal triglyceride transfer protein (MTTP), IPEC-1 cell lines containing a tetracycline-regulatable expression system were used to overexpress native swine apo A-IV and "piglike" human apo A-IV, a mutant human apo A-IV with deletion of the EQQQ-rich COOH-terminus, previously shown to upregulate basolateral triglyceride (TG) secretion 5-fold and 25-fold, respectively. Cells were incubated 24 h with and without doxycycline and oleic acid (OA, 0.8 mM). Overexpression of the native swine apo A-IV and piglike human apo A-IV increased MTTP lipid transfer activity by 39.7% (P = 0.006) and 53.6% (P = 0.0001), respectively, compared with controls. Changes in mRNA and protein levels generally paralleled changes in activity. Interestingly, native swine apo A-IV overexpression also increased MTTP large subunit mRNA, protein levels, and lipid transfer activity in the absence of OA, suggesting a mechanism not mediated by lipid absorption. Overexpression of piglike human apo A-IV significantly increased partitioning of radiolabeled OA from endoplasmic reticulum (ER) membrane to lumen, suggesting increased net transfer of membrane TG to luminal particles. These results suggest that the increased packaging of TG into nascent CMs in the ER lumen, induced by apo A-IV, is associated with upregulation of MTTP activity at the pretranslational level. Thus MTTP is regulated by apo A-IV in a manner to promote increased packaging of TG into the CM core, which may be important in neonatal fat absorption.