Faculty Bibliography

ApoB-crescent, an endoplasmic reticulum (ER)-lipid droplet amalgamation structure, is a useful marker to indicate aberrant lipidated apolipoprotein B accumulation in the hepatocyte ER. Blockade of the ER-to-Golgi transport by either vesicle transport inhibitors or dominant-negative Arf1 caused a significant increase in ApoB-crescents. However, a low concentration of Brefeldin A induced the same result without affecting protein secretion, suggesting ADP-ribosylation as an additional mechanism. ADP-ribosylation inhibitors not only suppressed the increase of ApoB-crescents, but also rapidly dissolved existing ApoB-crescents. These results implicate the involvement of ADP-ribosylation in the ApoB-crescent formation and maintenance process at the ER.

BACKGROUND:Clock is a key transcription factor that positively controls circadian regulation. However, its role in plasma cholesterol homeostasis and atherosclerosis has not been studied. METHODS AND RESULTS/RESULTS:We show for the first time that dominant-negative Clock mutant protein (Clock(Δ19/Δ19)) enhances plasma cholesterol and atherosclerosis in 3 different mouse models. Detailed analyses revealed that Clk(Δ19/Δ19)Apoe(-/-) mice display hypercholesterolemia resulting from the accumulation of apolipoprotein B48-containing cholesteryl ester-rich lipoproteins. Physiological studies showed that enhanced cholesterol absorption by the intestine contributes to hypercholesterolemia. Molecular studies indicated that the expression of Niemann Pick C1 Like 1, Acyl-CoA:Cholesterol acyltransferase 1, and microsomal triglyceride transfer protein in the intestines of Clk(Δ19/Δ19)Apoe(-/-) mice was high and that enterocytes assembled and secreted more chylomicrons. Furthermore, we identified macrophage dysfunction as another potential cause of increased atherosclerosis in Clk(Δ19/Δ19)Apoe(-/-) mice. Macrophages from Clk(Δ19/Δ19)Apoe(-/-) mice expressed higher levels of scavenger receptors and took up more modified lipoproteins compared with Apoe(-/-) mice, but they expressed low levels of ATP binding casette protein family A member 1 and were defective in cholesterol efflux. Molecular studies revealed that Clock regulates ATP binding casette protein family A member 1 expression in macrophages by modulating upstream transcription factor 2 expression. CONCLUSIONS:Clock(Δ19/Δ19) protein enhances atherosclerosis by increasing intestinal cholesterol absorption, augmenting uptake of modified lipoproteins by macrophages, and reducing cholesterol efflux from macrophages. These studies establish that circadian Clock activity is crucial in maintaining low plasma cholesterol levels and in reducing atherogenesis in mice.

We have shown previously that Clock, microsomal triglyceride transfer protein (MTP), and nocturnin are involved in the circadian regulation of intestinal lipid absorption. Here, we clarified the role of apolipoprotein AIV (apoAIV) in the diurnal regulation of plasma lipids and intestinal lipid absorption in mice. Plasma triglyceride in apoAIV(-/-) mice showed diurnal variations similar to apoAIV(+/+) mice; however, the increases in plasma triglyceride at night were significantly lower in these mice. ApoAIV(-/-) mice absorbed fewer lipids at night and showed blunted response to daytime feeding. To explain reasons for these lower responses, we measured MTP expression; intestinal MTP was low at night, and its induction after food entrainment was less in apoAIV(-/-) mice. Conversely, apoAIV overexpression increased MTP mRNA in hepatoma cells, indicating transcriptional regulation. Mechanistic studies revealed that sequences between -204/-775 bp in the MTP promoter respond to apoAIV and that apoAIV enhances expression of FoxA2 and FoxO1 transcription factors and their binding to the identified cis elements in the MTP promoter at night. Knockdown of FoxA2 and FoxO1 abolished apoAIV-mediated MTP induction. Similarly, knockdown of apoAIV in differentiated Caco-2 cells reduced MTP, FoxA2, and FoxO1 mRNA levels, cellular MTP activity, and media apoB. Moreover, FoxA2 and FoxO1 expression showed diurnal variations, and their expression was significantly lower in apoAIV(-/-) mice. These data indicate that apoAIV modulates diurnal changes in lipid absorption by regulating forkhead transcription factors and MTP and that inhibition of apoAIV expression might reduce plasma lipids.

The native capacity of adult skeletal muscles to regenerate is vital to the recovery from physical injuries and dystrophic diseases. Currently, the development of therapeutic interventions has been hindered by the complex regulatory network underlying the process of muscle regeneration. Using a mouse model of skeletal muscle regeneration after injury, we identified hexamethylene bisacetamide inducible 1 (HEXIM1, also referred to as CLP-1), the inhibitory component of the positive transcription elongation factor b (P-TEFb) complex, as a pivotal regulator of skeletal muscle regeneration. Hexim1-haplodeficient muscles exhibited greater mass and preserved function compared with those of WT muscles after injury, as a result of enhanced expansion of satellite cells. Transplanted Hexim1-haplodeficient satellite cells expanded and improved muscle regeneration more effectively than WT satellite cells. Conversely, HEXIM1 overexpression restrained satellite cell proliferation and impeded muscle regeneration. Mechanistically, dissociation of HEXIM1 from P-TEFb and subsequent activation of P-TEFb are required for satellite cell proliferation and the prevention of early myogenic differentiation. These findings suggest a crucial role for the HEXIM1/P-TEFb pathway in the regulation of satellite cell–mediated muscle regeneration and identify HEXIM1 as a potential therapeutic target for degenerative muscular diseases.

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.

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.