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Background & Aims: The nuclear receptor subfamily 1, group D, member 1 (Nr1d1, also called Rev-erbα) is highly expressed in the kidney, and its transcriptional activity is increased by the clock gene Bmal1 (ARNTL or MOP3). Here we studied the roles and functional interactions of Bmal1 and Nr1d1 in mouse kidney, particularly in regulating the expression of the glucose transporter SGLT2 (SLC5A2) and glucose reabsorption. Methods: We performed histologic and biochemical analyses of kidney tissues from male C57BL/6 and Bmal1-knockout mice and performed gene expression analyses to identify genes regulated by Bmal1. The effects of fasting and refeeding on renal expression of Sglt2 was examined in male C57BL/6, Bmal1-knockout, and Nr1d1-knockout mice. Some mice were given hemin to stimulate Nr1d1 gene expression (daily for 1 week) at 12:00 with 100 μg/kg/day; renal glucose reabsorption, and glucose and fatty acid levels were measured. Renal proximal tubule epithelial cells (RPTECs) were generated from C57BL/6 and Bmal1-knockout mice and glucose uptake determined. Luciferase reporter assays were performed using HK2 cell. Results: We found that kidney SGLT2 mRNA and protein expression and glucose absorption were increased in Bmal1-knockout mice compared with C57BL/6 mice. Expression of SGLT2 was affected by fasting and refeeding in C57BL/6 mice, but not in Bmal1-knockout or Nr1d1-knockout mice. Bmal1-knockout mice also presented with higher levels of renal fatty acids compared with C57BL/6 mice. Hemin injection reduced expression of SGLT2, decreased glucose re-absorption in RPTECs, and increased levels of fatty acid in kidney; these changes were not observed in Bmal1-knockout mice. Nuclear respiratory factor 1 (NRF1), which regulates glucose, activated transcription of Sglt2. Fasting caused MAPK signing pathway to phosphorylate Bmal1, which activated NR1d1 and inhibited NRF1 activity and thereby reduced Sglt2 expression. Conclusions: The data indicate that fasting activates Bmal1 and Nr1d1, which inhibits NRF1 and leads to repression of renal SGLT2 expression and glucose reabsorption. This is the first study that mechanistically links food intake to the regulation of kidney glucose transport through a circadian clock gene. NIH National Heart, Lung, and Blood Institute grant R56 HL137912-01, and American Heart Association grant-in-aid 16GRNT30960027 to PX; NIH R01DK112042 to VV This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The prevalence of non-alcoholic fatty liver disease (NAFLD) increases year by year, and as a consequence, NAFLD has become one of the most prevalent liver diseases worldwide. Unfortunately, no pharmacotherapies for NAFLD have been approved by the United States Food and Drug Administration despite promising pre-clinical benefits; this situation highlights the urgent need to explore new therapeutic targets for NAFLD and for the discovery of effective therapeutic drugs. The mouse is one of the most commonly used models to study human disease and develop novel pharmacotherapies due to its small size, low-cost and ease in genetic engineering. Different mouse models are used to simulate various stages of NAFLD induced by dietary and/or genetic intervention. In this review, we summarize the newly described patho-mechanisms of NAFLD and review the preclinical mouse models of NAFLD (based on the method of induction) and appraises the use of these models in anti-NAFLD drug discovery. This article will provide a useful resource for researchers to select the appropriate model for research based on the research question being addressed.

High plasma lipid/lipoprotein levels are risk factors for various metabolic diseases. We previously showed that circadian rhythms regulate plasma lipids and deregulation of these rhythms causes hyperlipidemia and atherosclerosis in mice. Here, we show that global and liver-specific brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal1)-deficient mice maintained on a chow or Western diet developed hyperlipidemia, denoted by the presence of higher amounts of triglyceride-rich and apolipoprotein AIV (ApoAIV)-rich larger chylomicron and VLDL due to overproduction.Bmal1 deficiency decreased small heterodimer partner (Shp) and increased microsomal triglyceride transfer protein (MTP), a key protein that facilitates primordial lipoprotein assembly and secretion. Moreover, we show that Bmal1 regulates cAMP-responsive element-binding protein H (Crebh) to modulate ApoAIV expression and the assembly of larger lipoproteins. This is supported by the observation that Crebh-deficient and ApoAIV-deficient mice, along with Bmal1-deficient mice with knockdown of Crebh, had smaller lipoproteins. Further, overexpression of Bmal1 in Crebh-deficient mice had no effect on ApoAIV expression and lipoprotein size.These studies indicate that regulation of ApoAIV and assembly of larger lipoproteins by Bmal1 requires Crebh. Mechanistic studies showed that Bmal1 regulates Crebh expression by two mechanisms. First, Bmal1 interacts with the Crebh promoter to control circadian regulation. Second, Bmal1 increases Rev-erbα expression, and nuclear receptor subfamily 1 group D member 1 (Nr1D1, Rev-erbα) interacts with the Crebh promoter to repress expression. In short, Bmal1 modulates both the synthesis of primordial lipoproteins and their subsequent expansion into larger lipoproteins by regulating two different proteins, MTP and ApoAIV, through two different transcription factors, Shp and Crebh. It is likely that disruptions in circadian mechanisms contribute to hyperlipidemia and that avoiding disruptions in circadian rhythms may limit/prevent hyperlipidemia and atherosclerosis.

High plasma levels of lipids and/or lipoproteins are risk factors for atherosclerosis, nonalcoholic fatty liver disease (NAFLD), obesity, and diabetes. These four conditions have also been identified as risk factors leading to the development of chronic kidney disease (CKD). Although many pathways that generate high plasma levels of these factors have been identified, most clinical and physiologic dysfunction results from aberrant assembly and secretion of lipoproteins. The results of several published studies suggest that elevated levels of low-density lipoprotein (LDL)-cholesterol are a risk factor for atherosclerosis, myocardial infarction, coronary artery calcification associated with type 2 diabetes, and NAFLD. Cholesterol metabolism has also been identified as an important pathway contributing to the development of CKD; clinical treatments designed to alter various steps of the cholesterol synthesis and metabolism pathway are currently under study. Cholesterol synthesis and catabolism contribute to a multistep process with pathways that are regulated at the cellular level in renal tissue. Cholesterol metabolism may also be regulated by the balance between the influx and efflux of cholesterol molecules that are capable of crossing the membrane of renal proximal tubular epithelial cells and podocytes. Cellular accumulation of cholesterol can result in lipotoxicity and ultimately kidney dysfunction and failure. Thus, further research focused on cholesterol metabolism pathways will be necessary to improve our understanding of the impact of cholesterol restriction, which is currently a primary intervention recommended for patients with dyslipidemia.

Macrophages play a critical role in atherosclerosis. We have previously shown that Clock mutant and global or liver-specific Bmal1 deficient mice show enhanced atherosclerosis. Yet, the role of macrophage Bmal1 in cholesterol transport and atherosclerosis remains unknown. Here, we used global and macrophage-specific ablation of Bmal1 to clarify the role of Bmal1 in macrophage function in atherosclerosis. We found that macrophage-specific Bmal1 deficient mice show increased atherosclerosis. Biochemical studies showed that Bmal1 deficient macrophages exhibit increased expression of Lox-1 and Cd36 and take up more oxLDL. Furthermore, they show decreased expression of Abca1 and Abcg1 and reduced cholesterol efflux as well as in vivo reverse cholesterol transport. Thus, Bmal1 regulates macrophage cholesterol metabolism by regulating uptake of oxidized lipoproteins and cholesterol efflux pathways. Molecular studies showed that Bmal1 indirectly regulates Cd36 and Abca1/Abcg1 by modulating Nr1d1 and Znf202, respectively. We provide evidence that Bmal1 collaborates with Clock and interacts with Nr1d1 promoter. Furthermore, our studies show that Bmal1 interacts with both Clock and Npas2 to regulate Znf202. Thus, Bmal1 regulates two different transcription factors to regulate uptake of oxidized lipoproteins and cholesterol efflux. In summary, our data show that Bmal1 is a key regulator of cholesterol metabolism in macrophages and atherosclerosis. It regulates macrophage function by modulating the expression of different transcription factors. It is likely that augmentation of Bmal1 may prevent atherosclerosis.

Ferritins are a large family of iron storage proteins, which are used by bacteria and other organisms to avoid iron toxicity and as a safe iron source in the cytosol. Agrobacterium tumefaciens, a phytopathogen, has two ferritin‐encoding genes: atu2771 and atu2477. Atu2771 is annotated as a Bfr‐encoding gene (Bacterioferritin, Bfr) and atu2477 as a Dps‐encoding gene (DNA binding protein from starved cells, Dps). Three deletion mutants (Δbfr, Δdps, and bfr‐dps double‐deletion mutant ΔbdF) of these two ferritin‐encoding genes were constructed to investigate the effects of ferritin deficiency on the iron homeostasis, oxidative stress resistance, and pathogenicity of A. tumefaciens. Deficiency of two ferritins affects the growth of A. tumefaciens under iron starvation and excess. When supplied with moderate iron, the growth of A. tumefaciens is not affected by the deficiency of ferritin. Deficiency of ferritin significantly reduces iron accumulation in the cells of A. tumefaciens, but the effect of Bfr deficiency on iron accumulation is severer than Dps deficiency and the double mutant ΔbdF has the least intracellular iron content. All three ferritin‐deficient mutants showed a decreased tolerance to 3 mM H2O2 in comparison with the wild type. The tumour induced by each of three ferritin‐deficient mutants is less than that of the wild type. Complementation reversed the effects of ferritin deficiency on the growth, iron homeostasis, oxidative stress resistance, and tumorigenicity of A. tumefaciens. Therefore, ferritin plays an important role in the pathogenesis of A. tumefaciens through regulating iron homeostasis and oxidative stress survival., Deficiency of two Agrobacterium tumefaciens ferritins, Bfr and Dps, not only affects the growth under iron stress, reduces the iron accumulation, and impairs the oxidative resistance, but also attenuates the virulence.

BiFeO3(BFO)/Sr2Bi4Ti5O18(SBT) bilayer composite thin films were prepared using the sol-gel method. The influence of the SBT transition layer thickness on the properties of BFO/SBT bilayer composite thin films was studied. The results showed that an SBT transition layer with the proper thickness can improve the BFO/SBT bilayer composite film performance. X-ray diffraction (XRD) analysis revealed that a bismuth layered perovskite structure is formed in all of the thin films. The scanning electron microscopy (SEM) result showed that the BFO/80 nmSBT sample exhibits good crystallization, an improved density and a uniform minimum grain size compared with the other samples. The X-ray photoelectron spectroscopy (XPS) result showed that the Fe2+ content and oxygen vacancy concentration are lowest in the BFO/80 nm SBT sample. This sample has good ferroelectric properties: the leakage current density has the lowest value of J = 5.21 × 10-5 A/cm2 among the samples at 350 kV/cm, 2Ec = 900 kV/cm, and 2Pr = 151 μC/cm2. At a low electric field (E 100 kV/cm). The relative dielectric constant (er) is 121, the dielectric loss (tanδ) is 0.024 at 105 Hz.

High plasma lipid and lipoprotein levels are risk factors for various metabolic diseases, such as diabetes, obesity, metabolic syndrome, and atherosclerosis. We previously showed that circadian rhythms regulate plasma lipids, and deregulation of these rhythms cause hyperlipidemia and atherosclerosis in mice. Here, we show that global and liver-specific Bmal1-deficient mice maintained on a chow or a Western diet developed hyperlipidemia, which was denoted by the presence of higher amounts of triglyceride- and apoAIV-rich larger chylomicron and very-low-density lipoprotein, due to overproduction. Bmal1 deficiency decreased Shp and increased MTP, a key protein that facilitates primordial lipoprotein assembly and secretion. Moreover, we show that Bmal1 regulates Crebh to modulate apoAIV expression and the assembly of larger lipoproteins. This is supported by the observation that Crebh- and apoAIV-deficient mice, along with Bmal1-deficient mice with knockdown of Crebh, had smaller lipoproteins. Further, overexpression of Bmal1 in Crebh-deficient mice had no effect on apoAIV expression and lipoprotein size. These studies indicate that regulation of apoAIV and assembly of larger lipoproteins by Bmal1 requires Crebh. Mechanistic studies showed that Bmal1 regulates Crebh expression by two mechanisms. First, Bmal1 interacts with the Crebh promoter to control circadian regulation. Second, Bmal1 increases Rev-erbα expression, and Rev-erbα interacts with the Crebh promoter to repress expression. In short, Bmal1 modulates both the synthesis of primordial lipoproteins and their subsequent expansion into larger lipoproteins by regulating two different proteins, MTP and apoAIV, via two different transcription factors, Shp and Crebh. It is likely that disruptions in circadian mechanisms contribute to hyperlipidemia, and synchronization of circadian rhythms may limit/prevent hyperlipidemia and atherosclerosis.

The basic helix-loop-helix-PAS transcription factor (CLOCK, Circadian locomotor output cycles protein kaput) was discovered in 1994 as a circadian clock. Soon after its discovery, the circadian clock, Aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL, also call BMAL1), was shown to regulate adiposity and body weight by controlling on the brain hypothalamic suprachiasmatic nucleus (SCN). Farther, circadian clock genes were determined to exert several of lipid metabolic and diabetes effects, overall indicating that CLOCK and BMAL1 act as a central master circadian clock. A master circadian clock acts through the neurons and hormones, with expression in the intestine, liver, kidney, lung, heart, SCN of brain, and other various cell types of the organization. Among circadian clock genes, numerous metabolic syndromes are the most important in the regulation of food intake (via regulation of circadian clock genes or clock-controlled genes in peripheral tissue), which lead to a variation in plasma phospholipids and tissue phospholipids. Circadian clock genes affect the regulation of transporters and proteins included in the regulation of phospholipid metabolism. These genes have recently received increasing recognition because a pharmacological target of circadian clock genes may be of therapeutic worth to make better resistance against insulin, diabetes, obesity, metabolism syndrome, atherosclerosis, and brain diseases. In this book chapter, we focus on the regulation of circadian clock and summarize its phospholipid effect as well as discuss the chemical, physiology, and molecular value of circadian clock pathway regulation for the treatment of plasma lipids and atherosclerosis.

The goal of this review was to seek a better understanding of the function and differential expression of circadian clock genes during the reproductive process. Through a discussion of how the circadian clock is involved in these steps, the identification of new clinical targets for sleep disorder-related diseases, such as reproductive failure, will be elucidated. Here, we focus on recent research findings regarding circadian clock regulation within the reproductive system, shedding new light on circadian rhythm-related problems in women. Discussions on the roles that circadian clock plays in these reproductive processes will help identify new clinical targets for such sleep disorder-related diseases.

Objectives Recently, long noncoding RNAs (lncRNAs) have captured much attention for their important roles in human diseases. Deregulation of lncRNA taurine-upregulated gene 1 (TUG1) has been reported to regulate cancer progression in many cancer types. However, how TUG1 contributes to renal cell carcinoma (RCC) remains elusive; we were eager to resolve the questions. Methods Tumor tissues and the matched adjacent normal tissues were collected from patients with RCC. Messenger RNA (mRNA) levels of TUG1, yes-associated protein (YAP), and microRNA (miR)-9 levels were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The regulation of YAP by TUG1 was investigated using Western blot analysis, RT-qPCR, and immunofluorescence. The oncogenic roles of TUG1 and YAP were studied using a cell proliferation assay and a wound healing assay. The interaction of TUG1-miR-9-YAP was analyzed in RCC cell lines. Results In the current study, we observed a positive correlation between TUG1 expression and YAP expression in RCC using the Gene Expression Omnibus database and tumor tissues collected from 58 patients with RCC. The TUG1 elevation enhanced YAP expression but did not alter the Hippo-signaling pathway activity or YAP protein distribution in cells. In addition, we found that TUG1 could bind to miR-9; therefore, TUG1 could positively control YAP expression via downregulation of miR-9 level. Furthermore, we observed that inhibition of cell proliferation and cell migration induced by TUG1 silencing could be reversed by overexpression of YAP in RCC cell lines. Conclusions Our findings indicated a pivotal role of TUG1 in driving RCC progression via regulation of miR-9/YAP, suggesting a potential therapeutic targeting role of TUG1 in RCC.

Nonalcoholic fatty liver disease (NAFLD) is becoming a major health issue as obesity increases around the world. We studied the effect of a circadian locomotor output cycles kaput (CLOCK) mutant (Clk(Δ19/Δ19)) protein on hepatic lipid metabolism in C57BL/6 Clk(wt/wt) and apolipoprotein E–deficient (Apoe(−/−)) mice. Both Clk(Δ19/Δ19) and Clk(Δ19/Δ19) Apoe(−/−) mice developed a full spectrum of liver diseases (steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma) recognized in human NAFLD when challenged with a Western diet, lipopolysaccharide, or CoCl(2). We identified induction of CD36 and hypoxia-inducible factor 1α (HIF1α) proteins as contributing factors for NAFLD. Mechanistic studies showed that WT CLOCK protein interacted with the E-box enhancer elements in the promoters of the proline hydroxylase domain (PHD) proteins to increase expression. In Clk(Δ19/Δ19) mice, PHD levels were low, and HIF1α protein levels were increased. When its levels were high, HIF1α interacted with the Cd36 promoter to augment expression and enhance fatty acid uptake. Thus, these studies establish a regulatory link among circadian rhythms, hypoxia response, fatty acid uptake, and NAFLD. The mouse models described here may be useful for further mechanistic studies in the progression of liver diseases and in the discovery of drugs for the treatment of these disorders.

The transcription factor aryl hydrocarbon receptor nuclear translocator-like protein-1 (BMAL1) is an essential regulator of the circadian clock, which controls the 24-h cycle of physiological processes such as nutrient absorption. To examine the role of BMAL1 in small intestinal glucose absorption, we used differentiated human colon adenocarcinoma cells (Caco-2 cells). Here, we show that BMAL1 regulates glucose uptake in differentiated Caco-2 cells and that this process is dependent on the glucose transporter sodium-glucose cotransporter 1 (SGLT1). Mechanistic studies show that BMAL1 regulates glucose uptake by controlling the transcription of SGLT1 involving paired-homeodomain transcription factor 4 (PAX4), a transcriptional repressor. This is supported by the observation that clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated endonuclease Cas9 (Cas9) knockdown of PAX4 increases SGLT1 and glucose uptake. Chromatin immunoprecipitation (ChIP) and ChIP-quantitative PCR assays show that the knockdown or overexpression of BMAL1 decreases or increases the binding of PAX4 to the hepatocyte nuclear factor 1-α binding site of the SGLT1 promoter, respectively. These findings identify BMAL1 as a critical mediator of small intestine carbohydrate absorption and SGLT1.

High plasma cholesterol levels are a major risk factor for atherosclerosis. Plasma cholesterol can be reduced by inhibiting lipoprotein production; however, this is associated with steatosis. Previously we showed that lentivirally mediated hepatic expression of microRNA-30c (miR-30c) reduced hyperlipidemia and atherosclerosis in mice without causing hepatosteatosis. Because viral therapy would be formidable, we examined whether a miR-30c mimic can be used to mitigate hyperlipidemia and atherosclerosis without inducing steatosis. Delivery of a miR-30c mimic to the liver diminished diet-induced hypercholesterolemia in C57BL/6J mice. Reductions in plasma cholesterol levels were significantly correlated with increases in hepatic miR-30c levels. Long term dose escalation studies showed that miR-30c mimic caused sustained reductions in plasma cholesterol with no obvious side effects. Furthermore, miR-30c mimic significantly reduced hypercholesterolemia and atherosclerosis in Apoe(-/-) mice. Mechanistic studies showed that miR-30c mimic had no effect on LDL clearance but reduced lipoprotein production by down-regulating microsomal triglyceride transfer protein expression. MiR-30c had no effect on fatty acid oxidation but reduced lipid synthesis. Additionally, whole transcriptome analysis revealed that miR-30c mimic significantly down-regulated hepatic lipid synthesis pathways. Therefore, miR-30c lowers plasma cholesterol and mitigates atherosclerosis by reducing microsomal triglyceride transfer protein expression and lipoprotein production and avoids steatosis by diminishing lipid syntheses. It mitigates atherosclerosis most likely by reducing lipoprotein production and plasma cholesterol. These findings establish that increasing hepatic miR-30c levels is a viable treatment option for reducing hypercholesterolemia and atherosclerosis.

Aims/hypothesis Recent studies have suggested that determination of HDL function may be more informative than its concentration in predicting its protective role in coronary artery disease (CAD). Apolipoprotein AI (apoAI), the major protein of HDL, is nitrosylated in vivo to nitrated apoAI (NT-apoAI) that might cause dysfunction. We hypothesized that NT-apoAI/apoAI ratio might be associated with diabetes mellitus (DM) in CAD patients. Methods We measured plasma NT-apoAI and apoAI levels in 777 patients with coronary artery disease (CAD) by ELISA. Further, we measured plasma cholesterol efflux potential in subjects with similar apoAI but different NT-apoAI levels. Results We found that median NT-apoAI/apoAI ratio was significantly higher in diabetes mellitus (DM) (n = 327) versus non-diabetic patients (n = 450). Further analysis indicated that DM, thiobarbituric acid-reactive substances and C-reactive protein levels were independent predictors of higher NT-apoAI/apoAI ratio. There was negative correlation between NT-apoAI/apoAI and use of anti-platelet and lipid lowering drugs. The cholesterol efflux capacity of plasma from 67 individuals with differing NT-apoAI but similar apoAI levels from macrophages in vitro was negatively correlated with NT-apoAI/apoAI ratio. Conclusions Higher NT-apoAI/apoAI ratio is significantly associated with DM in this relatively large German cohort with CAD and may contribute to associated complications by reducing cholesterol efflux capacity.

A series of BiFe0.98Mn0.02O3/Sr2Bi4Ti5O18(BFMO/SBT) double-layer composite films with different transition thickness were prepared via a sol-gel method, and the effect of the thickness of SBT as transition layer on the performance of the BFMO/SBT double-layer composite films was researched. According to the XRD and SEM analyses, the perovskite structure was formed in all film samples. The BS40 sample had good crystallization, good density, uniform and small grain size. It could be seen from the XPS results that the Fe2+ and ( V O 2 − ) • • concentrations were the lowest in this sample. In addition, the 40 nm transition layer contributed to enhancing the piezoelectric (d33∼121 pm/V) and ferroelectric properties (2Pr∼99 μC/cm2@1250 kV/cm, 2Ec∼846 kV/cm) and reducing the leakage current density (J∼5.21 × 10−5A/cm2@350 kV/cm) of the sample due to the coupling effect between SBT and BFMO layers and the action of stress. The magnetic properties of the samples decreased derived from the grain size effect after the SBT transition layer was introduced. Since the degree of aging depended on the rearrangement of the defect dipoles affected by the oxygen vacancy concentration, the samples with the transition layer were weak in the aging degree.

In most signal transduction systems, coupling or scaffold proteins establish crucial connections between receptors and histidine kinases. These connections are important for signal transduction. The bacterial chemotaxis system is a canonical signal transduction system that relies on coupling proteins. The coupling proteins in the chemotaxis system have two architectures: CheW or CheV. In a typical chemotaxis signal transduction system, two CheW coupling protein molecules bridge a histidine kinase CheA dimer and two chemoreceptor (also called as methyl-accepting chemotaxis protein, MCP) trimers of dimers to form a core signaling complex and couple CheA activity to chemoreceptor control. Although CheW is a small cytoplasmic protein, it plays multiple functions in chemotaxis. CheW also builds connections between core signaling complexes, which leads to the formation of large chemosensory arrays that are responsible for collecting and amplifying signals from various chemoreceptors. Another coupling protein, CheV, shares a largely redundant ability with CheW; however, the function of CheV is not identical to that of CheW in chemotaxis. In this article, we summarize the molecular mechanism of chemotaxis in Escherichia coli and review the recent advances in the structural details and functions of CheW and CheV. Furthermore, we focus on the diversity of coupling proteins and discuss the relationship among multiple coupling proteins in one organism.

Dietary fat triggers synthesis of OEA in enterocytes of the upper small intestine. OEA initiates a cascade of events to optimize fatty acid uptake and reduce feeding frequency. Effects of OEA have been studied after oral gavage and intraperitoneal injections. Here, we studied dose dependent effects of OEA on the assembly and secretion of lipoproteins in differentiated human colon carcinoma Caco-2, human hepatoma Huh-7 and HepG2 cells as well as in mouse primary enterocytes and hepatocytes. Differentiated Caco-2 cells were treated with OEA or oleic acid for 16 h. OEA increased synthesis of triglycerides in dose dependent manner compared with oleic acid:taurocholate micelles. Further, OEA increased secretion of triglycerides and apolipoprotein B (apoB) in chylomicrons. Expression and activity of microsomal triglyceride transfer protein (MTP) was increased by OEA in a dose dependent manner in these cells. Next, mouse primary enterocytes were incubated with OEA or oleic acid for 4 h. OEA increased MTP activity and secretion of triglycerides in these cells as well. These studies suggest that OEA enhances MTP expression, lipid synthesis and lipoprotein assembly in intestinal cells more than oleic acid. To study the effect of OEA on liver derived cells, these cells were exposed to OEA or no lipids for 16 h. OEA reduced secretion of triglycerides and apoB in Huh-7 and HepG2 cells without affecting the secretion of apoAI. Further, MTP expression and activity was reduced in these cells after OEA treatment compared to no OEA controls. Similarly, OEA reduced MTP expression and activity as well as secretion of triglycerides in mouse primary hepatocytes. These studies indicate that OEA reduces MTP expression, lipid synthesis and lipoproteins secretion in liver derived cells. Thus, OEA has differential effects on MTP expression and lipoprotein assembly and secretion in intestine and liver derived cells. It is likely that OEA regulates postprandial response to optimize exogenous lipid absorption and reduce endogenous lipid mobilization.

Among all the metabolites present in the plasma, lipids, mainly triacylglycerol and diacylglycerol, show extensive circadian rhythms. These lipids are transported in the plasma as part of lipoproteins. Lipoproteins are synthesized primarily in the liver and intestine and their production exhibits circadian rhythmicity. Studies have shown that various proteins involved in lipid absorption and lipoprotein biosynthesis show circadian expression. Further, intestinal epithelial cells express circadian clock genes and these genes might control circadian expression of different proteins involved in intestinal lipid absorption. Intestinal circadian clock genes are synchronized by signals emanating from the suprachiasmatic nuclei that constitute a master clock and from signals coming from other environmental factors, such as food availability. Disruptions in central clock, as happens due to disruptions in the sleep/wake cycle, affect intestinal function. Similarly, irregularities in temporal food intake affect intestinal function. These changes predispose individuals to various metabolic disorders, such as metabolic syndrome, obesity, diabetes, and atherosclerosis. Here, we summarize how circadian rhythms regulate microsomal triglyceride transfer protein, apoAIV, and nocturnin to affect diurnal regulation of lipid absorption.

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. Background: The role of apoAIV in the diurnal regulation of plasma lipids is unknown. Results: Plasma lipids, lipid absorption, and MTP, FoxO1, and FoxA2 levels are lower at night and at mealtime in apoAIV−/− mice. Conclusion: ApoAIV increases intestinal lipid absorption by modulating the expression of MTP involving FoxA2 and FoxO1. Significance: Inhibition of intestinal apoAIV expression might reduce plasma lipids.

Background: The anti-infammatory and cardioprotective effect of acetylcholine (ACh) has been reported; nevertheless, whether and how ACh exhibits an antioxidant property against ischemia/reperfusion (I/R)-induced oxidative stress remains obscure. Methods: In the present study, H9c2 rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) to mimic I/R injury. We estimated intracellular different sources of reactive oxygen species (ROS) by measuring mitochondrial ROS (mtROS), mitochondrial DNA (mtDNA) copy number, xanthine oxidase (XO) and NADPH oxidase (NOX) activity and expression of rac 1. Cell injury was determined by lactate dehydrogenase (LDH) release and cleaved caspase-3 expression. The siRNA transfection was performed to knockdown of M2 acetylcholine receptor (M2 AChR) expression. Results: 12-h hypoxia followed by 2-h reoxygenation resulted in an abrupt burst of ROS in H9c2 cells. Administration of ACh reduced the levels of ROS in a concentration-dependent manner. Compared to the H/R group, ACh decreased mtROS, recovered mtDNA copy number, diminished XO and NOX activity, rac 1 expression as well as cell injury. Co- treatment with atropine rather than hexamethonium abolished the antioxidant and cardioprotective effect of ACh. Moreover, knockdown of M2 AChR by siRNA showed the similar trends as atropine co-treatment group. Conclusions: ACh inhibits mitochondria-, XO- and NOX-derived ROS production thus protecting H9c2 cells against H/R-induced oxidative stress, and these benefcial effects are mainly mediated by M2 AChR. Our findings suggested that increasing ACh release could be a potential therapeutic strategy for treatment and prevention of I/R injury.

Circadian rhythms controlled by clock genes affect plasma lipids, known risk factors for atherosclerosis. We observed that global ablation of a critical clock gene, Bmal1 , in Apoe –/– and Ldlr –/– mice, and liver-specific ablation of Bmal1 in Apoe –/– (L -Bmal1 –/– Apoe –/– ) mice increases hyperlipidemia and atherosclerosis. In contrast, overexpression of BMAL1 in L-Bmal1 –/– Apoe –/– mice decreases hyperlipidemia and atherosclerosis. Physiologic studies showed that Bmal1 deficiency augments hepatic lipoprotein secretion and diminishes cholesterol excretion to the bile. Molecular studies identified that Bmal1 deficiency reduces expression of Shp, a repressor, and Gata4, an activator. Reductions in Shp increase Mtp expression and lipoprotein production, whereas reductions in Gata4 diminish Abcg5/Abcg8 expression and cholesterol excretion to the bile. SHP expression normalized lipoprotein secretion with no effect on cholesterol excretion to the bile, while GATA4 expression increased cholesterol excretion to the bile and reduced plasma lipids in L-Bmal1 –/– Apoe –/– mice. Taken together, our data indicate that Bmal1 modulates lipoprotein secretion to the plasma and cholesterol excretion to the bile by regulating the expression of Mtp and Abcg5/Abcg8 via Shp and Gata4, and that the loss of Bmal1 promotes hyperlipidemia and atherosclerosis.

Background: High plasma cholesterol levels are a major risk factor for atherosclerosis. Plasma cholesterol can be reduced by inhibiting lipoprotein production; however, this is associated with steatosis. Previously we showed that lentiviral mediated hepatic over-expression of microRNA-30c (miR-30c) reduced hyperlipidemia and atherosclerosis in mice without causing hepatosteatosis. Since viral therapy would be formidable, we tested the hypothesis that a miR-30c oligonucleotide mimic can mitigate hyperlipidemia and atherosclerosis without causing steatosis. Methods and results: Delivery of a miR-30c mimic to the liver diminished Western diet-induced hypercholesterolemia in C57BL/6J wild type mice. Reductions in plasma cholesterol levels were significantly correlated with increases in hepatic miR-30c levels. Long-term dose escalation studies showed that miR-30c mimic causes sustained reductions in plasma cholesterol without increasing hepatic lipids and plasma transaminases. Further, miR-30c mimic significantly reduced hypercholesterolemia and atherosclerosis in Apoe –/– mice without causing steatosis or increasing plasma transaminases. Mechanistic studies indicated that miR-30c mimic reduced hepatic lipoprotein production, in part by reducing microsomal triglyceride transfer protein (MTP) activity. This is supported by the observation that miR-30c did not lower plasma cholesterol in liver-specific MTP deficient mice but did reduce hepatic lipid synthesis. In order to understand why there was no increase in hepatic lipids, we carried out whole transcriptome studies by RNA-sequencing. Bioinformatic analysis of the data revealed that genes down-regulated by miR-30c mimic are most significantly enriched for lipid synthesis pathways. Consistent with this finding, we showed that miR-30c mimic reduced fatty acid and glycerolipid synthesis in the liver but had no effect on hepatic β-oxidation. Conclusions: We conclude that miR-30c reduces plasma cholesterol by targeting MTP and prevents hepatosteatosis by repressing lipid synthesis programs in the liver. These findings suggest that increasing hepatic miR-30c levels may be a viable treatment modality for hypercholesterolemia and atherosclerosis.

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.

To summarize the new knowledge about the regulation of dietary lipid absorption by circadian locomotor output cycles kaput (Clock) and Nocturnin.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.Clock and Nocturnin regulate lipid absorption involving different mechanisms. It is likely that other clock genes also modulate lipid absorption and plasma lipid levels.

Summary. 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: 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 ClockΔ19/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 ClockΔ19/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.

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.

Summary We examined the role of clock genes in the diurnal regulation of plasma triglyceride-rich apolipoprotein B-lipoproteins and their biosynthetic chaperone, microsomal triglyceride transfer protein (MTP). Clock mt/mt mice showed sustained hypertriglyceridemia and high MTP expression. CLOCK knockdown activated MTP promoter and reduced small heterodimer partner ( SHP , NROB2). CLOCK upregulated SHP by binding to its E box. SHP suppressed MTP expression by binding to the HNF4α/LRH-1 at the MTP promoter. Cyclic expression of MTP after serum shock was abrogated by siCLOCK and siSHP. Plasma triglyceride and MTP showed reduced diurnal variations in Shp −/− mice. Whereas peaks and nadirs in SHP expression were inversely correlated with those of MTP, these changes were reduced in Clock mt/mt mice. Expression of Shp abrogated hypertriglyceridemia in Clock mt/mt mice. Together, these studies describe a role of Clock/Shp in the diurnal regulation of MTP and plasma triglyceride and indicate that disruptions in circadian regulation might cause hyperlipidemia.

Serine palmitoyltransferase (SPT) is the key enzyme for the biosynthesis of sphingolipids. It has been reported that oral administration of myriocin (an SPT inhibitor) decreases plasma sphingomyelin (SM) and cholesterol levels, and reduces atherosclerosis in apoE knockout (KO) mice. We studied cholesterol absorption in myriocin-treated WT or apoE KO animals and found that, after myriocin treatment, the mice absorbed significantly less cholesterol than controls, with no observable pathological changes in the small intestine. More importantly, we found that heterozygous Sptlc1 (a subunit of SPT) KO mice also absorbed significantly less cholesterol than controls. To understand the mechanism, we measured protein levels of Niemann-Pick C1-like 1 (NPC1L1), ABCG5, and ABCA1, three key factors involved in intestinal cholesterol absorption. We found that NPC1L1 and ABCA1 were decreased, whereas ABCG5 was increased in the SPT deficient small intestine. SM levels on the apical membrane were also measured and they were significantly decreased in SPT deficient mice, compared with controls. In conclusion, SPT deficiency might reduce intestinal cholesterol absorption by altering NPC1L1 and ABCG5 protein levels in the apical membranes of enterocytes through lowering apical membrane SM levels. This may be also true for ABCA1 which locates on basal membrane of enterocytes. Manipulation of SPT activity could thus provide a novel alternative treatment for dyslipidemia.

This review summarizes recent advances about the role of microsomal triglyceride transfer protein in plasma and tissue lipid homeostasis.Microsomal triglyceride transfer protein emerged as a phospholipid transfer protein and acquired triacylglycerol transfer activity during evolution from invertebrates to vertebrates. These activities are proposed to participate in 'nucleation' and 'desorption' steps during the biosynthesis of primordial apoB-containing lipoproteins. Microsomal triglyceride transfer protein also transfers phospholipids to the glycolipid antigen presentation molecule CD1d. Under physiologic conditions, plasma apoB-containing lipoproteins and microsomal triglyceride transfer protein expression exhibit diurnal variations synchronized by food and light. Microsomal triglyceride transfer protein is regulated at the transcriptional level. HNF4alpha is critical for its transcription. Other transcription factors along with coactivators and corepressors modulate microsomal triglyceride transfer protein expression. Reductions in microsomal triglyceride transfer protein mRNA and activity are related to steatosis in HCV-3 infected patients. CCl4 induces steatosis by enhancing proteasomal degradation of microsomal triglyceride transfer protein and can be partially avoided by inhibiting this degradation. Chemical antagonists cause hepatosteatosis, but this was not seen in the absence of fatty acid binding protein.Microsomal triglyceride transfer protein is a target to lower plasma lipids and to reduce inflammation in certain immune disorders. More knowledge is required, however, regarding its regulation and its role in the biosynthesis of apoB-containing lipoproteins and CD1d.

Plasma lipids are maintained within a narrow physiologic range and exhibit circadian rhythmicity. Plasma triglyceride and cholesterol levels were high in the night due to changes in apolipoprotein B-lipoproteins in ad libitum fed rats and mice maintained in a 12-h photoperiod. Absorption of [(3)H]triolein or [(3)H]cholesterol was higher at 2400 h than at 1200 h, indicating that intestinal lipoprotein production shows diurnal variation. Moreover, intestinal microsomal triglyceride transfer protein (MTP) activity, protein, mRNA, and gene transcription showed diurnal variations and were high at 2400 h. Similar to the small intestine, hepatic MTP activity, protein, and mRNA levels also changed significantly within a day. MTP was induced in fasted animals soon after refeeding. When mice were subjected to restricted feeding, MTP expression was high at the expected time of food availability. In contrast, extended exposures to light and dark completely abolished rhythmicity in MTP expression and plasma lipid levels. These studies show that MTP expression and plasma lipid undergo diurnal regulation and exhibit peaks and nadirs at similar times and suggest that diurnal modulation of MTP is a major determinant of daily changes in plasma lipids. Furthermore, environmental factors, such as food and light, play an important role in MTP regulation.