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Regulation of Intestinal Lipid Absorption by Clock Genes M. Mahmood Hussain Departments of Cell Biology and Pediatrics, SUNY Downstate Medical Center, Brooklyn, New York 11203, and Virginia New York Harbor Healthcare System, Brooklyn, New York 11209; email: [email protected]

Annu. Rev. Nutr. 2014. 34:357–75

Keywords

The Annual Review of Nutrition is online at nutr.annualreviews.org

circadian rhythms, intestine, lipid absorption, triacylglycerol, MTP, clock genes

This article’s doi: 10.1146/annurev-nutr-071813-105322 c 2014 by Annual Reviews. Copyright  All rights reserved

Abstract Plasma levels of triacylglycerols and diacylglycerols, the lipoproteins that transport them, and proteins involved in their absorption from the intestinal lumen fluctuate in a circadian manner. These changes are likely controlled by clock genes expressed in the intestine that are probably synchronized by neuronal and humoral signals from the suprachiasmatic nuclei, which constitute a master clock entrained by light signals from the eyes and from the environment, e.g., food availability. Acute changes in circadian rhythms— e.g., due to nonsynchronous work schedules or a transcontinental flight— may trigger intestinal discomfort. Chronic disruptions in circadian control mechanisms may predispose the individual to irritable bowel syndrome, gastroesophageal reflux disease, and peptic ulcer disease. A more detailed understanding of the molecular mechanisms underlying temporal changes in intestinal activity might allow us to identify novel targets for developing therapeutic approaches to these disorders.

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Contents

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INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Circadian Clock and Clock-Controlled Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MODULATION OF THE INTESTINAL CLOCK BY SUPRACHIASMATIC NUCLEI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Food as a Modulator of Peripheral Circadian Clocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clock Genes in the Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intestinal Lipid Absorption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Circadian Patterns of Plasma Triacylglycerol Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Circadian Regulation of Lipid Absorption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clock Gene Dysfunction Deregulates Intestinal Lipid Absorption . . . . . . . . . . . . . . . . . Perspective and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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INTRODUCTION Animals spend much of their waking hours in anticipation of a meal and in digesting and absorbing food. The digestion and absorption of food are coordinated through changes in the expression of proteins, which is synchronized with the rising and setting of the sun. Mechanisms controlling the circadian expression of these proteins are not fully understood. Recent studies indicate that clock genes, which control circadian patterns of behavior and physiological activity, participate in the temporal control of intestinal lipid absorption. This review introduces the clock genes and their mode of action in determining the circadian rhythms of intestinal activities. In addition to briefly reviewing the process of intestinal lipid absorption, I examine the mechanisms underlying the role of food availability in altering the normal circadian pattern of intestinal activity, summarize evidence of diurnal changes in plasma triacylglycerol levels, review data indicating that disruptions in clock activity can alter diurnal variations in the expression of genes involved in intestinal absorption of fat and those affecting plasma triacylglycerol levels, and point to a few unresolved issues and to potential directions for future experiments.

The Circadian Clock and Clock-Controlled Genes In diurnal animals, waking and sleeping are synchronized with the rising and setting of the sun (for nocturnal animals, waking is synchronized with the onset of darkness) and, thus, exhibit a periodicity of approximately 24 hours. Arousal from sleep is associated with the physical activity required to gather food from the environment, whereas sleep is characterized by low locomotor activity and fasting. These daily routines are centrally controlled by two bilateral suprachiasmatic nuclei (SCN) in the anterior hypothalamus that receive day/night signals from the retina through the retinohypothalamic tract (18, 36, 52, 72). When these photic signals reach the SCN, they are processed to induce molecular events leading to changes in the expression of a set of core “clock genes”— Circadian locomotor output cycles kaput (Clock), Brain and muscle ARNT-like protein 1 (Bmal1), Period 1 (Per1), Per2, Cryptochrome 1 (Cry1), and Cry2—that code for transcription factors involved in the temporal regulation of behavior and physiology. These transcription factors form specific partnerships and constitute an autoregulatory transcription/translation feedback loop. A key step in the generation of the feed-forward transcriptional cascade is the heterodimerization of Clock and Bmal1. Clock is a histone acetyltransferase; its activity increases when it binds 358

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Clock genes (primary loop) Per

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Dbp

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Pparα

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Avp

Blood pressure regulation

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Shp

Plasma lipids

Drug metabolism Fatty acid oxidation

Figure 1 Regulation of clock and clock-controlled genes. (a) The Clock:Bmal1 heterodimer interacts with E-box enhancer elements in the promoter region of several clock genes to increase the transcription of genes involved in initiating the feed-forward loop. When concentrations of Per and Cry increase, these two proteins form heterodimers that can inhibit Bmal1 activity and repress their own expression. This autoregulatory transcriptional control constitutes the primary circadian loop, occurring with a periodicity of approximately 24 hours. (b) Bmal1 expression is regulated by Rorα and Rev-Erbα. Both of these transcription factors bind to the same RORE element in the promoter region of Bmal1. The binding of Rorα to the RORE enhancer element increases the expression of Bmal1, whereas the binding of Rev-Erbα represses it. Thus, Rorα and Rev-Erbα constitute a secondary feedback loop for circadian clocks. (c) Regulation of clock-controlled genes. The Clock:Bmal1 heterodimer interacts with E-box enhancer elements in the promoter region of the genes that code for many transcription factors that control different metabolic pathways. Abbreviations: Bmal1, brain and muscle ARNT-like protein 1; Clock, circadian locomotor output cycles kaput; Cry, cryptochrome; Dbp, D site–binding protein; Per, period; PPARα, peroxisome proliferator-activated receptor alpha; Rev-Erbα, reverse erythroblastosis virus α; Rorα, receptor-related orphan receptor α; RORE, retinoic acid–related orphan receptor response element; Shp, small heterodimer partner.

to Bmal1 (20). The Clock:Bmal1 heterodimer binds to E-box enhancer elements in the promoter region of two different sets of clock-controlled and clock genes and increases their transcription (Figure 1a). Activation of the clock-controlled genes sets into motion a cascade of transcriptional and translational events leading to the increased expression of critical transcription factors that enhance the expression of various output genes. Consequently, a signal is generated, amplified, and transmitted that affects metabolism throughout the body. The feed-forward loop initiated by Clock:Bmal1 is terminated by the enhanced expression of a set of genes that code for Per and Cry repressors. When Per and Cry levels rise, they form heterodimers that oppose the action of Clock:Bmal1. Thus, Per:Cry reduces the expression of Per and Cry, thereby constituting a negative feedback loop. This pattern of transcription is fine-tuned through the posttranslational phosphorylation of Per and Cry by casein kinases (CKIα, CKIδ, and CKIε), which results in the degradation of these proteins through an ubiquitin-proteasome www.annualreviews.org • Regulation of Intestinal Lipid Absorption

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PVN: paraventricular nucleus of the thalamus DMH: dorsomedial hypothalamus

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pathway. The consequential decrease in Per and Cry levels represses their inhibitory effects on Clock:Bmal1, which allows the feed-forward pattern of gene activation to resume (3, 18, 36, 52). An important feature of clock genes is that they need to be regularly exposed to external cues to maintain their periodic rhythmicity. In the absence of regular cues, the intensity of the circadian response shortens over time and eventually disappears. Circadian rhythms are also modulated by a secondary loop (Figure 1b) that regulates the expression of Bmal1. This loop consists of retinoic acid receptor-related orphan receptor α (Rorα) and reverse erythroblastosis virus α (Rev-erbα). The expression of these two proteins increases in the presence of Clock:Bmal1 heterodimers. In turn, each of these proteins regulates the expression of Bmal1 by interacting with retinoic acid–related orphan receptor response element (RORE) in the Bmal1 promoter region, although with opposite results: Rorα increases Bmal1 expression, and Rev-erbα represses it. Recent studies indicate that the expression of circadian clock genes is sensitive to changes in intracellular metabolic processes and metabolic end products such as oxidized nicotinamide adenine dinucleotide (NAD+ ), reactive oxygen species, the adenosine triphosphate:adenosine monophosphate (ATP:AMP) ratio, Ca+2 /cyclic AMP (cAMP) signaling, and glucose (3, 55). Because such metabolic end products may alter the expression of circadian clock genes, it may be worthwhile to investigate the pathways that generate these metabolites for their potential as novel mechanisms amenable to therapeutic intervention. Besides clock genes, Clock:Bmal1 increases the expression of several clock-controlled transcription factors to regulate different metabolic processes (Figure 1c). In addition to the SCN, clock genes and clock-controlled genes are expressed in almost all peripheral tissues. Unlike the SCN, however, peripheral tissues are not served by neurons that receive light cues from the eyes. Instead, they detect changes in hormone levels and neuronal activity that are generated and transmitted by the SCN clock in response to changes in environmental luminosity (Figure 2a) (8, 17, 33). The blood-borne factors that are critical for the transmission of SCN signals to peripheral tissues are unknown. It has been proposed that these factors interact with unknown receptors to activate the intracellular Rho-kinase pathway to induce the polymerization of G-actin to form F-actin (17). This polymerization enables the transport of a myocardin-related transcription factor to the nucleus, where it interacts with another transcription factor, serum response factor, to activate the expression of Per2. It is possible that this and other similar mechanisms entrain the expression of clock genes in peripheral tissues. Thus, clock genes appear to regulate cellular metabolism throughout the body through a hierarchical system that responds to light cues at a central location (the SCN), which generates and amplifies diurnal signals that are transmitted to peripheral tissues to control local biological functions. Such a system might have evolved to allow animals to anticipate changes in the environment and respond to these changes in a synchronized manner through appropriate biological, molecular, and metabolic events.

MODULATION OF THE INTESTINAL CLOCK BY SUPRACHIASMATIC NUCLEI The SCN clock may synchronize the activities of enteric clock genes through neuronal and humoral cues described in the following sections (4, 8, 13, 43, 46, 58, 65). The SCN is in contact with different parts of the brain that control nutrient absorption (Figure 2b). It may send rhythmic autonomic GABAergic signals to the paraventricular nucleus (PVN) in the thalamus and to the dorsomedial hypothalamus (DMH)—either directly or through the subparaventricular zone (SPZ) of the hypothalamus—which transmit them to the dorsal motor nucleus of the vagus (DMV) nerve that has autonomic inputs to the intestine. The SCN also modulates the activity of other 360

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Hormones

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Parasympathetic projections to the intestine

Autonomic nervous system

Food

Figure 2 Circadian regulation of intestinal function. (a) Intestinal functions are regulated by the brain through the autonomic nervous system and hormones. Additionally, they can be modulated by external cues. Food availability is a major modulator of intestinal function. (b) Schematic diagram of the possible paths of communication between different parts of the brain that are involved in the neuronal regulation of intestinal function by the SCN. The SCN, which is located in the hypothalamus, sends neuronal signals to the PVN in the thalamus, the DMH, and the SPZ of the hypothalamus. These signals are then relayed from the PVN and DMH to the DMV in the medulla. The DMV has parasympathetic neuronal projections to the intestine. Abbreviations: DMH, dorsomedial hypothalamus; DMV, dorsal motor nucleus of the vagus; PVN, paraventricular nucleus of the thalamus; SCN, suprachiasmatic nuclei; SPZ, subparaventricular zone.

regions of the brain by releasing vasoactive intestinal peptide and arginine-vasopressin, which have a considerable excitatory influence on the SPZ. In addition to the parasympathetic input from the brain, intestinal activity is also controlled through extrinsic efferent and afferent nerves from the spinal sympathetic nervous system (Figure 3) (4, 22, 38, 53, 65). The efferent neurons of the sympathetic and parasympathetic system are located in the thoraco-lumbar segments and the medulla oblongata, respectively. The major outflow of the parasympathetic system is through the vagus nerve, whereas the sympathetic outflow consists of several spinal nerves. The sensory afferent neurons of both systems consist of myelinated and nonmyelinated neurons. The cell bodies of the sympathetic and parasympathetic afferent neurons are present in the dorsal root ganglia and the nodose ganglion of the vagus nerve. The majority of the sensory afferent nerves are sensitive to capsaicin, an active ingredient found in hot peppers. Thus, most of the sympathetic and parasympathetic afferent neuronal communications can be severed by the systemic injection of high doses of capsaicin. However, partial parasympathetic deafferentation can be achieved by the topical application of capsaicin. Vagotomy (cutting of the vagus nerve) ablates parasympathetic afferent and efferent communications. Uptake of amino acids and peptides is controlled by vagal inputs. These inputs have stimulatory or inhibitory effects on amino acid and peptide absorption in different segments of the intestine. In contrast to carbohydrate and protein absorption, enteric and central nervous systems do not affect fat absorption, as vagotomy has no effect on early and late phases of jejunal fat absorption (32, 43). Furthermore, vagotomy does not affect the expression of microsomal triglyceride transfer www.annualreviews.org • Regulation of Intestinal Lipid Absorption

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Spinal cord (sympathetic system) Sympathetic ganglion Spinal nerve

Dorsal root ganglion

Brain stem (parasympathetic system) Nodose ganglion

Dorsal motor nucleus of the vagus Vagus nerve

Longitudinal muscle

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Myenteric plexus Circular muscle

Submucosal plexus

Columnar epithelium

Figure 3 Neuronal control of intestinal function. The intestine has two major enteric neuronal plexi: the myenteric plexus, which lies between the inner circular and outer longitudinal layers of smooth muscle; and the submucosal plexus, which lies underneath the mucosal epithelial layer. These two plexi communicate with each other through efferent, afferent, and association neurons. The intestine is also innervated by efferent and afferent neurons originating from the spinal cord (sympathetic system) and the brain stem (parasympathetic system). Various regions of the intestine (excluding the enterocytes) are innervated by both sympathetic and parasympathetic neurons.

protein (MTP), which is critical in chylomicron assembly and secretion (30). Nonetheless, enteric and central nervous systems play an important role in fat absorption by modulating pancreatic secretions, bile acid delivery, and intestinal motility. It has been shown that vagotomy has no effect on diurnal expression of clock genes and hexose and peptide transporters in the intestine (69). Furthermore, chemical sympathectomy achieved by high doses of capsaicin has no effect on the diurnal expression of clock genes (38). However, isoproterenol, a β-adrenergic agonist, shifts Per2 expression. This may suggest that vagal and spinal inputs might not be critical for the regulation of clock gene expression, and humoral factors might compensate for the loss of signals from the brain. More research is needed to elucidate the involvement of different pathways and their redundancy in the entrainment of intestinal functions. The SCN may exert control over intestinal function through the enteric nervous system as well. The enteric nervous system consists of two neural (external and submucosal) sheaths and three kinds of neurons (afferent, efferent, and association) (Figure 3) (43). The external neural sheath (myenteric plexus) is located between the outer longitudinal and inner circular smooth muscle layers of the intestine. The internal neuronal sheath (submucosal plexus) is in the submucosa between the muscularis mucosa and inner circular smooth muscle layer of the intestine. The cell bodies of the afferent enteric neurons are located in both plexi, and their axons project toward the spinal cord. These neurons sense mechanical and chemical stimuli. The efferent enteric neurons project to various regions of the intestine, communicating with the longitudinal and circular muscle layers, the muscularis mucosa, and the submucosa in each region with which it makes contact. The association enteric neurons project within the same plexus or between the two plexi.

MTP: microsomal triglyceride transfer protein

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The SCN may also synchronize other clocks using as yet unknown paracrine factors. Evidence for the existence of these factors has been provided in studies in which the SCN from one animal was transplanted into various parts of the brain in other animals (13, 72). Plasma glucocorticoid hormones demonstrate robust daily oscillations and clock-resetting properties and synchronize cellular clocks in culture (13). The cyclic changes induced by plasma glucocorticoids are driven by the SCN through the hypothalamic-pituitary-adrenal axis. Melatonin may also play a role in the synchronization of circadian patterns of intestinal activities (33). The SCN controls the release of melatonin by the pineal gland. Obviously, further investigation of the role of neuronal, hormonal, and other factors in clock control is necessary for us to understand how the observed circadian pattern of intestinal activity is controlled.

Food as a Modulator of Peripheral Circadian Clocks In addition to the neuronal and hormonal mechanisms described, the activities of intestinal clocks may be synchronized by changes in body temperature and feeding (65). Temporal food availability is a very strong synchronizer of intestinal activities (12, 15, 19, 26, 41, 53, 66, 67). Its ability to alter the circadian pattern of digestive activity is easily demonstrated by providing food for a few days during the day to rodents, which usually eat at night, without changing the normal light/dark cycle. Such food entrainment is characterized by a shift in peak food anticipatory activity (i.e., locomotor activity reflecting an intense search for food) and peak food-consuming activity (i.e., the expression of proteins involved in the digestion and absorption of nutrients) from night to day. Food entrainment is also characterized by changes in the expression of intestinal clock and clock-controlled gene products when food is available, but it does not alter rhythmic expression of SCN clock genes. These findings suggest that the SCN is entrained by light cues but does not respond to other external stimuli. By contrast, peripheral tissues, e.g., intestinal clock genes, are also susceptible to changes in environmental cues. Studies have shown that the changes that follow food entrainment occur in a circadian pattern that is sustained for some time after food entrainment has stopped. On the basis of this observation, it has been proposed that a “food-entrainable oscillator” exists. SCN ablation has no significant effect on responses characteristic of food entrainment; this indicates that SCN-independent loci might be involved in controlling the behavioral and molecular changes observed during food entrainment. The identity of this proposed oscillator is unknown. The food-entrained oscillator probably consists of a web of neuronal sites in the brain that interacts with signals from other parts of the body to produce a behavioral and physiological response that fosters optimal food consumption when food is available (4, 62). Although the SCN clock does not appear to influence food-entrainable oscillator functions, evidence indicates that peripheral clock genes participate in this response. For example, changes in the feeding schedule shift the timing of the expression of peripheral clock genes (46, 48, 67). Specifically, after food entrainment, the expression of clock genes peaks around the time of food availability (daytime) instead of at the normal peak expression time at night in rodents. Such changes require coordination with the SCN. In the absence of circadian light cues, clock gene expression in mice does not increase at or around the mealtime. Thus, the normal light-entrained response may be necessary for food entrainment. The involvement of clock genes in the food entrainment response has been evaluated in ClockΔ19/Δ19 mice, which express a dominant negative Clock protein (71), and in other mouse models deficient in different clock genes. ClockΔ19/Δ19 mice do not respond to food entrainment, and the expression of the clock genes is not induced at or around mealtime (47, 48). Per2−/− and Cry1−/− Cry2−/− double knockout mice display a delayed or reduced food-anticipatory response, www.annualreviews.org • Regulation of Intestinal Lipid Absorption

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whereas Bmal1−/− mice exhibit a robust food anticipatory response (13). These findings suggest that some, but not all, clock genes participate in the food entrainment process, but they do not explain the mechanism(s) by which these genes participate in this process. It has been suggested that Clock is directly involved in the food entrainment response (48). It is possible that ClockΔ19/Δ19 mice and other knockout mice are unable to sense the availability of food at a given time because of defects in the central clock. Experiments involving tissue-specific knockout mice that retain circadian SCN activity but are deficient in intestinal clock genes are needed to determine whether intestinal clock genes are required for the food entrainment response. There is also evidence that several humoral factors help induce some of the changes in intestinal function that are associated with food entrainment (3, 8, 13, 33, 46, 53, 65). For example, ghrelin (an orexigenic hormone produced in the stomach and intestine) may play a role in food-anticipatory activity, given that it is involved in the initiation of feeding and its plasma level increases prior to feeding. Support for its role has been provided by the observation that ghrelin receptor knockout mice exhibit considerably less food-anticipatory activity than do wild-type mice (35, 62). Other humoral factors that may participate in food entrainment include corticosterone and glucose (46). Thus, food entrainment may involve complex interactions among various brain loci and hormones secreted by peripheral tissue.

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Clock Genes in the Intestine Various intestinal activities—such as gastrointestinal motility, gastric emptying, DNA synthesis, epithelial cell renewal, food-anticipatory activity, and nutrient and electrolyte absorption—occur in a circadian pattern (21, 22, 26, 56, 64). It is well known that the major complaints of shift workers and transcontinental travelers are gastrointestinal disturbances (70). Voluntary sleep curtailment or the misalignment of the day/night cycle, as occurs in shift workers, might contribute to gastrointestinal discomfort by overriding neuroendocrine entrainment, disrupting intestinal rhythms, and altering behavioral and physiological functions. Indeed, chronic sleep disturbance in shift workers has been suspected of causing irritable bowel syndrome, gastroesophageal reflex disease, and peptic ulcer disease (22, 33). Clock genes in the jejunum (48) and colon (23, 63) show diurnal patterns of expression. The peaks and nadirs in the expression of their gene products are in phase with their expression in the liver but are phase-delayed compared with their expression in the SCN (67). It is possible that a temporal delay in the rhythmic activation of intestinal clock genes reflects the time needed for neuronal and hormonal signals generated in the SCN to reach the intestine.

Intestinal Lipid Absorption Before embarking on a discussion of factors responsible for the circadian pattern of intestinal activity, it is crucial to understand the processes involved in the absorption, resynthesis, packaging, and recirculation of intestinal lipids. Digestion of intestinal lipids. The predominant dietary lipids—triacylglycerols, phospholipids, and cholesterol esters—are digested in the intestinal lumen (Figure 4). Because lipids are water insoluble, an important step in their digestion is their emulsification with bile salts (44, 54). In this process, the hydrophobic lipids are incorporated into bile salt micelles, which render them water miscible. The digestion of triacylglycerols starts in the mouth through the action of lingual lipases, continues in the stomach with gastric lipases, and is completed in the small intestine with 364

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Bile acids Dietary fat

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Figure 4 Intestinal lipid absorption. Dietary fat is emulsified by bile acids and hydrolyzed by pancreatic lipases to release free fatty acids (FFAs) and monoacylglycerols (MAGs) in the intestinal lumen. These products are taken up by enterocytes using various transporters, including cluster of differentiation 36 (CD36) and the fatty acid transport proteins (FATPs) in the apical membrane of the cell. Similarly, dietary cholesterol esters (CEs) are hydrolyzed to generate free cholesterol (FC), which is taken up with the help of Niemann-Pick C1-like 1 (NPC1L1). FFA and MAG are used by diacylglycerol acyltransferase (DGAT) for the synthesis of triacylglycerols, whereas FC is esterified to CE by acyl-CoA:cholesterol acyltransferase (ACAT). These lipids are then packaged into apoB48-containing chylomicrons (CMs) with the assistance of microsomal triglyceride transfer protein (MTP) in the endoplasmic reticulum (ER) and then transported in specialized prechylomicron transport vesicles (PCTVs) to the Golgi to undergo further modification. They are then transported to the basolateral membrane of the cell using various transport vesicles and secreted. These particles are concentrated in the lymphatics and are delivered to the blood at the thoracic duct. FC taken up by the enterocytes may have two additional fates: (a) excretion back to the intestinal lumen through the ATP-binding cassette family G proteins 5 and 8 (ABCG5 and ABCG8) transporters or (b) secretion toward the basolateral side via the high-density lipoprotein (HDL) pathway involving ATP-binding cassette transporter family A member 1 (ABCA1) and apolipoprotein AI (apoAI). Abbreviations: AIV, apolipoprotein AIV; RE, retinyl ester; TG, triglyceride; Vit E, vitamin E. www.annualreviews.org • Regulation of Intestinal Lipid Absorption

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FFAs: free fatty acids NPC1L1: Niemann-Pick C1-like 1 ER: endoplasmic reticulum

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apoB48: apolipoprotein B48 HDL: high-density lipoprotein PCTVs: prechylomicron transport vesicles

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pancreatic lipases, which cause the release of free fatty acids (FFAs) and monoacylglycerols. Phospholipids are hydrolyzed by pancreatic phospholipases (mainly phospholipase A2) to yield FFAs and lysophospholipids. Cholesterol esters are hydrolyzed by cholesterol esterases to release free cholesterol and FFAs. Monoacylglycerols and FFAs enter the enterocyte by diffusion and through protein-mediated transport mechanisms (2, 29, 49). Diffusion across these epithelial cells occurs when FFA concentrations in the lumen exceed those inside the cell. When their extracellular concentration is lower than their intracellular concentration, FFAs can also be taken up by enterocytes with the help of various transport proteins, including cluster of differentiation 36 (CD36) and FA transport proteins (1, 2, 29, 49). Cholesterol uptake involves the Niemann-Pick C1-like 1 (NPC1L1) protein and possibly other transporters, such as scavenger receptor B1 and CD36. Resynthesis of hydrolyzed lipids. FFAs that enter the cell are transported by FA-binding proteins (40, 68) to various organelles for further processing. In the endoplasmic reticulum (ER), FAs are used to synthesize triacylglycerols, phospholipids, and cholesterol esters. Monoacylglycerols are used to synthesize triacylglycerols and phospholipids through the monoacylglycerol and glycerol-3-phosphate pathways. The monoacylglycerol pathway is the major pathway in the enterocyte. In this pathway, monoacylglycerols are esterified with an FFA by monoacylglycerol acyltransferases to form diacylglycerols, which are converted to triacylglycerols by diacylglycerol acyltransferases. Diacylglycerol can also be combined with choline and ethanolamine to synthesize phospholipids by choline and ethanolamine transferases. Free cholesterol taken up by the enterocytes is esterified in the ER by membrane-bound acyl-CoA:cholesterol acyltransferases. These conversions may enhance the diffusion gradient to favor the entry of hydrolyzed intraluminal products into the cell. Esterification may also prevent the accumulation of free cholesterol and FFA, which could have a deleterious effect on the cell. Packaging of resynthesized lipids for secretion. The lipids are then packaged into lipoproteins called chylomicrons, which are very large, spherical triacylglycerol-rich particles that also contain cholesterol and phospholipids. The chylomicron surface is covered with a phospholipid monolayer, and its core is rich in triacylglycerols and cholesteryl esters. The chylomicron particle is assembled around and held together by the large amphiphilic scaffolding protein apolipoprotein B48 (apoB48). In addition to the nonexchangeable apoB48, several exchangeable apolipoproteins can be found on the surface of the chylomicron, including apoAI, apoAIV, and apoCs. The apoB48-containing lipoproteins are synthesized in the intestine in a constitutive manner, but the amount of lipid transported with these particles changes dramatically during the postprandial state as a result of increased amounts of lipids being packaged into larger lipoprotein particles (25, 29). Chylomicron assembly begins with the translation of apoB48, which interacts with the inner phospholipid monolayer of the ER. In the absence of a sufficient supply of lipids or MTP, this nascent polypeptide is degraded by proteasomes. MTP can interact physically with and transfer lipids to the nascent apoB (27). In so doing, MTP helps apoB fold into a structural configuration that is conducive to accepting more lipids (31). The result is the formation of a smaller “primordial” particle that is similar in size to a high-density lipoprotein (HDL) (24, 25). During this step, phospholipids in the inner membrane of the ER associate with the nascent apoB48 along with a small contingent of neutral lipids. During the second step of “core expansion,” a large bolus of lipids, preferably newly synthesized triacylglycerols, is added to produce a larger lipoprotein (37). The mechanisms underlying the addition of the lipid bolus and the origin of these lipid droplets have not been elucidated. Chylomicron particles are then transported to the cis-Golgi by prechylomicron transport vesicles (PCTVs), which are larger than protein transport vesicles (5, 34, 39, 40). Liver FA-binding

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protein and protein kinase C isoform ζ are involved in the initiation of the budding of these PCTVs from the surface of the ER (45, 61). These PCTVs contain the unique vesicle-associated membrane protein 7 (60). Coat protein complex II–interacting proteins are not required for the formation of these transport vesicles, but they are important for the subsequent fusion of PCTVs with the cis-Golgi (59). Chylomicrons undergo further modifications in the Golgi that involve the addition of apoAI and glycosylation of apoB48. Chylomicrons exit the Golgi in various transport vesicles and are released across the basolateral side of the enterocyte. They become concentrated in the lacteals, mesenteric lymphatics, and thoracic duct and enter the general circulation at the level of the subclavian vein. The postprandial rise in triacylglycerol levels in the blood is due to the increased rate of entry of these particles into the general circulation. Cholesterol is another major constituent of the chylomicron. Significant amounts of cholesterol are transported in these particles. Cholesterol can also be absorbed through the HDL pathway (28, 73). The ATP-binding cassette transporter family A member 1 (ABCA1) protein plays a key role in this pathway, to the extent that an ABCA1 deficiency reduces cholesterol transport through the HDL pathway (7). Such a deficiency has no effect on cholesterol secretion through the chylomicron pathway, however. In summary, as a result of the absorption of hydrolyzed lipids from the intestine, followed by their resynthesis and packaging into chylomicrons within the enterocyte and the release of chylomicrons into the general circulation, the chylomicron is the major contributor to plasma triacylglycerol levels during the postprandial state.

ABCA1: ATP-binding cassette transporter family A member 1

Circadian Patterns of Plasma Triacylglycerol Levels In several human and animal studies, plasma triacylglycerols demonstrated robust circadian variations (6, 14, 16, 57), peaking when the animals were awake and consuming food. The increase in plasma triacylglycerol levels while feeding may be a consequence of the circadian pattern of eating during wakefulness and may not reflect an inherent circadian characteristic. Morgan and colleagues (42) studied the effects of endogenous clock and sleep/wake time on plasma triacylglycerol levels in female undergraduate students (ages 19–20 years) by subjecting them to two different sleep/wake patterns: (a) simulation of a normal circadian pattern and (b) simulation of a disrupted pattern similar to that of shift workers. In the first part of the experiment, the participants were allowed to sleep from 0000 hours to 0800 hours each day over 7 days. On “day” 8, they were awake for 26 hours, during which time they remained seated at a desk (except for toilet visits) under dim light. At each hour, they received a liquid test meal (all meals provided the same amount of calories), and plasma samples were collected to measure melatonin and triacylglycerol levels. The participants then followed a nonsynchronous sleep/wake schedule, sleeping 9 hours and then remaining awake for 18 hours. Thus, they experienced a 27-hour “day,” during which bedtime and mealtimes were delayed daily by 3 hours each day. After 27 days on this schedule, the participants resumed a 26-hour “day,” during which they were fed hourly in dim light. Plasma was again collected hourly and analyzed for melatonin and plasma triacylglycerol levels. The investigators found that disruptions in the wake/sleep cycle had no significant effect on diurnal variations in melatonin levels, which remained high during the night whether the participants were awake or sleeping. When they slept 8 hours a day at the same time each day and were fed at hourly intervals, their plasma triacylglycerol levels exhibited two peaks: the first appearing approximately 8 hours after awakening, and the second larger peak appearing with approximately 20 hours of wakefulness. When the participants were subjected to desynchronized sleep/wake cycles, the first peak was still seen 8 hours after waking, but the second peak was not observed for 20 hours after waking. These findings suggest that the endogenous clock determines the first peak (observed www.annualreviews.org • Regulation of Intestinal Lipid Absorption

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after 8 hours of arousal) and that the second larger peak, which is associated with wakefulness, is disrupted when the sleep/wake cycle is desynchronized. Thus, plasma triacylglycerol levels appear to be controlled by two factors: an internal clock and environmental factors. To determine whether changes in plasma metabolite levels are a result of eating and wakefulness, Dallmann and colleagues (11) performed an unbiased study of a metabolome of plasma and saliva collected over 40 hours at one-hour intervals from 10 healthy male volunteers who had been kept awake over that period of time in reclining beds in a room with constant light and temperature and fed an isocaloric meal and water every hour. Approximately 15% of the small molecular-weight metabolites studied displayed a circadian profile; more than 75% of these metabolites were lipids, mainly fatty acids. Approximately 15% of the metabolites in saliva also displayed circadian changes, but they were amino acids. These findings suggest a considerable degree of circadian variation in plasma levels of lipids compared with other metabolites. Interestingly, the lipid levels were highest around the subjective “lunch time” despite the hourly consumption of an isocaloric meal. This further suggests that changes in plasma lipid levels are most likely controlled by an internal clock. The origin and chemical composition of the FAs in this study were not clear. They could have been FFAs associated with albumin or the esterified FAs in glycerolipids (e.g., triacylglycerols and phospholipids), sphingolipids (e.g., sphingomyelin and ceramides), or cholesterol esters. Chua and colleagues (10) studied changes in 263 plasma lipid metabolites measured at 4-hour intervals over a 24-hour period in 20 healthy male volunteers aged 21 to 28 years. A group analysis showed that 35 of these metabolites (∼13%) exhibited circadian changes in plasma levels and that the majority of these metabolites were triacylglycerols and diacylglycerols. This finding provides additional support for triacylglycerols as the main plasma metabolite showing circadian rhythms. Triacylglycerols are transported in plasma with apoB-containing lipoproteins. Campos and colleagues (9) compared plasma apoB48 with levels of apoB100 (a normal constituent of human plasma that is manufactured in the liver) in healthy human subjects after an acute fat load. They found that plasma apoB48 levels increased sharply in larger lipoproteins within two hours following the fat-loading meal and that apoB100 levels remained relatively unchanged. This suggests that the assembly and secretion of intestinal apoB48-containing lipoproteins increases in response to dietary fat. These investigators also studied diurnal variations in plasma apoB48 and apoB100 levels in healthy subjects who were provided a standard diet for three weeks. On the day of the study, the participants had breakfast, lunch, dinner, and a snack at 0800, 1300, 1800, and 1830 hours, consuming 25%, 30%, 35%, and 10% of their total daily caloric intake at each meal, respectively. They found that apoB48 levels in the larger lipoproteins were low at 0800 hours but peaked at 1000 hours and again at 1400 hours. Another peak was observed between 1830 and 1900 hours; it dropped to a nadir at 0200 hours. Such daily changes were not evident for plasma apoB100 levels. These studies indicate that plasma apoB48 levels change several times during the day and that these changes might not be related to the amount of calories ingested. Thus, it is possible that the secretion of apoB48-containing lipoproteins by enterocytes is under the control of clock genes. Animal studies discussed elsewhere in this review provide additional evidence that the assembly and secretion of intestinal lipoproteins follow a diurnal pattern.

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Circadian Regulation of Lipid Absorption It has been suggested that the circadian variations in plasma triacylglycerol levels are related to daily changes in the production of the lipoprotein. The question then arises whether clock genes modulate lipoprotein production (which occurs mainly in the intestine and liver). Studying the effect of clock genes on intestinal lipid absorption and lipoprotein production is complicated 368

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because of the integrated nature of lipid metabolism and the dependence of different intestinal functions on circadian rhythms. For example, it is known that gastric emptying demonstrates diurnal variations. Therefore, it may be challenging to rule out the possibility that the circadian pattern of intestinal lipid absorption is secondary to changes in gastric emptying. Intestinal lipid absorption studies can be performed in the jejunum without interference from activities in other regions of the intestine. Lipid absorption can be studied in the jejunum using in situ intestinal loops or isolated enterocytes. In intestinal loop studies, jejunal segments are severed to block signals and secretions from the stomach. Radiolabeled precursors are injected into the loops, and lipid levels are measured in the plasma. In these experiments, the immediate effects of gastric emptying and pH changes on lipid absorption from the jejunum are minimized. Because other systemic controls remain intact in these loops, intestinal lipid absorption can be studied at the cellular level using primary enterocytes isolated from the jejunum. These enterocytes can be removed at different times, allowing the investigator to monitor lipid absorption at specific intervals of time. It has been shown that lipid absorption into the mouse enterocyte is maximal during the night and lowest during the day (47, 48, 50, 51). As described elsewhere in this review, lipid absorption involves the uptake of hydrolyzed lipid products into the cell, where they are assembled into chylomicrons, which are subsequently secreted from the cell. The uptake of these products is studied by incubating isolated enterocytes with radiolabeled lipids for short periods of time and analyzing the amount of radioisotope taken up by the cell. Lipid secretion can be studied by pulse-labeling the cells with radioisotopes and then monitoring the amount of label at various time points in the absence of radiolabeled precursors. In studies of the uptake and secretion of FAs and cholesterol by enterocytes, diurnal variations were observed in both processes (47, 48), which suggests that the uptake, packaging, and secretion of FAs are probably controlled by clock genes. Similar approaches have been used to study changes in the expression of various proteins involved in the uptake and packaging of FAs and cholesterol. The mRNA levels of most of the proteins examined thus far—which include apoB, MTP, apoAIV, DGAT2, FAS, and SCD-1— show diurnal patterns of expression (47). Additional studies have shown that the expression of these genes also changes in response to food entrainment (48). On the basis of circadian changes in gene expression observed in food-entrained mice compared with wild-type mice fed ad libitum, several genes involved in lipid absorption have been classified into three categories: food responsive, food and light responsive, and nonresponsive. Additional systemic studies are needed to determine whether the individual steps and proteins involved in lipid absorption are indeed regulated by circadian clock genes.

Clock Gene Dysfunction Deregulates Intestinal Lipid Absorption The role of Clock in the circadian control of lipid absorption has been derived from studies in mice that express dominant negative Clock proteins on a C57Bl/6J background that are arrhythmic, exhibiting longer periodicity (26–29 hours instead of 23–24 hours) in their locomotor activity (71). We studied the expression of clock genes and several nutrient transporters in mice that express normal and dominant negative Clock proteins and found that enterocytes express canonical clock genes in a circadian manner and that this pattern of activity changes after food entrainment. In ClkΔ19/Δ19 mice, however, these genes do not exhibit a circadian pattern of activity and are not entrained by food (47, 51). Thus, normal Clock activity appears to be important for both the circadian and food-entrained expression of intestinal clock genes. In situ intestinal loop and isolated enterocyte studies of lipid absorption have shown that FFA uptake and triacylglycerol secretion are significantly altered in ClkΔ19/Δ19 mice. The uptake of www.annualreviews.org • Regulation of Intestinal Lipid Absorption

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FFAs or secretion of triacylglycerol in the intestinal loops or in isolated enterocytes was not significantly different in these animals at midnight compared with midday, which suggests that diurnal variations in lipid absorption are lost with this mutation. Furthermore, the daytime pattern of lipid absorption in ClkΔ19/Δ19 mice was similar to the nighttime pattern seen in these mutant animals and in wild-type mice. Thus, the mutant animals appear to absorb large amounts of lipids throughout the day, and this may contribute to the hypertriglyceridemia observed in these animals. The absence of diurnal variations in nutrient absorption could be secondary to the inability of the mutant mice to sense feeding times, or it could be a result of molecular changes that affect lipid absorption. To examine the possibility that it may be a result of molecular changes, we measured the expression of various proteins involved in lipid transport. Their expression did not change throughout the day. The absence of a circadian pattern of expression might be due to alterations in molecular events that control the expression of the genes involved. In a study of the regulation of MTP by Clock in mutant and wild-type mice (47, 48, 51), we found that MTP does not follow a diurnal pattern of expression in ClkΔ19/Δ19 mice. Given that the mutant clock protein can have significant effects on gene expression, we investigated the possibility that Clock regulates MTP expression. Because MTP lacks an E-box enhancer element that is recognized by Clock:Bmal1, we looked for other transcription factors regulated by Clock that could modulate MTP expression. We reasoned that an increase in activators or a reduction in repressors might explain the increased MTP expression in ClkΔ19/Δ19 mice. SiRNA-mediated knockdown of Clock reduced or had no effect on several activator proteins, but it did reduce the expression of a repressor protein, small heterodimer partner (Shp) (51). Overexpression of SHP reduces MTP expression in human hepatoma cells, which indicates that it is indeed a repressor protein. Chromatin immunoprecipitation studies have shown that Shp interacts with several activators that associate with the microsomal triglyceride transfer protein (Mttp) promoter in mouse livers (Figure 5). These interactions occur most frequently during the day. This suggests that Shp is a clock-controlled gene that regulates lipid absorption and plasma triacylglycerols in mice. This hypothesis is further supported by the observation that Shp knockout mice show no diurnal variations in the expression of MTP and plasma triacylglycerols. In addition to hyperlipidemia, which has been observed in ClkΔ19/Δ19 mice, the mutant Clock protein has been associated with atherosclerosis in Apoe−/− mice (50). Mechanistic studies have revealed that ClkΔ19/Δ19 Apoe−/− mice absorb more lipids than do Apoe−/− mice and that macrophages in ClkΔ19/Δ19 Apoe−/− mice are defective in cholesterol efflux. Molecular studies have shown that Clock regulates cholesterol efflux in macrophages by modulating the expression of ABCA1. Thus, the mutant Clock protein increases the risk for atherosclerosis in Apoe−/− mice by increasing lipid absorption and reducing cholesterol efflux from macrophages. These studies suggest that Clockcontrolled circadian mechanisms are critical for normal lipid metabolism and that disruptions in these mechanisms increase susceptibility to hyperlipidemia and atherosclerosis.

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Perspective and Future Directions Mechanisms involved in the circadian regulation of intestinal lipid absorption are beginning to be explored. Studies have shown that some of the proteins involved in lipid absorption exhibit diurnal patterns of expression and that their expression is regulated by clock genes. Additional studies have shown that intestinal lipid absorption and the proteins involved in this process are regulated by a food-entrainable oscillator. These findings have pointed to the possibility that a coordinated interaction between light- and food-entrainable oscillators regulates intestinal lipid absorption. In the future, we need to identify the proteins involved in lipid absorption that exhibit diurnal variations and are affected by food entrainment and then determine how clock genes modulate the temporal 370

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Repression of MTP expression by Clock at midday CLOCK: BMAL1

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Shp HNF-1α

b

Nr0b2

Shp HNF-4α Mttp

Transcription at dawn

Transcription at midday

Derepression of MTP expression at night

Nr0b2

HNF-1α

HNF-4α Mttp

Transcription at night

Transcription at night

Figure 5 Temporal regulation of microsomal triglyceride transfer protein. (a) At dawn, the CLOCK:BMAL1 (circadian locomotor output cycles kaput:brain and muscle ARNT-like protein 1) heterodimer interacts with the E-box enhancer element in the promoter region of nuclear receptor subfamily 0, group B, member 2 (Nr0b2), which codes for small heterodimer partner (Shp). When Shp concentrations are high, it interacts with transcription factors such as hepatic nuclear factor (HNF)-1α and HNF-4α, which occupy the promoter region of microsomal triglyceride transfer protein (Mttp) to reduce expression at midday. (b) At night, Shp levels are probably low because of the reduced binding of Clock:Bmal1 to the Nr0b2 promoter region. Under these conditions, Shp might dissociate from the transcription factors that are involved in the enhanced expression of Mttp, thereby allowing increased expression of microsomal triglyceride transfer protein (MTP) and enhanced assembly and secretion of chylomicrons. Symbols: ↓, decrease; ↑, increase.

expression of these proteins. We will also need to identify critical molecules that resynchronize the expression of genes involved in lipid absorption after food entrainment and determine how regulatory proteins that are affected by food entrainment can override/resynchronize light-entrainment signals to temporally adjust intestinal activity to promote optimal lipid absorption at the time of food availability. It remains to be determined how neuronal and hormonal signals entrain intestinal functions in response to light and food. Identification of these pathways would enhance our understanding of the intricate molecular and neuronal communications between the brain and gut and might serve as a basis for understanding how the SCN regulates clocks in other tissues. The role of brain-specific and peripheral tissue–specific clock genes in the regulation of intestinal activities needs further investigation. This information can be deduced from studies carried out in tissue-specific gene-ablated mice. The effects of metabolic genes and their end products on the regulation of clock gene activities also require further elucidation. Studies in mice with wholebody knockout and tissue-specific ablation of genes involved in specific metabolic functions may provide clues to the regulation of central and peripheral clock genes by metabolic end products. In summary, behavioral and physiological changes are synchronized with light for optimal physiological functioning. Disruptions in central clock and irregularities in temporal food intake www.annualreviews.org • Regulation of Intestinal Lipid Absorption

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adversely affect intestinal activity. Such disruptions occur in our modern lifestyle and increase the risk for metabolic syndrome, obesity, diabetes mellitus, and atherosclerosis. By furthering our understanding of the regulation of circadian intestinal functions, we may be able to identify new targets for the development of therapeutic approaches to various metabolic disorders. SUMMARY POINTS 1. Intestinal activity is entrained by the central circadian clock and environmental stimuli involving neuronal and hormonal cues. 2. Several intestinal processes and proteins involved in lipid absorption demonstrate circadian rhythms and are regulated by clock genes. Annu. Rev. Nutr. 2014.34:357-375. Downloaded from www.annualreviews.org Access provided by University of California - San Diego on 11/18/14. For personal use only.

3. Triacylglycerols are the major constituents of plasma that exhibit circadian rhythms. 4. Clock dysfunction disrupts the circadian behavior of intestinal lipid absorption, thereby increasing the risk for hyperlipidemia and atherosclerosis in mice. 5. By understanding the mechanisms that regulate circadian patterns of intestinal activity, we may identify new ways to avoid the gastrointestinal disturbances associated with shift work and transcontinental flights.

DISCLOSURE STATEMENT The author is not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENT This work was supported in part by a grant (DK-81879) from the NIH and the VA Merit Award (BX001728).

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Contents

Annual Review of Nutrition Volume 34, 2014

Annu. Rev. Nutr. 2014.34:357-375. Downloaded from www.annualreviews.org Access provided by University of California - San Diego on 11/18/14. For personal use only.

Acyl-CoA Metabolism and Partitioning Trisha J. Grevengoed, Eric L. Klett, and Rosalind A. Coleman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 AMP-Activated Protein Kinase: Maintaining Energy Homeostasis at the Cellular and Whole-Body Levels D. Grahame Hardie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p31 Macrophages and the Regulation of Adipose Tissue Remodeling Gabriel Martinez-Santibanez ˜ and Carey Nien-Kai Lumeng p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p57 Bone Morphogenetic Proteins as Regulators of Iron Metabolism Nermi L. Parrow and Robert E. Fleming p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p77 Molecular Mediators Governing Iron-Copper Interactions Sukru Gulec and James F. Collins p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p95 Vitamin D as a Neurosteroid Affecting the Developing and Adult Brain Natalie J. Groves, John J. McGrath, and Thomas H.J. Burne p p p p p p p p p p p p p p p p p p p p p p p p p p 117 Breast Milk Oligosaccharides: Structure-Function Relationships in the Neonate Jennifer T. Smilowitz, Carlito B. Lebrilla, David A. Mills, J. Bruce German, and Samara L. Freeman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 143 Sulfur as a Signaling Nutrient Through Hydrogen Sulfide Omer Kabil, Victor Vitvitsky, and Ruma Banerjee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 171 Hypoxia and Adipocyte Physiology: Implications for Adipose Tissue Dysfunction in Obesity Paul Trayhurn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 207 Incretins and Amylin: Neuroendocrine Communication Between the Gut, Pancreas, and Brain in Control of Food Intake and Blood Glucose Matthew R. Hayes, Elizabeth G. Mietlicki-Baase, Scott E. Kanoski, and Bart C. De Jonghe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 237 Lipid-Metabolizing CYPs in the Regulation and Dysregulation of Metabolism David Bishop-Bailey, Scott Thomson, Ara Askari, Ashton Faulkner, and Caroline Wheeler-Jones p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 261

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Structure-Function of CD36 and Importance of Fatty Acid Signal Transduction in Fat Metabolism Marta Yanina Pepino, Ondrej Kuda, Dmitri Samovski, and Nada A. Abumrad p p p p p p p 281 The Emerging Role of microRNAs and Nutrition in Modulating Health and Disease Sharon A. Ross and Cindy D. Davis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 305 Epigenetic Mechanisms Affecting Regulation of Energy Balance: Many Questions, Few Answers Robert A. Waterland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 337 Annu. Rev. Nutr. 2014.34:357-375. Downloaded from www.annualreviews.org Access provided by University of California - San Diego on 11/18/14. For personal use only.

Regulation of Intestinal Lipid Absorption by Clock Genes M. Mahmood Hussain p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 357 Fifty Years of Human Space Travel: Implications for Bone and Calcium Research S.M. Smith, S.A. Abrams, J.E. Davis-Street, M. Heer, K.O. O’Brien, M.E. Wastney, and S.R. Zwart p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 377 Use and Applications of Systematic Reviews in Public Health Nutrition Patsy M. Brannon, Christine L. Taylor, and Paul M. Coates p p p p p p p p p p p p p p p p p p p p p p p p p p p p 401 Launching a New Food Product or Dietary Supplement in the United States: Industrial, Regulatory, and Nutritional Considerations John Weldon Finley, John Wescott Finley, Kathy Ellwood, and James Hoadley p p p p p p p p p 421 Indexes Cumulative Index of Contributing Authors, Volumes 30–34 p p p p p p p p p p p p p p p p p p p p p p p p p p p 449 Cumulative Index of Article Titles, Volumes 30–34 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 452 Errata An online log of corrections to Annual Review of Nutrition articles may be found at http://www.annualreviews.org/errata/nutr

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Annual Reviews It’s about time. Your time. It’s time well spent.

New From Annual Reviews:

Annual Review of Statistics and Its Application Volume 1 • Online January 2014 • http://statistics.annualreviews.org

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Editor: Stephen E. Fienberg, Carnegie Mellon University

Associate Editors: Nancy Reid, University of Toronto Stephen M. Stigler, University of Chicago The Annual Review of Statistics and Its Application aims to inform statisticians and quantitative methodologists, as well as all scientists and users of statistics about major methodological advances and the computational tools that allow for their implementation. It will include developments in the field of statistics, including theoretical statistical underpinnings of new methodology, as well as developments in specific application domains such as biostatistics and bioinformatics, economics, machine learning, psychology, sociology, and aspects of the physical sciences.

Complimentary online access to the first volume will be available until January 2015. table of contents:

• What Is Statistics? Stephen E. Fienberg • A Systematic Statistical Approach to Evaluating Evidence from Observational Studies, David Madigan, Paul E. Stang, Jesse A. Berlin, Martijn Schuemie, J. Marc Overhage, Marc A. Suchard, Bill Dumouchel, Abraham G. Hartzema, Patrick B. Ryan

• High-Dimensional Statistics with a View Toward Applications in Biology, Peter Bühlmann, Markus Kalisch, Lukas Meier • Next-Generation Statistical Genetics: Modeling, Penalization, and Optimization in High-Dimensional Data, Kenneth Lange, Jeanette C. Papp, Janet S. Sinsheimer, Eric M. Sobel

• The Role of Statistics in the Discovery of a Higgs Boson, David A. van Dyk

• Breaking Bad: Two Decades of Life-Course Data Analysis in Criminology, Developmental Psychology, and Beyond, Elena A. Erosheva, Ross L. Matsueda, Donatello Telesca

• Brain Imaging Analysis, F. DuBois Bowman

• Event History Analysis, Niels Keiding

• Statistics and Climate, Peter Guttorp

• Statistical Evaluation of Forensic DNA Profile Evidence, Christopher D. Steele, David J. Balding

• Climate Simulators and Climate Projections, Jonathan Rougier, Michael Goldstein • Probabilistic Forecasting, Tilmann Gneiting, Matthias Katzfuss • Bayesian Computational Tools, Christian P. Robert • Bayesian Computation Via Markov Chain Monte Carlo, Radu V. Craiu, Jeffrey S. Rosenthal • Build, Compute, Critique, Repeat: Data Analysis with Latent Variable Models, David M. Blei • Structured Regularizers for High-Dimensional Problems: Statistical and Computational Issues, Martin J. Wainwright

• Using League Table Rankings in Public Policy Formation: Statistical Issues, Harvey Goldstein • Statistical Ecology, Ruth King • Estimating the Number of Species in Microbial Diversity Studies, John Bunge, Amy Willis, Fiona Walsh • Dynamic Treatment Regimes, Bibhas Chakraborty, Susan A. Murphy • Statistics and Related Topics in Single-Molecule Biophysics, Hong Qian, S.C. Kou • Statistics and Quantitative Risk Management for Banking and Insurance, Paul Embrechts, Marius Hofert

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Regulation of intestinal lipid absorption by clock genes.

Plasma levels of triacylglycerols and diacylglycerols, the lipoproteins that transport them, and proteins involved in their absorption from the intest...
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