ApoA - IV : Current and emerging roles in intestinal lipid metabolism , glucose homeostasis , and 1 satiety 2 3

44 Apolipoprotein A-IV (apoA-IV) is secreted by the small intestine on chylomicrons into 45 intestinal lymph in response to fat absorption. Many physiological functions have been ascribed to 46 apoA-IV, including a role in chylomicron assembly and lipid metabolism, a mediator of reverse47 cholesterol transport, an acute satiety factor, regulator of gastric function, and finally, a modulator 48 of blood glucose homeostasis. The purpose of this review is to update our current view of intestinal 49 apoA-IV synthesis and secretion and the physiological roles of apoA-IV in lipid metabolism and 50 energy homeostasis, and to underscore the potential for intestinal apoA-IV to serve as a 51 therapeutic target for the treatment of diabetes and obesity related disease. 52 Introduction 53 Apolipoprotein A-IV (apoA-IV) was discovered more than 30 years ago as a major protein 54 component of lymph chylomicrons in the post-prandial state (31, 90, 99). Human apoA-IV is found 55 in the apoA-I/C-III/A-IV gene cluster on chromosome 11 (43). This gene cluster is essential for 56 lipoprotein metabolism and the maintenance of plasma lipid levels, and as such, is a modulator of 57 cardiovascular disease risk. In humans, apoA-IV is synthesized only by the small intestine; in rodents 58 a small amount is also synthesized by the liver (31, 51, 84). The jejunum is the major site of apoA59 IV synthesis, but it is also produced in the duodenum and ileum (42). 60 ApoA-IV is secreted into the lymph on chylomicrons in response to lipid feeding. During 61 subsequent metabolism of chylomicrons in the periphery, approximately 25% of the apoA-IV is 62 transferred to HDL in the plasma and the rest is found in the lipoprotein-free fraction (24, 69). 63 Therefore, the presence of apoA-IV in the periphery (on chylomicrons, circulating on HDL, and free 64 in the plasma) is linked to intestinal lipid absorption and chylomicron packaging. 65 Many physiological functions have been ascribed to apoA-IV, including a role in chylomicron 66 assembly and lipid metabolism, a mediator of reverse-cholesterol transport, an acute satiety factor, 67 regulator of gastric function, and finally, a modulator of blood glucose homeostasis. This review will 68 highlight the evidence for these physiological roles of apoA-IV, as well as emerging evidence of a 69 role for intestinal apoA-IV in mediating chronic disease. 70 71 Role of apoA-IV in chylomicron assembly and lipid absorption 72 Nearly 40 years after the first description of apoA-IV, a considerable body of evidence links 73 apoA-IV to intestinal triglyceride absorption and secretion. ApoA-IV is responsive to nutritional 74 status, and dietary fat absorption results in increased apoA-IV synthesis and secretion in a dose75 dependent manner (39–42). Dietary fat absorption also results in increased plasma apoA-IV levels 76 (32), and in patients with malabsorptive disorders (such as chronic pancreatitis) apoA-IV levels in 77 plasma do not rise in response to a fatty meal (45). ApoA-IV production requires chylomicron 78 synthesis, and is upregulated in response to dietary long chain fatty acids (the absorption of which 79 requires chylomicron synthesis), but not when short chain fatty acids are absorbed through the 80 portal blood (44). Blocking chylomicron secretion inhibits the rise in apoA-IV synthesis induced by 81 fat absorption (88). Finally, apoA-IV co-localizes with apo-B in the chylomicron secretory pathway 82 (50). 83 Despite this considerable evidence linking apoA-IV with triglyceride absorption and 84 chylomicron formation, in vivo models with gain or loss of apoA-IV (apoA-IV transgenic or KO 85 mice), do not result in a loss of triglyceride secretion, or in the secretion of triglyceride-deficient 86 chylomicron particles (1, 46, 47, 97). This dichotomy between the apparent role of apoA-IV in 87 intestinal triglyceride packaging and secretion and its lack of effect in genetically modified mice has 88 led to two prevailing conclusions: 1) that apoA-IV plays a substantive role in triglyceride secretion, 89 or 2) that its main physiological role may be extra-intestinal. We will discuss both. 90 Evidence supporting the role of apoA-IV in intestinal triglyceride absorption and packaging 91 is significant. In newborn swine enterocytes (IPEC-1 cells) the overexpression of apoA-IV greatly 92 enhances the secretion of triglyceride, cholesterol ester, and phospholipid in chylomicron/VLDL 93 particles (28, 62, 95, 100). This role of apoA-IV in IPEC-1 cells appears to rely on a region of apoA94 IV (residues 344-354), the presence of which stimulates the secretion of larger, more triglyceride95 rich lipoproteins in a dose-dependent manner (62). This role of apoA-IV may be due to its ability to 96 stabilize the expanding lipid/aqueous interface during lipoprotein synthesis, resulting in the 97 formation of larger triglyceride-rich chylomicrons. For apoA-IV to facilitate the expansion of 98 triglyceride-rich lipoproteins, it is likely that it would interact with apoB in the endoplasmic 99 reticulum (as part of the triglyceride secretory pathway). Studies in both COS and McA-RH7777 100 hepatoma cells have shown that the ER-retained apoA-IV (tagged with the ER retention signal 101 KDEL) inhibited the early stages of triglyceride-rich lipoprotein assembly (23) and that 102 overexpression of WT apoA-IV is able to modulate the secretory trafficking of apoB leading to an 103 enhancement of particle expansion and triglyceride secretion (95). In addition to this work in cell 104 culture, work by Simon and Weinberg suggests that apoA-IV expression mediates the expression of 105 other triglyceride packaging and nutrient sensing genes in the intestine, which may act to regulate 106 regional lipid absorption (83). 107 Despite this evidence, most in vivo studies show that the total loss of apoA-IV or the 108 transgenic overexpression of apoA-IV do not result in a loss or gain of triglyceride secretion, nor in 109 the secretion of triglyceride-deficient chylomicron particles. Using the conscious in vivo lymph 110 fistula model to directly assess intestinal lipoprotein secretion [Figures 1 and 2], apoA-IV KO mice 111 have been shown to have no change in dietary lipid absorption, or in the cumulative secretion of 112 radiolabeled triglyceride into lymph (under both an acute bolus acute dose or continuous 113 intraduodenal lipid infusion paradigms)(47). In mice expressing high levels of human apoA-IV, lipid 114 absorption was measured using a variety of labeled metabolic tracers and there was no difference 115 found (1). These data suggest apoA-IV is not required for normal dietary lipid absorption. 116 Though these in vivo studies show that apoA-IV may not be required for triglyceride 117 absorption (since the amount of triglyceride absorbed and secreted was the same between 118 models), the loss of apoA-IV in apoA-IV KO mice does result not only in the secretion of larger 119 chylomicrons from the intestine but these chylomicrons were less rapidly cleared from plasma than 120 WT chylomicrons [Figures 3 and 4](46). This suggests that apoA-IV has a previously unknown role 121 in regulating chylomicron metabolism and is therefore important in regulating plasma lipid 122 metabolism. It should also be noted the chylomicrons derived from the apoA-IV null animals also 123 metabolized more slowly than those derived from WT animals, suggesting that apoA-IV may not be 124 freely interchangeable between the free and the HDL-bound compartment with the circulating 125 chylomicrons. 126 This dichotomy in findings between cell culture and in vivo models is not only a conundrum, 127 but also remains an important unresolved issue especially in light of the importance of apoA-IV in 128 mediating cardiovascular disease risk. Over-expression of apoA-IV protects transgenic mice from 129 atherosclerosis caused by either apoE-deficiency, LDL receptor deficiency or cholesterol feeding (8, 130 16, 72). Interestingly, apoA-IV knock-out (KO) may also confer protection against diet-induced 131 atherosclerosis by decreasing the rise in plasma lipid levels following the feeding of high fat; most 132 notably by decreasing total triglyceride and cholesterol levels (70, 97). Since both the 133 overproduction of chylomicrons during chronic disease (such as diabetes)(12, 15) and their 134 subsequent clearance from plasma are both involved in the development of cardiovascular disease, 135 apoA-IV may be indeed be playing multiple important roles. ApoA-IV may be modulating the 136 retention of triglyceride-rich lipoproteins in plasma through its regulation of chylomicron clearance, 137 and may also regulate the production of particles (either by stimulating bulk loading or triglyceride 138 onto apoB-containing chylomicrons, or by modulating regional gene expression to regulate 139 regional lipid absorption)(46, 62, 83, 95). It is unknown how many apoA-IV molecules are secreted 140 from the intestine per chylomicron particle, or whether these changes during chronic disease states, 141 are due to the potential role of apoA-IV in chylomicron clearance and its major role in modulating 142 glucose homeostasis. Another point worth noting is the fact that apoA-IV protects lipoproteins 143 from oxidative processes [72] and so it may be an extremely complicated task to sort out the 144 relative importance of the various roles played by apoA-IV in lipid metabolism and cardiovascular 145 events associated with both the protection and the promotion of vascular diseases. 146 147 Role of ApoA-IV in reverse cholesterol transport 148 ApoA-IV is the largest member of the exchangeable apolipoprotein family. After its synthesis 149 in the enterocytes, it is subsequently secreted on the surface of chylomicrons, which are then 150 rapidly acted upon by lipoprotein lipase in the periphery. As chylomicrons undergo lipolysis in the 151 post-prandial state, apoA-IV dissociates from the chylomicrons as a result of the shrinking surface 152 and is then associated with HDL and also is found in the lipid-free fraction of plasma (24, 69). As 153 such, apoA-IV can promote cellular cholesterol efflux as a potent activator of lecithin-cholesterol 154 acyltransferase (86). In several cell types (including both fibroblasts and adipocytes) apoA-IV 155 containing particles are cholesterol acceptors. This role of apoA-IV as a mediator of cholesterol 156 efflux may partly explain the observation that apoA-IV transgenic mice have significantly reduced 157 atherogenesis than their WT counterparts (16). 158 Additional physiological functions have been attributed to intestinal apoA-IV expression, 159 including its role as an anti-oxidant and anti-inflammatory factor (69, 78, 85). Although these are 160 potentially important functions of apoA-IV, these functions are also carried out by other 161 apolipoprotein and are not unique to apoA-IV. 162 163 Role of ApoA-IV as a satiety factor 164 ApoA-IV has been reported to reduce food intake. When rodents receive an intravenous 165 administration of chylous lymph this significantly reduces food intake, whereas infusion of chylous 166 lymph missing apoA-IV does not recapitulate this effect (21). Furthermore, the administration of 167 native apoA-IV inhibits food intake (21, 22). Subsequent studies have determined that central and 168 peripheral administration of recombinant apoA-IV inhibits food intake, and is not toxic (21, 56, 61); 169 whereas administration of apoA-I at comparable doses has no effect on satiety (21). These 170 observations suggest that both intestinal apoA-IV (secreted in response to dietary lipids) and 171 exogenous apoA-IV injected in the periphery can regulate food intake. 172 173 ApoA-IV regulation of gastric emptying and secretion 174 Lipid, particularly long-chain triglyceride, inhibits gastric motor and secretory function (38). 175 Free fatty acids of chain length 12 or greater are much more effective in the inhibition of gastric 176 motility than C10 fatty acids (38). Intestinally absorbed long-chain fatty acids are re-synthesized 177 into triglyceride in the enterocytes and are subsequently secreted in chylomicrons (89). Mesenteric 178 lymph enriched with chylomicrons collected from lymph fistula rats reduces gastric motor function 179 (27). In contrast, when chylomicrons are removed from the chylous lymph, gastric motility 180 significantly increases. In subsequent studies, purified recombinant apoA-IV was shown to 181 significantly inhibits gastric motility (26, 27); in contrast, apoA-IV KO mice have significantly faster 182 gastric emptying and greater secretion of gastric acid after an ingested meal (98). It has been 183 demonstrated that this mechanism involves a negative feedback of apoA-IV containing 184 chylomicrons on gastric motility via cholecystokinin-1R (CCK-1R) on capsaicin-sensitive vagal 185 afferent nerve terminals (26). 186 187 Role of apoA-IV in glucose homeostasis and insulin secretion 188 Recently, studies in apoA-IV KO mice have revealed a novel role for apoA-IV in glucose 189 metabolism and insulin secretion (92). ApoA-IV KO mice are glucose intolerant, accompanied with 190 attenuated insulin secretion upon glucose challenge, suggesting that apoA-IV is essential for 191 physiological blood glucose control. Administration of exogenous apoA-IV (in an amount 192 equivalent to the amount secreted in response to a fatty meal) dramatically improved glucose 193 tolerance along with the restoration of insulin secretion (92). Therefore, it is likely that 194 physiologically, the apoA-IV secreted after a fat-rich meal could be rapid and sufficient enough to 195 stimulate insulin secretion. 196 As an endogenous regulator of insulin secretion, intestinal apoA-IV comprises one of the 197 components of the enteroinsular axis; i.e., the collective signaling pathways between the gut and 198 the pancreatic islets that control nutrient-dependent insulin secretion. ApoA-IV has a half-life of 199 about 7-8 hours, which is much longer than that of typical incretins such as GLP-1 and GIP (44) 200 whose half life is a few minutes. For example, the glucose-lowering effect of a single injection of 201 apoA-IV lasts about 12 hours in KKAy diabetic mice (92). 202 Experiments in isolated islets have examined the direct effect of apoA-IV on β-cell insulin 203 secretion. ApoA-IV induces a dose-dependent increase in insulin release when the islets are 204 exposed to 20 mM, but not 3 mM glucose. In addition, functional KATP and calcium channels are 205 required for the action of apoA-IV on insulin secretion, but apoA-IV does not act directly to 206 stimulate calcium influx (92). Membrane depolarization by closure of the KATP channels activates 207 voltage-gated Ca channels and lead to Ca influx, which triggers the exocytosis of insulin. 208 Instead, the action of apoA-IV seems to lie downstream of Ca influx and amplify insulin exocytosis 209 (92). While these data suggest apoA-IV acts on the late stages of insulin secretion, the precise 210 molecular mechanism remains to be elucidated. ApoA-IV exhibits high-affinity binding to isolated 211 human hepatocellular plasma membranes, which is saturable, reversible, and specific, supporting 212 the idea that a membrane protein is involved in binding (96). However, a specific receptor for 213 apoA-IV has not been discovered to date. However, apoA-IV is able to enter the hepatocyte and 214 interact with the transcription factor NR1D1 (52). ApoA-IV inhibits gluconeogenesis through 215 NR1D1. Thus through the insulinotropic and the anti-gluconeogenic action of apoA-IV, apoA-IV 216 plays an important role in glucose homeostasis following the ingestion of food. 217 We have proposed that it is the change in circulating apoA-IV levels between fasting and 218 feeding (rather than the absolute level of circulating level of apoA-IV) that stimulates insulin 219 secretion. This may be another reason why the loss of regulation in apoA-IV that occurs in 220 response to a chronic high fat diet correlates with poor glucose homeostasis (3, 41, 49, 55, 59, 94). 221 222 Actions of apoA-IV in the brain. 223 Compelling evidence suggests that central (rather than circulating) apoA-IV is important for 224 the control of food intake and body weight: 1) when I-labeled apoA-IV is injected IV in mice, this 225 apoA-IV does not cross the blood brain barrier (81); 2) intracerebroventricular (ICV) injection of 226 apoA-IV significantly and dose-dependently reduces food intake without eliciting signs of toxicity 227 (22); 3) apoA-IV mRNA and protein are present in the rat hypothalamus neurons (54); using 228 immunohistochemistry, apoA-IV distribution is found in brain areas involved in energy homeostasis, 229 including the arcuate (ARC) and ventromedial hypothalamic (VMH), the paraventricular (PVN), 230 dorsomedial (DMN) nuclei, and the nucleus of the solitary tract (NTS)(81); finally, 4) double-staining 231 immunohistochemistry with a neuronal marker revealed apoA-IV is largely present in neurons. 232 233 Circadian rhythm of hypothalamic apoA-IV. 234 To determine the roles of central apoA-IV in the regulation of daily food intake, Liu et al. 235 examined the diurnal patterns of hypothalamic apoA-IV gene and protein expression (58). In freely 236 feeding rats, the hypothalamic apoA-IV mRNA and protein levels were found to peak 3 h after 237 lights on, and with a nadir 3 h after lights off (the normal feeding period of rodents). To make 238 sure that it is not just a coincidence, we restricted the feeding period to four hours during the light 239 cycle. In the food-restricted rats, the daily patterns of the apoA-IV fluctuation were altered with a 240 marked decrease in hypothalamic apoA-IV mRNA and protein levels during the 4 h-feeding period 241 of the light phase. Although corticosterone (CORT) secretion temporally coincided with the 242 decreasing phase of apoA-IV in the hypothalamus, the diurnal expression of hypothalamic apoA-IV 243 is not regulated by CORT because depletion of CORT by adrenalectomy significantly decreased, 244 rather than increased, hypothalamic apoA-IV mRNA and protein levels (58). The observations that 245 apoA-IV levels in the hypothalamus were inversely related to food intake during the normal diurnal 246 cycle as well as in the period of restricted feeding implies that hypothalamic apoA-IV is involved in 247 the regulation of daily food intake. 248 249 Interaction of apoA-IV with other neuroand endocrine peptides in the regulation of food 250 intake and energy metabolism. 251 252 Neuropeptide Y. NPY is a hypothalamic neuropeptide with regulatory action on food intake. Liu, 253 et. al. have demonstrated there is an interaction of apoA-IV with neuropeptide Y (NPY) (57). ICV 254 injection of NPY alone significantly increased hypothalamic apoA-IV expression in a dose255 dependent manner (57). While intraduodenal infusion of lipid also increased hypothalamic apoA-IV 256 mRNA levels, there was no further significant increment with the combination of ICV injection of 257 NPY and lipid infusion, indicating that there is a lack of potentiation in the regulation of 258 hypothalamic apoA-IV gene expression by both NPY and lipid. One possibility to explain why 259 administered NPY increases apoA-IV gene expression is to maintain a balance between these two 260 opposing factors thereby regulating food intake, which could be further investigated in apoA-IV KO 261 mice. 262 263 Melanocortin system. Hypothalamic melanocortin system plays an important role in the regulation 264 of body weight. α-MSH (α-Melanocyte-stimulating hormone) derived from proopiomelanocortin 265 (POMC) neurons exerts a tonic inhibitory influence over feeding through melanocortin type 3 and 4 266 receptors (MC3/4-R) in the hypothalamus. Agouti-related protein (AgRP) is a peptide produced in 267 the ARC, which antagonizes MC3/4-R. Therefore, when administered centrally, AgRP elicits 268 hyperphagia (17). Consistent with this, it has been demonstrated that ICV administration into the 269 third ventricle (i3vt) of metallothionein-II (MT-II), a synthetic MC3/4-R agonist, potently reduces 270 feeding (57), whereas i3vt administration of SHU9119, a synthetic MC3/4-R antagonist, blocks the 271 anorectic effect of MT-II (33). To determine whether apoA-IV exerts its anorectic effect through the 272 melanocortin system, These data suggest that the brain apoA-IV system suppresses food intake by 273 stimulating the ARC POMC system (81). Gotoh et al. also found that ICV administration of MT-II 274 potentiated a subthreshold dose of apoA-IV suppression of 30-min feeding in rats, and the 275 anorectic effect of ICV apoA-IV was almost completely attenuated by a subthreshold dose of 276 SHU9119 (29). These data support a synergistic interaction between apoA-IV and melanocortins 277 that reduces food intake by acting downstream of the ARC. 278 279 Leptin. Leptin is a peptide synthesized and secreted by adipocytes (9, 20). Like apoA-IV, leptin 280 reduces food intake (35, 80) and it interacts synergistically with apoA-IV in this regard (14, 82). 281 Leptin is an important component of lipid homeostasis and its circulating level is directly correlated 282 with the amount of fat in the body (20). Thus, a high fat diet increases plasma leptin levels in obese 283 humans and rodents (5, 10, 20). Leptin directly acts on leptin receptors in intestinal cells (6, 32, 34, 284 71) and attenuates the lipid-induced stimulation of apoA-IV synthesis and secretion (14, 66). 285 Consumption of a high fat diet initially increases plasma apoA-IV levels in rodents, however chronic 286 consumption of a high fat diet results in an attenuation of this effect on apoA-IV (41, 59, 94). This 287 suggests that the elevation of circulating leptin induced by high fat feeding might attenuate 288 intestinal apoA-IV response to the consumption of a lipid meal. Animal studies also support this 289 notion. Intestinal apoA-IV levels are markedly increased in ob/ob mice, which lack the ability to 290 make leptin. Further studies are required to determine the molecular mechanism in the regulation 291 of intestinal apoA-IV by leptin, but it is possible that leptin decreases intestinal apoA-IV gene 292 expression to transport fewer lipids from the small intestine to the circulation. 293 Central administration of leptin decreases food intake and increases energy expenditure. 294 Shen et al. demonstrated that the hypothalamic apoA-IV mRNA levels is significantly lower in 295 leptin-deficient obese (ob/ob) mice than wild type controls (82). Intragastric infusion of a lipid 296 emulsion significantly stimulated hypothalamic apoA-IV gene expression in lean controls but not in 297 ob/ob mice. When leptin was ip administered daily for 5 d, it significantly stimulated apoA-IV 298 mRNA levels in the hypothalamus of ob/ob mice, compared to the pair-fed controls. Additionally, 299 the fasting-induced reduction of apoA-IV mRNA levels was also restored by centrally administered 300 leptin. Shen et al. also demonstrated that apoA-IV is present in leptin-sensitive phosphorylated 301 signal transducer and activator of transcription-3 (pSTAT3)-positive cells of the ARC as determined 302 by immunohistochemistry (82). The stimulatory effect of leptin on apoA-IV protein expression was 303 significantly attenuated by the suppression of STAT3 expression by small interfering RNA (siRNA) 304 in cultured primary hypothalamic neurons. These observations imply that leptin can regulate apoA305 IV gene and protein expression in the hypothalamus, and such effects are at least partially via the 306 STAT3 signaling pathway. More importantly, when subthreshold doses of leptin (1 μg) and apoA-IV 307 (0.5 μg) were ICV co-administered, it leads to a significant reduction of food intake in rats, 308 indicating the existence of a functional synergistic interaction between leptin and apoA-IV, leading 309 to suppression of food intake. The differential effect of leptin on apoA-IV expression in the 310 hypothalamus and the enterocytes is intriguing and deserve to be further investigated. 311 312 Cholecystokinin (CCK). The secretion of CCK by the intestinal L cells is stimulated by the 313 consumption of lipid and protein. The fat-induced stimulation is associated with the the 314 formation and secretion of chylomicrons (53, 74, 75). CCK stimulates gallbladder contraction and 315 pancreatic enzyme secretion, modulates intestinal motility, and inhibits food intake (13, 25, 104). 316 Exogenous administration of CCK reduces short-term food intake in experimental rats by reducing 317 meal size (25, 67) and this satiating effect is abolished by deactivation of vagal afferents with 318 capsaicin, vagotomy, or CCK-1R antagonist. Thus, the satiation signals are relayed via CCK-1R on 319 vagal afferent nerves (64, 74, 75, 77, 79). Secretion of CCK and apoA-IV are both induced by fat 320 absorption and this lipid-induced stimulation is dependent on chylomicron formation (30, 37, 74). 321 In addition, both act peripherally (through either intraperitoneal or intravenous administration) as 322 well as centrally to reduce food intake (22, 25, 63, 93). 323 We conducted a series of experiments to determine if there is interaction between CCK and 324 apoA-IV on the control of food intake and if this is mediated via CCK-1R on vagal afferent fibers. 325 Using sub-threshold doses of either apoA-IV or CCK a combination of both produces a short326 term satiation for 1 h and this satiation effect is attenuated by CCK-1R antagonist, lorglumide (61). 327 Increasing the doses of CCK and apoA-IV are increased, the satiating effect is prolonged. 328 Furthermore, we reveal that apoA-IV-elicited satiation is greatly attenuated in CCK-KO mice (60). In 329 contrast, CCK-induced satiating response is greater in apoA-IV KO mice due to increased CCK-1R 330 expression in the nodose ganglia and/or nucleus of the solitary tract (NTS)(102). These findings 331 suggest that endogenous apoA-IV and CCK interact with each other to reduce food intake via CCK332 1R and systemic apoA-IV requires an intact CCK system in order for it to work physiologically. This 333 working model was first proposed by Raybould and colleagues that the stimulation of apoA-IV 334 release induced by lipid absorption results in the stimulation of CCK secretion, followed by 335 activation of the vagal nerves via a CCK-1R-dependent pathway. 336 Circulating apoA-IV does not cross the blood-brain barrier (81), but is able to increase CCK337 elicited activity in vagal afferent fibers, which discharge via a CCK-1R-dependent pathway in vitro 338 (26). Blockade of CCK-1R using lorglumide significantly reduces apoA-IV-induced satiation, 339 suggesting that CCK-1R on vagal afferent nerves plays an important role in relaying apoA-IV340 induced anorectic signals to the brain (60). Subdiaphragmatic vagal deafferentation (SDA) is a 341 surgical procedure which eliminates all neuronal signals mediated via vagal afferent fibers from the 342 upper gut, including the liver, while leaving half of the vagal efferent fibers intact (4, 68). We have 343 demonstrated that Long-Evan rats with SDA have attenuated apoA-IV-elicited satiation effect (60). 344 These findings support the notion that systemic apoA-IV and CCK work co-dependently to 345 suppress food intake and peripheral apoA-IV requires an intact CCK system and CCK-1R on vagal 346 afferent nerves to exert its satiety effects in the brain. 347 348 Regulation of apoA-IV synthesis and secretion 349 ApoA-IV synthesis and secretion is stimulated by active lipid absorption and chylomicron 350 formation in the small intestine (36, 37). Interestingly, neither protein nor carbohydrate absorption 351 by the small intestine effects apoA-IV secretion. The stimulation of apoA-IV secretion occurs rapidly 352 (within half an hour following the beginning of active lipid absorption) (21). ApoA-IV synthesis and 353 secretion relies on chylomicron formation. This effect is abolished when chylomicron formation is 354 inhibited (37), and in studies in which short chain fatty acids are infused directly into the intestine 355 (which are directly secreted into the portal vein), there is no stimulation of apoA-IV synthesis and 356 secretion (42). 357 Fasting and refeeding modulates both circulating and central apoA-IV levels (54). Feeding 358 overnight (the normal feeding periods for rodents) significantly decreases apoA-IV gene expression 359 in both the jejunum and hypothalamus. Refeeding with low-fat chow in fasting rats for 4 h evokes 360 a pronounced increase of apoA-IV gene expression in jejunum, but not in the hypothalamus. 361 However, refeeding with lipid restores apoA-IV mRNA levels in both jejunum and hypothalamus in 362