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Thematic Review |
Department of Biology, Neurobiology and Behavior Program, Georgia State University, Atlanta, GA 30302-4010
Published, JLR Papers in Press, April 25, 2007.
1 To whom correspondence should be addressed. e-mail: bartness{at}gsu.edu
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ABSTRACT |
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Supplementary key words obesity • humans • viral tract tracing • melanocortin • melatonin • denervation • proliferation • adipocyte • hamster • rat • mouse • human
Abbreviations: BAT, brown adipose tissue; BrDU, bromodeoxyuridine; CGRP, calcitonin gene-related peptide; CNS, central nervous system; DRG, dorsal root ganglia; DWAT, dorsosubcutaneous white adipose tissue; EPI, epinephrine; EWAT, epididymal white adipose tissue; FCN, fat cell number; FCS, fat cell size; HIV, human immunodeficiency virus; HSL, hormone-sensitive lipase; HSV, herpes simplex virus; IBAT, interscapular brown adipose tissue; -ir, immunoreactivity; IWAT, inguinal white adipose tissue; LD, long day; MC4-R, melanocortin 4-receptor; MEL, melatonin; NE, norepinephrine; NETO, norepinephrine turnover; 6OHDA, 6-hydroxy-dopamine; PRV, pseudorabies virus; PSNS, parasympathetic nervous system; PVN, paraventricular nucleus; RWAT, retroperitoneal white adipose tissue; SCN, suprachiasmatic nucleus; SD, short day; SNS, sympathetic nervous system; TH, tyrosine hydroxylase; WAT, white adipose tissue
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INTRODUCTION |
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325,000 deaths per year (6) to as low as 26,000 (7), making overweight/obesity either the number two or number seven cause of death in adults in the United States, respectively. Considerable research effort has focused on determining the factors involved in the development of obesity in nonhuman animals; consequently, our understanding of these is substantial, but still far from complete. Somewhat surprisingly, less effort has focused on determining the factors involved in the reversal of obesity in nonhuman animals. We have contributed to these latter efforts by studying the reversal of a naturally occurring seasonal obesity in Siberian hamsters (Phodopus sungorus). In the process of conducting this work, we discovered the importance of the sympathetic nervous system (SNS) in lipid mobilization from white adipose tissue (WAT) as well as more recently realizing the possible importance of the sensory innervation of WAT. Several overviews of the role of the SNS in lipid mobilization were published recently (8–11). To give a better understanding of how we came to the realization that lipid mobilization occurs primarily through the sympathetic innervation of WAT, we will describe some background studies focused on the reversal of photoperiod-induced obesity that ultimately led us to define and test the SNS innervation of WAT. |
CHANGES IN THE PHOTOPERIOD (DAYLENGTH) TRIGGER A NATURALLY OCCURRING SUITE OF PHYSIOLOGICAL AND BEHAVIORAL RESPONSES |
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When Siberian hamsters are exposed to long days (LDs), they develop a severe obesity [50% body fat (17–19)], a level as extreme as that seen in genetically inbred strains of rats and mice [e.g., ob/ob mice (20) and Zucker fa/fa rats (21)]. The LD-induced obesity of Siberian hamsters is completely reversible by exposure to short days (SDs), with the decrease in body fat being most rapid during the first 5–6 weeks of SDs (17, 18, 22–24). Moreover, the body fat loss during this time occurs without a concomitant decrease in food intake but eventually (>6 weeks of SD exposure) is accompanied by an 30% decrease in food intake (17, 25, 26); thus, the initial body fat loss is attributable to increased energy expenditure. Unlike other photoperiod-induced obesity models (27), the SD-induced decrease in WAT is nonuniform, with the more internally located visceral fat pads mobilizing their lipid stores first and to a greater extent than the more externally located subcutaneous fat pads (18, 28, 29). This differential lipid mobilization across WAT depots also is a characteristic of body fat loss in exercising/dieting/fasting humans (30, 31), although the exact pattern and fat pads involved are not homologous. Nevertheless, this highlights another advantage of using this model to study obesity reversal. It was this differential pattern of lipid mobilization and, as we shall see, differential sympathetic drive to WAT, that fascinated us early in our studies and prompted us to delve into the mechanisms underlying this phenomenon.
How does the daylength cue get transduced into a biological signal? The photoperiod cue is received by the retina and transmitted through a multisynaptic pathway that includes the suprachiasmatic nucleus [SCN; the primary biological clock (32)], the hypothalamic paraventricular nucleus (PVN), and the intermedial lateral horn of the spinal cord that ultimately terminates in the pineal gland (33), where this photic information is transduced into an endocrine signal via the rhythmic pattern of secretion of its principal hormone, melatonin (MEL) (15). Specifically, MEL is only synthesized and secreted by pinealocytes at night; thus, the duration of night is faithfully coded by the duration of nocturnal MEL secretion, thereby triggering seasonal responses: long durations of the circulating MEL signal short "winter-like" days (SDs), and short durations of circulating MEL signal long "summer-like" days (LDs) (for reviews, see Refs. 15, 34). The MEL receptor subtype mediating photoperiodic responses is the MEL1a receptor [or the mt1 receptor (35)]. MEL itself does not directly trigger lipolysis; incubation of isolated white adipocytes in vitro with physiological or even "industrial-strength" doses of MEL does not increase lipolysis (36). We tested hormones that both changed seasonally in Siberian hamsters and either directly or indirectly affected lipolysis in these or other animals (e.g., insulin, prolactin, glucocorticoids, gonadal steroids, thyroid hormones). None of these hormones, at least singly, accounted for the SD-induced decrease in body fat, as tested in depletion-repletion studies (for review, see Ref. 37).
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THE SD (MEL)-INDUCED DECREASE IN BODY FAT IS VIA THE SNS INNERVATION OF WAT AND NOT THROUGH ADRENAL MEDULLARY EPINEPHRINE |
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Histological evidence for the SNS innervation of WAT has a long history beginning with Dogiel (45) in 1898, when he reported the staining of nerves innervating WAT. Confusion arose initially regarding which components of WAT received the SNS innervation, because there were descriptions of only the sympathetic innervation of blood vessels in WAT as well as other descriptions of the sympathetic innervation of the WAT parenchyma (for reviews, see Refs. 9, 11). It is now realized that the failures to detect neural fibers within the parenchymal space of WAT of normal ad libitum-fed laboratory rats was the result of the tight packing of the fully loaded, lipid-filled adipocytes in the WAT matrix. By fasting animals and thus inducing lipolysis, the consequent lipid mobilization causes decreases in fat cell size (FCS), thereby exposing more parenchymal space and revealing catecholaminergic (via histofluorescence) innervation of both the vasculature and white adipocytes (46–49). The nerves innervating white adipocytes are not as boutons juxtaposed closely to the fat cells; rather, they are of the en passant variety (50).
This histofluorescence labeling of catecholaminergic nerve fibers within WAT is not considered direct evidence of SNS innervation; tract tracing of the sources of input to the WAT pads is the sine qua non of direct innervation of a tissue. Therefore, we used fluorescent tract tracers (DiI, FluoroGold) to demonstrate bidirectionally direct neuroanatomical projections of postganglionic neurons residing in the sympathetic chain to WAT (26). In addition, we found evidence of largely, but not completely, separate postganglionic neurons within the sympathetic chain innervating inguinal white adipose tissue (IWAT) and epididymal white adipose tissue (EWAT) (26). Such segregation of populations of postganglionic neurons innervating individual WAT pads could provide the neuroanatomical underlying basis for the differential mobilization of lipid across individual WAT depots, as occurs with most lipolysis-promoting stimuli [e.g., starvation (51, 52), estradiol (52), and leptin (53)]. A similar conclusion was drawn more than a decade later (54) when one of two strains of the pseudorabies virus (PRV), a transneuronal tract tracer (see directly below), each possessing a distinct fluorescent reporter, was injected into mesenteric and subcutaneous WAT. This resulted in the identification of an additional segregation of neurons innervating these two WAT pads but located a synapse earlier than those we found in the sympathetic chain (26), in the intermediolateral horn of the spinal cord [i.e., the sympathetic preganglionic neurons (54)]. Although partially satisfying, the determination of the preganglionic and postganglionic sympathetic innervation of WAT left open to speculation the origins of the sympathetic outflow to WAT from the brain.
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CENTRAL NERVOUS SYSTEM ORIGINS OF THE SYMPATHETIC OUTFLOW TO WAT HAVE BEEN DEFINED USING VIRAL TRACT TRACING METHODOLOGIES |
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We retrogradely labeled the SNS outflow from brain to WAT (IWAT and EWAT) in laboratory rats and Siberian hamsters (62) and later to retroperitoneal white adipose tissue (RWAT) in Siberian hamsters (63), thereby identifying CNS-SNS-WAT circuitries (for review, see Ref. 11, 64). The general patterns of infection after WAT inoculation with PRV prominently show that WAT (62), as well as brown adipose tissue (BAT) (65), receives input from CNS cell groups that are part of the general SNS outflow from the brain [PVN, A5 of the noradrenergic lateral tegmental system, caudal raphe region, rostral ventrolateral medulla, ventromedial medulla (66), and many other areas as well (67)]. Some of these include the hindbrain: area postrema, nucleus of the solitary tract, and raphe regions (e.g., pallidus, obscurus, magnus, and dorsal raphe and the raphe cap); midbrain: periaqueductal gray and pontine regions; and forebrain: hypothalamic arcuate, preoptic, SCN, PVN and dorsomedial nuclei, and thalamic paraventricular and reuniens nuclei (for a complete list, see Ref. 67).
Although there were some differences in the degrees of infection (i.e., neurons participating in the circuit) at several sites across the neural axis among these WAT pads in Siberian hamsters, the general patterns of infection were more similar than different for the same WAT depots between Siberian hamsters and laboratory rats (62, 63). This should not be misconstrued as a dismissal of "viscerotopic" sympathetic innervation of WAT at some point(s) across the neuroaxis (see above for evidence of preganglionic and postganglionic viscerotopic patterns of sympathetic nerves innervating WAT); however, unequivocal demonstration of the viscerotopic organization of WAT circuitries requires careful studies using isogenic strains of the PRV, each with unique fluorescent or other reporter injected into separate WAT pads (54). From the perspective of shared circuits, we found some common neurons in the sympathetic outflow circuits innervating a WAT (IWAT) and a BAT depot [interscapular brown adipose tissue (IBAT)] in a preliminary experiment (61). It would not be surprising, therefore, to see some shared neurons at some level(s) of the neuroaxis for the sympathetic outflow to two distinct WAT pads, despite the differential sympathetic drives to WAT, differential degrees of lipid mobilization, and some shared circuits for WAT and BAT discussed above.
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THE EXACT CIRCUITS AND BRAIN SITES RESPONSIBLE FOR ORCHESTRATING THE PHOTOPERIOD-INDUCED CHANGES IN BODY FAT ARE NOT KNOWN, BUT SOME LIKELY CANDIDATES HAVE BEEN IDENTIFIED |
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Unlike humans, in which increases in energy expenditure and/or decreases in energy intake trigger physiological responses to counter these attempts at reductions in adiposity (for review, see Refs. 78, 79), Siberian hamsters respond to SDs with a suite of coordinated sympathetic responses that work together to decrease body fat. Thus, in addition to the SD-induced increase in WAT NETO (26) discussed above, the potency/efficacy of norepinephrine (NE)-triggered lipolysis increases in a temporally and fat pad-specific manner (80). That is, during the rapid decrease in body fat occurring during the first 5–6 weeks of SD exposure, the potency (sensitivity/EC50) and efficacy (maximal response asymptote) of NE-induced lipolysis was increased in isolated adipocytes from IWAT and EWAT compared with isolated adipocytes from their LD counterparts (80). These enhanced responses to NE were most prominent when lipid mobilization was greater (5 vs. 10 weeks of SD exposure) and were more marked in EWAT than in IWAT, paralleling the greater decrease in EWAT than in IWAT mass (80). Moreover, the SD stimulation of lipolysis was similar for NE and BRL 37344 (a specific ß3-adrenoceptor agonist), implying the primacy of this receptor subtype in this response (80) (see below for a brief discussion of ß- and 
-adrenoceptors in WAT). WAT ß3-adrenoceptor mRNA expression (and, thus, likely protein) also increases in SDs (81), suggesting that these increases in ß3-adrenoceptors could underlie the SD-induced increased NE sensitivity/efficacy. Collectively, therefore, there is a coordinated set of SD-triggered sympathetic responses that promote the seemingly effortless shift from the obese to the lean state in Siberian hamsters experiencing SDs.
To recapitulate, we have defined the likely sequence of events from environmental cue (change in photoperiod) to decreases in adipocyte size that result in the reversal of photoperiod-induced seasonal obesity in Siberian hamsters. Thus, with increasing durations of night (SDs), the peak duration of nocturnal MEL secretion by the pineal is lengthened, resulting in increases in MEL1a receptor stimulation, some of which are located in the circuits constituting the sympathetic outflow neurons to WAT. This increase in the sympathetic drive to WAT stimulates primarily ß3-receptors that have increased efficacy/potency to NE, the principal sympathetic postganglionic neurotransmitter, as a result of SD exposure. This, in turn, initiates the lipolytic cascade that ultimately results in decreases in total body fat of SD-exposed Siberian hamsters.
We do not believe, however, that MEL interacting with the SNS in humans is an important factor controlling lipid mobilization. This does not diminish the importance of these findings, because it led us and others to realize the importance of the SNS innervation of fat for lipid mobilization. Moreover, as will be discussed below, the sympathetic innervation of WAT appears to be the underlying mechanism that initiates lipolysis under a number of conditions in both human and nonhuman animals.
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DIFFERENTIAL SYMPATHETIC OUTFLOW TO PERIPHERAL TISSUES INCLUDING WAT IS THE RULE, NOT THE EXCEPTION |
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There have been several studies measuring the changes in the firing rate of sympathetic nerves innervating WAT, all by Niijima and associates (83–90), showing that a number of stimuli increase sympathetic drive to WAT, including odors, tastes, histamine, leptin, and other factors. This relative paucity of electrophysiological measures of sympathetic nerves to WAT contrasts with that for BAT (for reviews, see Refs. 91, 92), perhaps because the nerves to WAT are more difficult to identify and are smaller, making them more difficult to record from than the sympathetic nerves innervating BAT. In these studies of sympathetic nerve activity to WAT, this activity has not been measured in more than one WAT depot simultaneously (two EWAT pads have been measured simultaneously, but not, for example, EWAT and IWAT), and as we shall see, based on biochemical measures of sympathetic activity (NETO), differential sympathetic drive across WAT pads almost always occurs.
The deficit in our knowledge of sympathetic drives to WAT has been approached by measuring NETO as a proxy for direct electrophysiological measures. Most frequently, the -methyl-para-tyrosine method has been used, in which -methyl-para-tyrosine, a competitive inhibitor of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is exploited to allow the estimation of NETO by the rate of NE disappearance from the tissues of interest (93). Using this method, therefore, a measure of sympathetic drive can be compared across multiple tissue types and multiple adipose depots. When this is done, it is clear that there is differential NETO across WAT pads in response to many lipid-promoting stimuli. For example, as noted above, SD-exposed Siberian hamsters increase WAT NETO, with the more internally located WAT pads (RWAT and EWAT) showing the greatest increases compared with more externally located WAT pads (IWAT) (26). These SD-induced increases in NETO are accompanied by proportional decreases in WAT mass. Fasting does not significantly increase NETO differentially in laboratory rat RWAT or EWAT, although a trend for increased RWAT compared with EWAT NETO exists (94). By contrast, in 48 h fasted Siberian hamsters, NETO is significantly increased in IWAT but not in the other fat pads (M. Brito, N. Brito, C. K. Song, and T. J. Bartness, unpublished data). Similarly, although acute cold exposure does not produce significant differences in EWAT and RWAT NETO in laboratory rats, there is a trend toward increases in RWAT compared with EWAT NETO (95), and with acute cold exposure in Siberian hamsters, IWAT NETO is significantly greater than RWAT or EWAT NETO (no change) and dorsosubcutaneous white adipose tissue (DWAT) NETO (M. Brito, N. Brito, C. K. Song, and T. J. Bartness, unpublished data). Finally, another example of differential sympathetic drive across WAT pads occurs with the glucoprivic stimulus 2-deoxy-D-glucose: NETO is increased to several WAT pads (IWAT, RWAT, and DWAT) but not in EWAT (no change) in Siberian hamsters (9; M. Brito, N. Brito, C. K. Song, and T. J. Bartness, unpublished data). It should be noted that the relation between increases in NETO and decreases in WAT mass, as occurred in SD-exposed Siberian hamsters (26), does not always hold, as third ventricularly or peripherally administered leptin increases WAT NETO but is not always positively correlated with decreases in WAT mass (96). When such a disparity is seen, it may be because WAT pad mass is not a sensitive indicator of lipid mobilization when the degree of lipolysis is more subtle. Alternatively, receptor or post receptor events could be altered such that the sympathetic nerve activity is without effect. In summary, differential WAT NETO exists for several lipid-promoting stimuli, but our knowledge of the control of this and other trafficking of sympathetic drives to peripheral tissues is an important unsolved mystery that seems fundamental to solve for a deeper understanding of regulatory biology (for review, see Ref. 97).
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THE NEUROCHEMICAL IDENTITIES OF NEURONS IN THE SYMPATHETIC OUTFLOW CIRCUITS FROM BRAIN TO WAT ARE LARGELY UNKNOWN, BUT A MAJORITY OF THESE NEURONS HAVE GENE EXPRESSION FOR THE MELANOCORTIN 4-RECEPTOR |
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The melanocortins have been heavily implicated in the control of food intake and energy expenditure, largely through MC4-Rs (for review, see Refs. 99, 100). More specifically, MC4-Rs have been implicated in SNS-mediated WAT lipolysis because central application of the MC3/4-R agonist, melanotan-II, in laboratory rats decreases food intake and body fat (101). These decreases in body fat are greater than can be accounted for by the decreases in food intake, as revealed by pair-feeding, suggesting increases in energy expenditure (101). We tested whether these increases in lipid mobilization were attributable to stimulation of MC4-Rs located on neurons that make up the sympathetic outflow circuits innervating WAT and found extensive colocalization of MC4-R mRNA with PRV-labeled SNS outflow neurons across the neural axis, with a high incidence and percentage (60% or greater) for most brain areas showing PRV immunoreactivity (-ir) (67). These areas include the PVN, preoptic area, bed nucleus of the stria terminalis, and amygdala in the forebrain, periaqueductal gray in the midbrain, and the nucleus of the solitary tract, lateral paragigantocellular nucleus, lateral reticular area, rostroventrolateral medulla, and anterior gigantocellular nucleus in the brainstem, to name a few of the more predominant sites of colocalization. This extensive high level of colocalization (60%) suggests that MC4-Rs play a prominent role in the modulation of SNS outflow to WAT, either through stimulation by the endogenous melanocortin agonist -melanocyte-stimulating hormone and/or through inhibition by the naturally occurring MC3/4-R inverse agonist, agouti-related protein (102). We have found similar high levels of colocalization of PRV with MC4-R mRNA sympathetic outflow to BAT (61; C. K. Song, E. Keen-Rhinehart, C. H. Vaughan, D. Richard, and T. J. Bartness, unpublished data), and while that work was in progress, a similar high level of colocalization was found after PRV injections into IBAT of MC4-R-green fluorescent protein transgenic mice (103). These studies demonstrate the usefulness of PRV in delineating the neurochemical phenotype of the sympathetic outflow neurons to WAT, BAT, or other tissues.
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WAT DENERVATION STUDIES PROVIDE FUNCTIONAL EVIDENCE FOR THE ROLE OF THE SYMPATHETIC INNERVATION OF WAT IN LIPID MOBILIZATION |
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Complementary to the denervation studies are a few studies of sympathetic nerve stimulation (for review, see Ref. 11). In an ingenious yet simple study by Correll (118) conducted almost 45 years ago, the intact nerves innervating EWAT were electrically stimulated after the pads were removed with the nerves attached from laboratory rats and placed into a beaker of medium. Stimulation of the nerves markedly increased the FFA concentration in the incubation medium, suggesting lipolysis (118). This effect was blocked if the rats were sympathectomized 4–11 days before removal and the nerves to the pads were subsequently electrically stimulated in a similar manner (118). Moreover, adding dibenamine, a ß-adrenergic blocker, to the incubation medium before the initiation of electrical stimulation blocked these increases in FFAs (119). Using the same preparation, others repeated and extended these findings, showing that another adrenergic receptor blocking agent [1-(2',4'-dichlorophenyl)-1-hydroxyl-2-(t-butylamino)], a depletor of catecholamine stores (syrosingopine), and an inhibitor of NE release (BW 392C60) all blocked the electrical stimulation-induced increase in FFA concentration in the incubation medium (119). This electrical stimulation-triggered lipolysis is exaggerated by pargyline, a monoamine oxidase inhibitor (thus inhibiting NE degradation), and by theophylline, the phosphodiesterase inhibitor (119), the latter suggesting that endogenously released catecholamines are involved (119). In a relatively analogous in vivo preparation in humans, Dodt et al. (120–122) intraneurally stimulated the lateral femoral cutaneous nerve that innervates subcutaneous WAT and measured local lipolysis (changes in glycerol concentrations) via microdialysis of the interstitial subcutaneous WAT area receiving the stimulation. This stimulation increases interstitial glycerol concentrations in vivo in humans (120, 121). Thus, complementary electrical stimulation studies to the denervation studies discussed above also support a role of sympathetic innervation in initiating lipolysis in WAT.
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ADRENOCEPTORS ARE INTIMATELY INVOLVED IN THE CONTROL OF LIPOLYSIS IN WHITE ADIPOCYTES |
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, glucagon, and growth hormone (for reviews, see Refs. 123, 126). In addition to the adrenoceptor subtype 2-adrenoceptor (see directly below), other nonadrenergic factors that inhibit lipolysis also are important, such as insulin, the most potent of these (127, 128), as well as prostaglandin E2, adrenomedullin, adenosine (for review, see Ref. 123), and neuropeptide Y (129–132), the latter a neuropeptide that is often colocalized with NE in sympathetic nerves (133–135).
Pioneering work of Lafontan (136–139) and others (140–142) set the stage for our current knowledge of the role of adrenoceptors (adrenergic receptors) in lipolytic and other cell functions. In its simplest form, four subtypes of adrenoceptors have a role in catecholamine-stimulated lipolysis; these are the ß1-, ß2-, and ß3-adrenoceptor subtypes as well as the 2-adrenoceptor (for reviews, see Refs. 124, 143–146). Activation of the three ß-receptor subtypes stimulates lipolysis, but the participation of each subtype in lipolysis varies according to the fat pad, species, gender, age, and degree of obesity (147). By contrast, as noted above, activation of 2-adrenoceptors inhibits lipolysis (for review, see Ref. 144). These four adrenoceptors coexist in adipocytes. Because activation of the ß1-, ß2-, and ß3-adrenoceptors stimulates lipolysis, whereas activation of the 2-adrenoceptor inhibits lipolysis, it is not surprising that postreceptor events are different for each. Stimulation of the GTP binding protein Gs with ß1-, ß2-, and ß3-adrenoceptor agonism triggers increases in cAMP production by adenylyl cyclase that, in turn, stimulates protein kinase A, which, among other things, phosphorylates hormone-sensitive lipase (HSL) and perilipin (for review, see Refs. 123, 126, 148; see below for more on perilipin). By contrast, stimulation of the GTP binding protein Gi with 2-adrenoceptor agonism triggers decreases in cAMP as a result of the inhibition of adenylyl cyclase and thus the lack of stimulation of protein kinase A and, therefore, the lack of phosphorylation of HSL and perilipin (for reviews, see Refs. 123, 126, 148).
Once this stimulation of ß-adrenoceptors has begun, the process of hydrolyzing the stored triacylglycerol begins. Triacylglycerols in adipocytes are hydrolyzed in three steps, with HSL catalyzing triacylglycerol and diacylglycerol and monoglyceride lipase completing the process (for review, see Ref. 149). Perilipins interact with HSL and are positioned on the surface of lipid droplets within the adipocyte to block the translocation of HSL and thus inhibit catecholamine-induced lipolysis (for reviews, see Ref. 150, 151). With protein kinase A phosphorylation of perilipin and HSL, however, the phosphorylated HSL now has access to the lipid droplet and lipolysis proceeds (for reviews, see Ref. 126, 148). In addition, adipocyte lipid binding protein (or adipocyte fatty acid binding protein, A-FABP, 422 protein, aP2, and p15 protein) has been isolated, purified, and cloned in humans and rodents (152–155) and appears to assist in lipolysis. Adipocyte lipid binding protein accepts FFAs and activates HSL as well. Because adipocyte lipid binding protein removes FFAs from the cytosol, the well-known end product inhibition of lipolysis that occurs with accumulating FFAs seen in vitro (156) is negated in vivo, allowing lipolysis to proceed.
The preponderance of the data suggests that the balance between the lipolysis-promoting ß-adrenoceptor activation and lipolysis-inhibiting 2-adrenoceptor activation dictates the degree of lipolytic activity, all other factors being equal (for reviews, see Refs. 8, 145, 157). Thus, when ß-adrenoceptor activation predominates, lipolysis is stimulated; conversely, when 2-adrenoceptor activation predominates, lipolysis is inhibited (for reviews, see Refs. 143–145). The affinity of the naturally occurring agonists (EPI and NE) for these adrenoceptors is as follows, based on in vitro adipocyte membrane binding assays (145): for NE, 2 > ß1 
ß2 > ß3; for EPI, 2 > ß2 > ß1 > ß3. The presence of these receptors or their levels varies considerably across species, with humans, laboratory rats, and hamsters having ample 2-adrenoceptors and laboratory mice having none (132).
It has been a physiological conundrum to understand how adipocytes can have receptors that stimulate lipolysis and those that inhibit lipolysis yet share the same agonists. It has been suggested that because of the higher affinity of the 2-adrenoceptors compared with the ß-adrenoceptors for low naturally occurring (basal) concentrations of NE or EPI, 2-adrenoceptors are activated, thereby inhibiting lipolysis (148). This inhibition can be broken, however, with increases in concentrations of the agonists, especially NE released from the postganglionic sympathetic nerve terminals that impinge close to the adipocytes (148). Further evidence for the interplay between these receptors and a role of the 2-adrenoceptor in obesity was shown in a clever genetic experiment in which ß3-adrenoceptor knockout mice also had human 2-adrenoceptors expressed transgenically in their WAT (158). These animals became obese, but only with both high-fat diet feeding and the presence of the 2-adrenoceptors (158).
The challenge for a pharmacological approach to obesity reversal through the stimulation of ß3-adrenoceptors has been to find a WAT-specific ß3-adrenoceptor agonist (despite the low numbers of this receptor subtype in human WAT) that has a long-term, nonadapting stimulation of lipolysis without negative side effects, such as hypertension or stroke (for reviews, see Refs. 159, 160).
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SNS INNERVATION OF WAT CAN CONTROL WHITE ADIPOCYTE PROLIFERATION |
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We have since replicated this surgical denervation-induced increase in FCN several times in Siberian hamster WAT using the unilateral denervation model (63, 107, 109). A consistent finding of these studies is a denervation-induced increase in FCN but not FCS (63, 107). To more precisely determine whether it was the surgical denervation of WAT via the destruction of its sympathetic innervation or of its sensory innervation (see below), we surgically denervated WAT (positive control) or locally injected 6OHDA to kill only the sympathetic nerves and spare the sensory nerves or locally injected capsaicin to kill the sensory nerves and spare the sympathetics (107). Capsaicin is the pungent part of red chili peppers and also is a selective neurotoxin for unmyelinated sensory nerves (166, 167). We verified the destruction of the sympathetics by 6OHDA using immunohistochemistry for TH and the destruction of sensory innervation by capsaicin using immunohistochemistry for CGRP (107). Surgical denervation of WAT significantly decreased both TH-ir and CGRP-ir, as expected, and 6OHDA treatment significantly decreased TH-ir but not CGRP-ir, and both triggered an 3-fold increase in FCN. By contrast, capsaicin significantly decreased CGRP-ir but not TH-ir and did not alter FCN (107). Collectively, these results point to the sympathetic innervation of WAT as inhibiting FCN.
Although FCN is increased by destruction of the sympathetic innervation of WAT, this does not necessarily translate into a denervation-induced increase in fat cell proliferation. We measure cellularity using a modification of the osmium tetroxide fixation method combined with Coulter Counter quantification of fat cells (168). With this method, adipocytes having diameters of <20 µm are literally screened out to eliminate lipid droplets and cell debris. Thus, denervation could cause an apparent increase in FCN through increased accumulation of lipid in small adipocytes by the elimination of basal lipolysis, thereby allowing them to be counted. To test for bona fide proliferation by sympathetic denervation, we recently (109) injected 6OHDA or capsaicin locally in WAT to selectively destroy the sympathetic innervation and spare the sensory innervation or to selectively destroy the sensory innervation and spare the sympathetic innervation, respectively. Surgical denervation was included as a positive control. To label proliferating adipocytes, we also injected bromodeoxyuridine (BrDU; a nonradioactive method of identifying dividing cells), and to determine whether the BrDU-labeled cells were adipocytes, we double labeled the tissue for AD3-ir, a white adipocyte-specific membrane protein (169, 170). Surgical denervation significantly decreased TH-ir and CGRP-ir, whereas 6OHDA treatment only significantly decreased TH-ir, but both treatments were associated with 3- to 4-fold increases in BrDU+AD3-ir cells. By contrast, capsaicin treatment significantly decreased CGRP-ir but not TH-ir and was not associated with an increase in BrDU+AD3-ir (109). These results suggest that destruction of the sympathetic innervation of WAT stimulates preadipocyte proliferation (109). Collectively, it appears that increases in the sympathetic stimulation of ß-adrenoceptors inhibits fat cell proliferation, whereas decreases in sympathetic stimulation of ß-adrenoceptors promotes it.
Interestingly, the magnitude of the SNS denervation-induced increases in FCN (proliferation) appears to reflect the particular propensity of each WAT depot to grow by increasing FCN. Specifically, surgically denervated Siberian hamster IWAT achieves a greater percentage increase in FCN than similarly denervated RWAT (63), whereas EWAT surgical denervation does not affect FCN (107). This lack of denervation-induced increases in FCN by EWAT is completely consistent with the tendency of EWAT to increase its mass by increasing FCS, and the denervation-induced increases in FCN by RWAT and IWAT reflect the tendency of these pads to increase their masses primarily by increasing FCN, at least in the obesity associated with aging in Wistar rats (171). These effects of denervation also are consistent with the greater in vivo incorporation of radiolabeled thymidine into DNA in RWAT (a radioactive method of identifying dividing cells) than in EWAT from high-fat diet-fed laboratory rats (172). Therefore, it is easy to envision that a decrease in sympathetic drive to some of these WAT pads could promote fat cell proliferation and thereby make significant contributions to increases in adiposity.
The underlying mechanisms triggering the sympathetic denervation-induced increase in fat cell proliferation are unknown beyond the decreases in the activation of adipocyte ß-adrenoceptors (163). Mature adipocytes and their precursor cells have 2-adrenoceptors, the function of which in mature fat cells is to inhibit lipolysis when stimulated (see above). The 2-adrenoceptors, in turn, have been implicated in adipocyte proliferation (164, 173), because the surgical denervation of laboratory rat WAT is associated with increases in FCN that are preceded by increases in adipocyte 2-adrenoceptor number (165). Stimulation of these receptors in denervated WAT could occur by any remaining sympathetic nerves or by adrenal medullary catecholamines (primarily EPI). Either of these alternatives seems possible because 2-adrenoceptors show special affinity for low concentrations of EPI and to a lesser degree NE (see above). In neurally intact WAT, decreases in the sympathetic drive to WAT and consequent stimulation of fat cell proliferation seem to be associated with decreases in ß-adrenoceptor number and increases in 2-adrenoceptor number as the WAT pad mass expands (174). One way that 2-adrenoceptor stimulation might trigger fat cell proliferation is through the local release of lysophosphatidic acid that is elicited by WAT 2-adrenoceptor activation (173). Because lysophosphatidic acid added to preadipose cell lines triggers fat cell proliferation (173), one could imagine such an event occurring in vivo as well. This hypothesized role for 2-adrenoceptors in fat cell proliferation is bolstered by the genetic addition of these receptors in ß3-adrenoceptor knockout mice fed a high-fat diet, in which their obesity is solely attributable to increases in FCN (158). Finally, decreases in sympathetic drive may stimulate the release of the recently discovered white adipocyte paracrine factor autotoxin, a type II ectonucleotide pyrophosphatase phosphodiesterase, that in turn could stimulate proliferation by its ability to release lysophosphatidic acid (175). Collectively, this scenario provides a possible mechanism by which fat cell proliferation is stimulated with decreases or abolition of its sympathetic drive.
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THE PSNS INNERVATION APPEARS SPARSE AT BEST |
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For these and other reasons (72, 106, 177, 178), we sought PSNS nerve markers in WAT (72). We used three types of animals, a standard mouse strain (C57BL mice), a genetically obese mouse strain (ob/ob mice), and a standard laboratory rat strain (Sprague-Dawley rats), examined three WAT pads (IWAT, RWAT, and EWAT), and tested for three previously proven neurochemical markers of PSNS innervation in other tissues (vesicular acetylcholine transporter, vasoactive intestinal peptide, and neuronal nitric oxide synthase). There was only one result: no labeling in any animal in any WAT pad for any established PSNS marker (72). Next, we selectively and locally sympathetically denervated WAT with 6OHDA, as shown by significantly decreased WAT NE content and TH-ir, with no effect on sensory innervation (CGRP-ir), thereby sparing the putative PSNS innervation (72). PRV was then injected into the 6OHDA- or vehicle-injected WAT several days later. PRV did not infect the sympathetic chain, spinal cord, or brain in any animal with 6OHDA-treated WAT, but vehicle-injected WAT inoculated with PRV had typical sympathetic chain, spinal cord, and brain viral infection patterns (72). These data showing a lack of significant or no WAT PSNS innervation are supported by older biochemical data showing no acetylcholinesterase, an enzyme important in the degradation of acetylcholine (183). Collectively, these findings suggest that PSNS innervation either does not exist or is relatively minor in its extent. If, however, convincing data supporting PSNS innervation emerge in the future, this would be interesting, as it would afford WAT the fine control of metabolism that other tissues/glands have with dual autonomic nervous system innervation.
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WAT HAS SENSORY INNERVATION, THE FUNCTION OF WHICH REMAINS TO BE DEFINITIVELY IDENTIFIED |
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ABSTRACT
INTRODUCTION