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,2
* Departments of Medicine and
Molecular, Cell and Developmental Biology, The Mount Sinai School of Medicine, New York, NY 10029
2To whom correspondence and reprint requests should be addressed. E-mail: paul.berk{at}msnyuhealth.org.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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21 d) on adipocyte fatty acid uptake and transporter gene expression were examined. Mice were initially hyperinsulinemic (5.25 ± 1.57 nmol/L). Plasma insulin decreased by 55 ± 10% within 8 h of leptin infusion, declining progressively to normal by d 14. The Vmax for saturable adipocyte fatty acid uptake fell from 31.1 ± 5.6 to 25.2 ± 4.0 pmol/(s · 50,000 cells) (P < 0.05) by 24 h, and to a normal rate (8.0 ± 0.8 pmol/(s · 50,000 cells) by d 21 (P > 0.5 vs. normal C57BL/6J controls). Adipocyte mRNA levels for plasma membrane fatty acid binding protein and fatty acid translocase, putative fatty acid transporters that are up-regulated three- to fourfold in adipocytes from ob/ob mice, had also normalized by d 21. The initial changes in Vmax preceded decreases in food intake and body weight by at least 24 h. In pair-fed mice, insulin levels, Vmax and body weight all declined more slowly than in leptin-treated mice, and all remained significantly elevated compared with normal values at d 21. The data suggest that insulin up-regulates and leptin down-regulates adipocyte fatty acid uptake, leading to alterations in fatty acid partitioning that affect adiposity.
KEY WORDS: • obesity • nutrient partitioning • animal models • membrane transport
Although it is evident that the recently discovered hormone leptin (OB protein), encoded by the ob gene, plays an important role in the control of body weight, or at least of body fat mass (1–4), the precise mechanism(s) responsible for this effect are not entirely clear (5). Leptin undoubtedly functions as an appetite suppressant, an effect mediated through its regulation of the production of neuropeptide Y (6) and other hypothalamic peptides [reviewed in (7)]. However, the degree of weight loss that follows leptin administration in a number of settings exceeds the amount that can be attributed to decreased energy intake (3,8,9), indicating that some alteration of metabolism also contributes to leptin-mediated weight loss. Although the nature of this metabolic alteration remains to be fully defined, recent reports demonstrate that leptin controls a unique and complex program of gene-regulatory responses that is independent of its effects on food intake (5).
We and others have established that the cellular uptake of nonesterified long-chain fatty acids (LCFA)2 is accomplished by both facilitated (saturable) and diffusive (nonsaturable) processes (10–15). Facilitated uptake of LCFA is selectively upregulated in adipocytes from rodents with a number of genetic and dietary models of obesity, whereas LCFA uptake into liver and cardiac myocytes appears unaltered in these rodents (16,17). The result of this change is an alteration in nutrient partitioning (18–20) that tends to divert LCFA away from tissues in which they would be burned as fuel and preferentially into adipocytes, where they are stored as fat. Comparable up-regulation of LCFA uptake is also found in the adipocytes of obese human patients (unpublished data).
Several of the animal models studied previously exhibited defective leptin signaling, due either to a primary genetic deficiency of leptin production (ob/ob mouse) (1) or of the leptin receptor (rOB) (db/db mouse, Zucker fa/fa rat) (21–24), or to apparent leptin resistance (fat and tubby mice) (25–29). Data obtained in these earlier studies (16,17) led us to postulate that leptin might normally function as a selective down-regulator of adipocyte LCFA uptake. As a result, defective leptin signaling would increase partitioning of LCFA into adipose tissue fat stores and increase energy efficiency, and consequent obesity, independent of the loss of appetite suppression. In the current studies, we tested that hypothesis by examining the effects of continuous leptin infusion in ob/ob mice on food intake, body weight, adipocyte size, plasma insulin levels and adipocyte LCFA uptake kinetics, compared with saline-treated and pair-fed controls.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Recombinant mouse leptin was purchased from R & D Systems (Minneapolis, MN), [3H]-oleic acid from NEN Life Science Products (Boston, MA), fatty acid free bovine serum albumin (BSA) from Boehringer Mannheim (Indianapolis, IN) and type I collagenase from Sigma Chemical (St. Louis, MO).
Mice.
Male ob/ob and C57BL/6J mice (6–8 wk old) were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were housed in group cages in a temperature-controlled facility with a 12-h light:dark cycle, with free access to LabDiet 5001 (PMI, St. Louis, MO) and water, and weighed periodically until used.
Treatment protocol.
As ob/ob mice reached a body weight of 60–65 g (at a mean age of 14 wk), they were placed in individual cages and assigned randomly to one of three groups. Studies in C57BL/6J mice were conducted at a comparable age, to provide background data for comparison with those obtained in the mutant ob/ob strain. The leptin-treated ob/ob group had free access to the LabDiet 5001 and water while receiving a continuous subcutaneous infusion of recombinant leptin, dissolved in PBS, at 500 ng/h, for up to 21 d. Delivery was by an osmotic pump (Alzet, Palo Alto, CA) through an in-dwelling catheter inserted into the back of the neck. A saline-treated control group also had free access to the LabDiet 5001 and water while receiving a similarly delivered infusion of PBS. Weights and food consumption were recorded daily. A pair-fed control group had free access to water and received PBS infusion, but was offered a progressively decreasing amount of food, matched to the mean food intake of the leptin-treated mice on the corresponding day of the study.
Measurements.
Either immediately after insertion of the subcutaneous catheter (time 0), or after study periods of 8 h (0.33 d), or 1,2,4,7,14 or 21 d, mice were weighed and then killed by CO2 narcosis according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC), which also approved the treatment protocol. Epididymal fat pads were immediately removed and weighed before the rapid freezing of tissue aliquots for later RNA isolation (see below) and the isolation of adipocytes (30–32). Blood samples were taken for quantitation of plasma insulin and leptin by RIA, using commercial RIA kits (Mouse Leptin RIA Kit and Sensitive Rat Insulin RIA Kit; Linco, St. Charles, MO).
LCFA uptake studies.
Single-cell suspensions of adipocytes (
109/L), isolated from the excised fat pads after collagenase digestion (30–32), met previously established viability criteria (32). The distribution of cell diameters in each suspension was determined by direct microscopy, using a graduated eyepiece reticle, and the corresponding mean cell surface area (in µm2) calculated (33). Cell aliquots from each mouse were incubated at 37°C in 500 µmol/L BSA containing one of five different concentrations of [3H]-oleic acid, such that the oleic acid:BSA molar ratio (
) was 0.25, 0.5, 1.0. 1.5 or 2.0:1. The initial velocity of cellular oleate uptake from each test solution was determined by a standard, rapid filtration technique (30,32) from four samples obtained over the initial 30 s of incubation, during which uptake was a linear function of time (16,17,32).
Computations and data fitting.
The unbound oleate concentration ([OAu]) in each test solution was calculated from (34), using the LCFA:BSA binding constants of Spector et al. (35). Although recent reports based on alternative methods (36–38) suggest that these constants overestimate [OAu], the values for [OAu] computed according to these newer methods differ from one another by two or more orders of magnitude, none is totally free of methodologic criticism [e.g., (39)], and there is as yet no consensus concerning which of the alternatives most accurately approximates actual values for [OAu]. Furthermore, although use of more recently published binding constants to calculate [OAu] can modify computed values of certain of the kinetic constants defining LCFA uptake, they do not change the conceptual interpretation of these studies nor the calculated values of trans-membrane LCFA transfer rates (15). Therefore, we continue to use the Spector constants (35) to facilitate comparison of these studies with a large body of related earlier work. Based on prior analyses (15), measurements of initial oleate uptake velocity at values of from 0.25 to 2.0 were fitted to the sum of a saturable and a nonsaturable function of the corresponding [OAu], according to the equation:
 | (1) |
in which UT([OAu]) is the experimental measurement of uptake at the stipulated concentration of unbound oleic acid; Vmax and Km are the maximal uptake velocity of the saturable oleic acid uptake component and the value of [OAu] at one half the maximal uptake velocity, respectively; and k is the rate constant for nonsaturable uptake (10,15–17,32). Data fitting was accomplished using the SAAM II version of the Simulation, Analysis and Modeling (SAAM) program of Berman and Weiss (40) as modified for execution on a lap-top PC computer (41). SAAM uses an iterative, nonlinear algorithm to compute for each data set values of the Vmax [pmol/(s · 50,000 cells)] and Km (nmol/L) of the saturable uptake function, and the rate constant k [mL/(s · 50,000 cells)] for the nonsaturable uptake process, as well as their variances and covariances (42). To better elucidate the relationship between Vmax and adipocyte size, values for Vmax were also expressed relative to the experimentally determined mean adipocyte surface area [pmol/(s · µm2)].
RNA isolation and Northern hybridization.
Aqueous cellular contents were extracted from chilled adipose tissue samples (0.5–1 g) as described (16,17). RNA was isolated from the extracts using the Qiagen RNeasy Maxi kit (Qiagen, Valencia, CA) and used to determine steady-state levels of mRNAs encoding two putative LCFA transporters, i.e., plasma membrane fatty acid binding protein (FABPpm), which is identical to mitochondrial aspartate aminotransferase (mAspAT) (43) and fatty acid translocase (FAT/CD36) (44). Effects on fatty acid transport protein (FATP) (45) mRNA were not studied because this putative LCFA transporter is not appreciably upregulated in ob/ob mouse adipocytes (17,46). Gel electrophoresis and Northern hybridization were performed using the NorthernMax Kit (Ambion, Austin, TX). In the protocol used, RNA were separated on 1% agarose-formaldehyde gels and transferred to Hybond-N nylon membrane (Amersham Biosciences, Piscataway, NJ). Probes were labeled using DECAprime II (Ambion, Austin, TX) with 32P-dATP (Perkin Elmer Life Science, Boston, MA). Data were analyzed as described (16,17), except that lane loading was assessed using an 18S RNA template (Ambion) rather than ß-actin. In addition, the effects of leptin infusion on mRNA levels for leptin and lipoprotein lipase (LPL) were determined with a microarray developed by the Mount Sinai Microarray Core Facility (47).
Statistical methods.
The principal hypothesis being tested in these studies was that down-regulation of the Vmax for LCFA uptake by adipocytes preceded changes in weight loss and adipocyte cell size during leptin infusion in ob/ob mice. The choice of statistical methods employed therefore focused on those most appropriate to test this specific hypothesis. Measurements of the experimental variables at each time point within each treatment group were summarized initially in terms of mean, median and SD, using standard methods of descriptive statistics (48). The distributions of these data were further examined to determine whether a transformation, e.g., to logarithms, was required to achieve normality, as assumed for subsequent analyses. Logarithmic transformations were also used to accommodate disparities in the SD of some of the data sets (e.g., plasma insulin values). In view of the variability in the experimental SD, differences in mean values of experimental measurements in the leptin-treated mice killed on different days of treatment were tested by one-way ANOVA. These tests were all based on data obtained at the times of death from the same sets of mice. Dunnett’s test (one-tailed) was used to determine the significance of differences from baseline at succeeding time points (49). The Dunnett test internally corrects the significance level for multiple testing, thereby maintaining an overall level of significance at P = 0.05 for the set of tests of each time point vs. baseline. Differences are therefore reported simply as either significant or not significant. Primary or log-transformed data, as appropriate in each case, were used for these analyses. Between-group comparisons e.g., of the course of body weight over time are based on repeated measures of weight up to the time of death of the mice that were part of the kinetic studies, as well as of similar mice undergoing identical treatments in other studies. Slopes describing the change of weight over time were estimated for each such mouse with at least one weight measurement beyond d 1. These slopes were entered in a one-way ANOVA, and pairwise comparisons between groups were tested using the Bonferroni correction, using an algorithm that, again, reports differences simply as either significant or not significant. Evaluations of differences between groups at the end of the study (d 21) and time-independent comparisons of single variables (e.g., plasma leptin concentrations) between the current study and previously published information were done by two-tailed Student’s t tests. All data are presented as mean ± SD.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plasma leptin concentrations. Leptin was undetectable in plasma of untreated ob/ob mice with the RIA employed. Plasma leptin reached 101.9 ± 29.4 nmol/L within 8 h of starting leptin infusion and averaged 180.6 ± 104.3 nmol/L during the period from 1 to 21 d of infusion. This did not differ from values we reported (17) in db/db (251.9 ± 76.6 nmol/L, P > 0.1), fat (fat) (181.3 ± 98.1 nmol/L, P > 0.5) and tubby (tub) (221.9 ± 29.1 nmol/L, P > 0.1) mice but was higher (P < 0.001) than those we reported in C57BL/6J (26.3 ± 7.0 nmol/L) and C57BLKS/J (18.8 ± 5.4 nmol/L) mice (17), the background strains for the ob and tub mutations, and for db and fat mice, respectively.
Plasma insulin. On d 0, the total population of ob/ob mice were markedly hyperinsulinemic, with a plasma insulin concentration of 5.25 ± 1.57 nmol/L, a value similar to previous results in ob/ob males of similar age and weight (50) (Fig. 1). Examination by one-way ANOVA on the log-transformed data of each group, separately, showed that there were significant changes over time (P < 0.0001) in both the leptin and pair-fed groups. When plasma insulin concentrations at various times were compared with baseline, levels in the leptin-treated group had fallen 55 ± 10% by 8 h of treatment. That this rapid initial decline was not significant reflects the small number of mice available for study at that time point. Insulin levels were reduced significantly from baseline by d 1 in the leptin-treated group, and continued to decrease thereafter.
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Plasma insulin. Insulin levels declined much more slowly in pair-fed than in leptin-treated mice (Fig. 1). Concentrations at 8 and 24 h were 89 ± 28 and 85 ± 17% of baseline, respectively, and were more than double those in leptin-treated mice at 24 h. Although continuing to decline, they remained from two to four times the levels in leptin-treated mice throughout the study. In contrast to the leptin group, insulin levels in pair-fed mice were above the normal range throughout the study.
Body weight, and adipocyte cell size. The pair-fed mice consistently consumed the total quantity of food offered, which was based on intake of the leptin-treated mice during the corresponding study day (Fig. 2). Body weights fell linearly with time after d 1, but the rate of weight loss, defined as the slope of the weight loss curve, was significantly less in the pair-fed (-1.03 ± 0.28 g/d) than in the leptin-treated (-1.49 ± 0.47 g/d) mice (Fig. 3). The rates of weight change in both of these groups differed significantly from that in the saline-treated controls (+0.22 ± 0.13 g/d). Although adipocyte surface area in the pair-fed mice decreased linearly (r = 0.954) with time from d 4 through 21 of the study, the cells remained persistently larger than those in leptin-treated mice (Table 1).
Fatty acid uptake kinetics. The Vmax for saturable OA uptake in the pair-fed mice on d 1 was unchanged from its baseline value. It fell more slowly and more variably thereafter than in the leptin-treated mice (Table 1), reaching twice the rate in the leptin-treated group by d 21.
Correlations among variables
Vmax, body weight and plasma insulin. In the total study population of leptin-treated and pair-fed ob/ob mice and untreated C57BL/6J mice, mean group Vmax were linearly correlated with body weight (r = 0.88, P < 0.001) (Fig. 6A). By contrast, plasma insulin levels were exponentially correlated with weight (r = 0.90, P < 0.001) (Fig. 6B), a pattern consistent with the development of insulin resistance at higher weights. There was a linear correlation between plasma insulin and Vmax (r = 0.86, P < 0.001) (Fig. 6C), but this relationship was better described by expressing Vmax as a logarithmic function of the plasma insulin concentration (r = 0.94, P < 0.001) (Fig. 6D). Although correlative, these data nevertheless strongly suggest a dependence of saturable LCFA uptake on plasma insulin levels.
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 | (2) |
Equation 2 indicates that a correlation between Vmax and SA is to be expected even for a saturable uptake process. When saturable LCFA uptake was expressed per unit surface area, Vmax' fell by 35% (P < 0.025) over the first 2 d of leptin treatment, during which there was no change in cell size (Fig. 7). Over this same period, Vmax' and cell size were unchanged in both control groups. These data indicate that changes in Vmax in adipocytes from obese mice are not merely reflective of changes in cell size; rather, they reflect down-regulation of a membrane transport system mediating LCFA uptake.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The current studies confirm that weight loss in leptin-treated ob/ob mice exceeds that in pair-fed mice (3,8,9). One mechanism by which leptin administration leads to weight loss is through appetite suppression and a reduction in food intake (3,9,50,51). Although leptin-induced weight loss is clearly greater than can be accounted for solely by reduced energy intake (3,8,9), the mechanism(s) responsible for this additional weight loss remain ill-defined. The present studies may shed new light on this issue.
In these studies, leptin administration to ob/ob mice had the following sequential effects: 1) a decline in plasma insulin concentration for which the initial trend was noted by 8 h; 2) a reduction in the Vmax for LCFA uptake into adipocytes by d 1; 3) decreased food intake and body weight (d 3–4); 4) reductions in adipocyte cell size (d 4) and in the rate constant for nonsaturable LCFA uptake, with which surface area is highly correlated. The sequence is instructive. The earliest events were a rapid reduction in plasma insulin levels and down-regulation of both Vmax and Vmax' for saturable adipocyte LCFA uptake. Insulin promotes triglyceride storage by adipocytes (52), in part by inhibiting lipolysis (53). Leptin serves in several ways, including the promotion of lipolysis (7), and as an insulin counterregulatory hormone (54), possibly accomplished by the inhibition of insulin secretion from the pancreatic ß-cells (55,56). Hence, the early decrease in plasma insulin levels during leptin infusion is not surprising, nor is the observation that adipocyte LCFA uptake is highly correlated with insulin levels, which is consistent with the known role of insulin in promoting LCFA accumulation in these cells. That the decrease in plasma insulin preceded or accompanied the decrease in saturable LCFA uptake is a more novel finding.
The observation that leptin administration resulted in a rapid fall in Vmax', a measure of the rate of LCFA influx per unit of adipocyte surface area, before measurable changes in adipocyte size, clearly indicates that the decrease in saturable LCFA uptake reflects down-regulation of processes mediating the trans-membrane import of LCFA, and is not merely a consequence of alterations in cell size. That this down-regulation precedes weight loss at the very least raises the possibility that decreased LCFA import by adipocytes in obesity is a factor that contributes to subsequent weight reduction. In this regard, we have already shown that the Vmax for adipocyte LCFA uptake in preobese 19-d-old weanling Zucker fa/fa rats is up-regulated before overt expression of the fatty phenotype, and inferred from this that increased adipocyte LCFA uptake might contribute to the development of obesity (16). This inference remains valid even though studies in much larger numbers of fa/+ and fa/fa pups found that other evidence for evolution of an obese phenotype was already detectable in these and even younger rats (57). The striking, rapid up-regulation of adipocyte LCFA uptake in obesity-prone Osborne-Mendel rats fed a high fat diet and the absence of such up-regulation in the obesity-resistant S5B/PL strain fed the same diet (unpublished) also suggests a role for regulation of adipocyte LCFA uptake in the evolution of obesity. Collectively, the finding that down-regulation of saturable LCFA uptake in adipocytes precedes weight loss, decreased adipocyte size, and, by inference, triglyceride content (58), whereas up-regulation precedes weight gain, focuses attention on LCFA transport as a potentially key mediator of adiposity. Elucidation of the regulation of adipocyte LCFA uptake thus becomes an important goal.
The finding that obesity due to mutations in the leptin signaling system (ob and db mice, fa/fa rats), or associated with the phenomenon of leptin resistance (tub and fat mice), was accompanied by tissue-selective up-regulation of adipocyte LCFA uptake led us to postulate that leptin might function normally as a down-regulator of this transport process (16,17). The current studies, in which leptin administration to leptin-deficient ob/ob mice down-regulated adipocyte LCFA uptake before the predictable reduction in food intake, body weight or adipocyte cell size, support this hypothesis.
Up- or down-regulation of LCFA uptake presumably reflects regulation of the expression of one or more transporters. Several proteins reportedly function as plasma membrane LCFA transporters (10–13,15); of these, mAspAT, FAT/CD36 and FATP undergo tissue-specific regulation of their mRNA in response to physiologic perturbations including obesity (16,17,59–67). Two of these, mAspAT and FAT/CD36, are up-regulated in adipocytes from obese, adult ob/ob mice (17,46). Our data show that normalization of ob/ob adipocyte mAspAT and FAT/CD36 mRNA levels parallels the normalization of saturable LCFA uptake produced by leptin infusion in these mice, providing further evidence that expression of these proteins is related to facilitated LCFA uptake.
Regulation of LCFA transporter expression appears to be quite complex and is incompletely understood. Overall mAspAT expression in the HuH7 human hepatoma cell line is regulated mainly at a transcriptional level, but its cell surface expression is further regulated, during its intracellular trafficking, by partitioning between mitochondria and a brefeldin A-inhibitable vesicular pathway leading to the plasma membrane (unpublished). We also identified a population of small, mAspAT-rich vesicles beneath the plasma membrane in these cells. Although we did not find FATP to be up-regulated consistently in adipocytes from several mouse obesity models (17), insulin promotes rapid translocation of FATP1 from an intracellular pool to the plasma membrane of 3T3-L1 adipocytes, with a parallel increase in LCFA uptake (68). Finally, in rat cardiac myocytes, plasma membrane expression of both mAspAT and FAT/CD36, like that of glucose transporter 4, is regulated in the short term by transfer to the plasma membrane of preformed protein stored in submembrane vesicles (69); in rat skeletal muscle, leptin administration decreases plasma membrane FAT/CD36 and mAspAT protein content in parallel with decreased LCFA uptake (70). Thus, the current observations are consistent with an emerging pattern.
How leptin might effect the down-regulation of transporter expression is unknown. There are at least five splice variants of the leptin receptor (Ob-R) (71), of which the form with the long intracytoplasmic tail (Ob-Rb, Ob-R-L) has the most potent signaling capabilities (72). Early studies found Ob-Rb either to be absent or expressed at very low levels in adipocytes (71), arguing against major autocrine or paracrine effects. In addition, chronic administration of leptin into the intracerebral ventricles at 3 ng/h led to ablation of body adipose stores, whereas peripheral leptin administration required doses of 
500 ng/h to achieve the same result (73,74). This, too, suggested that leptin’s principal site of action was in the brain, rather than directly on adipose cells. Therefore, we initially expected the efferent signal to be a second hormone or an autonomic impulse, released from the brain in response to the binding of leptin to its hypothalamic receptors. Studies identifying Ob-Rb in white adipose tissue (75–78), and other data indicating a direct effect of leptin on isolated adipocytes (78–80), do not rule out these possibilities, but rather add a third, i.e., that leptin produced locally in response to increased adipocyte triglyceride content might inhibit further LCFA uptake and triglyceride accumulation by direct autocrine or paracrine effects. The significant correlation noted between plasma insulin levels and adipocyte LCFA uptake Vmax raises yet another possibility, namely, that insulin up-regulates adipocyte LCFA uptake, and that the negative effects of leptin on this process reflect at least in part its inhibition of insulin release or action. Finally, cellular LCFA efflux, as well as uptake, is mediated by mAspAT (81,82). Unger (83) reported that leptin-induced lipolysis in adipose tissue is associated with increased expression of genes involved in LCFA oxidation, so that glycerol, but not LCFA, is released from the cells. Decreased LCFA efflux during leptin-induced lipolysis may be one mechanism leading to a down-regulation of mAspAT expression. That more than one of these mechanisms may be active is not only possible, but likely.
Even if insulin and leptin have the up- and down-regulatory effects, respectively, on adipocyte LCFA uptake suggested by our data, the overall regulation of this critical process will undoubtedly prove to be far more complex than might be inferred from the current experiments. At the very least, other hormones such as glucocorticoids and triiodothyronine are likely modulators [reviewed in (7,84)]. Although the hyperinsulinemia in ob/ob mice is associated with an appreciable degree of insulin resistance (85,86), the prompt response of facilitated adipocyte LCFA uptake to leptin administration suggests that, at least at the onset of treatment, there was no resistance to the down-regulatory effects of leptin on this particular process in these mice. Indeed, the ob/ob mouse may be unique in lacking leptin resistance despite marked obesity. By contrast, in dietary models of rodent obesity, and in single-gene rodent obesities in which the primary defect is not within the leptin signaling system (e.g., tub and fat), plasma leptin concentrations similar to those achieved in the current experiments do not effectively down-regulate adipocyte LCFA uptake (17), indicating that leptin resistance accompanies the insulin resistance well described in these situations (87–89). Both hyperinsulinemia and hyperleptinemia are common in human obesity (90) and accompany the up-regulation of adipocyte LCFA uptake seen in obese patients (unpublished). Indeed, obese patients, like fat and tub mice, exhibit both insulin and leptin resistance. This suggests that if LCFA uptake is indeed regulated by a balance between insulin and leptin, the balance depends not on plasma levels, but on how much their respective regulatory effects are "discounted" by processes creating resistance to their actions. In this regard, it is important to clarify the roles of the newly identified adipocyte-produced peptides, resistin and AdipoQ/liponectin/Acrp30, in this complex situation (91,92).
The current studies thus suggest that selective regulation of adipocyte LCFA uptake may play an important the role in controlling body adiposity, perhaps serving as a component of the hypothetical "lipostat" (7,93,94) and that insulin and leptin are central to the regulation of this critical process. Full appreciation of the roles of insulin and leptin will require a more detailed understanding of the mechanisms underlying insulin and leptin resistance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BSA, bovine serum albumin; FABPpm, plasma membrane fatty acid binding protein; FAT/CD36, fatty acid translocase; FATP, fatty acid transport protein; LCFA, nonesterified long-chain fatty acid; mAspAT, mitochondrial aspartate aminotransferase; [OAu], concentration of unbound oleic acid in equilibrium with BSA; , LCFA:BSA molar ratio; SA, surface area; UT, uptake.
Manuscript received 7 January 2003. Initial review completed 6 February 2003. Revision accepted 22 June 2003.
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