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a Institute for Aging Research, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York
b Diabetes Research and Training Center, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York
c Division of Pediatric Endocrinology, Children's Hospital at Montefiore, Bronx, New York
Nir Barzilai, Institute for Aging Research, Belfer Building 701, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 E-mail: barzilai{at}aecom.yu.edu.
Decision Editor: Andrzej Bartke, PhD
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Abstract |
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50% decrease in food intake compared with saline controls, an 50% decrease in visceral fat, and improved hepatic (fourfold) and peripheral (30%) insulin action (euglycemic hyperinsulinemic clamp technique) compared with the pair-fed group (p < .001). Although the plasma leptin level was doubled in aging rats, leptin failed to produce a significant change in food intake, in fat mass and its distribution, and in hepatic and peripheral insulin action. Increasing plasma leptin levels failed to suppress leptin gene expression in aging rats as compared with the 50% suppression seen in young rats (p < .01). We propose that leptin resistance may play a causative role in the metabolic decline seen with aging. AGING is characterized by a decline in metabolic functions (1)(2). The hallmarks of the metabolic decline of aging are the development of obesity, alterations in body fat distribution, and insulin resistance (1)(2)(3). These conditions have been shown to be associated with a variety of age-related diseases (4)(5)(6)(7)(8). Chronic administration of leptin, a 16-kd fat-derived peptide, to young animals induces a decrease in food intake (9), fat mass (10), and visceral fat depots (11), and an improvement in hepatic (12)(13) and peripheral (13) insulin action. High plasma leptin levels demonstrated in aging animal models (14)(15) and humans (16)(17)(18) suggest that aging is a leptin-resistant state. Although increased fat mass could contribute to elevated plasma leptin levels in aging (19), the observed increase in plasma leptin levels is often disproportionate to the amount of fat. Impaired responses to pharmacological doses of leptin on food intake, energy expenditure, and fat distribution have been previously demonstrated in aged rats (20). We hypothesized that failure in leptin action may represent the primary defect in the metabolic syndrome of aging. To test this hypothesis, we experimentally increased plasma leptin levels in both young and aging rats within the physiological range, and we examined its effect on food intake, fat distribution, insulin action, and other markers of the metabolic syndrome of aging. We also studied the effects of elevated plasma leptin levels on leptin gene expression in subcutaneous adipose tissue.
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30 months) were housed in individual cages and subjected to a standard light (6 AM to 6 PM)–dark (6 PM to 6 AM) cycle. Rats were assigned to ad libitum feeding at the postpubertal age of 10 weeks, and they were studied at 4 months of age (young; n = 18) or 21 months of age (aging; n = 12). The chow consisted of 64% carbohydrate, 30% protein, and 6% fat with a physiological fuel value of 3.3 kcal/g of chow. One week before the in vivo studies, rats were anesthetized by inhalation of methoxyflurane, and indwelling catheters were inserted in the right internal jugular vein and in the left carotid artery. The venous catheter extended to the level of the right atrium, and the arterial catheter was advanced to the level of the aortic arch. In addition, all rats were implanted with subcutaneous minipumps that delivered either recombinant mouse leptin, (0.5 mg/kg)/d, or a similar volume of normal saline (NS) for 7 days. The surgery, utilizing this method of anesthesia, allows fast recovery and normal food consumption after 1 day (12)(13)(14)(21)(22). The young animals were subdivided into three groups. Group 1 consisted of NS rats with ad libitum food intake, with monitoring of daily food intake (n = 6). Group 2 consisted of leptin (Lep) rats with ad libitum food intake, with close monitoring of food intake to account for an associated decline in food intake (n = 6). Group 3 consisted of pair-fed (PF) rats to match the food intake to the Lep group (n = 6). The aging animals were subdivided into two groups. Group 1 consisted of NS rats with ad libitum food intake, with monitoring of daily food intake (n = 6). Group 2 consisted of Lep rats with ad libitum food intake, with close monitoring of food intake to account for an associated decline in food intake (n = 6). As the daily food intake of leptin-treated aging rats did not differ significantly from the controls, an additional PF group was not necessary. All the rats were studied after 6 hours of fasting, while they were awake and unstressed. The study protocol was reviewed and approved by the Animal Care and Use Committee of the Albert Einstein College of Medicine.
Body composition..-- Lean body mass and fat mass (FM) were calculated as described elsewhere (11)(12)(13)(19)(21)(22)(23). Briefly, rats received an intra-arterial bolus injection of 20 µCi of tritiated-labeled water (3H2O; New England Nuclear, Boston, MA), and plasma samples were obtained at 30-minute intervals. Steady-state conditions for plasma 3H2O-specific activity were achieved within 45 minutes in all studies. Five plasma samples (50 µl) obtained between 60 and 90 minutes were used to calculate the total body distribution of water. Epididymal, perinephric, and mesenteric fat depots were dissected and weighed at the end of each experiment. Hepatic triglyceride content was measured from frozen liver homogenate, extracted by chloroform methanol, and measured by a triglyceride kit (GPO-Trinder, Sigma Diagnostics, St. Louis, MO) (21).
Hyperinsulinemic euglycemic clamp..-- After samples were obtained for body composition, all rats were administered a primed continuous (15–40 µCi bolus, 0.4 µCi/min) infusion of [3H-3]-glucose purified by high-performance liquid chromatography (New England Nuclear) throughout the study. After 120 minutes, a primed continuous infusion of insulin at (3 mU/kg)/min and a variable infusion of 25% glucose solution were started, and periodically adjusted, to clamp the plasma glucose concentration at the basal level for an additional 120 minutes of the clamp. Somatostatin at (1.5 µg/kg)/min was infused to suppress endogenous insulin secretion (11)(12)(13)(21)(22).
Plasma samples (50 µl) for determination of 3H-glucose-specific activity were obtained at 10-minute intervals throughout the insulin infusion. Samples were also obtained for determination of plasma insulin, leptin, and free fatty acid (FFA) concentrations at 30-minute intervals throughout the study. The total volume of blood withdrawn was 3.0 ml/study; for volume depletion and anemia to be prevented, a solution (4:1 vol/vol) of 3.0 ml of fresh blood (obtained by heart puncture from a littermate of the test animal) and heparinized saline (10 U/ml) was infused at a constant rate throughout the study.
Whole body glycolysis and glycogen synthesis..-- The rate of glycolysis was estimated from the rate of conversion of [3H-3]-glucose to 3H2O as previously described (12)(21)(24). Because tritium on the C-3 position of glucose is lost to water during glycolysis, it can be assumed that plasma tritium is present either in 3H water or in glucose. Plasma-tritiated water-specific activity was determined by liquid scintillation counting of the protein-free supernatant (Somogyi filtrate) before and after evaporation to dryness. Whole body glycogen synthesis was estimated by subtracting whole body glycolysis from whole body glucose uptake (Rd) (24).
Leptin gene expression..-- Total RNA from subcutaneous fat depots was prepared by following Clontech's protocol with some modifications as previously described (22)(25). The total RNA was analyzed in 1% agarose gel containing 2.2M formaldehyde before use. The first strand complementary DNA (cDNA) was synthesized from 3 µg of total RNA in 20 µl of final incubation volume by using the SuperScript Preamplification System for First Strand cDNA Synthesis (Gibco BRL, Carlsbad, CA) with a random primer. The polymerase chain reaction (PCR) was carried out in a 50-µl reaction mixture containing 4 µl of this first strand cDNA, 5 µl of 10x PCR buffer (Mg+2 plus, Boehringer, Ridgefield, CT), 1 µl of 10mM dNTP (nucleotide triphosphate) mix, 4 pmol of each primer, and 2.5 U of Taq DNA polymerase (Gibco BRL). The sequence of the upstream primer is TCC TAT CTG TCC TAT GTT CAA GCT GTG; that of the downstream primer is CAA CTG TTG AAG AAT GTC CTG CAG AGA; and the expected reverse transcription-PCR product is 454 bps. The conditions for real-time PCR were 94°C for 45 seconds and 69°C for 2 minutes (42 cycles), using the GeneAmp PCR System 9600 (Perkin-Elmer, Boston, MA). Each assay was repeated for 10, 20, and 30 cycles to establish linearity. Each experiment was repeated three times for each individual animal. As a control we used ß-actin gene expression described in detail elsewhere (22)(24). Quantification of leptin signals was performed by scanning densitometry, normalized for the ß-actin signal that is not typically affected by insulin, to correct for loading irregularities.
Analytical procedures..-- Plasma glucose was measured by the glucose-oxidase method (Glucose Analyzer II, Beckman Instruments, Palo Alto, CA). Plasma insulin was measured by radioimmunoassay, with rat and porcine insulin standards. Plasma [3H]-glucose radioactivity was measured in duplicates in the supernatants of Ba (OH)2 and ZnSO4 precipitates of plasma samples (20 µl) which were evaporated to dryness to eliminate tritiated water (24).
Statistical analysis..-- All shown values are expressed as means ± standard error. Statistical analyses were performed by using an analysis of variance in multiple comparisons. When the main effect was significant, a post hoc (Tukey's) test was applied to determine individual differences between means. A value of p < .05 was considered to be statistically significant. All statistical analyses were performed by using SPSS for Windows (SPSS, Chicago, IL).
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Results |
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57% (p < .001). However, even doubling the plasma leptin levels to 40 ng/ml in the aging rats decreased the food intake by only 8%.
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10-fold higher VF content compared with their young counterparts. Because leptin commits FFA from hepatic triglyceride (TG) stores to ß-oxidation, we measured hepatic TG levels as a marker of fat depletion and leptin action. Infusion of leptin in aging rats failed to change the hepatic TG levels; in the young animals there was a significant reduction (p < .01).
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30% lower insulin-mediated glucose uptake compared with those of young rats (p < .01; Table 2 and Table 3 and Fig. 2 and Fig. 2). Leptin infusions did not result in any change in basal plasma insulin levels or insulin-stimulated glucose uptake in aging rats. This is in sharp contrast to young rats, in which leptin treatment resulted in an approximately twofold decrease in basal plasma insulin levels and an 30% increase in glucose uptake during the clamp compared with the PF rats (p < .001). Leptin also failed to change glycolysis and glycogen synthesis in aging leptin-treated rats. In young rats, leptin infusion resulted in a 100% increase in glycogen synthesis and an 20% increase in glycolysis (p < .01). The suppression of endogenous glucose production (EGP) in response to physiological hyperinsulinemia was diminished in aging rats compared with that in young rats (p < .05). Leptin treatment did not enhance the ability of insulin to suppress EGP in aging rats. However, EGP was suppressed more than threefold in the young leptin-treated group compared with the PF group. Plasma FFA levels in aging rats were higher at baseline and were less suppressed by insulin in terms of both percent and absolute decrease, in both leptin-treated and control groups, compared with the young (p < .01).
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Discussion |
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1 to 11 ng/ml between 2 and 8 months of age (21). However, at 20 months, though there was no significant increase in weight, leptin levels were almost doubled, with an average of 20 ng/ml. Furthermore, leptin is a regulator of body fat distribution, decreasing VF in greater proportion than any other fat depot (11). This regulation seems to fail with aging, and in ad libitum rat models the accumulation of VF mass has been shown to increase disproportionately to the body weight with aging (22). Thus, we hypothesize that failing leptin action may play a role in initiating the metabolic phenotype of aging. Young and aging control animals demonstrated significant differences in leptin levels, basal insulin levels, body weight, FM, fat distribution, and degree of suppression of plasma FFA and EGP during the insulin clamp. These changes reflect the typical metabolic consequences of aging, particularly the relative decrease in hepatic and peripheral insulin sensitivity. Even after the plasma leptin levels were doubled through infusions in both young and aging rats, the aging rats had a twofold higher food intake, three times higher FM, and ninefold higher VF than the young rats. In comparison with their age-matched controls, leptin infusion did not alter the FM, fat distribution, and peripheral insulin action in aging rats; in young rats, FM was decreased by half and VF by one third. In the young PF group, though the body weight and total fat values were similar to those of the young leptin-treated rats, the VF was more than twofold higher, emphasizing the specific role of leptin in decreasing VF. Our data support previous observations by Scarpace and colleagues (20) that aged rats show impaired leptin responsiveness with regard to food intake, energy expenditure, and fat distribution.
Insulin action, as demonstrated by suppression of EGP, glycogen synthesis, and glucose uptake, was also significantly reduced in aging rats compared with the young leptin-treated rats. Leptin had dramatic effects on young rats in suppressing EGP, in increasing insulin-mediated glucose uptake, and in promoting glycogen synthesis and glycolysis, as noted previously (11)(13). Among the young rats, the suppression of EGP, glucose uptake, and glycogen synthesis were significantly better in the leptin-treated groups compared with the PF group, and some of the effects could be attributed to the higher VF content in the PF group. The hepatic TG content was unaltered with leptin treatment in aging rats, whereas there was more than a threefold decrease in younger PF animals. These data clearly demonstrate a resistance to leptin's action in aging rats compared with that in young rats.
Leptin resistance is also reflected in the inability of aged rats to suppress their own leptin gene expression in response to high plasma leptin levels. This feedback suppression is preserved in young rats, in which leptin infusion significantly decreases leptin gene expression compared with that of controls. These data are supported by similar observations by Shek and Scarpace (26) on the effect of leptin on leptin gene expression; they showed that old rats failed to decrease leptin gene expression in adipose tissue in response to centrally administered leptin. This failure suggests that the leptin expression in adipose tissue is under a central feedback regulation that is dependent on leptin and that there is resistance to this feedback regulation in aged rats. Failure of action of leptin with aging is evidenced in the transgenic mice model that overexpresses leptin (27). In this model, although increased leptin levels prevented an increase in FM in young animals, they failed to prevent fat accretion with aging. Similarly, leptin fails to decrease VF in aging rats, and this may contribute to impaired insulin action (20)(21)(22)(25).
The mechanisms for leptin resistance are under intensive investigation. Decreased availability of leptin in the hypothalamus, impaired leptin action, or both have been proposed as mechanisms of leptin resistance in old age. When availability of leptin was enhanced at the level of the hypothalamus with an adenovirus vector, the metabolic changes characteristic of aging were delayed, suggesting that decreased availability of leptin could be the initiating factor and the blood–brain barrier a potential site of leptin resistance (28). It has been suggested that the biological properties of the blood–brain barrier limit the transfer of leptin to its receptors in the arcuate nucleus, beyond certain plasma levels (29). However, with gradually increasing plasma levels, leptin initially should be able to cross the blood–brain barrier, and resistance at the leptin transfer could be explained only when higher levels are achieved. Leptin transduction, as evidenced by signal transducers and activators of transcription-3 (STAT-3) phosphorylation and binding to transcriptional sites, is significantly reduced in old rats compared with that in young rats (30)(31). However, even when leptin was infused intracerebroventricularly in old rats, signal transduction improved compared with controls but did not reach the levels seen in younger rats (31). These findings suggest that impaired transport across the blood–brain barrier accounts for some, but not all, of the leptin resistance in aging.
A decreased number of leptin receptors has been demonstrated in the hypothalamic nuclei of old rats, which may account for the leptin resistance in spite of higher plasma leptin levels (31)(32). An attractive hypothesis (33)(34) is the paradoxical induction of the suppressor of cytokine signaling 3 (SOCS-3). When SOCS-3 expression was compared in the hypothalamus and white adipocytes of young and old ad libitum fed Zucker (+/+) rats before and after induction of hyperleptinemia, hypothalamic SOCS-3 messenger RNA (mRNA) was approximately three times higher in old rats (18). The observation that old leptin-treated rats failed to decrease their own gene expression is consistent with this hypothesis. Impaired feedback between the plasma leptin levels and the leptin gene expression in the adipose tissue may contribute to increasing levels of leptin and further suppression of its action. The resistance to leptin action could also be related to changes in regulation of neuropeptide-Y expression with aging (35), as described in obese aging rats.
Most people in the Western world are overweight when they reach middle age (1). With advancing age they exhibit a further increase in body weight, abdominal obesity, insulin resistance, and increased plasma leptin levels in proportion to their body weight (1)(2)(3)(16)(17)(18)(36)(37), which may be modulated when aging is associated with losses of lean body mass and subcutaneous fat (38). The consequence of obesity in humans is decreased life span caused by an increase in all causes of death (39)(40). Leptin's failure may be an important biological initiator of the events leading to obesity. The failure of leptin injections to decrease body weight adequately in middle-aged obese and diabetic subjects demonstrates such a resistance (41). Taken together, these data, spanning humans and animal models, suggest that youth is a leptin-sensitive state, and that resistance to leptin occurs with aging. Because human and animal aging is also characterized by hypothalamic and pituitary failure, followed by changes in motor and cognitive abilities, we propose that these central events are markers that are coupled with the metabolic consequences of leptin failure (42)(43).
The failure of leptin to regulate food intake, body fat and its distribution, and insulin action suggests that leptin resistance plays a major role in the metabolic syndrome that is typical of aging.
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Acknowledgments |
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Xiao Hui Ma and Radhika Muzumdar contributed equally to this manuscript.
Received December 24, 2001
Accepted March 8, 2002
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| Journals of Gerontology Series B: Psychological Sciences and Social Sciences | |
) compared with the appropriate control (100%; 
). * p < .01.