- Email: [email protected]
Serum leptin response to endogenous hyperinsulinemia in aging rats
Mechanisms of Ageing and Development 115 (2000) 101 – 106 www.elsevier.com/locate/mechagedev
Serum leptin response to endogenous hyperinsulinemia in aging rats Arshag D. Mooradian *, Joe M. Chehade Department of Internal Medicine, Di6ision of Endocrinology, Diabetes and Metabolism, Health Sciences Center, Saint Louis Uni6ersity School of Medicine, 1402 South Grand Boule6ard, St Louis, MO 63104, USA Received 26 January 2000; accepted 23 March 2000
Abstract To determine if aging is associated with altered serum leptin response to diet-induced changes in endogenous hyperinsulinemia, male Fisher 344 (F344) rats at different age groups were studied while on regular rat chow and following 10 days of experimental diets consisting of 60% of the weight as fructose or glucose. The serum leptin concentration (ng/ml) gradually increased from basal levels of 2.5 9 0.1 at age of 4 months to 3.7 9 0.1, 6.990.9, 9.490.3 and 8.991.1 at 6, 12, 18 and 24 months of age, respectively (PB 0.001). Hyperinsulinemia associated with 60% fructose diet was associated with increased serum leptin levels in 4, 12, and 24 month old rats to 5.19 0.8, 6.79 1.2, and 8.69 1.1, respectively (PB0.001). Feeding 60% glucose diet also was associated with increased serum leptin levels in 4, 12 and 24 month old rats to 7.6 90.6, 7.2 9 0.7, and 9.1 91.1, respectively (P B0.001). Restricting dietary intake to 60% of the calories consumed by control rats for 10 days resulted in a decrease in serum leptin to 1.0 9 0.02 in 4 month old rats and 2.5 9 0.4 in 24 month old rats (P B0.01). It is concluded that aging in F344 rats is associated with increased serum leptin concentrations. However, diet-related hyperinsulinemic effect on leptin is blunted in aging rats although leptin response to caloric restriction is maintained. The inability of aging rats to mount hyperleptinemic response to dietary changes may contribute to the age-related increase in adiposity. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; Leptin; Adiposity; Obesity; Hyperinsulinemia
* Corresponding author. Tel.: +1-314-5778458; fax: +1-314-7734567. 0047-6374/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 1 1 0 - X
102 A.D. Mooradian, J.M. Chehade / Mechanisms of Ageing and De6elopment 115 (2000) 101–106
1. Introduction Age-related increase in body adiposity is well recognized in both humans and rats (Masoro, 1980; Epstein and Higgins, 1992; Mooradian, 1993; Rikans et al., 1993; Ahern et al., 1997; Li et al., 1997; Wolden-Hanson et al., 1999). One of the regulators of body adiposity is the Ob gene product leptin (Zhang et al., 1994). It appears that serum leptin is a marker of fat depots in the body and is a regulator of body weight through its effects on satiety and energy expenditure (Zhang et al., 1994). Age-related increase in body adiposity could not be attributed to leptin deficiency. In one study of Fisher 344 (F344) and Brown Norway hybrid rats, aging was associated with an increase in leptin gene expression which was out of proportion to increasing adiposity (Li et al., 1997). In a subsequent study of male Brown Norway rats, the increase in serum leptin with age was related to increased total and peripheral but not visceral fat (Wolden-Hanson et al., 1999). However, in mice the age-related increase in plasma leptin strongly correlates with visceral fat (Ahern et al., 1997). Although the basal serum leptin levels are increased with age, it is not known whether leptin response to various physiological stimuli are maintained in aged rats. It is possible that a loss of modulation of leptin secretion in response to various nutritional or hormonal stimuli may limit the ability of aging rats to regulate body fat mass. To test this hypothesis, the serum leptin response to caloric restriction and to endogenous hyperinsulinemia induced with dietary means was measured in young and aging rats.
2. Materials and methods
2.1. Animals Male Fischer 344 rats at the age of 4, 6, 12, 18 and 24 months were obtained from Harlan Laboratories (Indianapolis, IN). After 1–3 weeks at our animal facility, rats were divided into the following dietary groups: control diet consisting of standard rat chow with :62% of calories as complex carbohydrates, 23% as proteins, and 14.9% as fat. The high-fructose or high-glucose diet consisted of 65.7% calories as fructose or glucose respectively, 21.9% as protein and 12.3% as fat. All rats were maintained on these test diets and had water ad libitum for 10 days. The 60% fructose or 60% glucose diet refers to the amount of fructose or glucose as a percentage of the food weight. Per kilogram weight, these test diets contained 200 g casein protein, 3 g of DL-methionine, 600 g of corn oil, 102 g of cellulose fiber and 45 g vitamin (Teklad cat. No. 40060) and mineral (Teklad cat. No. 90015) mix. These diets have similar caloric content of : 3.65 Kcal/g. Body weight and food intake were measured every other day during the study. Some of the body weight data and hormonal values of the 4-month old rats have also been used in previous communications (Mooradian et al., 1997; Mooradian and Albert, 1999; Mooradian et al., 2000). The rats were killed by exsanguination
A.D. Mooradian, J.M. Chehade / Mechanisms of Ageing and De6elopment 115 (2000) 101–106 103
through the abdominal aorta under sodium pentobarbital anesthesia (45 mg/kg intraperitoneally). The rats were killed between 8:00 and 10:00 h after ad libitum overnight feeding. The internal organs of all the animals were inspected for gross pathology. Animals bearing tumors were discarded from the analysis.
2.2. Hormone assays Serum was stored at −70°C until assays were performed on duplicate samples. Glucose levels were determined using the glucose oxidase technique with a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA). Serum insulin was measured with a commercial rat-specific insulin radioimmunoassay kit (RI-13K, Linco Research, St Louis, MO). The limit of sensitivity of this assay was 0.1 ng/ml and the intra-assay variability was 5 3%. Serum leptin was measured with a rat-specific leptin radioimmunoassay kit (RL-83K, Linco Research, St Louis, MO). The sensitivity of the leptin assay was 0.5 ng/ml and the intra-assay variability was 7.2%.
2.3. Statistical analysis All results are presented as the mean9 SEM Data were analyzed by analysis of variance (ANOVA). When significant differences were found, subgroup analysis was performed with the Newman–Keuls test, using the statistical package Statistica for Windows. (Statsoft, Tulsa, OK, 1997).
3. Results and discussion The study results are summarized in Table 1. It is clear that the serum leptin concentration in rats on regular rat chow increases with age. This is consistent with the known age-related increase in adiposity in this strain of rats (Bertrand et al., 1980). Another observation to note is that insulin resistance in rats fed the fructose enriched diet is reflected in the elevated serum glucose and insulin concentrations. In rats fed the glucose enriched diet, a state of relative insulin resistance is suggested by the finding of increased serum insulin level without a significant increase in serum glucose concentration. Similar conclusions as to possible insulin resistance seen in aging rats can be drawn based on increased serum insulin concentrations during regular chow feeding. Of interest is that in 4-month old rats, the serum leptin level in those fed a high fructose diet (5.19 0.8 ng/ml) was not as high as that observed in those fed a glucose enriched diet (7.69 0.6 ng/ml) (PB 0.01). A similar trend was seen in 12 and 24-month old rats. The cause of this apparent dissociation between the increased insulin and leptin levels is not known. Since free fatty acids (FFA) suppress leptin production (Rentsch and Chiesi, 1996), it is possible that the marked increase in serum FFA commonly found in rats fed fructose enriched diets may have somewhat suppressed serum leptin levels. Another possible explanation is that reduced caloric consumption of rats allocated to high fructose feeding may
Rat group
I. 4 months old Control diet (10) 60% fructose (10) 60% glucose (10)
Body weight (g) at day 1
Body weight (g) at day 10
Food intake (g/d/rat)
298.4 94.9
314.6 9 6.2
19.9 9 0.7
126.393.5
1.79 0.3
286.3 93.5
296.6 9 3.4
16.8 90.9*
178.0 94.5*
11.79 2.1*
292.4 95.1
306.2 97.3
19.6 90.9
129.0 9 4.1
7.99 3.1*
7.69 0.6*
442.3 97.4**
16.9 9 0.6**
137.8 9 3.0
2.79 0.2**
6.99 0.9**
445.09 6.7**
15.3 90.4*
185.49 4.8*
10.99 1.2*
6.7 9 1.2
451.1 98.2**
16.9 9 0.9**
134.6 9 4.6
8.39 1.5*
7.79 0.7
17.9 9 0.9**
132.1 9 4.8
3.1 9 0.4**
8.9 9 1.1**
454.6 95.5**
15.2 90.8*
172.1 9 6.6*
13.29 1.1*
449.9 9 5.9**
17.2 90.3**
138.19 5.8
9.69 1.2*
II. 12 months old Control diet 438.9 9 6.9** (10) 60% fructose 439.1 97.1** (10) 60% glucose 442.3 9 6.8** (10) III. 24 months old Control diet 447.9 9 9.8** (10) 60% fructose 452.8 96.6** (10) 60% glucose 448.4 96.1** (10)
448 9 11.4**
The (n) in each group is shown in parentheses. Mean 9SEM. * PB0.01 compared to control rats on standard rat chow ad libitum. ** PB0.01 compared to 4-month-old rats on the same diet.
a
Glucose (mg/dl)
Insulin (ng/ml)
Leptin (ng/ml)
2. 5 90.1 5.1 9 0.8*
8.6 9 1.1** 9.1 9 1.1
104 A.D. Mooradian, J.M. Chehade / Mechanisms of Ageing and De6elopment 115 (2000) 101–106
Table 1 The effect of various dietary interventions on serum leptin concentrations in rats at 4, 12 and 24 months of age. 60% fructose and 60% glucose diet had 60% of the total weight as fructose and glucose, respectivelya
A.D. Mooradian, J.M. Chehade / Mechanisms of Ageing and De6elopment 115 (2000) 101–106 105
have blunted leptin response. The reduced food intake in rats given the high fructose diet was apparent in all age groups and occurred immediately after introducing this diet. The precise cause for this reduced food consumption in rats on high fructose diet is not known. During the course of these studies it was noted that a subgroup of 24 month old rats have reduced caloric intake and weight loss and reduced adiposity. This subgroup of rats was excluded from the analysis since it was not clear if the changes are age-related and they may have been secondary to an underlying disease commonly found in aged rats. To determine the effect of short term reduced caloric intake on serum leptin, 4 and 24 month old rats were maintained for 10 days on calorically restricted diet to be equivalent to 60% of the calories consumed by age-matched healthy cohort. This short-term dietary restriction reduced serum leptin level in 4-month-old rats to 1.09 0.02 ng/ml compared to a level of 2.59 0.1 ng/ml in young controls (P B 0.01). In 24 month old rats, serum leptin was reduced with caloric restriction to 2.59 0.4 ng/ml compared to 8.99 1.1 ng/ml in 24 month old ad libitum fed rats (PB 0.001). Thus, dietary restriction resulted in : 3.6-fold reduction of serum leptin in 24-month-old rats and : 2.4-fold reduction in 4-month-old rats. This difference was not statistically significant suggesting that the mechanisms underlying the downregulation of leptin expression are intact in the aged rats. To determine if aging alters the capacity of the organism to upregulate serum leptin concentrations, rats at 4, 12, and 24 months of age were maintained on 60% fructose and 60% glucose diets. Rats maintained on these diets have been previously found to have increased serum leptin concentrations (Mooradian et al., 2000). The serum leptin was significantly increased in 4-month-old rats maintained on 60% fructose or 60% glucose diet (Table 1). It is apparent that serum leptin increase was related to hyperinsulinemia associated with high glucose or fructose feeding rather than insulin resistance associated with fructose feeding. This is in agreement with previous studies in human subjects where endogenous chronic hyperinsulinemia is implicated as the stimulus for increased leptin in obesity independent of insulin resistance associated with large body mass index or increased adiposity (Kim-Motoyama et al., 1997; Mohamed-Ali et al., 1997; Zimmet et al., 1998). In contrast to young 4-month-old rats, aging rats at 12 and 24 months of age failed to increase their serum leptin concentration in response to diet-induced hyperinsulinemia. The precise underlying mechanisms for this observation are not known. It is possible that the molecular pathways mediating the leptin-inducing effect of insulin are saturated by the chronic hyperinsulinemic state of aging rats such that any additional diet-induced hyperinsulinemia would be ineffective in mounting an incremental leptin production. Overall, it appears that aging in rats is associated with blunting of the hyperleptinemic response to dietary changes while their ability to downregulate leptin production in response to caloric restriction is preserved. These observations suggest that the primary role of leptin in aging rats is to fend the organism against caloric restriction or starvation by downregulating its production to enhance hunger and stimulate food-seeking behavior. However, the inability of aging rats to
106 A.D. Mooradian, J.M. Chehade / Mechanisms of Ageing and De6elopment 115 (2000) 101–106
mount hyperleptinemic responses to dietary changes may contribute to the age-related increase in adiposity.
References Ahern, B., Mansson, S., Gingerich, R.L., Havel, P.J., 1997. Regulation of plasma leptin in mice: influence of age, high fat diet and fasting. Am. J. Physiol. 273, R113 – R120. Bertrand, H.A., Lynd, F.T., Masoro, E.J., Yu, B.P., 1980. Changes in adipose mass and cellularity through the adult life of rats fed ad libitum or a life prolonging restricted diet. J. Gerontol. 35, 827–835. Epstein, F.H., Higgins, M., 1992. Epidemiology of obesity. In: Bjorntorp, P., Brodoff, B.N. (Eds.), Obesity. Lippincott, Philadelphia, pp. 330 – 342. Kim-Motoyama, H., Yamaguchi, T., Katakura, T., et al., 1997. Serum leptin levels are associated with hyperinsulinemia independent of body mass index but not with visceral obesity. Biochem. Biophys. Res. Comm. 239, 340–344. Li, H., Matheny, M., Nicolson, M., Tumer, N., Scarpace, P.H., 1997. Leptin gene expression increases with age independent of increasing adiposity in rats. Diabetes 46, 2035 – 2039. Masoro, E.J., 1980. Rats as models for the study of obesity. Exp. Aging Res. 6, 261 – 270. Mohamed-Ali, V., Pinkney, J.H., Panahloo, A., et al., 1997. Relationship between plasma leptin and insulin concentrations but not insulin resistance in non-insulin dependent (type 2) diabetes mellitus. Diab. Med. 14, 376–380. Mooradian, A.D., 1993. Biology of aging. In: Felsenthal, G., Garrison, S.G., Steinberg, G.U. (Eds.), Rehabilitation of the Aging and Elderly Patient. Williams and Wilkins, Baltimore, MD, pp. 3 – 10. Mooradian, A.D., Wong, N.C.W., Shah, G.N., 1997. Apolipoprotein A1 expression in young and aged rats is modulated by dietary carbohydrates. Metabolism 46, 1132 – 1136. Mooradian, A.D., Albert, S.G., 1999. The age-related changes in lipogenic enzymes: the role of dietary factors and thyroid hormone responsiveness. Mech. Ageing Devel. 108, 139 – 149. Mooradian AD, Chehade J, Hurd R, Haas MJ. Monosaccharide enriched diets cause hyperleptinemia without hypophagia, Nutrition (in press). Rentsch, J., Chies, M., 1996. Regulation of Ob gene mRNA levels in cultured adipocytes. FEBS Lett. 379, 55–59. Rikans, L.E., Venkataraman, P.S., Cai, Y., 1993. Evaluation by dual-energy X-ray absorptiometry of age-related changes in body composition of male rats. Mech. Ageing Dev. 71, 155 – 158. Wolden-Hanson, T., Marck, B.T., Smith, L., Matsumoto, A.M., 1999. Cross-sectional and longitudinal analysis of age-associated changes in body composition of male Brown Norway rats: association of serum leptin levels with peripheral adiposity. J. Gerontol. Biol. Sci. 54A, B99 – B107. Zhang, Y., Proenca, R., Maffei, M., et al., 1994. Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425– 432. Zimmet, P.Z., Collins, V.R., Decoulten, M.P., 1998. Is there a relationship between leptin and insulin sensitivity independent of obesity? a population-based study in the Indian Ocean nation of Mauritius. Mauritius NCD Study Group. Int. J. Obesity Rel. Metab. Disord. 72, 171 – 177.
.
Serum leptin response to endogenous hyperinsulinemia in aging rats Arshag D. Mooradian *, Joe M. Chehade Department of Internal Medicine, Di6ision of Endocrinology, Diabetes and Metabolism, Health Sciences Center, Saint Louis Uni6ersity School of Medicine, 1402 South Grand Boule6ard, St Louis, MO 63104, USA Received 26 January 2000; accepted 23 March 2000
Abstract To determine if aging is associated with altered serum leptin response to diet-induced changes in endogenous hyperinsulinemia, male Fisher 344 (F344) rats at different age groups were studied while on regular rat chow and following 10 days of experimental diets consisting of 60% of the weight as fructose or glucose. The serum leptin concentration (ng/ml) gradually increased from basal levels of 2.5 9 0.1 at age of 4 months to 3.7 9 0.1, 6.990.9, 9.490.3 and 8.991.1 at 6, 12, 18 and 24 months of age, respectively (PB 0.001). Hyperinsulinemia associated with 60% fructose diet was associated with increased serum leptin levels in 4, 12, and 24 month old rats to 5.19 0.8, 6.79 1.2, and 8.69 1.1, respectively (PB0.001). Feeding 60% glucose diet also was associated with increased serum leptin levels in 4, 12 and 24 month old rats to 7.6 90.6, 7.2 9 0.7, and 9.1 91.1, respectively (P B0.001). Restricting dietary intake to 60% of the calories consumed by control rats for 10 days resulted in a decrease in serum leptin to 1.0 9 0.02 in 4 month old rats and 2.5 9 0.4 in 24 month old rats (P B0.01). It is concluded that aging in F344 rats is associated with increased serum leptin concentrations. However, diet-related hyperinsulinemic effect on leptin is blunted in aging rats although leptin response to caloric restriction is maintained. The inability of aging rats to mount hyperleptinemic response to dietary changes may contribute to the age-related increase in adiposity. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; Leptin; Adiposity; Obesity; Hyperinsulinemia
* Corresponding author. Tel.: +1-314-5778458; fax: +1-314-7734567. 0047-6374/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 1 1 0 - X
102 A.D. Mooradian, J.M. Chehade / Mechanisms of Ageing and De6elopment 115 (2000) 101–106
1. Introduction Age-related increase in body adiposity is well recognized in both humans and rats (Masoro, 1980; Epstein and Higgins, 1992; Mooradian, 1993; Rikans et al., 1993; Ahern et al., 1997; Li et al., 1997; Wolden-Hanson et al., 1999). One of the regulators of body adiposity is the Ob gene product leptin (Zhang et al., 1994). It appears that serum leptin is a marker of fat depots in the body and is a regulator of body weight through its effects on satiety and energy expenditure (Zhang et al., 1994). Age-related increase in body adiposity could not be attributed to leptin deficiency. In one study of Fisher 344 (F344) and Brown Norway hybrid rats, aging was associated with an increase in leptin gene expression which was out of proportion to increasing adiposity (Li et al., 1997). In a subsequent study of male Brown Norway rats, the increase in serum leptin with age was related to increased total and peripheral but not visceral fat (Wolden-Hanson et al., 1999). However, in mice the age-related increase in plasma leptin strongly correlates with visceral fat (Ahern et al., 1997). Although the basal serum leptin levels are increased with age, it is not known whether leptin response to various physiological stimuli are maintained in aged rats. It is possible that a loss of modulation of leptin secretion in response to various nutritional or hormonal stimuli may limit the ability of aging rats to regulate body fat mass. To test this hypothesis, the serum leptin response to caloric restriction and to endogenous hyperinsulinemia induced with dietary means was measured in young and aging rats.
2. Materials and methods
2.1. Animals Male Fischer 344 rats at the age of 4, 6, 12, 18 and 24 months were obtained from Harlan Laboratories (Indianapolis, IN). After 1–3 weeks at our animal facility, rats were divided into the following dietary groups: control diet consisting of standard rat chow with :62% of calories as complex carbohydrates, 23% as proteins, and 14.9% as fat. The high-fructose or high-glucose diet consisted of 65.7% calories as fructose or glucose respectively, 21.9% as protein and 12.3% as fat. All rats were maintained on these test diets and had water ad libitum for 10 days. The 60% fructose or 60% glucose diet refers to the amount of fructose or glucose as a percentage of the food weight. Per kilogram weight, these test diets contained 200 g casein protein, 3 g of DL-methionine, 600 g of corn oil, 102 g of cellulose fiber and 45 g vitamin (Teklad cat. No. 40060) and mineral (Teklad cat. No. 90015) mix. These diets have similar caloric content of : 3.65 Kcal/g. Body weight and food intake were measured every other day during the study. Some of the body weight data and hormonal values of the 4-month old rats have also been used in previous communications (Mooradian et al., 1997; Mooradian and Albert, 1999; Mooradian et al., 2000). The rats were killed by exsanguination
A.D. Mooradian, J.M. Chehade / Mechanisms of Ageing and De6elopment 115 (2000) 101–106 103
through the abdominal aorta under sodium pentobarbital anesthesia (45 mg/kg intraperitoneally). The rats were killed between 8:00 and 10:00 h after ad libitum overnight feeding. The internal organs of all the animals were inspected for gross pathology. Animals bearing tumors were discarded from the analysis.
2.2. Hormone assays Serum was stored at −70°C until assays were performed on duplicate samples. Glucose levels were determined using the glucose oxidase technique with a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA). Serum insulin was measured with a commercial rat-specific insulin radioimmunoassay kit (RI-13K, Linco Research, St Louis, MO). The limit of sensitivity of this assay was 0.1 ng/ml and the intra-assay variability was 5 3%. Serum leptin was measured with a rat-specific leptin radioimmunoassay kit (RL-83K, Linco Research, St Louis, MO). The sensitivity of the leptin assay was 0.5 ng/ml and the intra-assay variability was 7.2%.
2.3. Statistical analysis All results are presented as the mean9 SEM Data were analyzed by analysis of variance (ANOVA). When significant differences were found, subgroup analysis was performed with the Newman–Keuls test, using the statistical package Statistica for Windows. (Statsoft, Tulsa, OK, 1997).
3. Results and discussion The study results are summarized in Table 1. It is clear that the serum leptin concentration in rats on regular rat chow increases with age. This is consistent with the known age-related increase in adiposity in this strain of rats (Bertrand et al., 1980). Another observation to note is that insulin resistance in rats fed the fructose enriched diet is reflected in the elevated serum glucose and insulin concentrations. In rats fed the glucose enriched diet, a state of relative insulin resistance is suggested by the finding of increased serum insulin level without a significant increase in serum glucose concentration. Similar conclusions as to possible insulin resistance seen in aging rats can be drawn based on increased serum insulin concentrations during regular chow feeding. Of interest is that in 4-month old rats, the serum leptin level in those fed a high fructose diet (5.19 0.8 ng/ml) was not as high as that observed in those fed a glucose enriched diet (7.69 0.6 ng/ml) (PB 0.01). A similar trend was seen in 12 and 24-month old rats. The cause of this apparent dissociation between the increased insulin and leptin levels is not known. Since free fatty acids (FFA) suppress leptin production (Rentsch and Chiesi, 1996), it is possible that the marked increase in serum FFA commonly found in rats fed fructose enriched diets may have somewhat suppressed serum leptin levels. Another possible explanation is that reduced caloric consumption of rats allocated to high fructose feeding may
Rat group
I. 4 months old Control diet (10) 60% fructose (10) 60% glucose (10)
Body weight (g) at day 1
Body weight (g) at day 10
Food intake (g/d/rat)
298.4 94.9
314.6 9 6.2
19.9 9 0.7
126.393.5
1.79 0.3
286.3 93.5
296.6 9 3.4
16.8 90.9*
178.0 94.5*
11.79 2.1*
292.4 95.1
306.2 97.3
19.6 90.9
129.0 9 4.1
7.99 3.1*
7.69 0.6*
442.3 97.4**
16.9 9 0.6**
137.8 9 3.0
2.79 0.2**
6.99 0.9**
445.09 6.7**
15.3 90.4*
185.49 4.8*
10.99 1.2*
6.7 9 1.2
451.1 98.2**
16.9 9 0.9**
134.6 9 4.6
8.39 1.5*
7.79 0.7
17.9 9 0.9**
132.1 9 4.8
3.1 9 0.4**
8.9 9 1.1**
454.6 95.5**
15.2 90.8*
172.1 9 6.6*
13.29 1.1*
449.9 9 5.9**
17.2 90.3**
138.19 5.8
9.69 1.2*
II. 12 months old Control diet 438.9 9 6.9** (10) 60% fructose 439.1 97.1** (10) 60% glucose 442.3 9 6.8** (10) III. 24 months old Control diet 447.9 9 9.8** (10) 60% fructose 452.8 96.6** (10) 60% glucose 448.4 96.1** (10)
448 9 11.4**
The (n) in each group is shown in parentheses. Mean 9SEM. * PB0.01 compared to control rats on standard rat chow ad libitum. ** PB0.01 compared to 4-month-old rats on the same diet.
a
Glucose (mg/dl)
Insulin (ng/ml)
Leptin (ng/ml)
2. 5 90.1 5.1 9 0.8*
8.6 9 1.1** 9.1 9 1.1
104 A.D. Mooradian, J.M. Chehade / Mechanisms of Ageing and De6elopment 115 (2000) 101–106
Table 1 The effect of various dietary interventions on serum leptin concentrations in rats at 4, 12 and 24 months of age. 60% fructose and 60% glucose diet had 60% of the total weight as fructose and glucose, respectivelya
A.D. Mooradian, J.M. Chehade / Mechanisms of Ageing and De6elopment 115 (2000) 101–106 105
have blunted leptin response. The reduced food intake in rats given the high fructose diet was apparent in all age groups and occurred immediately after introducing this diet. The precise cause for this reduced food consumption in rats on high fructose diet is not known. During the course of these studies it was noted that a subgroup of 24 month old rats have reduced caloric intake and weight loss and reduced adiposity. This subgroup of rats was excluded from the analysis since it was not clear if the changes are age-related and they may have been secondary to an underlying disease commonly found in aged rats. To determine the effect of short term reduced caloric intake on serum leptin, 4 and 24 month old rats were maintained for 10 days on calorically restricted diet to be equivalent to 60% of the calories consumed by age-matched healthy cohort. This short-term dietary restriction reduced serum leptin level in 4-month-old rats to 1.09 0.02 ng/ml compared to a level of 2.59 0.1 ng/ml in young controls (P B 0.01). In 24 month old rats, serum leptin was reduced with caloric restriction to 2.59 0.4 ng/ml compared to 8.99 1.1 ng/ml in 24 month old ad libitum fed rats (PB 0.001). Thus, dietary restriction resulted in : 3.6-fold reduction of serum leptin in 24-month-old rats and : 2.4-fold reduction in 4-month-old rats. This difference was not statistically significant suggesting that the mechanisms underlying the downregulation of leptin expression are intact in the aged rats. To determine if aging alters the capacity of the organism to upregulate serum leptin concentrations, rats at 4, 12, and 24 months of age were maintained on 60% fructose and 60% glucose diets. Rats maintained on these diets have been previously found to have increased serum leptin concentrations (Mooradian et al., 2000). The serum leptin was significantly increased in 4-month-old rats maintained on 60% fructose or 60% glucose diet (Table 1). It is apparent that serum leptin increase was related to hyperinsulinemia associated with high glucose or fructose feeding rather than insulin resistance associated with fructose feeding. This is in agreement with previous studies in human subjects where endogenous chronic hyperinsulinemia is implicated as the stimulus for increased leptin in obesity independent of insulin resistance associated with large body mass index or increased adiposity (Kim-Motoyama et al., 1997; Mohamed-Ali et al., 1997; Zimmet et al., 1998). In contrast to young 4-month-old rats, aging rats at 12 and 24 months of age failed to increase their serum leptin concentration in response to diet-induced hyperinsulinemia. The precise underlying mechanisms for this observation are not known. It is possible that the molecular pathways mediating the leptin-inducing effect of insulin are saturated by the chronic hyperinsulinemic state of aging rats such that any additional diet-induced hyperinsulinemia would be ineffective in mounting an incremental leptin production. Overall, it appears that aging in rats is associated with blunting of the hyperleptinemic response to dietary changes while their ability to downregulate leptin production in response to caloric restriction is preserved. These observations suggest that the primary role of leptin in aging rats is to fend the organism against caloric restriction or starvation by downregulating its production to enhance hunger and stimulate food-seeking behavior. However, the inability of aging rats to
106 A.D. Mooradian, J.M. Chehade / Mechanisms of Ageing and De6elopment 115 (2000) 101–106
mount hyperleptinemic responses to dietary changes may contribute to the age-related increase in adiposity.
References Ahern, B., Mansson, S., Gingerich, R.L., Havel, P.J., 1997. Regulation of plasma leptin in mice: influence of age, high fat diet and fasting. Am. J. Physiol. 273, R113 – R120. Bertrand, H.A., Lynd, F.T., Masoro, E.J., Yu, B.P., 1980. Changes in adipose mass and cellularity through the adult life of rats fed ad libitum or a life prolonging restricted diet. J. Gerontol. 35, 827–835. Epstein, F.H., Higgins, M., 1992. Epidemiology of obesity. In: Bjorntorp, P., Brodoff, B.N. (Eds.), Obesity. Lippincott, Philadelphia, pp. 330 – 342. Kim-Motoyama, H., Yamaguchi, T., Katakura, T., et al., 1997. Serum leptin levels are associated with hyperinsulinemia independent of body mass index but not with visceral obesity. Biochem. Biophys. Res. Comm. 239, 340–344. Li, H., Matheny, M., Nicolson, M., Tumer, N., Scarpace, P.H., 1997. Leptin gene expression increases with age independent of increasing adiposity in rats. Diabetes 46, 2035 – 2039. Masoro, E.J., 1980. Rats as models for the study of obesity. Exp. Aging Res. 6, 261 – 270. Mohamed-Ali, V., Pinkney, J.H., Panahloo, A., et al., 1997. Relationship between plasma leptin and insulin concentrations but not insulin resistance in non-insulin dependent (type 2) diabetes mellitus. Diab. Med. 14, 376–380. Mooradian, A.D., 1993. Biology of aging. In: Felsenthal, G., Garrison, S.G., Steinberg, G.U. (Eds.), Rehabilitation of the Aging and Elderly Patient. Williams and Wilkins, Baltimore, MD, pp. 3 – 10. Mooradian, A.D., Wong, N.C.W., Shah, G.N., 1997. Apolipoprotein A1 expression in young and aged rats is modulated by dietary carbohydrates. Metabolism 46, 1132 – 1136. Mooradian, A.D., Albert, S.G., 1999. The age-related changes in lipogenic enzymes: the role of dietary factors and thyroid hormone responsiveness. Mech. Ageing Devel. 108, 139 – 149. Mooradian AD, Chehade J, Hurd R, Haas MJ. Monosaccharide enriched diets cause hyperleptinemia without hypophagia, Nutrition (in press). Rentsch, J., Chies, M., 1996. Regulation of Ob gene mRNA levels in cultured adipocytes. FEBS Lett. 379, 55–59. Rikans, L.E., Venkataraman, P.S., Cai, Y., 1993. Evaluation by dual-energy X-ray absorptiometry of age-related changes in body composition of male rats. Mech. Ageing Dev. 71, 155 – 158. Wolden-Hanson, T., Marck, B.T., Smith, L., Matsumoto, A.M., 1999. Cross-sectional and longitudinal analysis of age-associated changes in body composition of male Brown Norway rats: association of serum leptin levels with peripheral adiposity. J. Gerontol. Biol. Sci. 54A, B99 – B107. Zhang, Y., Proenca, R., Maffei, M., et al., 1994. Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425– 432. Zimmet, P.Z., Collins, V.R., Decoulten, M.P., 1998. Is there a relationship between leptin and insulin sensitivity independent of obesity? a population-based study in the Indian Ocean nation of Mauritius. Mauritius NCD Study Group. Int. J. Obesity Rel. Metab. Disord. 72, 171 – 177.
.