Introduction. S Lin 1, TC Thomas 1, LH Storlien 1 and XF Huang 1 *

(2000) 24, 639±646 ß 2000 Macmillan Publishers Ltd All rights reserved 0307±0565/00 $15.00 www.nature.com/ijo Development of high fat diet-induced obesity and leptin resistance in C57Bl=6J mice S Lin 1, TC Thomas 1, LH Storlien 1 and XF Huang 1 * 1 Metabolic Research Center, Department of Biomedical Science, University of Wollongong, NSW 2522, Australia OBJECTIVE: To investigate the development of high fat diet-induced obesity and leptin resistance. DESIGN: Two experiments were carried out in this study. Firstly, we fed the mice with a high- or low-fat diet for up to 19 weeks to examine a progressive development of high fat diet-induced obesity. Secondly, we examined peripheral and central exogenous leptin sensitivity in mice fed high- or low-fat diets for 1, 8 or 19 weeks. SUBJECTS: A total of 168 C57BL=6J mice (3 weeks old) were used in this study. MEASUREMENTS: In the rst experiment, we measured the body weight, energy intake, adipose tissue mass, tibia bone length, and plasma leptin in mice fed either a high- or low-fat diet for 1, 8, 15 and 19 weeks. In the second experiment, body weight change and cumulative energy intake were measured at 6 h intervals for 72 h after leptin injection in mice fed a high- or low-fat diet for 1, 8 or 19 weeks. RESULTS: The results from the rst experiment suggested that the development of high fat diet-induced obesity in mice could be divided into early, middle and late stages. Compared with the mice fed a low-fat diet, the mice fed a high-fat diet showed a gradually increased body weight ( 5.2%), fat storage (epididymal plus perirenal; 6.7%) and plasma leptin ( 18%) at 1 week; 11.4%, 68.1%, and 223%, respectively, at 8 weeks; and 30.5%, 141%, and 458%, respectively, at 19 weeks. Energy intake of high fat diet-fed mice was equal to that of low fat diet-fed controls for the rst 3 weeks; it fell below control levels over the next 5 week period, but began to increase gradually after 8 weeks of high-fat diet feeding and then increased dramatically from 15 weeks to be 14% higher than that of controls after 19 weeks. The results from our second experiment showed that: (1) after 1 week of feeding, the mice fed a highfat diet were sensitive to a 2 mg=g (body weight) intraperitoneal (i.p.) injection of leptin, with no differences in body weight change or cumulative energy intake post-injection; (2) after 8 weeks of feeding, the mice fed a high-fat diet were insensitive to 2 mg=g (body weight) i.p. leptin, but were sensitive to a 0.1 mg intracerebroventricular (i.c.v.) injection of leptin; (3) after 19 weeks of feeding, the mice fed a high-fat diet were insensitive to 0.1 mg i.c.v. leptin, but were sensitive to a high dose of 2 mg i.c.v. leptin. CONCLUSIONS: The present study demonstrated that the development of high fat diet-induced obesity (19 weeks) in C57 B1=6J mice could be divided into three stages: (1) an early stage in response to high-fat diet that mice were sensitive to exogenous leptin; (2) a reduced food intake stage when mice had an increase in leptin production and still retained central leptin sensitivity; and (3) an increased food intake stage, accompanied by a reduction of central leptin sensitivity. (2000) 24, 639±646 Keywords: obesity; dietary fats; hyperleptinaemia; leptin; mice Introduction Obesity is de ned as increased adipose mass resulting from a chronic imbalance between energy intake and expenditure. 1±3 In rodents, a number of autosomal recessive gene mutations that result in the obese phenotype have been described, including ob=ob and db=db mice. 4,5 The products of the ob and db genes represent a hormone ± receptor pair (leptin, produced and released into the plasma by adipocytes, and its receptor, found in hypothalamic nuclei, respectively) that appears to be a component of a central negative feedback system involved in the regulation of fat mass, such that a change in the amount of lipid *Correspondence: XF Huang, Department of Biomedical Science, University of Wollongong, NSW 2522, Australia. E-mail: Xu_Feng_Huang@uow.edu.au Received 18 September 1998; revised 23 August 1999 and 8 November 1999; accepted 16 December 1999 stored triggers a response that opposes the change, so as to maintain adiposity within a narrow range. 6±9 Mice unable to produce leptin (ob=ob) or to respond to it (db=db) are characterized by hyperphagia and decreased energy expenditure, as well as other metabolic abnormalities such as hyperinsulinaemia, hyperlipidaemia, insulin resistance, glucose intolerance and diabetes. 9,10 Further, treatment with leptin inhibits feeding, increases energy expenditure, and subsequently reduces body fat in a dose-dependent fashion in both ob=ob and normal mice, but not in db=db mice. 11,12 The ef cacy of such murine monogenic models of obesity in determining the mechanisms underlying human obesity remains to be seen. There is no convincing evidence to indicate that mutations in the genes encoding either leptin or its receptor are a part of the aetiology of normal human obesity. 13,14 Indeed, plasma leptin concentrations are closely correlated to body mass index in humans, and ob mrna has been shown to be increased in obese humans

640 relative to nonobese humans. 6,15 ± 17 As such it has been suggested that obese humans may be insensitive, or `resistant' to the leptin signal. In support of this hypothesis, leptin resistance has recently been described in a rodent model of obesity that may more closely re ect the human condition, namely, diet-induced obese mice. 18 Further, the evidence indicated that the mice and rats fed a high-fat diet for weeks ( < 10 weeks) develop obesity and resistance peripherally. 18,19 Our study was therefore undertaken to investigate whether or not high fat dietinduced obesity is associated with a progressive development of leptin insensitivity peripherally and centrally, and whether the duration of high fat feeding may be a factor in the development of such leptin insensitivity. Materials and methods Animals One-hundred and sixty-eight 3-week-old C57BL=6J male mice were used in this study. The study contains two major experiments. In the rst experiment, 48 mice were used to compare body weight, food intake, fat storage and serum leptin concentration between the mice fed a high and low-fat diet. In the second experiment, 120 mice were used to examine peripheral and central effects of exogenous leptin following a high-fat diet for 1, 8 and 19 weeks. Animals were randomized and housed individually in environmentally controlled conditions (temperature 22 C, light cycle from 06:00 to 18:00 and dark cycle from 18:00 to 06:00), and were given ad libitum access to tap water throughout the study. Diet and experimental procedure Mice were obtained from the Animal Resource Centre (Western Australia) and were fed standard laboratory chow for the rst week to allow them to adjust to the new environment. Mice were subsequently randomly assigned to one of two groups: a high-fat diet fed group or a low-fat diet fed group. The composition of the respective diets is the same as used previously. 20 Diets were freshly made every week and stored at 4 C. Mice were fed using small plastic dishes just before the beginning of the dark cycle. Experiment 1. Forty-eight mice were divided into two groups. The mice in the rst group were fed with high-fat diet (HFF) and the mice in the second group were fed with low-fat diet (LFF). Six mice for each group were killed after being fed either high or low-fat diet for 1, 8, 15 and 19 weeks. The body weight and energy intake were measured every week for the 19- week feeding period. After the mice were killed at week 1, 8, 15 and 19, adipose tissues (epididymal, perirenal and inguinal fat masses) and tibial length were measured. Blood samples were obtained by puncturing its right ventricle of the heart and plasma leptin was measured using an RIA kit for mouse leptin (Linco Inc., St Louis, MO). Experiment 2. One-hundred and twenty mice were divided into HFF or LFF groups. In accordance with the observed variability in energy intake of HFF mice in the rst experiment, three time points were selected for the administration of leptin as representative of the three time periods during which rates of consumption differed, namely, weeks 1, 8 and 19 of the feeding protocol. After 1 week of high or low-fat diet, six mice from each group received an i.p. injection of 2.0 mg=g (body weight) recombinant murine leptin (R&D Systems Inc., USA) or saline as controls. Body weight change and cumulative energy intake were then measured every 6 h until 72 h following the injection. After 8 weeks of high or low-fat diet, six mice from each group received an intraperitoneal (i.p.) injection of 2.0 mg=g (body weight) leptin or saline as controls, while another six mice from each group received an intracerebroventricular (i.c.v.) injection of leptin (0.1 mg) or vehicle (5 ml of 10mM sodium acetate, ph 7.4) as controls. Needle placement was con rmed by visual examination of the lateral ventricle in the tissue sections. Body weight change and cumulative energy intake were then measured at every 6 h until 72 h following the injection. After 19 weeks of high or low-fat diet, two dosages of i.c.v. leptin were used for this part of the study. Six mice from each group received an i.c.v. injection of 0.1 mg leptin or vehicle, while another six mice from each group received a higher dosage (2 mg) of i.c.v. leptin or vehicle as controls. Body weight change and cumulative energy intake were then measured at 6 h interval until 72 h following the injection. Food consumption measurement Food consumption was estimated by subtracting the amount of food left in the plastic dishes and the amount of food spilled from the initial food weight. Food spillage was determined as follows: any food that spilled was collected on absorbent paper placed beneath the cages in which the mice were kept. After removing faeces and woodshavings from the paper, food spillage was collected and weighed. Statistical analysis All data are shown as mean s.e. for groups based on six mice in each group. The effect of leptin dose, differences in body weights and cumulative energy intake over time between HFF and LFF mice were analysed using a two-way analysis of variance (ANOVA). Analyses were performed using the JMP statistical package (SAS institute Inc., NC, USA,

1997). Differences in variables between HFF mice and LFF mice were analysed using Student's t-test. Results Experiment 1: measurements of body weight, calorie intake, fat storage and plasma leptin in mice fed a high- or low-fat diet for 1, 8, 15 or 19 weeks A signi cant increase in body weight gain in mice fed the high-fat diet was evident after only 2 weeks of high-fat feeding, and this trend continued throughout the dietary protocol (Figure 1A). After 8, 15 and 19 weeks feeding, body weight gain of the HFF group was 11.4%, 23.1% and 30.5%, respectively, higher than that of the LFF group (all P < 0.0001). In conjunction with the changes in body weight, the increases in weight of the epididymal and perirenal fat masses of the HFF group were signi cantly greater than those of the LFF controls after 8, 15 and 19 weeks of feeding (Figure 2A, B). The nal gains in epididymal and perirenal fat masses of the HFF group were 144% and 130%, respectively. The gain in inguinal fat mass of the HFF group was not signi cantly greater than that of the LFF controls at 1 and 8 weeks feeding, and was 33.6% greater in the HFF group at 19 weeks (Figure 2C). There was no signi cant difference in tibia bone length between the two groups (Figure 2E). In the HFF group, energy intake paralleled that of the LFF controls for 4 weeks. However, at this point, the energy intake of HFF mice began to decrease, and by the fth week of feeding, it had fallen signi cantly below that of the LFF mice. From 8 weeks, however, the energy intake of the HFF group began a gradual increase; then, at approximately 15 weeks, it increased dramatically, surpassing that of the LFF mice, and after 19 weeks of feeding, was 14.6% (P < 0.0001) greater than the energy intake of the LFF controls (Figure 1B). As it had been noted that there was a decreased energy intake in HFF group between week 5 and 8, the HFF mice still gained signi cantly more epididymal ( 54%) and perirenal ( 123%) fats compared with the LFF mice at 8 week. Plasma leptin concentration were signi cantly elevated in the HFF mice compared with LFF mice when measured at 8, 15 and 19 weeks of feeding (Figure 2D). Compared with the LFF mice, plasma leptin concentrations were increased 5-fold (P < 0.0001) after 19 weeks in the HFF mice. Experiment 2.1: peripheral effects of exogenous leptin in mice after feeding a high-fat diet for 1 week 2 mg=g body weight i.p. leptin. After an i.p. injection of leptin, HFF mice had a signi cant 9.3%, 9.1%, and 7.8% reduction in body weight at 24, 48 and 72 h, compared with saline treated controls. Similarly, after an i.p. injection of leptin, LFF mice had a signi cant 10.2%, 10.4%, and 9.7% reduction in body weight at 24, 48 and 72 h, compared with saline treatment controls (Figure 3A). Cumulative energy intake after 72 h leptin treatment was reduced by 14.7% (P < 0.0001) in HFF mice and 18.4% (P < 0.0001) in LFF mice, compared with saline treatment controls (Figure 4A). However, comparing the mice fed a high-fat diet with the mice fed a low-fat diet, there were no signi cant differences in body weight change at 24 h (F (1,20) ˆ 0.146, P ˆ 0.706), 48 h (F (1,20) ˆ 0.29, P ˆ 0.596) and 72 h (F (1,20) ˆ 0.796, P ˆ 0.383). No signi cant differences in cumulative energy intake at 72 h (F (1,20) ˆ 4.157, P ˆ 0.055), were found after i.p. injection of leptin, that is, there was no signi cant interaction between diet and injected treatment for body weight change at 24, 48 and 72 h, nor for cumulative energy intake at 72 h. 641 Experiment 2.2: peripheral and central effects of exogenous leptin in mice after feeding a high-fat diet for 8 weeks Figure 1 (A) Body weight in mice fed either a high-fat or low-fat diet for 19 weeks; (B) Energy intake in mice fed either a high-fat or low-fat diet for 19 weeks. results are mean s.e., *P < 0.05; comparisons between the mice fed a high and low-fat diet, n ˆ 6 per group. (A) 2 mg=g body weight i.p. leptin. After an i.p. injection of leptin until 72 h, HFF mice did not show leptin effects on body weight (P ˆ 0.9772) and cumulative energy intake (P ˆ 0.7842; Figures, 3B and 4B), compared with saline-treated controls. However, after an i.p. injection of leptin, compared with

642 Figure 2 Amount of fat storage in mice fed either a high-fat or low-fat diet for 1, 8, 15 and 19 weeks. (A) Epididymal fat mass; (B) perirenal fat mass; (C) inguinal fat mass; (D) plasma leptin concentration in mice fed a high-fat or low-fat diet for 1, 8, 15 and 19 weeks; (E) the length of tibia in mice fed either a high-fat or low-fat diet for 1, 8, 15 and 19 weeks. Results are mean s.e. *P < 0.05; comparisons between the mice fed a high and low-fat diet, n ˆ 6 per group. saline treated controls, LFF mice showed signi cant 4.5% (P < 0.0001), 3.5% (P ˆ 0.012) and 0.6% (P ˆ 0.045) reduction of body weight at 24, 48 and 72 h, respectively (Figure 3B). Cumulative energy intake after 72 h leptin treatment was also reduced by 7.0% (P < 0.0001; Figure 4B). (B) 0.1 mg i.c.v. leptin. After an i.c.v. injection of 0.1 mg leptin, HFF mice had a signi cant 3.1%, 0.9% and 1.1% reduction in body weight at 24, 48 and 72 h, compared with saline treated controls (Figure 3C). Similarly, after an i.c.v. injection of 0.1 mg leptin, LFF mice had 4.1%, 1.3% and 0.9% reduction in body weight at 24, 48 and 72 h, compared with salinetreated controls (Figure 3C). Cumulative energy intake after 72 h leptin treatment was reduced by 17.7% (P ˆ 0.0003) in HFF mice and 18.3% (P < 0.0001) in LFF mice, compared with salinetreated controls (Figure 4C). However, comparing

the mice fed a high-fat with the mice fed a low-fat diet, no signi cant differences in body weight change at 24 h (F (1,20) ˆ 1.264, P ˆ 0.2743), 48 h (F (1,20) ˆ 0.014, P ˆ 0.905) and 72 h (F (1,20) ˆ 0.922, P ˆ 0.348), and no signi cant differences in cumulative energy intake at 72 h (F (1,20) ˆ 0.848, P ˆ 0.368), were found after i.c.v. injection of leptin. Experiment 2.3: central effects of exogenous leptin in mice after feeding a high fat diet for 19 weeks (A) 0.1 mg i.c.v. leptin. This study showed that 0.1 mg i.c.v. leptin had no signi cant effects on body weight and cumulative energy intake in the HFF mice at 24, 48 and 72 h, compared with saline-treated controls 643 Figure 3 Peripheral and central effects of exogenous leptin on the body weight change of mice fed either a high-fat or low-fat diet for 1, 8, 19 weeks. Although body weight changes were measured at every 6 h, the data is presented at three time points (24, 48 and 72 h). (A) Effects of intraperitoneal injection (i.p.) of leptin (2 mg=g body weight) in mice, fed a high-fat or low-fat diet for 1 week, on body weight change at 24, 48 and 72 h. (B) Effects of i.p. injection of leptin (2 mg=g body weight) in mice, fed a high-fat or low-fat diet for 8 weeks, on body weight change at 24, 48 and 72 h. (C) Effects of intracerebroventricular injection (i.c.v.) of leptin (0.1 mg) in mice, fed a high-fat or low-fat diet for 8 weeks, on body weight change at 24, 48 and 72 h. (D) Effects of i.c.v. injection of leptin (0.1 mg) in mice, fed a high-fat or low-fat diet for 19 weeks, on body weight change at 24, 48 and 72 h. (E) Effects of i.c.v. injection of leptin (2.0 mg) in mice, fed a high-fat or low-fat diet for 19 weeks, on body weight change at 24, 48 and 72 h. Results are mean s.e., *P < 0.05, comparing the effects between the injections of leptin and saline within the same dietary group. HFFL, the mice fed a high-fat diet following leptin injection; HFFS, the mice fed a high-fat diet following saline injection; LFFL, the mice fed a low-fat following leptin injection; LFFS, the mice fed a low-fat diet following saline injection; n ˆ 6 per group.

644 (Figure 3D). However, LFF mice, after i.c.v. injection of 0.1 mg leptin, showed a signi cant reduction in body weight of 2.2% (P < 0.0001) and 1.0% (P ˆ 0.005) at 24 and 48 h, respectively, compared with saline-treated controls (Figure 3D). Cumulative energy intake after 72 h leptin treatment was reduced by 7.0% (P ˆ 0.0013) compared with saline treatment controls (Figure 4D). (B) 2 mg i.c.v. leptin. HFF mice, after an i.c.v. injection of 2 mg leptin, had a signi cant reduction Figure 4 Peripheral and central effects of exogenous leptin on the cumulative energy intake of mice fed either a high-fat or low-fat diet for 1, 8 and 19 weeks. (A) Effects of intraperitoneal injection (i.p.) of leptin (2 mg=g body weight) in mice, fed a high-fat or low-fat diet for 1 week, on cumulative energy intake at 6, 12, 18, 24, 48 and 72 h. (B) Effects of i.p. injection of leptin (2 mg=g body weight) in mice, fed a high-fat or low-fat diet for 8 weeks, on cumulative energy intake at 6, 12, 18, 24, 48 and 72 h. (C) Effects of intracerebroventricular injection (i.c.v.) of leptin (0.1 mg) in mice, fed a high-fat or low-fat diet for 8 weeks, on cumulative energy intake at 6, 12, 18, 24, 48 and 72 h. (D) Effects of i.c.v. injection of leptin (0.1 mg) in mice, fed a high-fat or low-fat diet for 19 weeks, on cumulative energy intake at 6, 12, 18, 24, 48 and 72 h. (E) Effects of i.c.v. injection of leptin (2.0 mg) in mice, fed a high-fat or low-fat diet for 19 weeks, on cumulative energy intake at 6, 12, 18, 24, 48 and 72 h. Results are mean s.e. *P < 0.05, comparing the effects between the injections of leptin and saline within the same dietary group. HFFL, the mice fed a high-fat following leptin injection; HFFS, the mice fed a high-fat diet following saline injection; LFFL, the mice fed a low-fat diet following leptin injection; LFFS, the mice fed a low-fat diet following saline injection; n ˆ 6 per group.

in body weight for 2.5% (P < 0.0001) and 0.7% (P ˆ 0.007) at 24 and 48 h, respectively, compared with saline-treated controls (Figure 3E). LFF mice, after an i.c.v. injection of 2 mg leptin, had 5.4% (P < 0.0001), 2.7% (P < 0.0001) and 1.1% (P < 0.0001) reduction in body weight at 24, 48 and 72 h, compared with saline-treated controls (Figure 3E). Compared with saline-treated controls, cumulative energy intake after 72 h leptin treatment was reduced by 6.7% (P ˆ 0.0019) in HFF mice and 16.6% (P < 0.0001) in LFF mice (Figure 4E). Compared with the LFF mice, the HFF mice, after an i.c.v. injection of 2 mg leptin, had a signi cant 2.9% (F (1,20) ˆ 32.47, P < 0.0001), 2.0% (F (1,20) ˆ 55.35, P < 0.0001) and 1.0% (F (1,20) ˆ 52.99 P < 0.0001) lower reduction in body weight at 24, 48 and 72 h, respectively. After an i.c.v. injection of 2.0 mg leptin, HFF mice had a signi cant 9.9% (F (1,20) ˆ 15.11, P ˆ 0.0009) lower cumulative energy intake than that of LFF mice at 72 h. Discussion The results presented here show that mice exposed to a high-fat diet for 19 weeks develop obesity and, progressively, peripheral and then central leptin insensitivity. The early stage of high fat diet-induced obesity in mice is characterized by relatively normal food intake but elevated body weight and fat gain. As shown previously, in a direct comparison between a high carbohydrate and a high-fat diet fed isoenergetically, a high-fat diet appeared to be handled more ef ciently, resulting in a greater increase in adipose mass in rats. 21,22 The present study shows, however, that the mice fed a high-fat diet for 1 week had similar response in body weight change and cumulative energy intake following i.p. leptin injection, indicating that no peripheral leptin resistance had occurred. The middle phase is perhaps the most fascinating. Here there seems to be an active attempt, possibly driven by increased plasma leptin concentration, to control the rate of excess fat gain by signi cantly reducing food intake. However, despite the hypophagia, excess fat still accumulates with apparent gains in energetic ef ciency at least partially thwarting this regulatory endeavour. Peripheral, but not central, leptin insensitivity is evident by 8 weeks of HFF. This is consistent with other work showing that central leptin sensitivity was still intact after 10 weeks of HFF diet. 18 The hypophagia could then be driven via activation of hypothalamic leptin receptors. By some 4 months of high-fat feeding, the energy intake of the HFF mice increased dramatically, and by 19 weeks their energy intake was 14.6% greater than that of LFF controls. Furthermore, the inhibitory effects of central leptin administration on energy intake and body weight were attenuated in HFF mice but not in LFF mice; a 0.1 mg injection of leptin had virtually no effects on the energy intake and body weight change, and there was signi cantly less of an effect from a 2 mg injection. Several theories have been proposed to account for the apparent leptin insensitivity. Although the mechanism causing central leptin insensitivity is not fully understood, some suggest that the presence of high levels of leptin may induce desensitization of the hypothalamic leptin receptor. 19,23 ± 25 Alternatively, it has been suggested that the reduction in leptin action may be due to down-regulation of leptin receptor density or even saturation of the receptors with endogenous leptin as a result of the elevated leptin output. 19 In addition, another explanation may be that a high-fat diet affects hypothalamic neural networks downstream from the leptin receptor, such as the a-melanocyte-stimulating hormone (a-msh) pathway. We have recently observed a reduction of the arcuate hypothalamic a- MSH immunoreactivity in long-term (19 weeks) high fat diet-induced obese mice (unpublished observations). 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