Exp. Anim. 61(4), 461–466, 2012

—Original— Characteristics of Himalayan Marmots and Their Response to an Atherogenic Diet

Yafeng Li1), Zhongdong Wang2), Yuanqing Tao2), Wei Fan2), Meng Li1), Bingqiao Huang1), Sihai Zhao1), Jianglin Fan1,3), and Enqi Liu1)

1) Research Institute of Atherosclerotic Disease, Xi’an Jiaotong University School of Medicine, 76 West Road of Yanta, Xi’an 710061, China 2) Laboratory Center, Qinghai Institute for Endemic Disease Prevention and Control, Zongzhai, Xining 811602, China 3) Department of Molecular Pathology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan

Abstract: The purpose of the present study was to characterize Himalayan marmot lipoprotein profiles and investigate their response to an atherogenic diet. Sixteen marmots were randomly divided into two groups. The control group was fed with a standard chow diet, and the other group was fed with a chow diet containing 0.3% cholesterol, 6.7% lard, and 3.3% corn oil (designated as HFCD) for 16 weeks. The plasma lipids were measured, and lipoprotein profiles were analyzed. With the chow diet, the major lipoproteins were high density lipoproteins. HFCD feeding increased not only plasma total cholesterol levels but also body weight compared with the control group (P<0.05). Plasma lipoprotein (a) was detected in marmots, and the plasma lipoprotein (a) levels were 4.5-fold higher after being fed HFCD for 16 weeks. However, atherosclerotic lesions were not found in the aorta of HFCD-fed marmots. This study suggested that marmots are HDL-rich and resistant to HFCD-induced atherosclerosis. Key words: atherosclerosis, cholesterol diet, lipids, marmots

Introduction tion). Marmots (Marmota) have been used as a disease model in the field of hepatitis since the discovery of Cardiovascular diseases (CVD) are the major causes marmot hepatitis virus in 1978 [13]. In English literature, of death in both developed and developing countries [7, one paper showed that an old marmot can develop 9]. Atherosclerosis plays a basic role underlying the Mönckeberg medial arteriosclerosis (medial calcifica- development of CVD. In order to investigate the patho- tion) [2]. In the current study, we characterized the logical mechanisms of atherosclerosis and find new plasma lipid features and wanted to examine whether therapeutic targets for CVD, ideal animal models are marmots are sensitive to an atherogenic diet or whether required. Until now, a variety of including the they can develop atherosclerosis. mouse, rat, hamster, rabbit, , swine, dog, and nonhuman primate have been used for the study of dif- Materials and Methods ferent aspects of atherosclerosis [12]. Recently, we ob- tained Himalayan marmots from the Qinghai-Tibet Animals and diets Plateau in Qinghai Province of China and characterized Himalayan marmots (Marmota himalayana) (Fig. 1) their genetic features (Chen et al., submitted for publica- were live-trapped in Qinghai Province, China. After

(Received 4 October 2011 / Accepted 23 February 2012) Address corresponding: E. Liu, Research Institute of Atherosclerotic Disease, Xi’an Jiaotong University School of Medicine, 76 West Road of Yanta, Xi’an 710061, China ©2012 Japanese Association for Laboratory Animal Science 462 Y. LI, ET AL.

were assessed by automated analyzers (Olympus AU 5400, Mishima, Japan) at the Medical Laboratory of Qinghai Red Cross Hospital, Qinghai, China.

Pathologic analysis The marmot aorta was removed, and adventitial tissue was cleared carefully from the aorta. After that, the arte- rial tree was fixed immediately in 10% neutral buffered formalin. Specimens of aorta arch were cut, embedded in paraffin and stained with hematoxylin and eosin (HE) Fig. 1. a picture of a Himalayan marmot. [15]. In addition, the heart, liver, spleen, lungs, and kid- neys were fixed in 10% neutral buffered formalin, cut and stained with HE, and examined under a Nikon light quarantine and 30 days of acclimation, they were di- microscope. vided randomly into two groups. One group (4 males and 4 females) was fed HFCD, which contained 0.3% Statistical analysis cholesterol, 6.7% lard, and 3.3% corn oil per weight in All quantitative data are expressed as means ± SEM. standard chow diet. The control group (4 males and 4 Statistical analysis was performed using either the Stu- females) was fed a standard chow. Food and water were dent’s t-test for data with an equal F value or Welch’s provided ad libitum for all marmots. The study was per- t-test when the F value was not equal, and two-way formed over 16 weeks. At the end of the experiment, all ANOVA was also used for comparisons between groups, animals were sacrificed by intravenous injection of an followed by a Bonferroni post hoc test. Values were overdose of sodium pentobarbital solution. The animal considered statistically significant when P<0.05. experiments were approved by the Animal Administra- tion Committee of Xi’an Jiaotong University and per- Results formed according to the Xi’an Jiaotong University Guidelines for Animal Experimentation. Body weight and plasma lipids HFCD-fed marmots started to gain body weight at 6 Plasma lipid and lipoprotein analysis weeks and reached the peak at 16 weeks (Fig. 2A). Con- Marmots were anesthetized with ketamine injected trol marmots, however, had a relatively low body weights intramuscularly in a dose of 45 mg/kg, and then blood compared with the HFCD group. The concentrations of samples were collected via a vein in a hind limb while plasma TC and HDL-C were markedly increased after the animals were anesthetic. Approximately 5 ml of feeding HFCD compared with the control group (Fig. blood was collected into tubes containing EDTA antico- 2B and 2C). However, there was no significant difference agulant. Blood samples were centrifuged at 950 g for 15 in the TG levels between the 2 groups (Fig. 2D). FPLC min at 4°C, and plasma was collected. Total cholesterol analysis revealed that normal marmots are HDL-rich (TC), triglycerides (TG), and high- density lipoprotein animals because their lipids are mainly distributed in cholesterol (HDL-C) were measured using commercial HDL fractions. Furthermore, increased plasma choles- assay kits (Wako Pure Chemical Industries, Osaka, Ja- terol in HFCD-fed marmots was distributed mainly in pan). HDL fractions, with small amounts of cholesterol in LDL In order to determine the distribution of plasma lipo- fractions (Fig. 3A and 3B). protein, 200 µl of pooled plasma from 5 marmots per group was analyzed by fast protein liquid chromatogra- Biochemical and hematological parameters phy (FPLC) with a Superose 6 column (Amersham Bio- Tables 1 and 2 summarize the biochemical and hema- sciences, Inc., Piscataway, NJ, USA) as described previ- tological parameters of the control and HFCD-fed mar- ously [5]. We collected 35 fractions of 0.5 ml and then mots. We found that HFCD-fed marmots had higher assayed enzymatically their TC and TG contents. levels of apolipoproteins AI and B than control marmots, Blood biochemical and hematological parameters which is consistent with the findings of FPLC (namely, RESPONSE TO ATHEROGENIC DIET OF MARMOTS 463

Fig. 2. Body weights of marmots (A), and plasma total cholesterol levels (B), HDL-C levels (C), and plasma triglyceride levels (D). Combined data of male and female marmots are expressed as means ± SEM. * P<0.05 vs. control. ** P<0.01 vs. control.

Fig. 3. Lipoprotein profiles of normal and HFCD-fed marmots by FPLC analysis. Pooled samples of 5 marmots were analyzed by FPLC as described in the Materials and Methods. Cholesterol (A) and triglyceride (B) contents of each fraction were quantified and plotted. VLDL, very low density lipoproteins; CM, chylomicrons; IDL, intermediate density lipoproteins; LDL, low density lipoproteins; HDL, high density lipoproteins.

increased HDL and LDL). In addition, like humans and Pathologic examinations primates, marmots have 1.8 mg/dl of plasma lipoproteins Grossly and microscopically, there was no lesion on (a). With HFCD, the lipoprotein (a) levels were 4.5-fold the aortic surface in either the HFCD-fed or control higher than those of the controls (Table 1). Furthermore, group (Fig. 4A–D). Histological examination of the the plasma concentrations of aspartate transaminase and heart, spleen, lungs, and kidneys did not find abnor- alanine transaminase were high in the HFCD-fed mar- malities. However, in the liver, focal chronic inflamma- mots, as shown in Table 1. tory cell infiltration was noted in 4 HFCD-fed marmots (data not shown). 464 Y. LI, ET AL.

Table 1. Biochemical data Normal group (n=7) HFCD group (n=8) P value Alanine transaminase (U/l) 3.429 ± 0.4 9.125 ± 2.5 Aspartate transaminase (U/l) 27.57 ± 3.8 44.00 ± 11.6 Aspartate transaminase/alanine transaminase 8.129 ± 0.8 5.100 ± 0.4 0.0032 Total protein (g/l) 65.97 ± 1.7 69.31 ± 1.1 Albumin (g/l) 26.37 ± 0.9 26.71 ± 0.8 Globulin (g/l) 39.60 ± 1.8 42.60 ± 1.3 Albumin/globulin 0.6714 ± 0.1 0.6375 ± 0.03 Total bilirubin (μmol/l) 0.8286 ± 0.1 0.5625 ± 0.2 Direct bilirubin (μmol/l) 2.071 ± 0.1 1.900 ± 0.1 Indirect bilirubin (μmol/l) –1.243 ± 0.2 -1.338 ± 0.1 Alkaline phosphatase (U/l) 67.57 ± 9.9 110.4 ± 21.0 γ-glutamyl-transpeptidase (U/l) 1.143 ± 0.3 1.750 ± 0.4 Urea nitrogen (mmol/l) 6.950 ± 0.6 4.945 ± 0.2 0.0036 Creatinine (μmol/l) 91.57 ± 4.4 87.63 ± 3.1 Uric acid (μmol/l) 5.571 ± 0.7 5.875 ± 1.9 Glucose (mmol/l) 8.294 ± 0.5 7.670 ± 0.5 Total cholesterol (mmol/l) 4.604 ± 0.2 11.15 ± 1.8 0.0045 Tolal triglyceride (mmol/l) 1.393 ± 0.2 1.289 ± 0.2 Apolipoprotein A1 (g/l) 0.05286 ± 0.002 0.05875 ± 0.001 0.0180 Apolipoprotein B (g/l) 0.07286 ± 0.005 0.1063 ± 0.01 0.0347 Apolipoprotein A1/apolipoprotein B 0.7400 ± 0.04 0.5950 ± 0.06 Lipoprotein a (mg/l) 18.29 ± 5.4 81.63 ± 21.9 0.0207 Date are expressed as means ± SEM, and n=7 and n=8 for the control and HFCD-fed groups, respectively.

Table 2. Hematological data Normal group (n=7) HFCD group (n=8) P value White blood cell (*109/l) 5.833 ± 0.9 5.55 ± 0.5 Red blood cell (*1012/l) 5.262 ± 0.3 5.38 ± 0.1 Hematoglobin (g/l) 105.9 ± 16.7 117.1 ± 6.6 Hematocrit (%) 43.1 ± 2.3 42.59 ± 1.8 Platelet count (*109/l) 332.3 ± 63.6 399.9 ± 44.0 Plateletcrit (*10−2/l) 0.2507 ± 0.04 0.3031 ± 0.04 Lymphocyte percentage (%) 22.52 ± 2.261 27.84 ± 3.264 Monocyte percentage (%) 3.983 ± 0.7 5.55 ± 0.6 Neutrophil percentage (%) 73.03 ± 2.8 66.39 ± 3.3 Mean corpuscular volume (fl) 82.03 ± 1.3 79.31 ± 3.0 Mean corpuscular hemoglobin (pg) 23.18 ± 0.5 21.78 ± 1.1 Mean corpuscular hemoglobin concentration (g/l) 282.3 ± 2.5 274.3 ± 8.0 Platelet distribution (%) 15.77 ± 0.3 15.23 ± 0.1 0.0404 Absolute lymphocyte (*109/l) 1.35 ± 0.3 1.525 ± 0.2 Absolute monocyte (*109/l) 0.25 ± 0.1 0.3113 ± 0.05 Absolute neutrophil (*109/l) 4.217 ± 0.6 3.7 ± 0.4 Absolute eosinophilic (*109/l) 0.4 ± 0.2 0.1875 ± 0.1 Abosolute basocyte (*109/l) 0.06667 ± 0.03 0.0375 ± 0.02 Eosinophilic percentage (fl) 7.583 ± 0.2 7.638 ± 0.3 Red blood cell distribution width-SD (fl) 42.72 ± 0.6 41.73 ± 1.8 Red blood cell distribution width-CV (%) 14.75 ± 0.4 16.24 ± 1.0 Date are expressed as means ± SEM, and n=7 and n=8 for the control and HFCD-f means ed groups, respectively.

Discussion marmota caudata, marmota himalayana, and marmota sibirica [14]. In the current study, we used Himalayana Marmots belong to the division of the burrowing marmots (marmota himalayana) and studied the lipo- species and are distributed throughout Northern protein features and their response to an atherogenic diet. Asia, Europe, and Northern America. There are 4 species The lipoprotein profile of marmots is HDL-rich, indicat- of marmots in China, including the marmota baibacina, ing that plasma cholesterol is transferred by HDL as in RESPONSE TO ATHEROGENIC DIET OF MARMOTS 465

Fig. 4. Representative aorta pictures of marmots. Sudan IV-stained aortic surface (A and B) and micrographs of HE-stained sections (C and D).

. In addition, we found that marmots also have from a subclinical or acute transient infection to chron- low levels of lipoprotein (a), a unique lipoprotein that ic infection progressing to hepatocellular carcinoma; was thought to only be present in humans, old world therefore, marmots were regarded as an good model to primates and hedgehogs [1]. This suggests that marmots evaluate therapeutic vaccination against hepadnaviral may become a good model for the study of lipoprotein infection [10]. Compared with rats, marmots appear to (a) in the future. When marmots were fed HFCD, they be more resistant to induction of hepatic injury by a li- developed hyperlipidemia accompanied by increased potrope-deficient diet, and less sensitive to induction of body weight. FPLC analysis revealed that the main li- fatty liver by lipotrope deficiency [3, 4]. Furthermore, poproteins after HFCD feeding were still HDLs, and marmots can develop congestive cardiomyopathy [11]. only small amounts of cholesterol were distributed in Plasma derived from hibernating marmots is an effective LDL particles. This may explain why these animals were neuroprotective agent in a mouse model [6] and protects resistant to HFCD-induced atherosclerosis even though against hypoxia/reperfusion injury in nonhibernators’ their body weights were increased. Of note, the marmots skeletal muscles [8]. fed normal chow had lower body weights and higher In summary, we characterized the lipoprotein profiles plasma TG concentrations after the start of the experi- of Himalayan marmots and investigated their response ments. We speculated that this may have been caused by to an atherogenic diet. Marmots are HDL-rich rodents environmental or seasonal changes (such as living in and are resistant to high fat and cholesterol diet feeding. cages, eating a commercial chow diet, accumulation of Interestingly, these animals have lipoprotein (a) in their lipids for ), since they were originally wild. plasma, suggesting that they could be used as a model Marmots have been used for study of hepatitis. For for the study of lipoprotein metabolism in the future. example, marmot hepatitis virus infection in marmots is similar to hepatitis B virus infection in humans, ranging 466 Y. LI, ET AL.

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