Effects of I.C.V. Administration of Leptin on Copulatory and Ingestive Behavior in STZ-Induced Diabetic Male Rats

Exp. Anim. 53(5), 445–451, 2004 Effects of i.c.v. Administration of Leptin on Copulatory and Ingestive Behavior in STZ-induced Diabetic Male Rats Toru R. SAITO1), Takayuki TATSUNO1), Akira TAKEDA1), Haruo HASHIMOTO1), Mitsuro SUZUKI1), Misao TERADA1), Shinobu AOKI-KOMORI2), Minoru TANAKA3), Lajos KORANYI4), and Gyorgy M. NAGY5) 1)Behavioral Neuroscience Laboratory, Graduate School of Veterinary Medicine, 2)Division of Veterinary Surgery, 3)Division of Animal Physiology, Nippon Veterinary and Animal Science University, 1–7–1 Kyonan-cho, Musashino-shi, Tokyo 180-8602, Japan, 4)National Institute of Laboratory Medicine, Szabolcs U. 33-35, Budapest, and 5)Neuroendocrine Research Laboratory, Department of Human Morphology and Developmental Biology, Semmelweis University, Tuzolto U. 58, Budapest, Hungary Abstract: It is well known that circulating leptin concentrations correlate with adiposity in both humans and rodents and decrease after fasting, energy restriction, or weight loss. The goal of the present study was to confirm whether the decreases of copulatory behavior and the increases of ingestive behavior in STZ-induced diabetic male rats could be reversed by i.c.v. administration of leptin. Adult male Wistar-Imamichi rats aged 9 weeks were used for the studies. Males received a single injection of STZ (60 mg/kg, i.p.) and vehicle. During the experiment, individual body weight, and food and water intake were measured. The copulatory and ingestive behaviors in STZ-induced diabetic males were observed at 2 and 4 weeks after STZ. At 6 weeks after STZ, leptin (10 µg/10 µl) or aCSF (artificial cerebrospinal fluid) was injected through a lateral ventricle cannula and the above two behaviors were observed again. The i.c.v. leptin injection to STZ-induced diabetic males resulted in a significant increase of ejaculation frequencies (3.6 ± 0.26 vs. 2.9 ± 0.30 times) and a significant decrease in amount of food ingested (36.2 ± 1.93 vs. 23.2 ± 3.76 g), compared with the aCSF-injected control (p<0.01). These findings suggest that the copulatory and ingestive behaviors in i.c.v. leptin-injected STZ diabetic males were restored to levels equivalent to those in control males. Key words: copulation, diabetic rats, ingestion, leptin, streptozotocin Introduction dysfunction [11, 15–17]. STZ-induced diabetes results in marked insulin deficiency. It is considered that in- In general, animal models of insulin-deficient diabe- sulin deficiency is a main cause of STZ-induced tes mellitus are induced using streptozotocin (STZ) and hyperphagia because insulin administration plays a sup- are characterized by marked hyperphagia [3] and sexual pressive role in food intake [12, 21]. Steger et al. [15] (Received 25 March 2004 / Accepted 30 June 2004) Address corresponding: T.R. Saito, Behavioral Neuroscience Laboratory, Faculty of Veterinary Medicine, Nippon Veterinary and Animal Science University, 1–7–1 Kyonan-cho, Musashino-shi, Tokyo 180-8602, Japan 446 T.R. SAITO, ET AL. demonstrated that male rats with STZ-induced diabetes buffer (pH 4.5). Control animals received injections of provide an interesting and relevant model to study the citrate buffer only. Male blood glucose was determined effects of diabetes on male reproduction, since they in blood sampled from the tail vein. Diabetes was exhibit several deficits in reproductive function similar confirmed 24 h after STZ administration, and male to those in diabetic men. In diabetic men, erection blood glucose by Super Gluco Card II (Arkray Inc., problems are usually attributed to pelvic autonomic neu- Kyoto, Japan) was also determined at 4 and 7 weeks ropathy, although a significant correlation between sex after STZ. problems and neuropathy does not demonstrate a causal relationship [19]. Body weights, food and water intake measures Leptin, the obese (ob) gene product [22], has been Body weights, and food and water consumptions were proposed as an adipose-related satiety factor that re- measured daily until one week after STZ and then twice duces food intake and enhances energy expenditure, a week during the study. leading to reduction of fat storage [8]. Leptin may act as a signal, from peripheral adipose stores to the cen- Copulatory behavior test tral nervous system, to decrease food intake, increase Male rats were tested individually for copulatory be- energy expenditure, and limit adiposity [5]. Inversely, havior with hormonally induced receptive female rats low leptin levels after fasting or energy restriction may in circular (30- cm diameter) plexiglas arenas 1 h after act to increase food intake and decrease energy expen- the onset of darkness. Behavioral testing was conducted diture [5]. Nevertheless, a positive association between at 2 and 4 weeks after STZ. In addition, copulatory leptin and copulatory behavior in experimental animals behavior in STZ-induced diabetic males with leptin was has not been demonstrated. observed at 40 days after STZ. The occurrence of each The present study investigated the effects of a single mount, intromission, and ejaculation was recorded on administration of leptin on the increased ingestive and an event recorder. To identify ejaculation, the male’s decreased copulatory behavior of STZ-induced diabetic 22-kHz vocalization was monitored, using two Mini male rats. Bat-Detectors (QMC, Ltd., London, England) tuned to a range of 20 to 30 kHz. The behavior categories scored Materials and Methods included the following: mounting frequency (MF) - number of mounts without intromission during 30 min; Experimental animals intromission frequency (IF) - number of mounts with Adult male and female Wistar-Imamichi rats (The intromission during 30 min; ejaculation frequency (EF) - Imamichi Institute for Reproduction, Tsuchiura, Ibaraki, number of ejaculations during 30 min; mounting la- Japan), aged 9 weeks were used for the studies. The tency (ML) - elapsed time between introduction of the animals were maintained individually in an air-condi- female and the first display of mounting; intromission tioned, temperature-controlled (22–26°C) colony room latency (IL) - elapsed time between introduction of the in which the lights were on between 05:00 and 19:00. female and first display of intromission; ejaculation la- The rats were allowed free access to a standard pellet tency (EL) - elapsed time from first intromission of the rat diet (MF, Funabashi Farm, Chiba, Japan) and water. female to time of ejaculation; and postejaculatory inter- All procedures were in accordance with the NIH Guide val (PEI) - elapsed time from ejaculation to the next for the Care and Use of Laboratory Animals and were intromission. approved by the Institutional Animal Care and Use Committee of the Nippon Veterinary and Animal Sci- Surgery for implantation with a cannula ence University. Two weeks prior to observation of copulatory and ingestive behavior with leptin, diabetic rats were anes- Induction of diabetes thetized with pentobarbital (Nembutal, 40 mg/kg, Diabetes was induced with injections of freshly pre- intraperitoneally). A 21-gauge stainless steel guide can- pared streptozotocin (STZ, Sigma, St. Louis, MO, USA) nula was placed in the right lateral ventricle of the at a dose of 60 mg/kg in ice-cold 0.5 mol/L citrate brain, 1.0 mm posterior and 1.4 mm lateral to the DIABETIC RAT COPULATION AND INGESTION BY LEPTIN 447 bregma and 3.5 mm ventrally from the surface of the Results skull and anchored to the skull using dental cement. Our previous study suggested that the results of copu- Blood glucose levels, Body weights, food and water latory and ingestive behavior were unaffected by this consumptions surgery for a cannula implantation [1]. All 7 males in 60 mg/kg STZ group were diabetic with blood glucose >350 mg/dl within one day after Leptin administration STZ. The high value of blood glucose was maintained Rat leptin (Morinaga Biological Research Ltd., throughout the experimental period. In the vehicle con- Kawasaki, Kanagawa, Japan) was dissolved in sterile trol, all animals had normal blood glucose <100 mg/dl. artificial cerebrospinal fluid (aCSF): NaCl 8.98 g, KCl The results are shown in Fig. 1. Body weight gain 0.25 g, CaCl2 0.14 g, MgCl2 0.11 g, NaH2P04 0.07 g, of the STZ group was significantly inhibited after 3 urea 0.13 g, and glucose 0.61 g dissolved in 1,000 ml days (p<0.05–0.01). Also, the STZ group showed a triple-distilled water to a concentration of 1 µg/µl (pH significant increase in food consumption after 4 days 7.29). Leptin was administered to STZ-induced dia- (p<0.01), compared with the vehicle control. The betic males through the lateral ventricle cannula. The amount of water ingested by the STZ group after one dose of leptin was chosen on the basis of previous work day was two to four folds as much as that of the ve- [1]. hicle control. Ingestive behavior test Copulatory behavior The pellets were removed at 14:00, but water was Table 1 shows behavioral frequencies and latencies available at all time during the 45 days after STZ. Tests in the STZ and vehicle control groups at 2 and 4 weeks were conducted 6 h later, i.e., 1 h after the lights were after STZ or vehicle, respectively. The mean EF re- switched off. The STZ-induced diabetic males were corded 2 and 4 weeks after STZ was significantly lower injected with leptin or aCSF. Food was returned im- than that of the vehicle control group (p<0.05). The mediately and food intake was measured over the next STZ group at 2 and 4 weeks exhibited significantly 24 h after the leptin injection. longer EL and PEI than the vehicle control group (p<0.05). Except for the above items no significant Plasma leptin determination differences were found between the two groups. Individual blood samples were collected from the vena cava caudalis using a heparinized syringe under Copulatory behavior with leptin ether anesthesia between 09:00 and 10:00 at 7 weeks As shown in Table 2, the i.c.v.

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