Circulating leptin response to feeding and exogenous infusion of insulin in sheep exposed to thermoneutral and cold environments

Circulating leptin response to feeding and exogenous infusion of insulin in sheep exposed to thermoneutral and cold environments

Comparative Biochemistry and Physiology Part A 134 (2003) 329–335 Circulating leptin response to feeding and exogenous infusion of insulin in sheep e...

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Comparative Biochemistry and Physiology Part A 134 (2003) 329–335

Circulating leptin response to feeding and exogenous infusion of insulin in sheep exposed to thermoneutral and cold environments S. Asakuma, H. Morishita, T. Sugino, Y. Kurose, S. Kobayashi, Y. Terashima* Laboratory of Animal Nutrition, Faculty of Animal Science, Kitasato University, Towada-shi, Aomori 034-8628, Japan Received 10 July 2002; received in revised form 3 October 2002; accepted 5 October 2002

Abstract Leptin has been shown to regulate feed intake and energy expenditure. Insulin stimulates leptin secretion in rodents, but its action on leptin secretion is still obscure in ruminants. If insulin stimulates leptin secretion in ruminants, circulating leptin concentrations may change during exposure to cold, because of fluctuating insulin secretion and action in the cold environment. The present experiment was designed to determine whether feeding or exogenous administration of insulin affects circulating leptin levels in sheep exposed to thermoneutral and cold environments. Suffolk rams that were shorn and fed a diet once daily were subjected to a thermoneutral (20 8C) or cold (0 8C) environment for at least 1 week. Overall mean concentrations of plasma leptin in the feeding experiment were lower (P-0.05) in the cold environment than in the thermoneutral environment. Plasma leptin levels remained relatively unchanged after feeding in both environments, though plasma insulin response to feeding in both environments increased (P-0.01). The euglycemic clamps (insulin infusion rate: 4 mU kgBWy1 miny1 for 2 h) increased (P-0.01) circulating leptin concentrations in the thermoneutral, but not in the cold environment. These results suggest that lower circulating leptin levels in ruminants exposed to the cold environment could be partly due to the depressed insulin action on leptin secretion. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Cold; Feeding; Glucose infusion; Insulin infusion; Leptin; Radioimmunoassay; Sheep

1. Introduction Insulin has been shown to stimulate leptin secretion from adipose tissue in vivo and in vitro in rats and humans (Barr et al., 1997; Boden et al., 1997; Cheng et al., 2000; Levy et al., 2000), though conflicting results exist in human adipose tissue (Considine et al., 1997). Leptin secretion stimulated by insulin has been explained as a result of insulin action on glucose uptake of adipose tissue (Mueller et al., 1998). Plasma leptin *Corresponding author. Tel.: q81-176-23-4371; fax: q81176-23-8703. E-mail address: [email protected] (Y. Terashima).

levels in sheep, however, have been reported not to be responsive to short term (2 h) changes in blood glucose or insulin (Kauter et al., 2000). The reasons for this difference among animal species are not clear. As ruminants are less sensitive to insulin compared with those of mono-gastric animals (Sasaki and Takahashi, 1983), leptin secretion by adipose tissue of sheep may be less susceptible to the action of insulin. On the other hand, circulating leptin levels declined in rat exposed to cold environments (Bing et al., 1998). Previous studies have indicated that tissue responsiveness to insulin in sheep is enhanced by cold exposure (Sano et al., 1993; Weekes et al., 1983). If insulin stimulates leptin secretion in ruminants,

1095-6433/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 2 . 0 0 2 6 9 - 6

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circulating leptin levels may be influenced through changes in insulin secretion and action during cold exposure. However, the combined effects of insulin and cold exposure on circulating plasma leptin in ruminants still remain unclear. The present experiment was conducted to investigate whether feeding or continuous infusion of insulin influences plasma leptin concentrations in sheep exposed to thermoneutral and cold environments. We expected that circulating leptin levels in ruminants exposed to the cold environment may be influenced by insulin action on leptin secretion. 2. Materials and methods 2.1. Animals and diets Eight Suffolk rams, aged 2–3 years, that were shorn and weighing approximately 50.0"1.1 kg (mean"standard error (S.E.)) were used in the present study. They were surgically prepared with a skin loop enclosing the left carotid artery as described previously (Achmadi et al., 2001). Animals were kept in metabolism crates in a controlled environment chamber at 20 8C, and were fed alfalfa hay cubes at 2.0% of body weight and 100 g of ground corn once daily at 16:00 to meet approximately 120% of the daily metabolizable energy requirements for 2 weeks before starting the experiments (NRC, 1985). Mineral blocks and water were available at all times. 2.2. Plasma leptin response to feeding and euglycemic clamp experiments After an adaptation period of 2 weeks, each sheep was randomly subjected to a thermoneutral (20 8C: four animals) or cold (0 8C: four animals) environment. Animals were rapidly transferred to each environment room, and exposed to each environmental temperature for the entire experimental period. Plasma leptin response to feeding was determined on day 8, then euglycemic clamps (EGC) were performed on day 9–10 of each environmental treatment to determine plasma leptin responses to feeding and insulin infusion in both thermoneutral and cold environments. As the mean body weights of both groups were almost same, body mass of animals were not determined when the feeding and EGC experiments were performed.

Procedures for these experiments, including catheterization, site of infusion, and blood collection were similar to those reported previously (Sano et al., 1993). Implanted jugular vein and carotid artery catheters were used for infusion and blood collection, respectively. In the feeding experiment to determine plasma response to feeding, sheep were fed once daily at 16:00 (displayed as 0 min in Fig. 1), and blood collection started at 15:00 (1 h before feeding), and continued until 21:00 (5 h after feeding) at 1h intervals for the 6-h experimental period. In EGC, a sterile solution of insulin (Actrapid monocomponent porcine insulin; Novo Industry, Denmark) was given by primed continuous infusion during a 2-h period from 10:00 to 12:00 at the rate of 4.0 mU kgBWy1 miny1 into the infusion catheter using a peristaltic pump. The amounts of insulin infused as a priming dose were 1.5 times the continuous infusion rate during the initial 10 min of each infusion period and were given in a logarithmical decreasing manner (Achmadi et al., 2001). Euglycemia (basal glucose concentrations) was achieved throughout the 2-h insulin infusion period and the consecutive 2-h period after completion of insulin infusion by monitoring the level of blood glucose and adjusting the glucose infusion rate (GIR) at 5-min intervals. 2.3. Chemical analyses Blood samples taken during the experiments were immediately placed into heparinized tubes and centrifuged for 10 min (4 8C). Harvested plasma was stored at y80 8C until assay. Plasma glucose concentrations were determined by using a glucose analyzer (Glu-1, Erma Inc., Tokyo, Japan) based on the glucose oxidase reaction. Plasma insulin was assayed by a radioimmunoassay (RIA) kit (IRI, ‘Eiken’ Chemical Co. Ltd, Tokyo, Japan) based on the double antibody RIA method. Intra- and inter-assay coefficients of variation (CV) were 5.7 and 9.0%, respectively. Plasma leptin was determined using a RIA kit (Multi-Species Leptin RIA Kit, Linco Research, St. Charles, MO), that is suitable for sheep plasma (Delavaud et al., 2000; Bocquier et al., 1998). The intra- and inter-assay CV were 4.3 and 5.1%, respectively. 2.4. Statistical analysis The glucose, insulin, and leptin concentrations in plasma at each blood sampling time in the

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Fig. 1. Changes in mean plasma glucose, insulin and leptin concentration in thermoneutral (open circles) and cold (solid squares) during the feeding experiments in sheep. Values are the means and vertical lines represent the S.E. The effect of sampling time in plasma insulin was significant (P-0.01). Means with different superscripts are significantly different within a environmental treatment (abP-0.05; ABP-0.05). The effect of environmental treatment in plasma leptin was significant (P-0.05). Significant differences in each sampling time between environmental treatments are shown by *P-0.05.

feeding experiment and the insulin infusion (EGC) experiment are expressed as a mean of four animals with S.E. for thermoneutral and cold environments. Data were analysed with a mixed model analysis using restricted maximum likelihood of the JMP program package (version 4.05 for windows computer system, SAS Inst. Inc., Cary, NC). Fixed effects in the model were main effects of environmental treatment (thermoneutral or cold), sampling time, and the interaction between environmental treatment and sampling time. Sheep was used as a random effect. Mean values were compared using Student’s t-test, and its significant main effects were observed. Mean values are presented as least squares means"S.E. A probability value less than 0.05 was considered to the significant.

3. Results 3.1. Plasma glucose, insulin and leptin responses to feeding Changes in plasma glucose, insulin, and leptin concentrations after feeding in sheep exposed to each environment are presented in Fig. 1. Plasma glucose concentrations were not influenced (Ps 0.15) by feeding in either environment. Plasma insulin concentrations gradually increased (P0.01) after the start of feeding in both environments and peaked at 2–3 h after feeding. They then decreased to basal levels at approximately 4– 5 h after feeding. There is no significant difference in the mean of plasma glucose and insulin concen-

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Fig. 2. Changes in mean plasma glucose, insulin and GIR in thermoneutral (open circles or bar) and cold (solid squares or bar) during the EGC experiments in sheep. Values are the means and vertical lines represent the S.E.

trations between the thermoneutral and cold environments. Plasma leptin levels were significantly lower (P-0.05) in the cold environment than in the thermoneutral environment over the whole experimental period. Plasma leptin levels remained unchanged (Ps0.69) during the experimental period, and were not influenced by feeding in either environment. 3.2. Euglycemic clamp experiment

Plasma leptin concentrations were always lower (P-0.05) in the cold environment than in the thermoneutral environment during the EGC experimental period (Fig. 3). Plasma leptin levels gradually increased (P-0.01) during and after the EGC in the thermoneutral environment. No interaction (Ps0.89) of environmental treatment and sampling time was found in plasma leptin concentrations. In contrast, plasma leptin levels did not change (Ps0.86) during the whole EGC experiment in the cold environment (Fig. 3). 4. Discussion

The summary of the EGC experiment is presented in Fig. 2. During the 4-h experimental period that consisted of a 2-h insulin infusion period and a 2-h post-insulin infusion period, plasma glucose concentrations in both environments were successfully maintained at very close to the initial levels by varying the rates of glucose infusion.

Leptin production and secretion by adipocytes has been well-known to be under complex regulation such as endocrine factors and sympathetic nervous activity, and to act on the hypothalamus to diminish appetite and to increase energy expenditure (Houseknecht et al., 1998). Ruminants exposed to a cold environment must increase heat

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Fig. 3. Changes in mean plasma leptin concentration in thermoneutral (open circles) and cold (solid squares) during the EGC experiments in sheep. Values are the means and vertical lines represent the S.E. The effects of environmental treatment and sampling time was significant (P-0.01, respectively). Sampling time with different superscript letters are significantly different (a,b,cP-0.05).

production to maintain homeothermy (Sano et al., 1995). The need is met primarily by an increase in food intake in the animals exposed to a cold environment (Forbes, 1986), but the role played by leptin in the hyperphagia induced by a cold environment is still unknown. The objective of the present study was to determine the effects of both feeding and insulin infusion on circulating leptin concentrations in sheep exposed to thermoneutral and cold environments. The results of the feeding and EGC experiments indicate that plasma leptin concentrations are lower in a cold environment than in a thermoneutral. Plasma leptin has been shown to be lower in cold environment (Ricci et al., 2000; Kokkonen et al., 2002). The lower plasma leptin may be one factor to promote the hyperphagia of a cold environment. Leptin secretion has been shown to be reduced by low temperatures in experimental animals, and this effect has been explained as an adaptive mechanism to cold environments (Bing et al., 1998). Cold exposure activates a series of physiological events to ensure survival, notably increases heat production that is controlled by the sympathetic nervous system (SNS). It has been reported that plasma leptin decreased along with

an increase in plasma noradrenalin in humans, suggesting that activation of the SNS decreases leptin secretion (Gettys et al., 1996). Activation of the SNS such as administration of b-adrenergic receptor agonists and cold exposure reduced plasma leptin and leptin mRNA in the white adipose tissue of rodents (Trayhurm et al., 1998). Moreover, infusion of b-adrenergic agonist decreased plasma leptin in humans (Donahoo et al., 1997). Our study showed that EGC did not influence plasma leptin concentration in the cold environment, but increased it in the thermoneutral environment. Insulin has an action on leptin release and leptin mRNA production in vitro and in vivo (Leroy et al., 1996; Koopmans et al., 1998; Peino et al., 2000). However, the response on leptin secretion was delayed after the single injection of insulin in sheep (Kauter et al., 2000). Results in the present study suggest that insulin action on leptin secretion would be depressed in the cold environment. Therefore, lower plasma leptin concentrations in the cold environment could be partly due to depressed insulin sensitivity on leptin secretion in the cold environment. Cold exposure enhanced circulating glucose utilization induced by insulin action (Sano et al., 1992). However, leptin secretion by insulin action in the cold

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environment was not observed in our present study. Further studies are needed to explain the decreased action of insulin on leptin secretion in the cold environment. Leptin concentrations in sheep measured by the Multi-Species Leptin RIA Kit were considerably lower than those obtained by the bovine RIA (Ehrhardt et al., 2000). However, estimates of leptin concentrations obtained by both assays were positively correlated in sheep plasma (Ehrhardt et al., 2000). Therefore, the data on plasma leptin in the present study should be valid. Results of the present study indicate that cold exposure results in decreased circulating leptin concentrations in sheep, and that cold exposure emphasizes the depressed leptin secretion induced by insulin action. Although the mechanism of depressed leptin secretion to insulin infusion in the cold environment could not be explained in the present experiments, the altered insulin action on leptin secretion may be involved in changes of circulating leptin concentrations in ruminants exposed to cold environments. References Achmadi, J., Sano, H., Terashima, Y., 2001. Effect of hypomagnesemia and cold exposure on tissue responsiveness to insulin in sheep given a low magnesium and high potassium diet. Domest. Anim. Endocrinol. 20, 101–108. Barr, V.A., Malide, D., Zarnowski, M.J., Taylor, S.I., Cushman, S.W., 1997. Insulin stimulates both leptin secretion and production by rat white adipose tissue. Endocrinology 138, 4463–4472. Bing, C., Frankish, H.M., Pickavance, L., Wang, Q., 1998. Hyperphagia in cold-exposed rats is accompanied by decreased plasma leptin but unchanged hypothalamic NPY. Am. J. Physiol. 274, R62–R68. Bocquier, F., Bonnet, M., Faulconnier, Y., Martin, G.P., Chilliard, Y., 1998. Effect of photoperiod and feeding level on perirenal adipose tissue metabolic activity and leptin synthesis in the ovariectomized ewe. Reprod. Nutr. Dev. 38, 489–576. Boden, G., Chen, X., Kolaczynski, J.W., Polansky, M., 1997. Effects of prolonged hyperinsulinemia on serum leptin in normal human subjects. J. Clin. Invest. 100, 1107–1113. Cheng, J.T., Liu, I.M., Chi, T.C., et al., 2000. Role of adenosine in insulin-stimulated release of leptin from isolated white adipocytes of Wistar rats. Diabetes 49, 20–24. Considine, R.V., Nyce, M.R., Kolaczynski, J.W., et al., 1997. Dexamethasone stimulates leptin release from human adipocytes: unexpected inhibition by insulin. J. Cell. Biochem. 65, 254–258.

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