129 Chronic heat stress up-regulates leptin and adiponectin secretion and expression and improves leptin, adiponectin and insulin sensitivity in mice Patrizia Morera, Loredana Basirico` , Kenji Hosoda1 and Umberto Bernabucci Department of Agriculture, Forestry, Nature and Energy, University of Tuscia, via San Camillo De Lellis, SNC 01100 Viterbo, Italy 1National Institute of Livestock and Grassland Sciences, National Agriculture and Food Research Organization, Tochigi, Japan (Correspondence should be addressed to U Bernabucci; Email: [email protected]) Abstract Heat stress (HS) induces adaptive responses that are responsible for alterations of carbohydrate and lipid metabolism. This study aimed to evaluate the effects of chronic heat treatment on the expression and secretion of leptin and adiponectin, important regulators of energy homeostasis, food intake and insulin action. C57BL/6 mice were subdivided into three groups (24 mice each). The first group was kept under control conditions (C: 22G2 8C). The second group was exposed to HS (35G1 8C). The third group was kept under control conditions and was food restricted (FR). The HS group had higher rectal temperature than the C and FR groups and lower food intake than the C group. Hspa1 (Hspa1a) gene expression in adipose tissue, muscle and liver was higher under HS than FR and C. Heat treatment resulted in decreased blood glucose and non-esterified fatty acids; increased leptin, adiponectin and insulin secretion; and greater glucose disposal. Leptin, adiponectin, leptin and adiponectin receptors, insulin receptor substrate-1 and glucose transporter mRNAs were up-regulated in HS mice. This study provides evidence that HS improves leptin and adiponectin signalling in adipose tissue, muscle and liver. Heat stress was responsible for improving insulin sensitivity and glucose uptake in peripheral tissues, probably mediated by adipokines. Changes in the adipokine levels and sensitivity to them may be considered as an adaptive response to heat. Journal of Molecular Endocrinology (2012) 48, 129–138 Introduction HS rodents exhibit increased basal insulin levels, greater insulin sensitivity and glucose uptake and lower Under a hot environment, the body’s thermoregulatory plasma glucose (Torlinska et al. 1987, Gupte et al. 2009, system adjusts a variety of physiological mechanisms 2011). These results agree with the findings in dairy through a combination of dry heat exchange and cows that chronic HS leads to hypoglycaemia and evaporative heat loss to attain a balance between the increases basal insulin levels despite marked reductions heat produced within the body and the heat lost to in nutrient intake (Wheelock et al. 2010). the environment. Profound alterations at the cellular An increase in triglyceride content is observed in the level also occur upon heat stress (HS). liver of young rats (Ando et al. 1998) and dairy cows These alterations encompass changes in gene (Basirico` et al. 2011) exposed to a hot environment. In expression and biochemical adaptive responses, charac- addition, decreased plasma cholesterol, phospholipid terised by the impairment of major cellular functions and non-esterified fatty acid (NEFA) contents are and reprogramming of cellular metabolism (Lindquist found in HS dairy cows (Sano et al. 1983, Ronchi et al. 1986). 1999). Ronchi et al. (1999) concluded that the In contrast to acute HS, the effects of chronic HS on alterations of carbohydrate and lipid metabolism in gene expression and cellular metabolism have been HS cattle are due to the high ambient temperature studied only to a limited extent (Zhang et al. 2002, rather than indirect factors, such as the reduction of Bhusari et al. 2008). Acute and chronic HS pose two feed intake. distinct challenges to animals. Acute HS (animals Two adipokines, leptin and adiponectin, are meta- exposed to 40–42 8C for up to 24 h) results in bolically relevant in coordinating energy homeostasis hyperthermia and can lead to death if subjects are (Ailhaud 2006). Leptin affects energy homeostasis by exposed to it for an excessive length of time. In decreasing food intake and by up-regulating fatty acid contrast, chronic HS (33–35 8C) can be tolerated for a oxidation and down-regulating lipogenesis in periph- longer period, and although it alters animal physiology eral tissues (Rabe et al. 2008). Furthermore, leptin (Gordon 1993, Horowitz 2002), it is non-lethal. increases insulin sensitivity. Adiponectin regulates Journal of Molecular Endocrinology (2012) 48, 129–138 DOI: 10.1530/JME-11-0054 0952–5041/12/048–129 q 2012 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/27/2021 09:05:21AM via free access 130 P MORERA and others $ Heat stress and adipokines feeding behaviour through peripheral and central used to perform a glucose tolerance test (GTT). At the mechanisms and serves as a ‘starvation’ signal (Hoyda beginning (day 0) and after 2 and 5 days of the et al. 2011). Studies conducted on rodents demonstrate experimental period, six animals from each group were that adiponectin increases whole-body insulin sensi- killed via diethyl ether inhalation followed by cervical tivity, decreases hepatic glucose production and dislocation, and blood and tissue samples were increases fat oxidation in liver and muscle, leading to collected immediately. Blood was collected into vials an overall decrease in fat deposition (Ailhaud 2006, containing heparin and then centrifuged. The plasma Lafontan & Viguerie 2006). In addition to its insulin- was stored at K20 8C until analyses. Subcutaneous white sensitising effect, adiponectin may modify glucose adipose tissue (WAT), gastrocnemius skeletal muscle metabolism by stimulating pancreatic insulin secretion (M) and liver (L) samples were placed in RNA-Later (Rabe et al. 2008). solution (Ambion, Inc.) and frozen at K80 8C until Few data are available on the interactions between total RNA isolation. Small intestine tissue samples were HS and adipokine expression and secretion. To our fixed in glutaraldehyde and paraformaldehyde for knowledge, only such studies concern the effects of histological examination. HS on leptin gene expression and secretion in birds (Al-Azraqi 2008, Dridi et al. 2008) and in lactating pigs (Renaudeau et al. 2003) and on leptin and adiponectin HS treatment gene expression and secretion in mouse adipocytes The HS protocol lasted for 5 days and was designed to (Bernabucci et al. 2009). induce moderate chronic stress, i.e. mice were continu- On the basis of our previous results (Bernabucci G 8 et al. 2009), we conducted a study to evaluate whether ously exposed to an ambient temperature of 35 1 C. chronic HS rather than a reduction in voluntary food Hyperthermia was induced via an infrared ceramic heat intake is associated with changes in the expression and lamp (100 W; Ceramicx Ireland Ltd., Cork, Ireland). secretion of leptin and adiponectin and in insulin Therewerefourmicepercage(Metaboliccage sensitivity and glucose metabolism in peripheral tissues. 3700M022, Tecniplast, Varese, Italy), and the lamps were positioned above each cage at a distance of 10 cm. The air temperature within the cage was checked every hour. The RT of the mice was measured every 2 h Materials and methods during the daytime (from 0800 to 1800 h) using a thermistor probe (Temp JKT; Oakton Instruments, Animals and experimental design Vernon Hills, IL, USA). The use of an infrared heat lamp to induce All animal procedures were reviewed by the Animal Care hyperthermia is safe: does not emit any type of light at Ethics Committee of the University of Tuscia, Viterbo, the wavelengths to which murine retinal cells are Italy, approved by Italian Government Authorities, and sensitive and provides the opportunity to maintain the were in accordance with European Guidelines. Seventy- body temperature at any desired level for some time two male C57BL/6 mice (Charles River Laboratories, (Sharma 2007). Milan, Italy), 7 weeks old, were used. After an adaptation period of 14 days under control conditions (22G2 8C; photoperiod: 12 h light:12 h darkness) and with ad libitum Biochemical analysis access to a standard chow diet (VRF1 diet, Charles River Laboratories), the animals were subdivided into three Plasma indices of metabolic status were determined via groups of 24 mice each. One group was kept under an automatic analyser (IL-300 Plus, Instrumentation control conditions and fed ad libitum (C group; negative Laboratory, Lexington, MA, USA). Plasma glucose control). The second group was exposed to high ambient (kit from Instrumentation Laboratory) and NEFAs temperature (HS group; 35G1 8C; photoperiod: 12 h (NEFA-C kit; Wako Fine Chemical Industries, Dallas, light:12 h darkness) for 5 days continuously and fed TX, USA) were measured by enzymatic colorimetric ad libitum. The third group was kept under control methods. Plasma leptin, adiponectin and insulin were conditions and was food restricted (FR group; positive measured using commercial ELISA kits. The assays control) for 5 days. The level of food restriction were conducted in 96-well microplates according to the corresponded to 80% of the dietary intake by control manufacturer’s instructions, with a Spectra-I microplate mice, which is a normal response of mice exposed to HS. spectrophotometer (SLT Lab-Instruments GmbH, To avoid the influence of diurnal cycling, each Grodig/Salzburg, Austria). Mouse leptin (sensitivity: experiment was started between 0800 and