The FASEB Journal article fj.201601224R. Published online February 22, 2017. THE
JOURNAL
• RESEARCH •
www.fasebj.org
Changes in ambient temperature elicit divergent control of metabolic and cardiovascular actions by leptin Jussara M. do Carmo,*,1 Alexandre A. da Silva,*,‡ Damian G. Romero,† and John E. Hall*
*Department of Physiology and Biophysics, Mississippi Center for Obesity Research, Cardiovascular–Renal Research Center, and †Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi, USA; and ‡Centro Universit´ario Barão de Mau´a, Ribeirão Preto, São Paulo, Brazil
Interactions of hypothalamic signaling pathways that control body temperature (BT), blood pressure (BP), and energy balance are poorly understood. We investigated whether the chronic BP and metabolic actions of leptin are differentially modulated by changes in ambient temperature (TA ). Mean arterial pressure (MAP), heart rate (HR), BT, motor activity (MA), and oxygen consumption (VO 2) were measured 24 h/d at normal laboratory TA (23°C), at thermoneutral zone (TNZ, 30°C) for mice or during cold exposure (15°C) in male wild-type mice. After control measurements, leptin (4 mg/kg/min) or saline vehicle was infused for 7 d. At TNZ, leptin reduced food intake (211.0 6 0.5 g cumulative deficit) and body weight by 6% but caused no changes in MAP or HR. At 15°C, leptin infusion did not alter food intake but increased MAP and HR (8 6 1 mmHg and 33 6 7 bpm), while VO2 increased by ∼10%. Leptin reduced plasma glucose and insulin levels at 15°C but not at 30°C. These results demonstrate that the chronic anorexic effects of leptin are enhanced at TNZ, while its effects on insulin and glucose levels are attenuated and its effects on BP and HR are abolished. Conversely, cold T A caused resistance to leptin’s anorexic effects but amplified its effects to raise BP and reduce insulin and glucose levels. Thus, the brain circuits by which leptin regulates food intake and cardiovascular function are differentially influenced by changes in T A .—Do Carmo, J. M., da Silva, A. A., G. Romero, D. G., Hall, J. E. Changes in ambient temperature elicit divergent control of metabolic and cardiovascular actions by leptin. FASEB J. 31, 000–000 (2017). www.fasebj.org
ABSTRACT:
KEY WORDS:
blood pressure
•
energy expenditure
•
food intake
Leptin, a peptide hormone produced by adipose tissue in proportion to body fat mass, acts in the CNS to reduce appetite while increasing energy expenditure, sympathetic nerve activity (SNA), and blood pressure (BP). A key role for leptin in regulating body weight and cardiovascular function is evident from the observation that loss of leptin secretion or leptin receptor function in experimental animals and humans leads to hyperphagia, reductions in energy expenditure, and normal or reduced SNA and BP despite morbid obesity, ABBREVIATIONS: AgRP, agouti-related peptide; ARC, arcuate nucleus of the
hypothalamus; BP, blood pressure; BT, body temperature; CART, cocaine and amphetamine–regulated transcript; HR, heart rate; MA, motor activity; MAP, mean arterial pressure; NPY, neuropeptide Y; POMC, proopiomelanocortin neurons; qPCR, quantitative PCR; RAS, renin–angiotensin system; SNA, sympathetic nerve activity; TA, ambient temperature; TNZ, thermoneutral zone; VO2, oxygen consumption; WT, wild-type 1
Correspondence: Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 North State St., Jackson, MI 39216, USA. E-mail: [email protected]
doi: 10.1096/fj.201601224R
0892-6638/17/0031-0001 © FASEB
•
heart rate
which would normally increase sympathetic activity and BP (1–5). The CNS circuitry involved in leptin’s actions on metabolic and cardiovascular functions includes activation of proopiomelanocortin (POMC) neurons and inhibition of neurons expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus (ARC) (4–7). Leptin receptor activation in other neurons of the hypothalamus, as well as the nucleus of the solitary tract and dorsal motor nucleus of the vagus, has also been suggested to mediate part of leptin’s effects on appetite and SNA (8, 9). Leptin also regulates thermogenesis and locomotor activity. Mice with leptin deficiency (ob/ob mice) are cold intolerant and exhibit decreases in body temperature (BT) and motor activity (MA) compared to lean control mice (10, 11). Leptin may also link obesity with increased SNA and hypertension (12). In the hypothalamus, leptin activates centers that play a major role in modulating SNA and BP as well as temperature regulation. Previous studies
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showed that CNS injections of leptin stimulate SNA to peripheral tissues, activate uncoupling protein 1 (UCP-1) in brown adipose tissue, and increase energy expenditure, suggesting that increased thermogenesis contributes to leptin’s effects to reduce body fat (13, 14). In addition, Haltiner et al. (15) suggested that a warmer environment alters leptin’s effects to reduce fat depots in mice fed a high-fat diet, but not in lean control mice. It is well known that the hypothalamus regulates energy expenditure and BT (16), and some investigators have suggested that these functions may be coupled (17). Previous studies also showed that overweight subjects may have decreased food-induced thermogenesis and reduced metabolic responses to mild and moderate cold exposure compared to lean controls (18, 19). Although the hypothalamus is recognized as a critical integrating center for regulation of BT, energy balance, and cardiovascular function, the interactions of these signaling pathways in coordinating cardiometabolic functions is still unclear. Exposure to cold ambient temperature (TA) increases metabolic rate via nonshivering thermogenesis and increases SNA and BP, whereas warmer temperatures, in the thermoneutral zone (TNZ, 30°C), reduce BP and metabolic rate (20). However, to our knowledge, there have been no previous studies that have examined whether changes in TA modulate the chronic effects of peripheral hormonal signals, such as leptin, that are important for cardiometabolic regulation. Therefore, in the present study, we examined the chronic actions of leptin on metabolic and cardiovascular function at cold (15°C) and warm (TNZ, 30°C) temperatures compared to the usual laboratory temperature (23°C). Our observations indicate that the chronic anorexic effects of leptin are enhanced at TNZ, although its effects to reduce body weight, plasma insulin, and glucose levels are attenuated and its effects to increase BP and heart rate (HR) are completely abolished. Conversely, at 15°C, leptin caused a greater reduction in body weight, mainly as a result of increased metabolic rate, as it caused only a transient reduction in food intake, whereas its effects to raise BP and reduce insulin and glucose levels were amplified. Thus, TA has profound effects on leptin’s chronic cardiovascular and metabolic effects, where reducing TA causes resistance to the anorexic effects of leptin while enhancing its effects on BP, similar to the differential effects of leptin on BP and metabolic function observed in obesity (21). Our observations provide new insights into the interactions of temperature regulation with control of BP and metabolic function by leptin and have important implications for rodent experimental studies, which may be conducted at different TA points in various laboratory animal facilities. MATERIALS AND METHODS The experimental procedures and protocols for these studies followed the Guide for the Care and Use of Laboratory Animals, National Institutes of Health, Bethesda, MD, USA) and were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center. 2
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Animals Male wild-type (WT) C57BL/6J mice (n = 26), 22 wk old, were purchased from The Jackson Laboratories (Sacramento, CA, USA). Telemetry probe implantation Mice were anesthetized with 2% isoflurane. Under sterile conditions, a telemetry probe (TA11PA-C10; Data Science, New Brighton, MN, USA) was implanted in the left carotid artery for determination of mean arterial pressure (MAP) and HR 24 h/d using computerized methods for data collection as previously described (22, 23). Daily MAP and HR were obtained from the average of 24 h of recording using a sampling rate of 1000 Hz with duration of 10 s every 10-min period. A BT probe was implanted intraperitoneally (Mini Emitter 4000; Respironics, Bend, OR, USA) to measure BT 24 h/d using computerized data collection. Experimental design Food and water were offered ad libitum throughout the experiment, and TA was maintained at 23°C during recovery from surgery. The mice were allowed to recover for 8 to 10 d after the surgery before baseline measurements were taken. After an 8- to 10-d postsurgery recovery period and stable control measurements at 23°C, mice were divided into 3 groups: 1) TA was lowered from 23 to 15°C; 2) TA was increased from 23 to 30°C, the TNZ for mice; and 3) TA was maintained at 23°C throughout the experiment. Each TA was maintained for 22 consecutive days (5-d adaptation, 5-d baseline, 7-d vehicle or leptin infusion, 5-d recovery periods). After 5 d of baseline measurements at 15 or 30°C, the mice from each group were divided into 2 subgroups: one group was implanted with osmotic minipumps (1007; Durect, Cupertino, CA, USA) to infuse leptin (R&D Systems, Minneapolis, MN, USA) at 4 mg/kg/min as previously described (24), and the other group was implanted with osmotic minipumps to deliver isotonic saline vehicle (pH 7.4) for 7 d. Leptin was also infused for 7 consecutive days in group 3. The rate of leptin infusion was chosen on the basis of previous studies showing that in mice, this dose raises plasma leptin levels to those found in severe obesity (22). We have also shown that this rate of leptin infusion reduces food intake and increases BP in rodents at typical laboratory conditions (23°C) (22, 23). To prevent and alleviate pain, carprofen (1 mg/kg, s.c.) was provided immediately after surgery and once daily for 48 h after surgery. After the 7-d treatment regimen, the mice were followed for an additional 5-d posttreatment (recovery) period. Blood samples (100 ml) were taken via tail snip on the last day of the control period, on d 7 of leptin or vehicle infusion, and on the last day of the recovery period after 5 h of food withdrawal. Oxygen consumption, MA, and BT measurements Male WT mice aged 22 to 24 wk were placed individually in metabolic cages (Comprehensive Lab Animal Monitoring System, Columbus, OH, USA) equipped with an oxygen sensor to measure oxygen consumption (VO2) and receivers to measure BT. Animal MA was determined by infrared light beams mounted in the cages in the x- and y-axes. VO2 and BT were measured for 2 min at 10-min intervals continuously 24 h/d. After the mice were acclimatized to the new environment for at least 5 d, VO2, MA, and BT were measured for 5 consecutive days; then leptin or saline was infused for 7 d at
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15°C, 23°C, and TNZ temperatures. Additional measurements were taken for 5 d after leptin or saline infusion was stopped, as previously described, and blood samples were taken to measure plasma hormones. POMC, NPY, cocaine and amphetamine–regulated transcript, and AgRP mRNA levels in the hypothalamus Additional groups of mice (n = 5 per group) were kept at 15°C or TNZ for 5 consecutive days and then treated with leptin or saline injected intraperitoneally (5 mg/kg/d, 150 ml) for 5 consecutive days. One hour after the last injection, mice not denied food were humanely killed with an overdose of isoflurane, the brains were quickly removed, and the hypothalamus was dissected on an icecold platform. Tissue samples were frozen immediately by immersion in liquid nitrogen and stored at 280°C. Total RNA was extracted with Tri Reagent (Molecular Research Center, Cincinnati, OH, USA), resuspended in nuclease-free water, treated with DNase (Turbo DNA-Free Kit; Thermo Fisher Scientific, Waltham, MA, USA), and quantified by spectrophotometry. Four micrograms of total RNA was reverse transcribed with 0.5 mg of T20VN primer and Superscript III (Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed as previously described (25) and standardized against Rpl13a (26, 27). Briefly, qPCR reactions were performed with 1 ml reverse transcription product, 1 ml Titanium Taq DNA polymerase (Clontech Laboratories, Mountain View, CA, USA), 1:20,000 dilution SYBR Green I (Molecular Probes, Eugene, OR, USA), 0.2 mM dNTPs, and 0.2 mM each primer in a final volume of 25 ml. Cycling conditions were 1 min at 95°C, 50 cycles for 15 s at 95°C, 15 s at annealing temperature, and 1 min 68°C. Realtime data were obtained during the extension phase, and Ct values were obtained at the log phase of each gene amplification. PCR product quantification was performed by the relative quantification method (28, 29) and standardized against Rpl13a1. PCR product specificity was confirmed by postrun melting curve analysis and high-resolution agarose gel electrophoresis using NuSieve 3:1 agarose (Lonza, Rockland, ME, USA). Results are expressed as arbitrary units and normalized against Rpl13a1 mRNA expression. The 18s rRNA expression was assayed TaqMan qPCR using Eukaryotic 18S rRNA Endogenous Control (Thermo Fisher Scientific) and TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) following the manufacturer’s suggested protocol. The primer sequences were selected from previous studies (30–32). Rpl13a1 was selected as the housekeeping gene for gene expression analysis after analyzing the coefficient of variation (CV%) of the expression (arbitrary units) across all experimental conditions in a set of 12 housekeeping genes (data not shown). Analytical methods Plasma leptin and insulin levels were measured with ELISA kits (respectively, R&D Systems, Minneapolis, MN, USA, and Crystal Chem, Downers Grove, IL, USA), and plasma glucose concentrations were determined using the glucose oxidation method (Glucose Analyzer 2; Beckman Coulter, Brea, CA, USA). Statistical analyses The results are expressed as means 6 SEM. The data obtained were analyzed by paired Student’s t test or 1-way ANOVA with repeated measures followed by Dunnett’s post hoc test for comparison between control and experimental values within each group when appropriate. Comparisons between different groups were done by unpaired Student’s t test or 1-way ANOVA followed by Dunnett’s post hoc test when appropriate. Statistical significance was accepted at a level of P , 0.05. TEMPERATURE AND LEPTIN
RESULTS Impact of changes in TA on body weight and food intake, and responses to chronic leptin infusion Changing TA from a standard housing temperature (23°C) to either 15°C or TNZ did not significantly alter body weight in any of the groups (Fig. 1A–C). However, reducing TA from 23 to 15°C increased food intake from 3.2 6 0.5 to 4.5 6 0.5 g/d (Fig. 1D), whereas increasing TA from 23 to 30°C decreased food intake from 4.0 6 1.0 to 2.8 6 0.5 g/d (Fig. 1E). At 23°C, food intake averaged 3.2 6 0.4 g/d (Fig. 1F). Chronic leptin infusion for 7 d at 15°C caused a modest reduction in food intake and a cumulative deficit of only 26.5 6 0.5 g during the 7 d treatment period, but significantly reduced body weight (;14%) (Fig. 1A). Leptin infusion at TNZ caused a more substantial decrease in food intake (211.1 6 1.8 g cumulative deficit), which was associated with a weight loss of ;8% (Fig. 1B) compared to baseline. Leptin infusion at 23°C reduced body weight by 3 6 1 g (Fig. 1C) and significantly reduced food intake (Fig. 1F), causing a 28.2 6 1.1 g cumulative food deficit. These data indicate that leptin has a greater effect in reducing food intake despite a lesser effect to reduce body weight when TA is increased to TNZ compared to an TA of 15°C. Impact of changes in TA on VO2, MA, and BT, and responses to chronic leptin infusion Changing TA from 23 to 15°C increased VO2 by ;50% (Fig. 2A). Conversely, at TNZ, VO2 was reduced by ;50% compared to 23°C TA (Fig. 2B). Chronic leptin infusion at 15°C caused a transient small reduction in VO2 followed by a gradual increase in VO2 (Fig. 2A). At TNZ, however, no significant change in VO2 was observed during leptin infusion (Fig. 2B). When leptin infusion was stopped, VO2 increased slightly, but not significantly, in mice housed at TNZ (Fig. 2B). The increase in VO2 during the recovery posttreatment period may be partly due to the rebound hyperphagia that occurred in mice housed at TNZ after leptin treatment was stopped (baseline: 4.1 6 0.8 vs. 6.0 6 1.1 g posttreatment). We observed a small, but statistically insignificant (P = 0.06), increase in VO2 when leptin was infused at 23°C (Fig. 2C). No significant alterations were observed in BT during changes in TA or during leptin infusion (Fig. 2D–F). Although MA was slightly reduced during leptin infusion at TNZ, no significant changes were observed during leptin infusion at 15°C or 23°C (Fig. 3). Impact of changes in TA on plasma glucose, insulin, and leptin levels, and responses to chronic leptin infusion There were no significant differences in baseline plasma levels of leptin, insulin, or glucose in mice housed at TA ranging from 15 to 30°C (Table 1). Chronic leptin infusion, however, significantly increased plasma leptin to 42 to 67 ng/ml, which are levels similar to those
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Figure 1. Body weight and food intake responses to chronic leptin or saline infusion. A) Body weight when TA was reduced from 23 to 15°C. B) Body weight when TA was increased from 23 to 30°C. C ) Body weight when TA was maintained at 23°C (leptin group only). D) Food intake when TA was reduced from 23 to 15°C. E ) Food intake when TA was increased from 23 to 30°C. F ) Food intake when TA was maintained at 23°C (leptin group only).*P , 0.05 compared to saline group; #P , 0.05 compared to 23°C; $P , 0.05 compared to baseline at 23°C.
found in severe obesity. The difference in plasma leptin levels between the groups housed at 15 and 30°C may reflect the greater weight loss during chronic leptin infusion in mice housed at 15°C. Leptin infusion caused a significant reduction in fasting plasma glucose and insulin levels at 15 and 23°C, but not at 30°C. After stopping leptin infusion, plasma leptin and glucose levels returned to baseline values. These results indicate that leptin’s effects to reduce insulin and glucose levels are markedly attenuated at 30°C, even though the anorexic effect of leptin was impaired at TA of 15°C. 4
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Effect of changes in TA on BP and HR, and responses to chronic leptin infusion MAP increased by ;6 mmHg in the groups where TA was reduced from 23 to 15°C. Conversely, MAP decreased by ;11 mmHg in the groups where TA was increased from 23 to TNZ (Table 2). Thus, the difference in MAP between mice housed at 15°C and TNZ was ;17 mm Hg. Chronic leptin infusion at 15°C caused an additional gradual increase in MAP of ;8 mmHg (average of the last 3 d of treatment) and ;5 mmHg in mice housed at 23°C
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Figure 2. VO2 and BT responses to chronic leptin or saline infusion. A) TA reduced from 23 to 15°C. B) TA increased from 23 to 30°C. C) TA maintained at 23°C (leptin group only). D) BT when TA was reduced from 23 to 15°C. E) BT when TA was increased from 23°C to TNZ. F) BT when TA was maintained at 23°C (leptin group only). *P , 0.05 compared to saline group; #P , 0.05 compared to 23°C.
(Fig. 4A, C), while no change in MAP was observed at 30°C (Fig. 4B). No significant changes in MAP were observed after 7 d of vehicle infusion at 15°C or TNZ (Fig. 4A, B). On the first day after implantation of the osmotic minipump, there were significant increases in MAP in mice housed at 23°C and TNZ, which may be due to surgery for minipump placement, as MAP rapidly returned to control values on day 2 of leptin infusion. Changing TA from 23 to 15°C or from 23 to TNZ also caused profound alterations in HR. Reducing TA to 15°C increased HR by 110 to 130 bpm, whereas raising TA to 30°C markedly decreased HR by approximately ;120 bpm (Fig. 4D, E). Moreover, similar to what was observed for MAP, housing the mice at 15°C enhanced leptin’s effect TEMPERATURE AND LEPTIN
to raise HR (;20 bpm), while at TNZ leptin had no sustained effect on HR (Fig. 4D, E). We also did not observe a sustained effect of leptin to raise HR at 23°C (Fig. 4F). Vehicle infusion caused no sustained effect on HR at either TA (Fig. 4D, E). These results indicate that the chronic effects of leptin on BP and HR are markedly attenuated at TNZ and enhanced at 15°C. Expression of NPY, POMC, CART, and AgRP in the hypothalamus of WT mice after leptin infusion Decreasing TA and activation of NPY/AgRP neurons are well-known stimuli for increasing food intake, whereas
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observed in control mice at TNZ (Fig. 5B). Leptin treatment did not alter POMC mRNA at TNZ (Fig. 5B). No significant differences were observed for AgRP or CART mRNA levels between mice housed at 15°C or TNZ, and these levels were also not affected by leptin treatment at either temperature (Fig. 5C, D). DISCUSSION The results of the present study indicate that changes in TA not only have major effects on control of BP and metabolism but also greatly influence leptin’s metabolic and cardiovascular actions. Compared to the TA normally maintained in most laboratory animal facilities (23°C), a warmer TA (30°C, the TNZ for mice) enhanced the appetite-suppressing actions of leptin while attenuating its effects to raise BP and HR and to reduce glucose and insulin levels. In contrast, at cold TA, leptin’s effects on BP, HR, and insulin and glucose levels were exacerbated while its anorexic effects were attenuated. These findings are summarized in Fig. 6. These observations have important implications for cardiovascular and metabolic studies in rodents that may be housed at different TA points in various laboratory animal facilities and are consistent with previous studies showing the effects of TA on metabolic and cardiovascular function in rodents (34–36). In addition, our findings provide new insights into the interaction of brain circuits that regulate energy balance, BP, and BT. Varying TA may be a useful tool in revealing the mechanisms responsible for differential control of BP and metabolic functions by leptin, a topic of considerable interest for understanding the factors that contribute to selective leptin resistance in pathophysiological conditions such as obesity. Effect of changes in TA on food intake, VO2, MA, and responses to leptin
Figure 3. MA response to chronic leptin or saline infusion when TA was altered. A) MA when TA was reduced from 23 to 15°C. B) MA when TA was increased from 23°C to TNZ. C ) MA when TA was maintained at 23°C (leptin group only).
leptin is thought to suppress appetite by activating POMC/ cocaine and amphetamine–regulated transcript (CART) neurons and inhibiting NPY/AgRP neurons (33). We hypothesized that leptin may have differential effects on these pathways depending on TA. NPY mRNA levels in control saline-treated mice were higher at 15°C compared to TNZ (Fig. 5A), which parallels the changes in appetite evoked by housing the animals at these temperatures. Leptin infusion did not alter NPY mRNA in mice housed at 15°C, but surprisingly, leptin infusion at TNZ raised NPY mRNA levels to values comparable to that observed at 15°C (Fig. 5A). Also, POMC mRNA was reduced at TNZ in vehicle-treated mice, and leptin reduced POMC mRNA in mice housed at 15°C to the same level 6
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In the present study, as in previous studies (37, 38), reducing TA markedly increased food intake and energy expenditure while body weight and BT remained relatively constant. Conversely, increasing TA decreased food TABLE 1. Plasma glucose, leptin, and insulin levels in WT mice during control, leptin infusion, and recovery periods at different TA Characteristic Leptin (ng/ml) Glucose (mg/dl) Insulin (mU/ml)
15°C Control Leptin Recovery 30°C Control Leptin Recovery 23°C Control Leptin Recovery
461 42 6 3* 361
176 6 16 93 6 14* 160 6 10
12 6 3 4 6 1* 761
261 67 6 9* 361
167 6 19 149 6 24 172 6 10
13 6 3 10 6 2 761
361 57 6 5* 462
157 6 10 102 6 25* 162 6 12
Data are expressed as means 6 0.05 compared to control period.
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SEM.
962 4 6 2* 861
n = 5–6 in each group. *P ,
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TABLE 2. MAP, HR, and VO2 responses to changes in TA in WT mice Characteristic
MAP (mmHg) HR (bpm) VO2 (ml/kg/h)
23°C
15°C
23°C
30°C
106 6 1 532 6 18 3102 6 286
113 6 2* 650 6 20* 4588 6 195*
105 6 2 548 6 15 3134 6 152
98 6 2# 420 6 12# 2050 6 58#
*P , 0.05 compared to baseline at 23°C; #P , 0.05 compared to baseline at 23°C.
intake and energy expenditure while causing no substantial changes in body weight or BT. A novel finding of these experiments is that changes in TA markedly alter the metabolic effects of leptin. At cold TA, leptin’s anorexic effect was attenuated by approximately 40%, whereas its effect on reducing body weight was amplified compared to the responses observed at TNZ. Body weight decreased by approximately 10 g during leptin infusion at 15°C TA despite only a 6 g decrease in cumulative food intake and no major changes in MA. Thus, we speculate that an increase in VO2 during leptin treatment may have contributed to this reduction in body weight. At 30°C TA, leptin infusion for 7 d decreased body weight by only about 5 g despite causing a decrease in cumulative food intake of over 10 g and no significant change in VO2; however, leptin infusion at 30°C TA also reduced MA, which may have contributed to the maintenance of a relatively constant body weight despite a large decrease in food intake. Our studies also showed, surprisingly, that at cold TA, leptin did not increase POMC expression or decrease NPY mRNA, effects that are believed to mediate much of leptin’s anorexic effects under normal conditions (33). These findings suggest that at cold TA, the anorexic effects of leptin due to increased POMC activity and decreased NPY are limited by other factors responsible for stimulating appetite, although the mechanisms involved are still unknown. Changes in TA alter leptin’s effects on plasma glucose and insulin levels Central or peripheral leptin infusion markedly enhances peripheral tissue glucose utilization via insulin-dependent and -independent mechanisms (39). We previously showed that leptin receptor deletion in POMC neurons completely abolished the effects of leptin to reduce insulin and glucose levels, suggesting that the effects of leptin to stimulate peripheral glucose utilization are mediated largely by activation of POMC neurons (22). In the present study, we found that leptin’s effects to reduce glucose and insulin levels were enhanced at 15°C TA and completely abolished at TNZ, suggesting that the chronic effects of leptin on glucose homeostasis are greatly altered by changes in TA; higher TA impairs leptin’s ability to lower plasma insulin and glucose levels, while cooler TA enhances leptin’s effects on glucose regulation Although we found that leptin’s effects on glucose regulation were greatly attenuated at TNZ, the molecular mechanisms responsible are unclear. Our previous studies suggest that Shp2 signaling in forebrain neurons mediates most of the chronic antidiabetic TEMPERATURE AND LEPTIN
effects of leptin to reduce plasma glucose (23). Whether changing TA alters leptin signaling via changes in Shp2 responsiveness or through other pathways, such as Stat3 and Irs2 signaling, is still unclear. Effect of changes in TA on BP and HR, and responses to chronic leptin infusion The results of our study confirm previous studies showing that cold TA raises BP and HR while warm TA reduces BP and HR (36, 40, 41). The rise in BP may be an adaptive response that contributes to enhanced circulatory function needed for increases in metabolic rate and nonshivering thermogenesis, thereby helping to maintain BT (40). Although the mechanisms responsible for cold-induced hypertension are not completely understood, increases in SNA, especially renal SNA, appear to play a major role because bilateral denervation of the kidneys prevents development of coldinduced hypertension (37, 42). Increased renal SNA during cold exposure activates the renin–angiotensin system (RAS), and blockade of the RAS also abolishes cold-induced hypertension (40). Thus, activation the RAS and sympathetic nervous system together contribute importantly to the rise in BP that occurs during chronic exposure to cold TA. Chronic exposure to a warm TA of 30°C, the TNZ for mice, lowered BP and HR by approximately 11 mm Hg and 120 bpm, respectively, compared to values measured at 23°C, the usual TA of most laboratories. The mechanisms responsible for the decrease in BP with increasing TA have not been widely studied but are likely related to decreases in SNA and RAS activity (36). These observations emphasize the close associations among control of BP, metabolism, and BT, and have important implications for experimental studies in rodents. A novel finding of our study is that changes in TA altered the BP, HR, and metabolic responses to leptin in divergent ways. We and others have previously shown that at normal TA leptin administration increases SNA and causes small increases in BP (43). Moreover, leptinmediated increases in BP can be completely prevented by adrenergic blockade, indicating that they are due to sympathetic activation (12, 44). We have also provided evidence that leptin may link obesity with increased SNA and hypertension (12, 21, 43). Humans and mice that are leptin deficient have normal or even reduced BP and SNA despite severe obesity and many characteristics of the metabolic syndrome, suggesting that a functional leptin system may be necessary for obesity to increase SNA and BP (43). In the present study, we found, surprisingly, that leptin’s effects on BP were completely abolished at TNZ and enhanced by cold exposure. Thus, the neural circuits
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Figure 4. Changes in MAP and HR during chronic leptin or saline infusion at various TA. A) MAP changes at 15°C. B) MAP changes at TNZ. C ) MAP changes at 23°C (leptin group only). D) HR changes at 15°C. E ) HR changes at TNZ. F ) HR changes at 23°C (leptin group only). *P , 0.05 compared to saline group; #P , 0.05 compared to 23°C; $P , 0.05 compared to baseline at 23°C.
for leptin-mediated sympathetic activation and BP control interact closely with those involved with BT regulation. These circuits appear to be distinct from those that mediate leptin’s anorexic effects because changing the TA had opposite effects on the BP and food intake responses to leptin, with cold exposure attenuating leptin’s anorexic effect while amplifying its BP effect. However, the specific neural circuits and signaling pathways that mediate these divergent interactions of leptin and TA on regulation of food intake and BP are still poorly understood. 8
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On the basis of the sustained anorexic effect of leptin at TNZ, we hypothesized that the POMC/ CART pathway would exhibit higher endogenous expression in leptin-treated compared to salinetreated animals. However, the relative abundance of mRNA levels of POMC and CART were not different between groups. Although the anorexic effect of leptin was only transient at 15°C TA, the relative abundance of POMC mRNA levels was reduced, suggesting that this may be one of the mechanisms responsible for the impaired food intake response to leptin. Despite the
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Figure 5. Relative mRNA abundance quantified by qPCR in hypothalamus fractions of WT mice after 5 d of leptin or saline infusion and exposure to 15°C and TNZ temperatures for NPY (A), POMC (B), AgRP (C ) , and CART (D). *P , 0.05 compared to saline-treated group.
fact that leptin infusion significantly reduced food intake at TNZ, the NPY mRNA was increased in the leptin-treated compared to the saline-treated group. In addition, at TNZ, the POMC mRNA level was similar between saline- and leptin-treated groups, but increased at 15°C TA. A likely explanation for these unexpected results is that POMC neurons may already be activated at cold TA as a result of simultaneous activation of leptin receptors and the hypothalamic temperature regulation center. Another possible explanation for these surprising findings is that leptin does not have equal effects on all neuronal subtypes or downstream pathways during changes in TA. For instance, acute leptin injection activates POMC, but not NPY neurons, in the ARC as assessed by increased c-Fos protein, but suppressor of cytokine signaling-3 (SOCS3) mRNA was increased in both neuronal subtypes (45). Thus, further studies are needed to unravel the changes in gene expression and cell signaling pathways activated by leptin and TA. Our results therefore suggest that alterations in TA are associated with divergent control of metabolic and cardiovascular functions in responses to chronic leptin infusion. Changes in TA may be a useful tool to dissect the areas of the CNS and signaling pathways that permit leptin to independently control cardiovascular and metabolic functions, and they also may be useful to understand the interactions of the ARC and hypothalamic temperature center in regulating energy balance and BP. Implications for physiologic studies in rodents Our understanding of human physiology and mechanisms of disease depends heavily on translating results TEMPERATURE AND LEPTIN
from experiments in nonhuman animals, especially rodents. For many areas of research, murine models have increasingly been used largely because of their short gestation times and the molecular tools that are available to manipulate gene expression. Genetically modified mouse models have been especially important for investigating CNS pathways that contribute to regulation of food intake, energy expenditure, and the pathogenesis of obesity and associated metabolic disorders. In most of these studies, however, little attention is paid to the TA of the laboratory when phenotypes are assessed. The results of our study indicate that mice housed at the usual laboratory TA of 23°C have metabolic and cardiovascular phenotypes that differ considerably from those obtained at the TNZ in mice of the same genotype. Moreover, changes in TA markedly altered the cardiovascular and metabolic responses to leptin, sometimes in opposite directions. For example, increasing TA from 23 to 30°C abolished the increases BP and HR during leptin infusion while amplifying the anorexic effects of leptin. These results further emphasize the need to consider carefully the TA when conducting studies in mice. As noted by Maloney et al. (37), it could be argued that much of what we know about metabolism, obesity, and cardiovascular physiology of rodents comes from chronically hypertensive, hypermetabolic, and obesity-resistant animals because we usually study them at a TA that is comfortable for investigators but cold for rodents. It is clear that meaningful translation of results from experimental studies in rodents to understanding human physiology and pathophysiology will require us in the future to pay much more attention to the effects of environmental stressors such as TA. Our observations also
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University of Mississippi Medical Center) for technical assistance. The authors declare no conflicts of interest.
AUTHOR CONTRIBUTIONS J. M. do Carmo and A. A. da Silva designed the research, analyzed data, performed research, and wrote the paper; D. G. Romero performed gene expression analysis; and J. E. Hall contributed to analyzing the data and revising the article. REFERENCES
Figure 6. Comparison of cumulative food intake (A), changes in MAP (DMAP; B), and changes in blood glucose (C ) after 7 d of leptin infusion at T A of 15, 23, or 30°C. All values are expressed as changes relative to values measured during control period before starting leptin infusion.
provide new insights into the interaction of temperature regulation and control of BP and metabolism by leptin. ACKNOWLEDGMENTS This work was supported, in part, by the U.S. National Institutes of Health (NIH) National Heart, Lung, and Blood Institute (Grant PO1 HL51971), the NIH National Institute of General Medical Sciences (Grant P20 GM104357), and by an American Heart Association Scientist Development grant (to J.M.D.C. and D.G.R.). The authors thank H. Zhang, S. E. Ebaady, P. O. Sessums, C. Torrey, and J. P. Ball (all from the 10
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32. Tian, D. R., Li, X. D., Shi, Y. S., Wan, Y., Wang, X. M., Chang, J. K., Yang, J., and Han, J. S. (2004) Changes of hypothalamic alpha-MSH and CART peptide expression in diet-induced obese rats. Peptides 25, 2147–2153 33. Morrison, C. D., Morton, G. J., Niswender, K. D., Gelling, R. W., and Schwartz, M. W. (2005) Leptin inhibits hypothalamic Npy and Agrp gene expression via a mechanism that requires phosphatidylinositol 3-OH-kinase signaling. Am. J. Physiol. Endocrinol. Metab. 289, E1051–E1057 34. Cannon, B., and Nedergaard, J. (2011) Nonshivering thermogenesis and its adequate measurement in metabolic studies. J. Exp. Biol. 214, 242–253 35. Speakman, J. R., and Keijer, J. (2012) Not so hot: optimal housing temperatures for mice to mimic the thermal environment of humans. Mol. Metab. 2, 5–9 36. Swoap, S. J., Overton, J. M., and Garber, G. (2004) Effect of ambient temperature on cardiovascular parameters in rats and mice: a comparative approach. Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R391–R396 37. Maloney, S. K., Fuller, A., Mitchell, D., Gordon, C., and Overton, J. M. (2014) Translating animal model research: does it matter that our rodents are cold? Physiology (Bethesda) 29, 413–420 38. Overton, J. M. (2010) Phenotyping small animals as models for the human metabolic syndrome: thermoneutrality matters. Int. J. Obes. 34 (Suppl 2), S53–S58 39. Da Silva, A. A., Tallam, L. S., Liu, J., and Hall, J. E. (2006) Chronic antidiabetic and cardiovascular actions of leptin: role of CNS and increased adrenergic activity. Am. J. Physiol. Regul. Integr. Comp. Physiol. 291, R1275–R1282 40. Sun, Z. (2010) Cardiovascular responses to cold exposure. Front. Biosci. (Elite Ed.) 2, 495–503 41. Swoap, S. J., Li, C., Wess, J., Parsons, A. D., Williams, T. D., and Overton, J. M. (2008) Vagal tone dominates autonomic control of mouse heart rate at thermoneutrality. Am. J. Physiol. Heart Circ. Physiol. 294, H1581–H1588 42. Sun, Z., Fregly, M. J., and Cade, J. R. (1995) Effect of renal denervation on elevation of blood pressure in cold-exposed rats. Can. J. Physiol. Pharmacol. 73, 72–78 43. Carlyle, M., Jones, O. B., Kuo, J. J., and Hall, J. E. (2002) Chronic cardiovascular and renal actions of leptin: role of adrenergic activity. Hypertension 39, 496–501 44. Shek, E. W., Brands, M. W., and Hall, J. E. (1998) Chronic leptin infusion increases arterial pressure. Hypertension 31, 409–414 45. Elias, C. F., Aschkenasi, C., Lee, C., Kelly, J., Ahima, R. S., Bjorbaek, C., Flier, J. S., Saper, C. B., and Elmquist, J. K. (1999) Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area. Neuron 23, 775–786 Received for publication November 10, 2016. Accepted for publication January 30, 2017.
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Changes in ambient temperature elicit divergent control of metabolic and cardiovascular actions by leptin Jussara M. do Carmo, Alexandre A. da Silva, Damian G. Romero, et al. FASEB J published online February 22, 2017 Access the most recent version at doi:10.1096/fj.201601224R
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• RESEARCH •
www.fasebj.org
Changes in ambient temperature elicit divergent control of metabolic and cardiovascular actions by leptin Jussara M. do Carmo,*,1 Alexandre A. da Silva,*,‡ Damian G. Romero,† and John E. Hall*
*Department of Physiology and Biophysics, Mississippi Center for Obesity Research, Cardiovascular–Renal Research Center, and †Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi, USA; and ‡Centro Universit´ario Barão de Mau´a, Ribeirão Preto, São Paulo, Brazil
Interactions of hypothalamic signaling pathways that control body temperature (BT), blood pressure (BP), and energy balance are poorly understood. We investigated whether the chronic BP and metabolic actions of leptin are differentially modulated by changes in ambient temperature (TA ). Mean arterial pressure (MAP), heart rate (HR), BT, motor activity (MA), and oxygen consumption (VO 2) were measured 24 h/d at normal laboratory TA (23°C), at thermoneutral zone (TNZ, 30°C) for mice or during cold exposure (15°C) in male wild-type mice. After control measurements, leptin (4 mg/kg/min) or saline vehicle was infused for 7 d. At TNZ, leptin reduced food intake (211.0 6 0.5 g cumulative deficit) and body weight by 6% but caused no changes in MAP or HR. At 15°C, leptin infusion did not alter food intake but increased MAP and HR (8 6 1 mmHg and 33 6 7 bpm), while VO2 increased by ∼10%. Leptin reduced plasma glucose and insulin levels at 15°C but not at 30°C. These results demonstrate that the chronic anorexic effects of leptin are enhanced at TNZ, while its effects on insulin and glucose levels are attenuated and its effects on BP and HR are abolished. Conversely, cold T A caused resistance to leptin’s anorexic effects but amplified its effects to raise BP and reduce insulin and glucose levels. Thus, the brain circuits by which leptin regulates food intake and cardiovascular function are differentially influenced by changes in T A .—Do Carmo, J. M., da Silva, A. A., G. Romero, D. G., Hall, J. E. Changes in ambient temperature elicit divergent control of metabolic and cardiovascular actions by leptin. FASEB J. 31, 000–000 (2017). www.fasebj.org
ABSTRACT:
KEY WORDS:
blood pressure
•
energy expenditure
•
food intake
Leptin, a peptide hormone produced by adipose tissue in proportion to body fat mass, acts in the CNS to reduce appetite while increasing energy expenditure, sympathetic nerve activity (SNA), and blood pressure (BP). A key role for leptin in regulating body weight and cardiovascular function is evident from the observation that loss of leptin secretion or leptin receptor function in experimental animals and humans leads to hyperphagia, reductions in energy expenditure, and normal or reduced SNA and BP despite morbid obesity, ABBREVIATIONS: AgRP, agouti-related peptide; ARC, arcuate nucleus of the
hypothalamus; BP, blood pressure; BT, body temperature; CART, cocaine and amphetamine–regulated transcript; HR, heart rate; MA, motor activity; MAP, mean arterial pressure; NPY, neuropeptide Y; POMC, proopiomelanocortin neurons; qPCR, quantitative PCR; RAS, renin–angiotensin system; SNA, sympathetic nerve activity; TA, ambient temperature; TNZ, thermoneutral zone; VO2, oxygen consumption; WT, wild-type 1
Correspondence: Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 North State St., Jackson, MI 39216, USA. E-mail: [email protected]
doi: 10.1096/fj.201601224R
0892-6638/17/0031-0001 © FASEB
•
heart rate
which would normally increase sympathetic activity and BP (1–5). The CNS circuitry involved in leptin’s actions on metabolic and cardiovascular functions includes activation of proopiomelanocortin (POMC) neurons and inhibition of neurons expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus (ARC) (4–7). Leptin receptor activation in other neurons of the hypothalamus, as well as the nucleus of the solitary tract and dorsal motor nucleus of the vagus, has also been suggested to mediate part of leptin’s effects on appetite and SNA (8, 9). Leptin also regulates thermogenesis and locomotor activity. Mice with leptin deficiency (ob/ob mice) are cold intolerant and exhibit decreases in body temperature (BT) and motor activity (MA) compared to lean control mice (10, 11). Leptin may also link obesity with increased SNA and hypertension (12). In the hypothalamus, leptin activates centers that play a major role in modulating SNA and BP as well as temperature regulation. Previous studies
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showed that CNS injections of leptin stimulate SNA to peripheral tissues, activate uncoupling protein 1 (UCP-1) in brown adipose tissue, and increase energy expenditure, suggesting that increased thermogenesis contributes to leptin’s effects to reduce body fat (13, 14). In addition, Haltiner et al. (15) suggested that a warmer environment alters leptin’s effects to reduce fat depots in mice fed a high-fat diet, but not in lean control mice. It is well known that the hypothalamus regulates energy expenditure and BT (16), and some investigators have suggested that these functions may be coupled (17). Previous studies also showed that overweight subjects may have decreased food-induced thermogenesis and reduced metabolic responses to mild and moderate cold exposure compared to lean controls (18, 19). Although the hypothalamus is recognized as a critical integrating center for regulation of BT, energy balance, and cardiovascular function, the interactions of these signaling pathways in coordinating cardiometabolic functions is still unclear. Exposure to cold ambient temperature (TA) increases metabolic rate via nonshivering thermogenesis and increases SNA and BP, whereas warmer temperatures, in the thermoneutral zone (TNZ, 30°C), reduce BP and metabolic rate (20). However, to our knowledge, there have been no previous studies that have examined whether changes in TA modulate the chronic effects of peripheral hormonal signals, such as leptin, that are important for cardiometabolic regulation. Therefore, in the present study, we examined the chronic actions of leptin on metabolic and cardiovascular function at cold (15°C) and warm (TNZ, 30°C) temperatures compared to the usual laboratory temperature (23°C). Our observations indicate that the chronic anorexic effects of leptin are enhanced at TNZ, although its effects to reduce body weight, plasma insulin, and glucose levels are attenuated and its effects to increase BP and heart rate (HR) are completely abolished. Conversely, at 15°C, leptin caused a greater reduction in body weight, mainly as a result of increased metabolic rate, as it caused only a transient reduction in food intake, whereas its effects to raise BP and reduce insulin and glucose levels were amplified. Thus, TA has profound effects on leptin’s chronic cardiovascular and metabolic effects, where reducing TA causes resistance to the anorexic effects of leptin while enhancing its effects on BP, similar to the differential effects of leptin on BP and metabolic function observed in obesity (21). Our observations provide new insights into the interactions of temperature regulation with control of BP and metabolic function by leptin and have important implications for rodent experimental studies, which may be conducted at different TA points in various laboratory animal facilities. MATERIALS AND METHODS The experimental procedures and protocols for these studies followed the Guide for the Care and Use of Laboratory Animals, National Institutes of Health, Bethesda, MD, USA) and were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center. 2
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Animals Male wild-type (WT) C57BL/6J mice (n = 26), 22 wk old, were purchased from The Jackson Laboratories (Sacramento, CA, USA). Telemetry probe implantation Mice were anesthetized with 2% isoflurane. Under sterile conditions, a telemetry probe (TA11PA-C10; Data Science, New Brighton, MN, USA) was implanted in the left carotid artery for determination of mean arterial pressure (MAP) and HR 24 h/d using computerized methods for data collection as previously described (22, 23). Daily MAP and HR were obtained from the average of 24 h of recording using a sampling rate of 1000 Hz with duration of 10 s every 10-min period. A BT probe was implanted intraperitoneally (Mini Emitter 4000; Respironics, Bend, OR, USA) to measure BT 24 h/d using computerized data collection. Experimental design Food and water were offered ad libitum throughout the experiment, and TA was maintained at 23°C during recovery from surgery. The mice were allowed to recover for 8 to 10 d after the surgery before baseline measurements were taken. After an 8- to 10-d postsurgery recovery period and stable control measurements at 23°C, mice were divided into 3 groups: 1) TA was lowered from 23 to 15°C; 2) TA was increased from 23 to 30°C, the TNZ for mice; and 3) TA was maintained at 23°C throughout the experiment. Each TA was maintained for 22 consecutive days (5-d adaptation, 5-d baseline, 7-d vehicle or leptin infusion, 5-d recovery periods). After 5 d of baseline measurements at 15 or 30°C, the mice from each group were divided into 2 subgroups: one group was implanted with osmotic minipumps (1007; Durect, Cupertino, CA, USA) to infuse leptin (R&D Systems, Minneapolis, MN, USA) at 4 mg/kg/min as previously described (24), and the other group was implanted with osmotic minipumps to deliver isotonic saline vehicle (pH 7.4) for 7 d. Leptin was also infused for 7 consecutive days in group 3. The rate of leptin infusion was chosen on the basis of previous studies showing that in mice, this dose raises plasma leptin levels to those found in severe obesity (22). We have also shown that this rate of leptin infusion reduces food intake and increases BP in rodents at typical laboratory conditions (23°C) (22, 23). To prevent and alleviate pain, carprofen (1 mg/kg, s.c.) was provided immediately after surgery and once daily for 48 h after surgery. After the 7-d treatment regimen, the mice were followed for an additional 5-d posttreatment (recovery) period. Blood samples (100 ml) were taken via tail snip on the last day of the control period, on d 7 of leptin or vehicle infusion, and on the last day of the recovery period after 5 h of food withdrawal. Oxygen consumption, MA, and BT measurements Male WT mice aged 22 to 24 wk were placed individually in metabolic cages (Comprehensive Lab Animal Monitoring System, Columbus, OH, USA) equipped with an oxygen sensor to measure oxygen consumption (VO2) and receivers to measure BT. Animal MA was determined by infrared light beams mounted in the cages in the x- and y-axes. VO2 and BT were measured for 2 min at 10-min intervals continuously 24 h/d. After the mice were acclimatized to the new environment for at least 5 d, VO2, MA, and BT were measured for 5 consecutive days; then leptin or saline was infused for 7 d at
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15°C, 23°C, and TNZ temperatures. Additional measurements were taken for 5 d after leptin or saline infusion was stopped, as previously described, and blood samples were taken to measure plasma hormones. POMC, NPY, cocaine and amphetamine–regulated transcript, and AgRP mRNA levels in the hypothalamus Additional groups of mice (n = 5 per group) were kept at 15°C or TNZ for 5 consecutive days and then treated with leptin or saline injected intraperitoneally (5 mg/kg/d, 150 ml) for 5 consecutive days. One hour after the last injection, mice not denied food were humanely killed with an overdose of isoflurane, the brains were quickly removed, and the hypothalamus was dissected on an icecold platform. Tissue samples were frozen immediately by immersion in liquid nitrogen and stored at 280°C. Total RNA was extracted with Tri Reagent (Molecular Research Center, Cincinnati, OH, USA), resuspended in nuclease-free water, treated with DNase (Turbo DNA-Free Kit; Thermo Fisher Scientific, Waltham, MA, USA), and quantified by spectrophotometry. Four micrograms of total RNA was reverse transcribed with 0.5 mg of T20VN primer and Superscript III (Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed as previously described (25) and standardized against Rpl13a (26, 27). Briefly, qPCR reactions were performed with 1 ml reverse transcription product, 1 ml Titanium Taq DNA polymerase (Clontech Laboratories, Mountain View, CA, USA), 1:20,000 dilution SYBR Green I (Molecular Probes, Eugene, OR, USA), 0.2 mM dNTPs, and 0.2 mM each primer in a final volume of 25 ml. Cycling conditions were 1 min at 95°C, 50 cycles for 15 s at 95°C, 15 s at annealing temperature, and 1 min 68°C. Realtime data were obtained during the extension phase, and Ct values were obtained at the log phase of each gene amplification. PCR product quantification was performed by the relative quantification method (28, 29) and standardized against Rpl13a1. PCR product specificity was confirmed by postrun melting curve analysis and high-resolution agarose gel electrophoresis using NuSieve 3:1 agarose (Lonza, Rockland, ME, USA). Results are expressed as arbitrary units and normalized against Rpl13a1 mRNA expression. The 18s rRNA expression was assayed TaqMan qPCR using Eukaryotic 18S rRNA Endogenous Control (Thermo Fisher Scientific) and TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) following the manufacturer’s suggested protocol. The primer sequences were selected from previous studies (30–32). Rpl13a1 was selected as the housekeeping gene for gene expression analysis after analyzing the coefficient of variation (CV%) of the expression (arbitrary units) across all experimental conditions in a set of 12 housekeeping genes (data not shown). Analytical methods Plasma leptin and insulin levels were measured with ELISA kits (respectively, R&D Systems, Minneapolis, MN, USA, and Crystal Chem, Downers Grove, IL, USA), and plasma glucose concentrations were determined using the glucose oxidation method (Glucose Analyzer 2; Beckman Coulter, Brea, CA, USA). Statistical analyses The results are expressed as means 6 SEM. The data obtained were analyzed by paired Student’s t test or 1-way ANOVA with repeated measures followed by Dunnett’s post hoc test for comparison between control and experimental values within each group when appropriate. Comparisons between different groups were done by unpaired Student’s t test or 1-way ANOVA followed by Dunnett’s post hoc test when appropriate. Statistical significance was accepted at a level of P , 0.05. TEMPERATURE AND LEPTIN
RESULTS Impact of changes in TA on body weight and food intake, and responses to chronic leptin infusion Changing TA from a standard housing temperature (23°C) to either 15°C or TNZ did not significantly alter body weight in any of the groups (Fig. 1A–C). However, reducing TA from 23 to 15°C increased food intake from 3.2 6 0.5 to 4.5 6 0.5 g/d (Fig. 1D), whereas increasing TA from 23 to 30°C decreased food intake from 4.0 6 1.0 to 2.8 6 0.5 g/d (Fig. 1E). At 23°C, food intake averaged 3.2 6 0.4 g/d (Fig. 1F). Chronic leptin infusion for 7 d at 15°C caused a modest reduction in food intake and a cumulative deficit of only 26.5 6 0.5 g during the 7 d treatment period, but significantly reduced body weight (;14%) (Fig. 1A). Leptin infusion at TNZ caused a more substantial decrease in food intake (211.1 6 1.8 g cumulative deficit), which was associated with a weight loss of ;8% (Fig. 1B) compared to baseline. Leptin infusion at 23°C reduced body weight by 3 6 1 g (Fig. 1C) and significantly reduced food intake (Fig. 1F), causing a 28.2 6 1.1 g cumulative food deficit. These data indicate that leptin has a greater effect in reducing food intake despite a lesser effect to reduce body weight when TA is increased to TNZ compared to an TA of 15°C. Impact of changes in TA on VO2, MA, and BT, and responses to chronic leptin infusion Changing TA from 23 to 15°C increased VO2 by ;50% (Fig. 2A). Conversely, at TNZ, VO2 was reduced by ;50% compared to 23°C TA (Fig. 2B). Chronic leptin infusion at 15°C caused a transient small reduction in VO2 followed by a gradual increase in VO2 (Fig. 2A). At TNZ, however, no significant change in VO2 was observed during leptin infusion (Fig. 2B). When leptin infusion was stopped, VO2 increased slightly, but not significantly, in mice housed at TNZ (Fig. 2B). The increase in VO2 during the recovery posttreatment period may be partly due to the rebound hyperphagia that occurred in mice housed at TNZ after leptin treatment was stopped (baseline: 4.1 6 0.8 vs. 6.0 6 1.1 g posttreatment). We observed a small, but statistically insignificant (P = 0.06), increase in VO2 when leptin was infused at 23°C (Fig. 2C). No significant alterations were observed in BT during changes in TA or during leptin infusion (Fig. 2D–F). Although MA was slightly reduced during leptin infusion at TNZ, no significant changes were observed during leptin infusion at 15°C or 23°C (Fig. 3). Impact of changes in TA on plasma glucose, insulin, and leptin levels, and responses to chronic leptin infusion There were no significant differences in baseline plasma levels of leptin, insulin, or glucose in mice housed at TA ranging from 15 to 30°C (Table 1). Chronic leptin infusion, however, significantly increased plasma leptin to 42 to 67 ng/ml, which are levels similar to those
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Figure 1. Body weight and food intake responses to chronic leptin or saline infusion. A) Body weight when TA was reduced from 23 to 15°C. B) Body weight when TA was increased from 23 to 30°C. C ) Body weight when TA was maintained at 23°C (leptin group only). D) Food intake when TA was reduced from 23 to 15°C. E ) Food intake when TA was increased from 23 to 30°C. F ) Food intake when TA was maintained at 23°C (leptin group only).*P , 0.05 compared to saline group; #P , 0.05 compared to 23°C; $P , 0.05 compared to baseline at 23°C.
found in severe obesity. The difference in plasma leptin levels between the groups housed at 15 and 30°C may reflect the greater weight loss during chronic leptin infusion in mice housed at 15°C. Leptin infusion caused a significant reduction in fasting plasma glucose and insulin levels at 15 and 23°C, but not at 30°C. After stopping leptin infusion, plasma leptin and glucose levels returned to baseline values. These results indicate that leptin’s effects to reduce insulin and glucose levels are markedly attenuated at 30°C, even though the anorexic effect of leptin was impaired at TA of 15°C. 4
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Effect of changes in TA on BP and HR, and responses to chronic leptin infusion MAP increased by ;6 mmHg in the groups where TA was reduced from 23 to 15°C. Conversely, MAP decreased by ;11 mmHg in the groups where TA was increased from 23 to TNZ (Table 2). Thus, the difference in MAP between mice housed at 15°C and TNZ was ;17 mm Hg. Chronic leptin infusion at 15°C caused an additional gradual increase in MAP of ;8 mmHg (average of the last 3 d of treatment) and ;5 mmHg in mice housed at 23°C
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Figure 2. VO2 and BT responses to chronic leptin or saline infusion. A) TA reduced from 23 to 15°C. B) TA increased from 23 to 30°C. C) TA maintained at 23°C (leptin group only). D) BT when TA was reduced from 23 to 15°C. E) BT when TA was increased from 23°C to TNZ. F) BT when TA was maintained at 23°C (leptin group only). *P , 0.05 compared to saline group; #P , 0.05 compared to 23°C.
(Fig. 4A, C), while no change in MAP was observed at 30°C (Fig. 4B). No significant changes in MAP were observed after 7 d of vehicle infusion at 15°C or TNZ (Fig. 4A, B). On the first day after implantation of the osmotic minipump, there were significant increases in MAP in mice housed at 23°C and TNZ, which may be due to surgery for minipump placement, as MAP rapidly returned to control values on day 2 of leptin infusion. Changing TA from 23 to 15°C or from 23 to TNZ also caused profound alterations in HR. Reducing TA to 15°C increased HR by 110 to 130 bpm, whereas raising TA to 30°C markedly decreased HR by approximately ;120 bpm (Fig. 4D, E). Moreover, similar to what was observed for MAP, housing the mice at 15°C enhanced leptin’s effect TEMPERATURE AND LEPTIN
to raise HR (;20 bpm), while at TNZ leptin had no sustained effect on HR (Fig. 4D, E). We also did not observe a sustained effect of leptin to raise HR at 23°C (Fig. 4F). Vehicle infusion caused no sustained effect on HR at either TA (Fig. 4D, E). These results indicate that the chronic effects of leptin on BP and HR are markedly attenuated at TNZ and enhanced at 15°C. Expression of NPY, POMC, CART, and AgRP in the hypothalamus of WT mice after leptin infusion Decreasing TA and activation of NPY/AgRP neurons are well-known stimuli for increasing food intake, whereas
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observed in control mice at TNZ (Fig. 5B). Leptin treatment did not alter POMC mRNA at TNZ (Fig. 5B). No significant differences were observed for AgRP or CART mRNA levels between mice housed at 15°C or TNZ, and these levels were also not affected by leptin treatment at either temperature (Fig. 5C, D). DISCUSSION The results of the present study indicate that changes in TA not only have major effects on control of BP and metabolism but also greatly influence leptin’s metabolic and cardiovascular actions. Compared to the TA normally maintained in most laboratory animal facilities (23°C), a warmer TA (30°C, the TNZ for mice) enhanced the appetite-suppressing actions of leptin while attenuating its effects to raise BP and HR and to reduce glucose and insulin levels. In contrast, at cold TA, leptin’s effects on BP, HR, and insulin and glucose levels were exacerbated while its anorexic effects were attenuated. These findings are summarized in Fig. 6. These observations have important implications for cardiovascular and metabolic studies in rodents that may be housed at different TA points in various laboratory animal facilities and are consistent with previous studies showing the effects of TA on metabolic and cardiovascular function in rodents (34–36). In addition, our findings provide new insights into the interaction of brain circuits that regulate energy balance, BP, and BT. Varying TA may be a useful tool in revealing the mechanisms responsible for differential control of BP and metabolic functions by leptin, a topic of considerable interest for understanding the factors that contribute to selective leptin resistance in pathophysiological conditions such as obesity. Effect of changes in TA on food intake, VO2, MA, and responses to leptin
Figure 3. MA response to chronic leptin or saline infusion when TA was altered. A) MA when TA was reduced from 23 to 15°C. B) MA when TA was increased from 23°C to TNZ. C ) MA when TA was maintained at 23°C (leptin group only).
leptin is thought to suppress appetite by activating POMC/ cocaine and amphetamine–regulated transcript (CART) neurons and inhibiting NPY/AgRP neurons (33). We hypothesized that leptin may have differential effects on these pathways depending on TA. NPY mRNA levels in control saline-treated mice were higher at 15°C compared to TNZ (Fig. 5A), which parallels the changes in appetite evoked by housing the animals at these temperatures. Leptin infusion did not alter NPY mRNA in mice housed at 15°C, but surprisingly, leptin infusion at TNZ raised NPY mRNA levels to values comparable to that observed at 15°C (Fig. 5A). Also, POMC mRNA was reduced at TNZ in vehicle-treated mice, and leptin reduced POMC mRNA in mice housed at 15°C to the same level 6
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In the present study, as in previous studies (37, 38), reducing TA markedly increased food intake and energy expenditure while body weight and BT remained relatively constant. Conversely, increasing TA decreased food TABLE 1. Plasma glucose, leptin, and insulin levels in WT mice during control, leptin infusion, and recovery periods at different TA Characteristic Leptin (ng/ml) Glucose (mg/dl) Insulin (mU/ml)
15°C Control Leptin Recovery 30°C Control Leptin Recovery 23°C Control Leptin Recovery
461 42 6 3* 361
176 6 16 93 6 14* 160 6 10
12 6 3 4 6 1* 761
261 67 6 9* 361
167 6 19 149 6 24 172 6 10
13 6 3 10 6 2 761
361 57 6 5* 462
157 6 10 102 6 25* 162 6 12
Data are expressed as means 6 0.05 compared to control period.
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SEM.
962 4 6 2* 861
n = 5–6 in each group. *P ,
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TABLE 2. MAP, HR, and VO2 responses to changes in TA in WT mice Characteristic
MAP (mmHg) HR (bpm) VO2 (ml/kg/h)
23°C
15°C
23°C
30°C
106 6 1 532 6 18 3102 6 286
113 6 2* 650 6 20* 4588 6 195*
105 6 2 548 6 15 3134 6 152
98 6 2# 420 6 12# 2050 6 58#
*P , 0.05 compared to baseline at 23°C; #P , 0.05 compared to baseline at 23°C.
intake and energy expenditure while causing no substantial changes in body weight or BT. A novel finding of these experiments is that changes in TA markedly alter the metabolic effects of leptin. At cold TA, leptin’s anorexic effect was attenuated by approximately 40%, whereas its effect on reducing body weight was amplified compared to the responses observed at TNZ. Body weight decreased by approximately 10 g during leptin infusion at 15°C TA despite only a 6 g decrease in cumulative food intake and no major changes in MA. Thus, we speculate that an increase in VO2 during leptin treatment may have contributed to this reduction in body weight. At 30°C TA, leptin infusion for 7 d decreased body weight by only about 5 g despite causing a decrease in cumulative food intake of over 10 g and no significant change in VO2; however, leptin infusion at 30°C TA also reduced MA, which may have contributed to the maintenance of a relatively constant body weight despite a large decrease in food intake. Our studies also showed, surprisingly, that at cold TA, leptin did not increase POMC expression or decrease NPY mRNA, effects that are believed to mediate much of leptin’s anorexic effects under normal conditions (33). These findings suggest that at cold TA, the anorexic effects of leptin due to increased POMC activity and decreased NPY are limited by other factors responsible for stimulating appetite, although the mechanisms involved are still unknown. Changes in TA alter leptin’s effects on plasma glucose and insulin levels Central or peripheral leptin infusion markedly enhances peripheral tissue glucose utilization via insulin-dependent and -independent mechanisms (39). We previously showed that leptin receptor deletion in POMC neurons completely abolished the effects of leptin to reduce insulin and glucose levels, suggesting that the effects of leptin to stimulate peripheral glucose utilization are mediated largely by activation of POMC neurons (22). In the present study, we found that leptin’s effects to reduce glucose and insulin levels were enhanced at 15°C TA and completely abolished at TNZ, suggesting that the chronic effects of leptin on glucose homeostasis are greatly altered by changes in TA; higher TA impairs leptin’s ability to lower plasma insulin and glucose levels, while cooler TA enhances leptin’s effects on glucose regulation Although we found that leptin’s effects on glucose regulation were greatly attenuated at TNZ, the molecular mechanisms responsible are unclear. Our previous studies suggest that Shp2 signaling in forebrain neurons mediates most of the chronic antidiabetic TEMPERATURE AND LEPTIN
effects of leptin to reduce plasma glucose (23). Whether changing TA alters leptin signaling via changes in Shp2 responsiveness or through other pathways, such as Stat3 and Irs2 signaling, is still unclear. Effect of changes in TA on BP and HR, and responses to chronic leptin infusion The results of our study confirm previous studies showing that cold TA raises BP and HR while warm TA reduces BP and HR (36, 40, 41). The rise in BP may be an adaptive response that contributes to enhanced circulatory function needed for increases in metabolic rate and nonshivering thermogenesis, thereby helping to maintain BT (40). Although the mechanisms responsible for cold-induced hypertension are not completely understood, increases in SNA, especially renal SNA, appear to play a major role because bilateral denervation of the kidneys prevents development of coldinduced hypertension (37, 42). Increased renal SNA during cold exposure activates the renin–angiotensin system (RAS), and blockade of the RAS also abolishes cold-induced hypertension (40). Thus, activation the RAS and sympathetic nervous system together contribute importantly to the rise in BP that occurs during chronic exposure to cold TA. Chronic exposure to a warm TA of 30°C, the TNZ for mice, lowered BP and HR by approximately 11 mm Hg and 120 bpm, respectively, compared to values measured at 23°C, the usual TA of most laboratories. The mechanisms responsible for the decrease in BP with increasing TA have not been widely studied but are likely related to decreases in SNA and RAS activity (36). These observations emphasize the close associations among control of BP, metabolism, and BT, and have important implications for experimental studies in rodents. A novel finding of our study is that changes in TA altered the BP, HR, and metabolic responses to leptin in divergent ways. We and others have previously shown that at normal TA leptin administration increases SNA and causes small increases in BP (43). Moreover, leptinmediated increases in BP can be completely prevented by adrenergic blockade, indicating that they are due to sympathetic activation (12, 44). We have also provided evidence that leptin may link obesity with increased SNA and hypertension (12, 21, 43). Humans and mice that are leptin deficient have normal or even reduced BP and SNA despite severe obesity and many characteristics of the metabolic syndrome, suggesting that a functional leptin system may be necessary for obesity to increase SNA and BP (43). In the present study, we found, surprisingly, that leptin’s effects on BP were completely abolished at TNZ and enhanced by cold exposure. Thus, the neural circuits
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Figure 4. Changes in MAP and HR during chronic leptin or saline infusion at various TA. A) MAP changes at 15°C. B) MAP changes at TNZ. C ) MAP changes at 23°C (leptin group only). D) HR changes at 15°C. E ) HR changes at TNZ. F ) HR changes at 23°C (leptin group only). *P , 0.05 compared to saline group; #P , 0.05 compared to 23°C; $P , 0.05 compared to baseline at 23°C.
for leptin-mediated sympathetic activation and BP control interact closely with those involved with BT regulation. These circuits appear to be distinct from those that mediate leptin’s anorexic effects because changing the TA had opposite effects on the BP and food intake responses to leptin, with cold exposure attenuating leptin’s anorexic effect while amplifying its BP effect. However, the specific neural circuits and signaling pathways that mediate these divergent interactions of leptin and TA on regulation of food intake and BP are still poorly understood. 8
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On the basis of the sustained anorexic effect of leptin at TNZ, we hypothesized that the POMC/ CART pathway would exhibit higher endogenous expression in leptin-treated compared to salinetreated animals. However, the relative abundance of mRNA levels of POMC and CART were not different between groups. Although the anorexic effect of leptin was only transient at 15°C TA, the relative abundance of POMC mRNA levels was reduced, suggesting that this may be one of the mechanisms responsible for the impaired food intake response to leptin. Despite the
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Figure 5. Relative mRNA abundance quantified by qPCR in hypothalamus fractions of WT mice after 5 d of leptin or saline infusion and exposure to 15°C and TNZ temperatures for NPY (A), POMC (B), AgRP (C ) , and CART (D). *P , 0.05 compared to saline-treated group.
fact that leptin infusion significantly reduced food intake at TNZ, the NPY mRNA was increased in the leptin-treated compared to the saline-treated group. In addition, at TNZ, the POMC mRNA level was similar between saline- and leptin-treated groups, but increased at 15°C TA. A likely explanation for these unexpected results is that POMC neurons may already be activated at cold TA as a result of simultaneous activation of leptin receptors and the hypothalamic temperature regulation center. Another possible explanation for these surprising findings is that leptin does not have equal effects on all neuronal subtypes or downstream pathways during changes in TA. For instance, acute leptin injection activates POMC, but not NPY neurons, in the ARC as assessed by increased c-Fos protein, but suppressor of cytokine signaling-3 (SOCS3) mRNA was increased in both neuronal subtypes (45). Thus, further studies are needed to unravel the changes in gene expression and cell signaling pathways activated by leptin and TA. Our results therefore suggest that alterations in TA are associated with divergent control of metabolic and cardiovascular functions in responses to chronic leptin infusion. Changes in TA may be a useful tool to dissect the areas of the CNS and signaling pathways that permit leptin to independently control cardiovascular and metabolic functions, and they also may be useful to understand the interactions of the ARC and hypothalamic temperature center in regulating energy balance and BP. Implications for physiologic studies in rodents Our understanding of human physiology and mechanisms of disease depends heavily on translating results TEMPERATURE AND LEPTIN
from experiments in nonhuman animals, especially rodents. For many areas of research, murine models have increasingly been used largely because of their short gestation times and the molecular tools that are available to manipulate gene expression. Genetically modified mouse models have been especially important for investigating CNS pathways that contribute to regulation of food intake, energy expenditure, and the pathogenesis of obesity and associated metabolic disorders. In most of these studies, however, little attention is paid to the TA of the laboratory when phenotypes are assessed. The results of our study indicate that mice housed at the usual laboratory TA of 23°C have metabolic and cardiovascular phenotypes that differ considerably from those obtained at the TNZ in mice of the same genotype. Moreover, changes in TA markedly altered the cardiovascular and metabolic responses to leptin, sometimes in opposite directions. For example, increasing TA from 23 to 30°C abolished the increases BP and HR during leptin infusion while amplifying the anorexic effects of leptin. These results further emphasize the need to consider carefully the TA when conducting studies in mice. As noted by Maloney et al. (37), it could be argued that much of what we know about metabolism, obesity, and cardiovascular physiology of rodents comes from chronically hypertensive, hypermetabolic, and obesity-resistant animals because we usually study them at a TA that is comfortable for investigators but cold for rodents. It is clear that meaningful translation of results from experimental studies in rodents to understanding human physiology and pathophysiology will require us in the future to pay much more attention to the effects of environmental stressors such as TA. Our observations also
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University of Mississippi Medical Center) for technical assistance. The authors declare no conflicts of interest.
AUTHOR CONTRIBUTIONS J. M. do Carmo and A. A. da Silva designed the research, analyzed data, performed research, and wrote the paper; D. G. Romero performed gene expression analysis; and J. E. Hall contributed to analyzing the data and revising the article. REFERENCES
Figure 6. Comparison of cumulative food intake (A), changes in MAP (DMAP; B), and changes in blood glucose (C ) after 7 d of leptin infusion at T A of 15, 23, or 30°C. All values are expressed as changes relative to values measured during control period before starting leptin infusion.
provide new insights into the interaction of temperature regulation and control of BP and metabolism by leptin. ACKNOWLEDGMENTS This work was supported, in part, by the U.S. National Institutes of Health (NIH) National Heart, Lung, and Blood Institute (Grant PO1 HL51971), the NIH National Institute of General Medical Sciences (Grant P20 GM104357), and by an American Heart Association Scientist Development grant (to J.M.D.C. and D.G.R.). The authors thank H. Zhang, S. E. Ebaady, P. O. Sessums, C. Torrey, and J. P. Ball (all from the 10
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Changes in ambient temperature elicit divergent control of metabolic and cardiovascular actions by leptin Jussara M. do Carmo, Alexandre A. da Silva, Damian G. Romero, et al. FASEB J published online February 22, 2017 Access the most recent version at doi:10.1096/fj.201601224R
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