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Ciliary Neurotrophic Factor^Sub Ax15^ Alters Energy Homeostasis, Decreases Body Weight, and Improves Metabolic Control in Diet- Induced Obese and UCP1-DTA Mice

Posted on: Tuesday, 16 November 2004, 03:00 CST

Ciliary neurotrophic factor (CNTF) potently reduces appetite and body weight in rodents and humans. We studied the short- and long- term effects of CNTF^sub Ax15^, a second-generation CNTF analog, in diet-induced obese C57BL/6J mice and brown adipose tissue (BAT)- deficient obese UCP1-DTA (uncoupling protein 1-diphtheria toxin A) mice. CNTF^sub Ax15^ administration (0.1, 0.3, or 1.0 g . g^sup -1^ . day^sup -1^ s.c.) for 3 or 7 days reduced food intake and body weight (mainly body fat mass). The effect of CNTF^sub Ax15^ on food intake and body weight was more pronounced in CNTF^sub Ax15^- treated diet-induced obese C57BL/6J mice compared with pair-fed controls and was associated with suppressed expression of hypothalamic neuropeptide Y and agouti gene-related protein. Moreover, CNTF^sub Ax15^ increased uncoupling protein 1 mRNA expression in BAT and energy expenditure in diet-induced obese C57BL/ 6J mice. Longitudinal observations revealed a sustained reduction in body weight for several days post-CNTF^sub Ax15^ treatment of CNTF^sub Ax15^-treated but not pair-fed mice, followed by a gradual regain in body weight over 28 days. Finally, CNTF^sub Ax15^ administration improved the metabolic profile in both diet-induced obese C57BL/6J and UCP1-DTA mice and resulted in a significantly improved glycemic response to oral glucose tolerance tests in CNTF^sub Ax15^-treated UCP1-DTA compared with pair-fed mice of similar body weight. These data suggest that CNTF^sub Ax15^ may act through a pathway downstream of the putative point responsible for leptin resistance in diet-induced obese C57BL/6J and UCP1-DTA mice to alter food intake, body weight, body composition, and metabolism. CNTF^sub Ax15^ has delayed and persistent eifects in diet-induced obese C57BL/6J mice, which account for a reduction in body weight over and above what would be expected based on decreased foot intake alone. Diabetes 53:2787-2796, 2004

Administration of ciliary neurotrophic factor (CNTF), a neuronal growth factor studied in amyotrophic lateral sclerosis, results in anorexia and weight loss in humans (1,2) and mice (3,4). CNTF acts via both leptin-like and -independent mechanisms, with previous studies showing that CNTF decreases body weight and food intake in leptin-deficient ob/ob (4) and leptin-resistant db/db and diet- induced obese AKR/J mice (5). These findings suggest that CNTF acts, at least in part, downstream of the abnormally spliced leptin receptor of db/db mice and of the putative point of leptin resistance in mouse models of leptin resistance and insulin resistance. We studied the short-and long-term actions of CNTF^sub Ax15^ (Axokine, a second-generation CNTF analog) in insulin- and leptin-resistant diet-induced obese C57BL/6J and UCP1-DTA (uncoupling protein 1-diphtheria toxin A) mice. Diet-induced obese C57BL/6 mice are the closest mouse model to human obesity (6), whereas UCP1-DTA mice develop deficits in energy expenditure and thermogenesis caused by ablation of brown adipose tissue (BAT) (7- 9).

In this study, we investigated whether obese hyperleptinemic and hyperinsulinemic diet-induced obese C57BL/6J and UCP1-DTA mice, which are resistant to the effects of endogenous or exogenous leptin on body weight, food intake, and glucose and insulin levels (9,10), would also be resistant to the effects of CNTF^sub Ax15^. We also sought to investigate whether 1) the effect of CNTF^sub Ax15^ is primarily mediated by decreased food intake and/or by an increase in energy expenditure, 2) CNTF^sub Ax15^ improves insulin resistance beyond what would be expected on the basis of weight loss alone, and 3) the effects of CNTF^sub Ax15^ persist after discontinuation of treatment.

RESEARCH DESIGN AND METHODS

Male C57BL/6J and female UCP1-DTA mice (The Jackson Laboratories, Bar Harbor, ME) were individually caged, maintained in a room with an automatically controlled 12-h lights-on (7:00 A.M.)/lights-off (7:00 P.M.) cycle with a temperature of 69-74F and humidity of 40- 60%. All animals were handled in accordance with the principles and guidelines established by the National Institutes of Health (6,10- 12). Regular mouse chow (Purina Rodent Chow 5008, caloric content 3.48 kcal/g, with 17.3% of calories from fat, 55.1% from carbohydrate, and 27.6% from protein; Ralston-Purina, St. Louis, MO) and water were available to all animals ad libitum, unless noted otherwise. To obtain diet-induced obese mice, C57BL/6J mice received a western diet (TD 88137, calorie content 4.53 kcal/g, with 42.2% of calories from fat, 42.8% from carbohydrates, and 15.0% from protein; Teklad, Madison, WI) for 12 weeks. Before the experiments, mice from each strain were divided into groups of equal mean body weight.

FIG. 1. The effect of short-term administration of CNTF^sub Ax15^ to reduce food intake and body weight in diet-induced obese C57BL/ 6J mice. Diet-induced obese C57BL/6J mice were administered the indicated doses of CNTF^sub Ax15^ for 3 consecutive days (arrows indicate beginning and end of treatment period), and food intake and body weight were assessed daily. Pair-feeding was discontinued 24 h after the last injection. Dual-energy X-ray absorptiometry (DEXA) scans were performed at baseline as well as 24 h and 6 days after the last injection as shown. A: Percent change in body weight (from starting body weight) compared with baseline levels. B: Caloric intake over time. Analyses were performed using unpaired t test and are expressed as the means SE. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. the vehicle-treated group; #P < 0.05, ##P < 0.01, P < 0.001, and P < 0.0001 vs. the pair-fed group; ^P < 0.05 between both CNTF^sub Ax15^-treated groups. PF, pair-fed group (n = 10); V, vehicle (PBS) treated.

CNTF^sub Ax15^ (Axokine), provided by Regeneron Pharmaceuticals (Tarrytown, NY), is cleared by glomerular filtration: C^sub max^ is ~45 min after subcutaneous injection in mice. Thus, negligible, if any, amounts of the medication are present in circulation 24 h after injection (~30 half-lives after C^sub max^) (unpublished data, Regeneron, Tarrytown, NY).

Experimental procedures

Effects of CNTF^sub Ax15^ administration for 3 days in diet- induced obese C57BL/6J mice. Food intake and body weights were measured daily in diet-induced obese C57BL/6J mice injected subcutaneously with CNTF^sub Ax15^ at 0.1 or 1.0 g . g^sup -1^ . day^sup -1^ between 8:00 and 9:00 A.M. for 3 days, mice pair-fed to CNTF^sub Ax15^ 1.0 g . g^sup -1^ . day^sup -1^, and vehicle-treated controls (n = 10/group). For pair-feeding, the amount of food consumed by the CNTF^sub Ax15^-treated group during the past 24 h was measured at 8:00 A.M. the following morning, and a corresponding amount of pellets was given to the pair-fed group during a 24-h period.

As we sought to investigate potential long-term effects of CNTF^sub Ax15^, including differences in posttreatment body weight regain, measurements were taken for 28 days after treatment was discontinued. Dual-energy X-ray absorptiometry scans were performed at baseline (day -3), day 1, and day 6 of CNTF^sub Ax15^ treatment to study the effect of CNTF^sub Ax15^ on in vivo body composition. In a separate cohort of diet-induced obese C57BL/6J mice treated with the same doses of CNTF^sub Ax15^ (0.1 or 1.0 g . g^sup -1^ . day^sup -1^ for 3 days), including the respective pair-fed or PBS- treated control groups (n = 10/ group), mice were killed, serum was collected, and tissues were isolated 4 days after the last injection.

Effects of CNTF^sub Ax15^ administration for T days in diet- induced obese C57BL/6J mice. Two cohorts of diet-induced obese C57BL/ 6J mice treated with CNTF^sub Ax15^, at 0.1 and 0.3 g . g^sup -1^ . day^sup -1^ s.c. for 7 days were killed either 24 h after the last injection (cohort 1) or 4 days after the last injection (cohort 2), and outcomes were compared with the effect of PBS treatment or pair- feeding as described above (n = 5/group). To investigate a potential effect of CNTF^sub Ax15^ on energy expenditure and activity levels during treatment and in the posttreatment period, indirect calorimetry was performed during treatment (days 2-4, cohort 1) and after discontinuation of treatment (days 8-10, cohort 2). Blood and tissues were collected at the time of death.

Effects of CNTF^sub Ax15^ administration in UCP1-DTA mice. To study the potential role of uncoupling protein 1 (UCP1) in mediating the effect of CNTF^sub Ax15^ on food intake, body weight, and serum hormone concentrations, UCP1-DTA mice were injected with CNTF^sub Ax15^ (0.1 or 0.3 g . g^sup -1^ . day^sup -1^ s.c.) and compared with PBS-treated and pair-fed control groups as described above (n = 5/group).

Effects of CNTF^sub Ax15^ administration on insulin sensitivity and glucose tolerance in UCP1-DTA mice. To investigate whether insulin sensitivity and glucose tolerance improves with CNTF^sub Ax15^ administration to UCP1-DTA mice, intraperitoneal insulin tolerance tests and oral glucose tolerance tests (OGTTs) were performed in these mice (n = 5/group) and compared with their control heterozygote littermates, which were either vehicle treated or pair-fed as described above. CNTF^sub Ax15^ (0.1 g/g s.c.) was injected daily for 10 days. The OGTTs were condu\cted starting at 10:00 A.M. in the morning after mice had been fasted for 16-18 h before gavaging with a standard glucose bolus, as previously outlined (5). In nonfasted mice, intraperitoneal insulin tolerance tests were performed with 0.75 units insulin/kg body wt and results were expressed as a percentage of starting blood glucose.

Physiological characterization. Individual body weights and total amount of food per cage were measured daily between 8:00 and 10:00 A.M. with an analytical balance, with the differences in food between two measurements assumed to represent the amount of food consumed (6,11,12). For whole-body composition analysis, mice were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg) and scanned with a PIXImus densitometer (Lunar, Madison, WI) (13).

TABLE 1

In vivo body composition in diet-induced obese mice at baseline, 24 h, and 6 days after CNTF^sub Ax15^ administration for 3 days (0.1 and 1.0 g . g^sup -1^ . day^sup -1^, respectively)

Indirect calorimetry and activity. To assess potential changes in energy expenditure, in vivo indirect open-circuit calorimetry using a comprehensive laboratory animal monitoring system (CLAMS; Columbus Instruments, Columbus,OH) was performed in three groups of diet- induced obese C57BL/6J mice (PBS, CNTF^sub Ax15^ 0.3, and pair-fed; n = 5/group) during and after treatment with CNTF^sub Ax15^, as described above. All animals were individually housed in metabolic cages over a 48-h period and had free access to food and water, except the pair-fed-group. After a 6-h acclimation period, oxygen and carbon dioxide gas fractions were monitored (two 12-h dark and two 12-h light phases), and oxygen consumption (VO^sub 2^) and respiratory quotient (ratio of VCO^sub 2^ to VO^sub 2^) were calculated as described previously (14,15). Locomotor activity was measured using passive infrared motion detectors (Columbus Instruments, Columbus, OH). Each movement of the animals was recorded as a 3-s impulse, which was regarded as one event. For all indirect calorimetry measurements, no changes in diurnal rhythmicity were noted.

Tissue collection and expression analysis. Animals were killed with carbon dioxide asphyxiation, and tissues (epididymal white adipose tissue, BAT and hypothalami) were collected, immediately frozen in liquid nitrogen, and stored at -80C. Total tissue RNA was purified using the RNA STAT-60 total RNA/mRNA isolation reagent according to the manufacturer's instructions (Tel-Test, Friendswood, TX). Hormone levels were measured, and hypothalamic neuropeptide mRNA expression (proopiomelanocortin [POMC], neuropeptide Y [NPY], agouti gene-related protein [AgRP], orexin) was analyzed using mouse- specific primers, as previously described (6,10,11,14). UCP1 mRNA was amplified in a 20-1 reaction mixture using SYBR-Green master mix (Applied Biosystems). The following mouse specific primers were used for the amplification of UCP1-DTA: 5'-ttffcctggcagatatcatca-3', 5'tgcattctgaccttcacgac-3'. All samples were quantified in triplicate, and averages were used for statistical analyses.

Statistics. Data are expressed as the means SE. Statistical significance was assessed by standard Student's t tests or two- tailed t tests as well as ANOVA with post hoc corrections, at the conventional P < 0.05 value. All statistical analyses were performed using StatView (Abacus, Berkley, CA) and SPSS 8 (Texas Instruments, Chicago, IL). Graphs were created with Prism (GraphPad, San Diego, CA).

RESULTS

Effects of CNTF^sub Ax15^ on body weight and food intake in diet- induced obese C57BL/6J mice

Short-term experiment. We investigated the effects of short- and long-term treatment of CNTF^sub Ax15^, on food intake and body weight in diet-induced obese C57BL/6J mice, particularly focusing on the posttreatment period. Consistent with studies in other strains of mice, CNTF^sub Ax15^ treatment for 3 days resulted in significantly reduced food intake and body weight (starting body weights: PBS group: 44.1 1.0 g; CNTF^sub Ax15^ 0.1 group: 44.1 1.2 g; CNTF^sub Ax15^ 1.0 group: 44.1 0.8 g; pair-fed group: 44.1 0.8 g) (Fig. LA). Caloric intake was significantly decreased in both CNTF^sub Ax15^-treated diet-induced obese C57BL/6J groups (CNTF^sub Ax15^ 1.0 group: 9.7 0.9 kcal; CNTF^sub Ax15^ 0.1 group: 17.9 0.9 kcal) when compared with PBS-treated controls (41.1 1.6 kcal, P < 0.01 for both treatment groups) (Fig. IB). By experimental design, food intake of the pair-fed group did not differ from the CNTF^sub Ax15^ 1.0-treated group (9.7 0.03 kcal) (Fig. 1B). After discontinuation of pair-feeding, food intake of mice from both control groups (PBS and pair-fed) rapidly increased above baseline (Fig. IE), resulting in an attenuation of body weight loss within 4 days (Fig. LA). Consistent with previous data (3), food intake of animals from both CNTF^sub Ax15^-treated groups was significantly decreased even 3 days after discontinuation of treatment, but it eventually returned to baseline. Body weight loss of CNTF^sub Ax15^- treated mice was maintained until the end of the observation period (Fig. 14). More importantly, body weight reduction was more pronounced in CNTF^sub Ax15^-treated than pair-fed mice (Fig. LA), an effect not previously observed in other diet-induced obese mouse models (e.g., AKR/J) (3-5). Consistent with results from other strains, administration of CNTF^sub Ax15^ for 3 days to diet- induced obese C57BL/6J mice significantly decreased body fat mass (3), an effect still evident 6 days after discontinuation of treatment, when food intake had already returned to baseline (Table 1). Moreover, CNTF^sub Ax15^ treatment significantly reduces abdominal fat mass bordered cranially by the xiphoid apophysis and caudally by the sacrum (P < 0.01 vs. PBS-treated mice) (Table 1), even when total fat mass was not significantly decreased (Table 1).

FIG. 2. The effect of long-term administration of CNTF^sub Ax15^ to reduce food intake and body weight in diet-induced obese C57BL/ 6J mice. Diet-induced obese C57BL/6J mice were administered the indicated doses of CNTF^sub Ax15^ for 7 consecutive days (arrows indicate beginning and end of treatment period), and food intake and body weight was assessed daily. Pair-feeding was continued until the end of the study. Indirect calorimetry using a comprehensive laboratory animal monitoring system (CLAMS) was performed on days 2- 4 and 8-10. A: Percent change in body weight (from starting body weight) compared with baseline levels. B: Caloric intake over time. Analyses were performed using an unpaired t test and are expressed as means SE. *P < 0.05, **P < 0.01, and ***p < 0.001 vs. the vehicle-treated group; ⋁P < 0.05 and &8897;P >< 0.01 between both CNTF^sub Ax15^-treated groups; #P = 0.06 vs. the pair- fed group. PF, pair-fed group (n = 5); V, vehicle (PBS) treated.

Long-term experiment. To expand the above observations in a more prolonged treatment regimen and using an intermediate CNTF^sub Ax15^ dose, a cohort of diet-induced obese C57BL/6J mice were treated with CNTF^sub Ax15^ for 7 days (0.1 and 0.3 g . g^sup -1^ . day^sup - 1^), and food intake and body weights were measured (starting body weights: PBS group: 44.0 2.3 g; CNTF^sub Ax15^ 0.1 group: 44.2 2.2 g; CNTF^sub Ax15^ 0.3 group: 44.2 1.8 g; pair-fed group: 44.3 2.6 g) (Fig. 2A and B). Importantly, pair-feeding was continued beyond the treatment period and until death to directly differentiate between an effect of reduced food intake versus increased energy expenditure. Similar to the short-term studies, after 7 days of treatment, cumulative food intake was significantly reduced in both treatment groups compared with PBS-treated mice (P < 0.01 for both treatment groups vs. PBS-treated mice) (Fig. 2E), whereas weight loss was significantly reduced in the CNTF^sub Ax15^ 0.3 g . g^sup - 1^ . day^sup -1^-treated group compared with the pair-fed group (Fig. 2A). Interestingly, gains in body weight were still significantly more pronounced in mice that continued to be pair-fed the same amount of food that the formerly CNTF^sub Ax15^-treated group consumed (P < 0.05 on day 10 vs. the pair-fed group) (Fig. 2A), indicating an effect of CNTF^sub Ax15^ to increase energy expenditure.

Effects of CNTF^sub Ax15^ on energy expenditure and metabolic rate. To address the effects of CNTF^sub Ax15^ administration on energy expenditure, we analyzed diet-induced obese C57BL/6J mice in metabolic chambers during treatment and in the posttreatment period. Interestingly, there were no changes in total energy expenditure while on treatment (Fig. 3A). After discontinuation of treatment, however, formerly CNTF^sub Ax15^-treated animals display a significant increase in energy expenditure versus PBS-treated and pair-fed controls, with VO^sub 2^ consumption in the CNTF^sub Ax15^- treated group being significantly increased during both the day and night periods compared with both control groups (P < 0.01 vs. pair- fed and PBS-treated mice) (Fig. 3B and online appendix). Consistent with observed reductions in abdominal fat, we observed a significant reduction in respiratory quotient in CNTF^sub Ax15^-treated mice, which remained reduced after discontinuation of treatment (Fig. 3C and D and online appendix [available from http:// diabetes.diabetesjournals.org]). The reduction in respiratory quotient in CNTF^sub Ax15^-treated mice was even more pronounced when data were analyzed separately for the light and dark phases (Fig. 3 and online appendix).

FIG. 3. Indirect calorimetry in diet-induced obese C57BL/6J mice immediately after initiating or terminating CNTF^sub Ax15^ for 7 days. Indirect calorimetry, using a comprehensive laboratory animal monitoring system, was performed on separate cohorts on days 2-4 of CNTF^sub Ax15^ treatment (0.3 g . g^sup -1^ . day^sup -1^) and on days 1-3 after completing 7 consecutive days of CNTF^sub Ax15^ treatment (0.3 g .g^sup -1^ . day^sup -1^), presented as single time points, measured over a 24-h period. A: Energy expenditure (VO^sub 2^) on treatment. B: Energy expenditure (VO^sub 2^) after discontinuation of treatment. C: Respiratory ratio (respiratory quotient) on treatment. D: Respiratory ratio (respiratory quotient) after discontinuation of treatment. E: Locomotor activity on treatment. F: Locomotor activity after discontinuation of treatment. RER, respiratory quotient. [black triangle up], PBS group; [black square], CNTF^sub Ax15^-treated group.

Assessment of total activity revealed a significant increase in the pair-fed group, a result consistent with a state of "food- seeking behavior" in response to food deprivation. This was evident in both the treatment and posttreatment periods (activity in the pair-fed group increased by 223.7 47.2% of baseline on treatment and remained elevated at 159.3 18.6% after cessation of treatment; P < 0.05 vs. the PBS-treated group) (Fig. 3 and online appendix). In contrast, total activity remained remarkably stable in the CNTF^sub Ax15^-treated mice, suggesting that CNTF^sub Ax15^-treated mice do not demonstrate a comparable drive to seek food, despite decreased caloric intake (Fig. 3E). There is only a minor effect of CNTF^sub Ax15^ on VO^sub 2^ consumption in diet-induced obese C57BL/6J mice (cohort 1, day 3 of CNTF^sub Ax15^ treatment), suggesting that there is probably a reduction in the basic metabolic rate. In addition, there is only a small difference in VO^sub 2^ consumption between 0.3-g . g^sup -1^ . day^sup -1^ CNTF^sub Ax15^-treated mice and pair- fed mice for the first 24 h after the last treatment (cohort 2, days 7-8) (Fig. 3 and online appendix). After this period of time (24 h after discontinuation of treatment or pair-feeding, i.e., after CNTF^sub Ax15^ washes out), however, there is a dramatic separation in VO^sub 2^, with a significant increase in the formerly CNTFAx15- treated group. Similarly, when resting VO^sub 2^ was computed, we did not detect any significant difference between the PBS- and CNTF^sub Ax15^-treated groups while on treatment, and resting VO^sub 2^ was significantly higher in the formerly CNTF^sub Ax15^-treated group (P < 0.001 unpaired two-tailed t test). Based on these data, it is reasonable to assume that thermogenesis might be increased in the long term after CNTF^sub Ax15^ treatment; therefore, an assessment of UCP-1 expression in BAT as well as in vivo studies using UCP1-DTA mice were performed.

Effects of CNTF^sub Ax15^ on UCP-1 expression and thermogenesis. To confirm that the effects of CNTF^sub Ax15^ are, at least in part, mediated by altered thermogenesis, we quantified UCP1 mRNA expression in BAT of diet-induced obese C57BL/6J mice treated with CNTF^sub Ax15^ for 3 days. At 4 days after the last injection of CNTF^sub Ax15^, when mice had already returned to baseline food intake levels, UCP1 mRNA expression in BAT was significantly and dose-dependently elevated compared with PBS-treated controls (P < 0.05 and P < 0.01, respectively) (Fig. 4). This increase in UCP1 mRNA expression corresponds to a concomitant increase in energy expenditure in the CNTF^sub Ax15^-treated group, which was not seen in the pair-fed group (Fig. 3A and 4). Although UCP1 mRNA expression was not significantly different between formerly treated and pair- fed animals, a trend was noted (P = 0.09, Fig. 4). However, the observed significant increase in formerly CNTF^sub Ax15^-treated compared with PBS-treated mice may be of physiological importance because the time point studied represents a time when food intake is already normalized in formerly pair-fed animals and is still reduced (whereas energy expenditure remains still elevated) in formerly CNTF^sub Ax15^-treated mice.

Effects of CNTF^sub Ax15^ on body weight and food intake in UCP1- DTA mice. To further determine the contribution CNTF^sub Ax15^ may have on energy expenditure and thermogenesis, we performed experiments in the BAT-ablated UCP1-DTA rodent model of obesity. Similar to the diet-induced obese C57BL/6J mice studies, 7 days of CNTF^sub Ax15^ treatment reduced caloric intake in a dose-dependent manner in UCP1-DTA mice (P < 0.05) (Fig. 5B), but more importantly, decreases in body weight in CNTF^sub Ax15^-treated animals precisely overlapped decreases observed in pair-fed controls (starting body weights: PBS-treated group: 28.9 1.8 g; CNTF^sub Ax15^ 0.1 group: 29.0 1.5g; CNTF^sub Ax15^ 0.3 group: 28.9 1.7; pair-fed group: 29.0 1.8 g) (Fig. 5A). Similar data were obtained in an independent experiment, as seen below (initial body weights: PBS-treated group: 45.3 3.2 g, CNTF^sub Ax15^ 0.3 group: 45.9 + 1.6 g; and pair-fed group: 45.1 1.4 g; final body weights: PBS group: 43.4 3.2 g; CNTF^sub Ax15^ 0.3 group: 38.6 1.0 g; pair-fed group: 36.5 2.8 g).

Effects of CNTF^sub Ax15^ on metabolic control in UCP1-DTA mice. Plasma insulin and glucose concentrations were significantly and dose dependently decreased in CNTF^sub Ax15^-treated UCP1-DTA mice compared with PBS-treated mice (Table 2), but not when compared with pair-fed mice. In the second, separate but similar experiment, we demonstrate, using more sophisticated methods to assess metabolic control, that treatment of CNTF^sub Ax15^ for 7 days not only decreases body weight to a similar degree in treated and pair-fed mice (Fig. 5A) but also improves glucose tolerance (Fig. 6A and .B), which is more obvious in UCP1-DTA mice than in pair-fed controls. After receiving 0.75 units/kg body wt of insulin, CNTF^sub Ax15^- treated and pair-fed UCP1-DTA mice show an approximate 50% decrease in blood glucose levels after 30 min, whereas vehicle-treated control mice failed to significantly decrease their glucose levels for 120 min (Fig. 6-B); however, although the effect of insulin administration was significant (P < 0.001), no significant difference between groups could be detected (P = 0.3). In addition, OGTTs demonstrate that CNTF^sub Ax15^ treatment tended to result in improved glucose tolerance in CNTF^sub Ax15^-treated compared with pair-fed animals (mean differences by ANOVA: effect of time P < 0.01, effect of group P < 0.01, and effect of time and group interaction P = 0.07; and area under the curve measurements: CNTF^sub Ax15^, 0.3: 5.51 9.2 vs. PBS-treated: 17.12 6.24 [P < 0.05], pair-fed: 8.85 2.24 [P = 0.09 vs. PBS treated and P = 0.12 vs. 0.3 g . g^sup -1^ . day^sup -1^ CNTF^sub Ax15^]) (Fig. 6A).

FIG. 5. CNTF^sub Ax15^ reduces food intake and body weight in UCP1-DTA mice. In a manner similar to Fig. 1, UCP1-DTA mice were administered the indicated doses of CNTF^sub Ax15^ for 7 consecutive days (arrows indicate treatment period), and food intake and body weight were assessed daily. Pair-feeding was continued until the end of the study. Separate cohorts of similarly treated mice were killed at day 8. A: Percent change in body weight compared with baseline levels. B: Caloric intake over time. A separate group of UCP-1-DTA mice received a daily subcutaneous injection of vehicle or CNTF ^sub Ax15^ at 0.1 g . g^sup -1^ . day^sup -1^ or were provided with the same amount of chow as that eaten by the group treated with CNTF^sub Ax15^ at 0.3 g . g^sup -1^ . day^sup -1^. All data are expressed as the means SE, analyzed by unpaired t test (n = 5). *P < 0.05 between the CNTF^sub Ax15^, 0.3-g . g^sup -1^ . day^sup -1^-treated and the PBS-treated group; #P < 0.05 between the pair-fed and PBS- treated group; @P < 0.05 between the CNTF^sub Ax15^ 0.1-g . g^sup - 1^ . day^sup -1^-treated and the PBS-treated group. [white square], PBS group; , group treated with CNTF^sub Ax15^ at 0.1 mg . kg^sup - 1^ . day^sup -1^; [black triangle down], group treated with CNTF^sub Ax15^, at 0.3 g . g^sup -1^ . day^sup -1^; [diamonds], group pair- fed to same amount as group treated with CNTF^sub Ax15^ at 0.3 g . g^sup -1^ . day^sup -1^.

TABLE 2

Effect of CNTF^sub Ax15^ administration for 3 days (0.1 and 1.0 g . g^sup -1^ . day^sup -1^) or 7 days (0.1 and 0.3 g . g^sup -1^ . day^sup -1^) on circulating insulin and blood glucose concentrations in diet-induced obese C57BL/6J and UCP1-DTA mice

Effects of CNTF^sub Ax15^ on circulating glucose and insulin levels in diet-induced obese C57BL/6J mice. CNTF^sub Ax15^ treatment for 3 days to diet-induced obese C57BL/6J mice significantly decreased serum insulin and plasma glucose concentrations, which were similar in CNTF^sub Ax15^-treated and pair-fed mice (Table 2). In accordance with these findings, CNTF^sub Ax15^ treatment for 7 days significantly decreased serum insulin levels in diet-induced obese C57BL/6J mice, but again, the decline was similar between treated and pair-fed-mice when measured 24 h or 4 days after the last treatment (Table 2). Plasma glucose concentrations were significantly and dose-dependently lower in CNTF^sub Ax15^-treated and pair-fed diet-induced obese C57BL/6J mice compared with PBS- treated mice (Table 2).

FIG. 6. Treatment with CNTF^sub Ax15^) improves glucose tolerance. Groups of UCP1-DTA mice received a daily subcutaneous injection of vehicle, CNTF^sub Ax15^ at 0.1 g . g^sup -1^ . day^sup - 1^, or were provided with the same amount of chow eaten by the 0.1- g . g^sup -1^ . day^sup -1^ CNTF^sub Ax15^ treatment group. A: An OGTT was conducted 24 h after the last treatment and serum glucose was measured at the indicated times after oral gavage. All data are expressed as the means SE (n = 5), analyzed by ANOVA. B: In nonfasted mice an intraperitoneal insulin tolerance test was performed with 0.75 units insulin/kg body wt. Results are expressed as a percentage of starting blood glucose (means SE, n = 5, analyzed by ANOVA with post hoc). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the PBS-treated group; ⋁0.07 < P < 0.05, #P < 0.05, ##P < 0.01, and P < 0.001 vs. the pair-fed group. PF, pair-fed group.

These data indicate that CNTF^sub Ax15^ tr\eatment has a favorable effect on the metabolic profile that proves to be more pronounced than pair-feeding when more sophisticated methods for assessing insulin resistance are used (as seen above). Thus, the effects of CNTFA^sub Ax15^ on improving insulin resistance and metabolic control as well as the underlying mechanisms need to be studied further.

Effects of CNTF^sub Ax15^ on expression of hypothalamic neuropeptides regulating food intake. Given the suppressive effect of CNTF^sub Ax15^ on food intake, the expression pattern of hypothalamic neuropeptides known to be involved in energy homeostasis (the orexigenic molecules NPY and AgRP and the anorexigenic molecules POMC and orexin) was studied. After food deprivation, NPY mRNA expression increased to 173% of baseline levels in the pair-fed group (P < 0.05 vs. the PBS-treated group) (data not shown). The compensatory increase of NPY normally seen with caloric restriction was also observed immediately after the 3- day treatment period with CNTF^sub Ax15^ in diet-induced obese C57BL/ 6J mice. However, after a 7-day treatment period, NPY mRNA expression in CNTF^sub Ax15^-treated mice was lower than in the pair- fed mice and comparable to levels seen in the PBS-treated controls (data not shown). The expression pattern of AgRP was not significantly different between groups in the short-term studies because AgRP expression is altered only after more prolonged food deprivation. Accordingly, after 7 days of CNTF^sub Ax15^ treatment, AgRP mRNA expression followed the pattern of NPY and was significantly increased in the pair-fed, but not the CNTF^sub Ax15^- treated, group compared with the PBS-treated controls (data not shown). Finally, no significant changes in POMC or orexin mRNA expression were observed, suggesting that these molecules do not play a role of major significance in mediating the effects of CNTF^sub Ax15^ on food intake (data not shown).

DISCUSSION

We show that CNTF^sub Ax15^ has effects on energy homeostasis, body weight, body composition, and metabolism during active treatment and after discontinuation of treatment. Specifically, we show that CNTF^sub Ax15^ reduces food intake and body weight through a pathway downstream of the putative point responsible for leptin resistance in both diet-induced obese C57BL/6J and UCP1-DTA mice and that it increases energy expenditure in dietinduced obese C57BL/6J mice, in part by upregulating UCP1 expression. Even more importantly, these effects persist several days after discontinuation of treatment.

Previous studies have shown that food intake is significantly reduced after peripheral CNTF administration to DIO-AKR/J or 06/06 mice (3) as well as central (intracerebroventricular) administration to Sprague-Dawley rats (4). We extend these findings by showing that CNTF^sub Ax15^ eifectively decreases food intake in diet-induced obese C57BL/6J and UCP1-DTA mice and that decreased caloric intake in UCP1-DTA mice is comparable to what is observed in diet-induced obese C57BL/6J mice. The resulting weight loss is less pronounced, however, probably because of the impaired thermogenesis/energy expenditure in UCP-DTA mice (16).

When food and water intake were measured using the CLAMS apparatus, water intake closely paralleled food intake, and no specific effect of the drug was observed. Activity levels of mice, oxygen consumption, carbon dioxide production, as well as respiratory quotients were measured in metabolic chambers. Behavioral effects or taste aversion phenomenon were not specifically studied; conditioned taste aversion response was previously assessed in diet-induced obese mice, and no adverse effects were found (3).

We have shown that CNTF^sub Ax15^ promotes weight loss in obese diet-induced obese C57BL/6J and UCP1-DTA mice and that reduced body weight in diet-induced obese C57BL/6J mice is mainly caused by decreased body fat mass. More importantly, we demonstrate that weight loss in CNTF^sub Ax15^-treated diet-induced obese C57BL/6J mice is significantly higher than what is observed in pair-fed controls, supporting our hypothesis that CNTF^sub Ax15^ exerts its effects not only by reducing food intake but also by increasing thermogenesis. CNTF^sub Ax15^ administration has resulted in significant decreases of both fat and lean body mass, but changes of the latter were much less pronounced than changes in fat mass. Similar changes have been observed in response to all weight- reducing methods in the past; future studies are needed to address in more detail the CNTF^sub Ax15^-induced alterations in body composition.

To gain a better understanding of the mechanisms driving CNTF- induced decreases in body weight and increases in thermogenesis, we investigated the effects of CNTF^sub Ax15^ in BAT-deficient UCP1- DTA mice and directly measured UCP1 expression and energy expenditure in diet-induced obese C57BL/6J mice. BAT is responsible for nonshivering thermogenesis, a major component of facultative thermogenesis in newborns and mammals, which is mediated by UCPl. The hypothesis of BAT playing an important role in the regulation of energy expenditure has been supported by the creation of BAT- deficient mice (UCP1-DTA mice), which are characterized by marked obesity and insulin resistance (7-9). Moreover, UCP1-DTA mice raised at thermoneutrality (35C), a temperature that would lead to total inactivation of BAT, completely prevents the development of obesity in these mice (16). UCP1-DTA mice were therefore used herein to support our hypothesis that CNTF^sub Ax15^ acts, at least in part, by altering energy expenditure. We show that weight loss in CNTF^sub Ax15^-treated diet-induced obese C57BL/6J mice is significantly higher than in pair-fed controls, whereas weight loss in CNTF^sub Ax15^-treated UCP1-DTA mice precisely parallels and overlaps the weight loss seen in their pair-fed controls. Thus, the effects of CNTF^sub Ax15^ to induce anorexia by altering hypothalamic neuropeptide expression are intact in these mice, but the peripheral effects mediating energy expenditure are disrupted.

Similar to leptin, CNTF-induced weight loss and appetite suppression is mediated through neurons in the medial arcuate nucleus, but unlike leptin, no evidence of CNTF action in other regions of the hypothalamus could be found (17,18). In addition, anorectic effects of CNTF are in part mediated by altered hypothalamic NPY expression (17,18), and CNTF induces expression of hypothalamic inhibitors of leptin signaling (19). We confirm these findings by showing that CNTF^sub Ax15^ exerts anorectic effects on diet-induced obese C57BL/6J mice, at least in part, by altering hypothalamic NPY and AgRP mRNA expression, and we extend previous findings by showing that POMC and orexin expression do not play a role of comparable significance. The question of whether changes in those neuropeptides are also responsible for the anorectic effects of CNTF^sub Ax15^ in UCP1-DTA mice needs to be addressed in future studies. Based on the data presented herein, we speculated that CNTF^sub Ax15^ mediates its effects in diet-induced obese C57BL/6J mice, at least in part, by altering energy expenditure, possibly through increased thermogenesis, demonstrating directly that CNTF^sub Ax15^, increases energy expenditure in diet-induced obese C57BL/6J mice. The fact that body fat mass and respiratory quotient are still decreased while energy consumption is significantly elevated in the posttreatment period implies that, similar to leptin, CNTF^sub Ax15^ promotes energy expenditure and possibly fatty acid oxidation, but that, in contrast to leptin, this effect persists after cessation of treatment and beyond the half-life of the medication. In accordance, we show herein that UCP1 mRNA expression in BAT is significantly elevated in CNTF^sub Ax15^- treated but not PBS-treated or pair-fed mice after cessation of treatment. Possibilities of posttranscriptional regulation of UCP-1 as well as the question of whether the effect of CNTF^sub Ax15^ on UCP1 mRNA expression is specific or secondary to other metabolic changes needs to be addressed in future studies.

The CNTF^sub Ax15^-induced weight loss significantly decreased circulating insulin and glucose levels, which were similar in CNTF^sub Ax15^-treated and pair-fed mice 24 h after the last treatment. However, 4 days after cessation of treatment, formerly CNTF^sub Ax15^-treated mice displayed significantly lower insulin concentrations than formerly pair-fed controls, which at that point showed an overshoot in food intake. In the long-term studies, when pair-feeding was maintained until the end of the experiments, circulating insulin levels were similar between formerly CNTF^sub Ax15^-treated and pair-fed mice, indicating that the persistent effect of CNTF^sub Ax15^ to reduce food intake in the long term may have beneficial effects on the metabolic profile. Conclusions derived by measuring serum insulin concentrations were further supported and extended by using more detailed methods for assessment of metabolic milieu, i.e., insulin tolerance test and OGTT in UCP1- DTA mice. CNTF^sub Ax15^-treated mice responded with a relatively lower excursion of blood glucose levels in response to an OGTT, suggesting that CNTF^sub Ax15^ treatment might exert a beneficial effect on metabolism of exogenously administered glucose. These observations need to be confirmed and the underlying mechanisms further studied by future experiments.

In summary, we demonstrate that CNTF^sub Ax15^ exerts central and peripheral effects in regulating energy homeostasis, body weight, body composition, and metabolism, and that these effects persist even after discontinuation of treatment. CNTF^sub Ax15^ reduces food intake and body weight in diet-induced obese C57BL/6J and UCP1-DTA mice through a pathway downstream of the putative point responsible for leptin and insulin resistance in these mouse models of obesity. An increas\e in energy expenditure is an additional mechanism driving weight loss in diet-induced obese C57BL/6J mice. In conclusion, CNTF^sub Ax15^ may be a valuable drug target toward obesity, insulin resistance/diabetes, and associated metabolic disorders.

ACKNOWLEDGMENTS

This study was supported in part by a discretionary grant from Beth Israel Deaconess Medical Center (to C.S.M) and Grant P30 DK 57521-3 from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. C.S.M. is supported by Grant ROI NIDDK 58785 and E.M.-F. by Grants DK 56113 and DK 56116 from the National Institutes of Health.

We thank the Metabolic Physiology Core at Beth Israel Deaconess Medical Center and Harvard Medical School for providing expert assistance with body composition measurements (dual-energy X-ray absorptiometry scans; NIDDK/NIH P30 DK 57521-3), as weU as energy expenditure measurements. We are especially grateful to Dr. Odile Peroni for expertise and valuable discussions regarding dual-energy X-ray absorptiometry measurements. We would also like to thank A. Borbely for excellent technical assistance in performing the calorimetry measurements and Dr. E. Bachman for valuable discussions about the energy expenditure data.

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Susann Blher,1 Stergios Moschos,1 John Bullen, Jr.,1 Efi Kokkotou,2 Eleftheria Maratos-Flier,2 Stanley J. Wiegand,3 Mark W. Sleemann,3 and Christos S. Mantzoros1

From the 1 Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; the 2 Joslin Diabetes Center, Harvard Medical School, Research Division, Boston, Massachusetts; and 3 Regeneron Pharmaceuticals, Tarrytown, New York.

Address correspondence and reprint requests to Susann Blher, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Ave., Boston, MA 02215. E-mail: sblueher@caregroup.harvard.edu.

Received for publication 11 February 2004 and accepted in revised form 8 July 2004.

Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.

AgRP, agouti gene-related protein; BAT, brown adipose tissue; CNTF, ciliary neurotrophic factor; NPY, neuropeptide Y; OGTT, oral glucose tolerance test; POMC, proopiomelanocortin; UCP1, uncoupling protein 1.

2004 by the American Diabetes Association.

Copyright American Diabetes Association Nov 2004

Source: Diabetes

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