The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 55:B588-B592 (2000)
© 2000 The Gerontological Society of America
Uncoupling Proteins 2 and 3 With Age
Regulation by Fasting and ß3-Adrenergic Agonist Treatment
Philip J. Scarpacea,
Monica V. Kumara,
Hua Lia and
Nihal Tümera
a Geriatric Research, Education and Clinical Center, Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida, and Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville
Philip J. Scarpace, GRECC (182), Department of Veterans Affairs Medical Center, Gainesville, FL 32608-1197 E-mail: scarpace{at}ufl.edu.
Decision Editor: John A. Faulkner, PhD
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Abstract
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In rodents, adaptive thermogenesis in brown adipose tissue (BAT) serves both to regulate body mass after hyperphagia and to conserve energy during food deprivation. In addition to uncoupling protein 1 (UCP1), UCP3 and possibly UCP2 may have a role in energy homeostasis in BAT. We examined basal levels of UCP2 and UCP3 mRNA with age and regulation of UCP1, UCP2, and UCP3 mRNA by two conditions known to modulate energy homeostasis: fasting and ß3-adrenergic agonists. UCP1, UCP2, and UCP3 mRNA levels were unchanged between 3, 24, and 31 months of age in BAT, and UCP2 and UCP3 mRNA levels were unchanged between 6 and 24 months of age in retroperitoneal white adipose tissue (RTWAT). Following a 2-day fast, there were sizable reductions in BAT UCP1 and UCP3 mRNA, but these decreases with fasting were significantly less in the older compared with the young rats. Fasting had no effect on UCP2 mRNA levels at any age. The ß3-adrenergic agonist, CL316,243, increased BAT UCP1 and UCP3 mRNA equally in both young and old rats. The ß3-adrenergic agonist did not increase UCP2 mRNA in BAT but did increase expression in RTWAT of both young and old rats. In summary, these data indicate that the expression of the three uncoupling proteins is unchanged with age. Although the upregulation of these uncoupling proteins by ß3-adrenergic agonist treatment is maintained with age, the downregulation by fasting is diminished with age. The parallel regulation of UCP1 and UCP3 expression in BAT suggests that UCP3, like UCP1, may have a role in energy homeostasis in BAT. The diminished downregulation of UCP1 and UCP3 expression in BAT by fasting suggests that energy conservation in response to food deprivation is impaired with age, and this may contribute to an inability of older animals to maintain body mass during periods when food is limited.
OBESITY is the most prevalent nutritional disorder in Western societies
(1). Moreover, adults tend to gain weight as they age until early senescence, after which body mass declines
(2). The gain in body mass is associated with an increase in adiposity demonstrated by an increase in mean body mass index with age up to age 60 followed by a decline in later life
(2). Similarly, rats tend to gain weight as they age. We demonstrated that the Fischer 344 x brown norway (F-344 x BN) rat may be a useful rodent model for late-onset obesity
(3). This rat strain, similar to humans, demonstrates a steady increase in body mass and adiposity into early senescence (from 3 to 24 months), followed by a decline from 24 to 30 months
(3).
In rodents, an important mechanism that serves both to regulate body mass after hyperphagia and to conserve energy during food deprivation is adaptive thermogenesis
(4). This process has been best characterized in brown adipose tissue (BAT). Thermogenesis in BAT is primarily mediated by sympathetically innervated ß3-adrenergic receptors and is facilitated by the presence of a specific mitochondrial uncoupling protein (UCP1)
(4). Sympathetic stimulation accelerates lipolysis, and the liberated fatty acids serve as substrates for mitochondrial oxidation and may provide the signal to activate UCP1. This protein uncouples mitochondria, producing high rates of substrate oxidation and an increase in heat production without the phosphorylation of adenosine diphosphate
(5). These processes are activated during periods of dietary excess and deactivated when the food supply is insufficient
(4).
The expression of UCP1 is limited to BAT, and there is little BAT in humans who normally reside in an externally maintained thermoneutral environment. However, recently, two additional uncoupling proteins, UCP2 and UCP3, have been identified
(6)
(7)
(8). These uncoupling proteins have 59% and 57% homology, respectively, with UCP1 and 73% homology with each other
(7)
(8). Similar to UCP1, both UCP2 and UCP3 can partially uncouple mitochondrial respiration in yeast
(7)
(8). The expression of UCP2 and UCP3, unlike UCP1, are not limited to BAT. UCP3 is expressed mainly in BAT and skeletal muscle, whereas UCP2 is widely expressed in many tissues, including white adipose tissue (WAT), heart, and muscle in both rodents and humans
(7)
(8). The identification of these two new uncoupling proteins has renewed interest in adaptive thermogenesis, especially because these uncoupling proteins are found in humans. However, whether UCP2 or UCP3 are involved in nonshivering or diet-induced thermogenesis is unclear in either rats or humans
(9).
The present study was designed to determine (i) whether the basal levels of UCP2 and UCP3 mRNA in BAT and retroperitoneal white adipose tissue (RTWAT) are decreased with age, and (ii) whether the regulation of UCP1, UCP2, and UCP3 mRNA by fasting and ß3-adrenergic agonist treatment (two conditions known to modulate energy homeostasis) are impaired by age. To this end, we examined UCP1, UCP2, and UCP3 mRNA in BAT after a 48-hour fast in rats of three ages and UCP2 and UCP3 mRNA in BAT and RTWAT in young and old rats following treatment with the ß3-adrenergic agonist, CL316,243, by osmotic minipumps for 7 days.
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Methods
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Animals
Male F-344 x BN rats, 3, 6, 24, or 31 months of age were obtained from Harlan Sprague-Dawley (Indianapolis, IN). Upon arrival, rats were examined and remained in quarantine for 1 week. Animals were cared for in accordance with the principles of the Guide to the Care and Use of Experimental Animals. Rats were housed individually with a 12:12 hour light-dark cycle (7 AM to 7 PM). Ambient temperature was 26°C, thermoneutrality for these rats
(10).
Experiment One: Fasting
Rats of 3, 24, and 31 months of age (8 per age) were acclimated at thermoneutrality and then fasted for 48 hours prior to sacrifice. Water was available ad libitum.
Experiment Two: ß3-Adrenergic–Agonist Administration
Rats of 6 and 24 months of age were acclimated at thermoneutrality prior to insertion of osmotic mini pumps. Osmotic mini pumps (Alzet model 2001, Alza, Palo Alto, CA) were implanted subcutaneously along the rat's back while under enflurane anesthesia. The pumps delivered saline or CL316,243 (American Cyanamid, Pearl River, NY), 1 mg/kg per day for 7 days.
Tissue Harvesting
Rats were sacrificed by cervical dislocation under 85 mg/kg pentobarbital anesthetic. Blood samples were collected by heart puncture, and serum harvested by a 30-minute centrifugation in serum separator tubes. The circulatory system was perfused with 20 ml of cold saline, and BAT and RTWAT were excised.
UCP1, UCP2, and UCP3 mRNA Levels
Total cellular RNA was extracted using a modification of the method of Chomczynski and Sacchi
(11). The integrity of the isolated RNA was verified using agarose gels (1%) stained with ethidium bromide. The RNA was quantified by spectrophotometric absorption at 260 nm using multiple dilutions of each sample.
The UCP1 probe (a full length cDNA clone) was obtained from Dr. Leslie Kozak, Jackson Laboratory, Bar Harbor, Maine
(12). The cDNA probe (IMAGE 389584) to detect UCP2 was provided by Craig Warden
(7), and the UCP3 cDNA was provided by J.-P. Giacobino
(6). UCP1, UCP2, and UCP3 mRNA levels were detected using dot blot analysis as described previously
(13). In some cases, nylon membranes probed for UCP1, UCP2, or UCP3 mRNA were stripped by brief exposure to boiling water and rehybridized with probes for ß-actin mRNA.
Statistical Analysis
All data are expressed as means ± standard error of measurement. Data were analyzed by two-way analysis of variance (ANOVA). When the main effect was significant, Fisher's protected least significant differences test was applied to determine individual differences between means. If there was significant interaction, further one-way ANOVA was applied and Fisher's protected least significant differences test was used to examine post-hoc comparisons. A p value of .05 or less was considered to be significant.
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Results
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UCP1, UCP2, and UCP3 mRNA in BAT With Age and Fasting
We previously reported that UCP1 mRNA levels in BAT are unchanged with age
(3). Similarly in the present study, UCP1 mRNA levels in BAT were unchanged between 3, 24, and 31 months of age (
Fig. 1). However, following a 48-hour fast, there was a substantial 79% decrease in UCP1 mRNA levels in the young rats. Both groups of older rats also demonstrated significant reductions of 59% and 53%, but these decreases with fasting were significantly less than that observed in the young rats (
Fig. 1).
Similar to UCP1 mRNA, UCP2 mRNA levels in BAT were unchanged with age (Table 1 ). However, in contrast to the fasting-induced decrease in UCP1 mRNA, fasting did not alter UCP2 mRNA levels in rats of any age.
Similar to both UCP1 and UCP2 mRNA, there were no age-related changes in basal UCP3 mRNA levels in BAT (
Fig. 2). Following a two-day fast, there was a 54% decrease in UCP3 mRNA in the young rats with 35% and 31% decreases, respectively, in the 24-month- and 31-month-old rats. The fasting-induced decrease in the two groups of older rats was significantly less than in the young rats (
Fig. 2).
UCP2 and UCP3 mRNA Following Treatment With CL316,243 in Young and Old Rats
We previously reported that a 7-day treatment with the ß3-adrenergic agonist, CL316,243, induced a greater than two-fold increase in UCP1 mRNA levels in BAT from both young and older rats
(14). Similarly, with CL316,243 treatment in our present report, there was nearly a 50% increase in UCP3 mRNA in both young and old rats (
Fig. 3). In contrast to UCP3 mRNA, there were no changes in UCP2 mRNA levels with ß3-adrenergic agonist treatment in rats of either age (Table 1 ).
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Figure 3. UCP3 mRNA levels in brown adipose tissue in control (open bars) and after treatment with CL316,243 for 7 days (hatched bars) in young and old rats. Data represent means ± standard error (SE) from eight rats in each group. UCP3 mRNA levels are expressed in arbitrary units/µg RNA, with the levels in saline-treated young rats set to 100 and SE adjusted proportionally. *p = .0001 for differences with CL316,243 treatment as the main effect. By post-hoc analysis, p = .001 (young rats) and p = .0003 (old rats) for differences between CL316,243 and saline treatment.
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The levels of UCP2 and UCP3 mRNA were also examined in RTWAT. There were no changes with age in either UCP2 or UCP3 mRNA basal levels (
Fig. 4). Following CL316,243 treatment, there was a 2.5-fold increase in UCP3 mRNA in RTWAT of young rats. However, the CL316,243-induced increase of 75% in older rats, although substantial, is significantly less than the increase in the younger rats (
Fig. 4, top). The exact opposite pattern of increase was observed for UCP2 mRNA, CL316,243 increased UCP2 mRNA in RTWAT to a greater extent in old compared with young rats (
Fig. 4, bottom).
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Figure 4. UCP3 mRNA levels (top) and UCP2 mRNA levels (bottom) in retroperitoneal white adipose tissue in control (open bars) and after treatment with CL316,243 for 7 days (hatched bars) in young and old rats. Data represent means ± SE from eight rats in each group. UCP2 and UCP3 mRNA levels are expressed in arbitrary units/µg RNA, with the levels in saline-treated young rats set to 100 and SE adjusted proportionally. * or  p = .0001 for differences with CL316,243 treatment and p = .095 for difference with age as the main effect. By post-hoc analysis, p = .0001 (UCP3), p = 0.006 (UCP2, young rats), and p = .0002 (UCP2, old rats) for differences between CL316,243 and saline treatment. p = .0008 (UCP3) and p = .003 (UCP2) for differences with age among CL316,243-treated rats.
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Discussion
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The recent identification of two new uncoupling proteins, UCP2 and UCP3, has renewed interest in adaptive thermogenesis, especially because these uncoupling proteins are found in humans
(15). Moreover, the presence of UCP2 and UCP3 in a variety of tissues suggests a role for UCP-induced mitochondrial futile cycles beyond that of heat production and compensation for dietary excess. For example, the uncoupling action of UCP2 may play a role in the maintenance of low levels of oxygen and oxygen radicals
(15), or UCP3 may regulate mitochondrial futile cycles in skeletal muscle in response to energy demands
(15). Moreover, other evidence suggests that these putative uncoupling proteins may not even uncouple mitochondria and may have other functions. For example, UCP3 may serve as a free fatty acid (FFA) carrier or facilitate the use of FFA as a skeletal muscle mitochondrial energy substrate under conditions of FFA excess
(9).
Despite these controversies, there is evidence that UCP3 and possibly UCP2 may have at least a secondary role in energy homeostasis in BAT. The regulation of the expression of UCP3, and sometimes UCP2, in BAT varies like that of UCP1
(15). Cold exposure increases UCP1, UCP2, and UCP3 mRNA levels in BAT that are reversed by thermoneutrality
(15). Fasting decreases UCP1 and UCP3 expression whereas refeeding restores UCP1 and UCP3 mRNA levels. In contrast, UCP2 mRNA levels are unaltered by food restriction or excess
(15). Sympathetic innervation of BAT is necessary to maintain normal UCP1 and UCP3 mRNA levels
(16), and ß3-adrenergic agonists increase expression of UCP1 and UCP3
(13)
(15)
(17). Thyroid hormone increases all three UCP transcripts
(15). In addition, leptin increases UCP1, UCP2, and UCP3 in BAT, however, the increase in UCP1 is dependent on sympathetic innervation, whereas the leptin induction of UCP2 and UCP3 are not
(13)
(16).
Very little is known about the expression or regulation of UCP2 or UCP3 with age. Rats demonstrate a steady increase in body mass with age
(3). We demonstrated that the F-344 x BN rat, similar to humans, demonstrates a steady increase in body mass and adiposity into early senescence (from 3 to 24 months), followed by a decline from 24 to 30 months
(3). Thus, this rat strain is a useful rodent model for late-onset obesity observed in humans
(2). The increase in body mass with age is most likely multifactorial. We reported previously that with age ß3-adrenergic agonist-stimulated thermogenesis in BAT is diminished
(18)
(19), ß3-adrenergic agonist signal transduction in BAT is impaired
(20), leptin responsiveness is diminished
(21), and leptin signal transduction in the hypothalamus is impaired
(22). Each of these may contribute to reduced energy expenditure with age and consequently lead to a gain in body mass.
The present study was designed to determine if the expression or regulation of UCP2 or UCP3 by fasting or ß3-adrenergic agonist treatment is altered with aging or senescence. We found that the basal expression of UCP1, UCP2, or UCP3 was unaltered with age in BAT and that the basal expression of UCP2 and UCP3 were unaltered with age in RTWAT. Furthermore, our data indicating that fasting diminishes UCP1 and UCP3 but not UCP2 mRNA in BAT support previous studies
(15). With age, the fasting-induced decrease in UCP1 and UCP3, although substantial, was less compared to the extent of the decrease in young rats. Fasting is a condition of whole body energy conservation, including diminished energy use in BAT. These data support the concept that UCP3, like UCP1, has a role in energy conservation, at least in BAT. Furthermore, the impaired fasting regulation of UCP1 and UCP3 in older rats suggests that energy conservation in response to fasting may be impaired with senescence. This may diminish the metabolic tolerance to periods of food deprivation and diminish the resistance to weight loss when food supplies are insufficient for senescent animals.
The present study also demonstrates that the ß3-adrenergic agonist CL316,243 upregulates UCP3 mRNA in BAT and RTWAT. This is in contrast to an earlier report that found no increase in UCP3 in BAT following administration of the ß3-adrenergic agonist, CL316,243
(23), but consistent with reports demonstrating an increase in UCP3 mRNA in BAT with the ß3-adrenergic agonist, Ro 16-8714
(15), and the ß3-adrenergic agonist, CGP-12177
(13)
(17). In the older rats, the pattern of regulation of UCP3 in BAT by the ß3-adrenergic agonist was similar to that in the young rats. Activation of the ß3-adrenergic receptor in BAT is the single most potent stimulus for mitochondrial uncoupling and thermogenesis
(4). The observation of parallel increases in UCP1 and UCP3 with ß3-adrenergic agonist stimulation in BAT is consistent with a role for UCP3 in energy homeostasis in BAT. Whether that role involves uncoupling of mitochondria is yet to be determined. However, the maintenance of ß3-adrenergic agonist induction of UCP3 mRNA in older rats suggests that this is not a factor in the diminished ß3-adrenergic agonist stimulation of thermogenesis with age
(18)
(19) or in the increase in body mass with age in these animals
(3).
The present data demonstrating no increase in UCP2 in BAT with ß3-adrenergic agonist treatment is consistent with other reports
(15) and suggest that UCP2 may not have a role in energy homeostasis in BAT. In contrast to BAT, ß3-adrenergic agonist treatment increased UCP2 mRNA in RTWAT, and this effect was enhanced in the older rats. The role of UCP2 in white adipose tissue is unclear, but the pattern of regulation does not suggest it is an important factor in the increase in body mass with age.
In summary, these data indicate that the expression of the three uncoupling proteins is unchanged with age. However, the upregulation of these uncoupling proteins by ß3-adrenergic agonist treatment is maintained with age, and the downregulation by fasting is diminished with age but not severely impaired. The parallel regulation of UCP1 and UCP3 expression in BAT suggests that UCP3, like UCP1, has a role in energy homeostasis in BAT. However, the nature of that role is unknown. The diminished downregulation of UCP1 and UCP3 expression in BAT by fasting suggests that energy conservation in response to food deprivation is impaired with age, and this may contribute to an inability of older animals to maintain body mass during periods when food is limited.
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Acknowledgments
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This study was supported by the Medical Research Service of the Department of Veterans Affairs and National Institute on Aging Grant AG-17047.
Received March 15, 2000
Accepted June 7, 2000
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References
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Abstract
Methods
