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Keywords:

  • Dicrostonyx groenlandicus;
  • mitochondria;
  • oxygen consumption;
  • Northern blots;
  • uncoupling protein 3

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Objective: To determine the gene sequence and tissue distribution of uncoupling protein 3 (Ucp3) in the collared lemming, we quantified mRNA expression of Ucp3 under known states of altered energy expenditure (photoperiod-induced weight gain and loss, cold exposure, and fasting) and measured mitochondrial oxygen consumption to assess possible functional changes in energy expenditure.

Research Methods and Procedures: The Ucp3 gene sequence information was obtained using the reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods. Northern blots were used to determine mRNA expression levels. Respirometry was used to measure oxygen consumption rates in isolated mitochondria.

Results: The lemming Ucp3 gene has a 97% sequence similarity with other published Ucp3 sequences at the amino acid level. Ucp3 mRNA is expressed in muscle, heart, and brown adipose tissue of collared lemmings. Long-photoperiod lemmings have a higher expression of Ucp3 mRNA than short-photoperiod lemmings (p < 0.001) in both muscle and brown adipose tissue. Transferring lemmings from long to short photoperiods (inducing weight gain) significantly decreased Ucp3 mRNA expression (p < 0.01), whereas transferring lemmings from short to long photoperiods (inducing weight loss) significantly increased Ucp3 expression (p < 0.001). Muscle Ucp3 mRNA expression was significantly decreased by 10 days of mild (10 °C) cold exposure (p < 0.001). Muscle Ucp3 mRNA expression was significantly increased by fasting (p < 0.01) and was correlated to plasma free fatty acid levels (r = 0.7). Photoperiod transfer did not alter mitochondrial coupling.

Discussion: These data suggest that UCP3 may not be involved in energy expenditure in the collared lemming.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Over the past 25 years, the physiological role of uncoupling protein 1 (UCP1)1 has been well characterized. It is known that UCP1 functions to “uncouple” the process of oxidative phosphorylation from electron transport producing heat instead of ATP (1). The more recently discovered uncoupling protein 3 (Ucp3) was thought to function like UCP1 by virtue of its close sequence homology with Ucp1 and its ability to decrease membrane potential in yeast (2,3). Although Ucp3 has been the subject of much research attention, the physiological function of UCP3 remains unknown. A number of studies have shown Ucp3 to be related to resting energy expenditure and changes in body weight, but these findings need further verification (4,5,6).

The collared lemming (Dicrostonyx groenlandicus) provides an interesting model to investigate the role of the uncoupling proteins in body weight dynamics. The collared lemming is a non-hibernating Arctic rodent. As a result of seasonal cues in nature or manipulations of photoperiod in the laboratory, these animals alter their body mass and composition (7,8,9). Lemmings transferred from a summer-like long photoperiod [long day (LD)] conditions (22 hours light:2 hours dark) to a winter-like short photoperiod [short day (SD)] conditions (8 hours light:16 hours dark) nearly double their body weight and fat mass in 10 weeks. When transferred from SD to LD conditions, lemmings decrease body weight and fat mass to levels of LD controls (10). Unlike other animal models of obesity, in the collared lemming, food intake and physical activity are not significantly altered before or after changes in body weight and, therefore, do not seem to be causative factors (11).

Recent data from our laboratory have shown that lemmings transferred from LD to SD conditions for 10 days have significantly lower resting energy expenditure (REE) than LD controls. The lowered REE was shown to be associated with a decreased expression of brown adipose tissue (BAT) Ucp1 mRNA (11). Because a change in REE was associated with a change in Ucp1 mRNA expression, we hypothesized that UCP3 could also be associated with the changes in REE observed after transfer to SD.

The purpose of this study was to examine the gene sequence and tissue distribution of Ucp3 in the collared lemming, quantify mRNA expression of Ucp3 under known states of altered energy expenditure (photoperiod induced weight gain and loss, cold exposure, and fasting), and measure mitochondrial oxygen consumption to assess possible functional changes in energy expenditure.

Research Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Effect of Photoperiod-Induced Weight Gain and Loss on Ucp3 Expression

A total of 20 7-month-old male lemmings were used for this experiment. The animals were divided into four groups: lemmings raised from weaning under either LD or SD conditions, LD lemmings transferred to SD conditions, and SD lemmings transferred to LD conditions (n = 5/group). Animals were housed two or three per cage with free access to food (LM-485; Harlan Teklad, Madison, WI) and water. Ten days after group assignment or photoperiod transfer, the animals were killed by decapitation, and gastrocnemius muscle and BAT were collected and frozen in liquid nitrogen for later processing.

Effect of Cold Exposure on Ucp3 Expression

A total of 19 6- to 7-month-old female LD and SD lemmings were used for this experiment. The animals were divided into four groups: LD 18 °C, SD 18 °C, SD 10 °C, and LD 10 °C (n = 4–5/group). Animals were housed two or three per cage with free access to food (LM-485; Harlan Teklad) and water. After 10 days of exposure to the respective temperature, the animals were killed by decapitation, and gastrocnemius muscle and BAT were collected and frozen in liquid nitrogen for later processing.

Effect of Fasting on Ucp3 Expression

A total of 32 8- to 10-week-old female LD lemmings were used for this experiment. The animals were divided into four groups: ad libitum fed and 6-, 12-, or 18-hour fast (n = 8/group). Animals were killed by decapitation, and blood and gastrocnemius muscle were collected.

Respirometry Experiment Animals

A total of 24 10-week-old female lemmings were used for this experiment. The animals were divided into four groups: lemmings raised from weaning under either LD or SD conditions, LD lemmings transferred to SD conditions, and SD lemmings transferred to LD conditions (n = 4–7/group). Animals were housed two or three per cage with free access to food (LM-485; Harlan Teklad) and water. After 10 days under these photoperiod conditions, the animals were killed by cervical dislocation, and gastrocnemius and quadriceps muscles were removed from both hindlimbs.

Polymerase Chain Reaction—based cDNA Cloning

Total RNA was obtained from lemming BAT, and first-strand cDNA was synthesized by SuperScript Choice System for cDNA Synthesis (Invitrogen, Carlsbad, CA). Polymerase chain reaction (PCR) was performed in a 50-μL reaction volume using 1 μL of the first-strand cDNA reaction. Two pairs of primers were designed based on the homologous sequence of Ucp3 among different species. The first pair of primers for amplifying 5′-end sequence was (forward) 5′-CATGGTTGGACTTCAGCCATCAGA-3′ and (reverse) 5′-GACAGAGTCGTAGAGGCCAATTCG-3′. The second pair of primers for amplifying 3′-end sequence was (forward) 5′-GCCTACAGAACCATCGCCAGGGAG-3′ and (reverse) 5′-TGTTCAAAARGGAGATTCCCGCAG-3′. PCR conditions were 94 °C for 4 minutes; 35 cycles of 94 °C for 1 minute, 62 °C for 30 seconds, 72 °C for 1 minute; and a final extension of 72 °C for 7 minutes. PCR reactions were analyzed by electrophoresis in 1% agarose gels with ethidium bromide staining. DNA products were purified and cloned into a TOPO TA cloning vector (Invitrogen, Applied Biosystems, Foster City, CA). Plasmids with insert were sequenced on an ABI377 automated DNA sequencer. Sequence analyses and comparisons were accomplished using NCBI sequence analysis tools at their web site (http:www.ncbi.nlm.nih.gov) and the on-line web site servers at http:www.expasy.chcontact.html and http:searchlauncher.bcm.tmc.edu.

Northern Blot Analysis

Total mRNA was extracted from tissues using TRIzol (Life Technologies, Carlsbad, CA) following the manufacturer's protocol. Once extracted, 15 μg of total RNA were electrophoresed in a 1.2% agarose-formaldehyde gel. The RNA was transferred to a Hybond-XL nylon membrane (Amersham, Piscataway, NJ) using the turboblotter apparatus (Schleicher and Schuell, Keene, NH). The membrane was hybridized for at least 16 hours using the Super Hyb kit (Molecular Research Center, Cincinnati, OH). The blots were used to expose Kodak Biomax MS film (Eastman Kodak, Rochester, NY). The autoradiographs were quantitated using densitometric analysis with an Alpha Innotech Fluorchem 8000 imaging system (Alpha Innotech, San Leandro, CA). Differences in loading were adjusted for by normalization of data to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

The probes used for the Northern blots were made by reverse transcription-PCR using the following primers: Ucp3 (forward) 5′-GTTGGACTTCAGCCATCAGAAGTG-3′ and (reverse) 5′-TGTATGCTGAAGATGGTGGCGCAG-3′ and GAPDH (forward) 5′-CAAAATGGTGAAGGTCGGTGTGA-3′ and (reverse) 5′-GAAGGTGGAAGAGTGGGAGTTGCT-3′.

Isolation of Mitochondria

Mitochondria were isolated following slightly modified protocols of Rasmussen et al. (12) and Tonkgonogi and Sahlin (13). Muscles were dissected clean of contaminating tissues and placed in chilled isolation buffer (100 mM sucrose, 100 mM KCl, 50 mM Tris HCl, 0.1 mM EGTA, and 0.2% bovine serum albumin, pH 7.4). The tissue was passed through a “garlic press”–like apparatus and placed in 2 mL of ice-cold isolation buffer containing 0.2 mg/mL Nagarse (Sigma, St. Louis, MO) for 2 minutes. The tissues were homogenized in a motor-driven Potter-Elvejhem glass homogenizer with a teflon pestle (clearance of 0.3 mm) fitted with a collar to center the pestle in the mortar. The tissues were homogenized on ice two times for 30 seconds each at a speed of 1550 rpm with a 30-second rest in between. The homogenate was transferred to a centrifuge tube, and the homogenization tube and pestle were washed three times with 1 mL of isolation buffer. The homogenate was centrifuged for 10 minutes at 500g. The supernatant was transferred to a clean tube and centrifuged for 10 minutes at 9000g. The resulting pellet was washed two times with a mitochondrial re-suspension buffer (225 mM mannitol, 75 mM sucrose, and 10 mM Tris HCl, pH 7.4) and centrifuged for 4 minutes at 5500g. The final pellet was re-suspended in a volume of re-suspension buffer equal to one-fifth of the initial tissue weight. Protein concentrations were assessed using the Lowry assay (Sigma) following the manufacturer's protocol.

Measurement of Mitochondrial Oxygen Consumption

Mitochondrial oxygen consumption was measured with a Clark-type oxygen electrode (model 1302; Strathkelvin, Glasgow, United Kingdom) maintained at 28 °C. The electrode was calibrated using air-saturated respiration buffer (225 mM mannitol, 75 mM sucrose, 10 mM Tris HCl, 10 mM KCl, 10 mM K2HPO4, and 40 nM EDTA, pH 7.4), which was assumed to contain 457 nmol of oxygen/mL at 28 °C (14). Approximately 0.1 to 0.25 mg/mL of mitochondrial protein was injected into the respirometry chamber. Pyruvate (5 mM) and malate (2 mM) were used as substrates. ADP (480 μM) was used to initiate state 3 respiration. The respiratory control ratio was defined as the rate of oxygen consumption after the addition of ADP divided by the rate after exhaustion of ADP. The data were acquired and analyzed with the model 782 oxygen meter (Strathkelvin) using version 3 of the software.

Measurement of Blood Metabolites

Blood glucose and triglycerides were determined spectrophotometrically using the Vitros DTS60II system (Ortho-Clinical Diagnostics, Rochester, NY). The glucose was oxidized by glucose oxidase to form hydrogen peroxide and gluconate, followed by oxidative coupling catalyzed by peroxidase in the presence of dye precursors to produce a red dye. Triglycerides were hydrolyzed by lipase to glycerol and fatty acids, the former of which was further reacted with l-α-glycerophosphate oxidase in the presence of dye precursors. Blood free fatty acids (FFAs) were measured using the NEFA-C kit (Wako Chemicals, Richmond, VA). This assay converted nonesterified fatty acids to their copper salts, which were then complexed with a dye for the purposes of colorimetric measurements. Blood β-hydroxybutyric acid (β-BHA) levels were measured using a β-BHA kit (Sigma) that oxidized β-BHA to acetoacetate, and absorbance was measured at 340 nm.

Statistics

Student's t test or ANOVA was used to determine statistical significance among groups. The level of significance was set at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Cloning and Sequencing of Lemming Ucp3

The full-length cDNA for lemming Ucp3 was cloned by reverse transcription-PCR from lemming BAT. The complete nucleotide and predicted amino acid sequences are shown in Figure 1. The open reading frame of lemming Ucp3 cDNA spans 940 nucleotides and encodes a protein of 313 amino acids. Sequence analysis indicated that the identity of UCP3 among lemming (GenBank 33114696), hamster (GenBank 11320975), mouse (GenBank 6678495), and rat (GenBank 7110732) was ∼ 97% at the amino acid level, and the identity between lemming UCP3 and human UCP3 (GenBank 2497983) was ∼84%. Analysis of the deduced amino acid sequence of lemming UCP3 and UCP1 (GenBank 21340400) revealed a 54% identity, and these two proteins share a highly conserved motif of mitochondrial energy transfer protein signature (11). A multiple tissue blot revealed that Ucp3 mRNA expression is limited to BAT, skeletal muscle, and heart (Figure 2).

image

Figure 1. Nucleotide sequence of lemming Ucp3 cDNA and its deduced amino acids sequences. The shaded amino acids indicate the three motifs of mitochondrial energy transfer proteins signature.

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image

Figure 2. Northern blot showing the tissue distribution of lemming Ucp3 mRNA. WAT, white adipose tissue.

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Effect of Photoperiod-Induced Weight Gain and Weight Loss on Ucp3 Expression

When SD-SD animals were compared with LD-LD animals, Northern blot analysis showed that Ucp3 mRNA expression was 20% and 42% lower in muscle and BAT, respectively, in SD animals (p < 0.001). When LD animals were transferred to SD conditions for 10 days, Ucp3 mRNA expression was reduced by 14% and 26% in muscle and BAT, respectively (p < 0.01). Animals that were transferred from SD to LD conditions for 10 days increased Ucp3 mRNA expression by 14% and 40% in muscle and BAT, respectively (p < 0.001; Figure 3).

image

Figure 3. Effect of photoperiod on Ucp3 mRNA expression in (A) skeletal muscle and (B) BAT. A total of 20 7-month-old male lemmings were used for this experiment. The animals were divided into four groups: lemmings raised from weaning under either LD or SD conditions (LD-LD, SD-SD), LD lemmings transferred to SD conditions for 10 days, or SD lemmings transferred to LD conditions for 10 days (LD-SD and SD-LD; n = 5/group). *Significant difference with photoperiod transfer. #Significant difference between photoperiod conditions (p < 0.05). Data were normalized to the expression of GAPDH.

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Effect of Cold Exposure on Ucp3 Expression

The results showed that Ucp3 mRNA expression in skeletal muscle was decreased by 16% in LD lemmings and 23% in SD lemmings by 10 days of mild cold exposure (p < 0.001). In BAT, Ucp3 mRNA expression was decreased by 36% (p < 0.05; Figure 4) in cold-exposed LD lemmings, whereas no difference was noted in SD lemmings (p = 0.25).

image

Figure 4. Effect of cold exposure on Ucp3 mRNA expression in (A) skeletal muscle and (B) BAT. A total of 19 6- to 7-month-old female LD and SD lemmings were used for this experiment. Animals were housed at two ambient temperatures for 10 days, thereby forming four groups: LD 18 °C, SD 18 °C, SD 10 °C, and LD 10 °C (n = 4–5/group). *Significant difference of cold exposure within a photoperiod (p < 0.05). Data were normalized to the expression of GAPDH.

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Effect of Fasting on Ucp3 Expression

The measured blood metabolites of the fed and fasted lemmings are shown in Table 1. Fasting significantly reduced blood glucose and triglycerides, whereas FFA and β-HBA were significantly increased (p < 0.001). Skeletal muscle Ucp3 expression was increased 60% after 6 hours of fasting and was further increased to 100% of control values after 12 hours (p < 0.01; Figure 5). The expression of Ucp3 mRNA during the fast was significantly correlated to FFA concentration (r = 0.70, p < 0.01).

Table 1.  Effect of hours of fasting on measured blood metabolites
Metabolite0 hours6 hours12 hours18 hours
  1. Different letters denote significant difference (p < 0.05).

  2. Values are means ± SE (n = 8/group).

Glucose (mg/dL)122 ± 4.7A77 ± 3.7B80 ± 4.5B64 ± 3.9C
FFA (mEq/L)0.80 ± 0.05A2.59 ± .015B3.65 ± 0.48C3.01 ± 0.31B,C
Triglycerides (mg/dL)219 ± 39A78 ± 3.7B80 ± 4.5B65 ± 3.9B
B-HBA (mM)0.29 ± 0.04A0.82 ± 0.05B1.15 ± 0.15C0.95 ± 0.10B,C
image

Figure 5. Effect of hours of fasting on skeletal muscle Ucp3 mRNA expression. A total of 32 8- to 10-week-old female LD lemmings were used for this experiment. The animals were divided into four groups: ad libitum fed or 6-, 12-, or 18-hour fast (n = 8/group). Different letters denote significant difference (p < 0.05). Data were normalized to the expression of GAPDH.

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Mitochondrial Oxygen Consumption Results

There were no significant differences in the respiratory control ratio of LD lemmings switched for 10 days to SD conditions or for SD lemmings switched to LD conditions (Figure 6).

image

Figure 6. Effect of photoperiod transfer on mitochondrial respiratory control ratio. A total of 24 10-week-old female lemmings were used for this experiment. The animals were divided into four groups: lemmings raised from weaning under either LD or SD conditions, LD lemmings transferred to SD conditions, or SD lemmings transferred to LD conditions (n = 4–7/group). After 10 days under these photoperiod conditions, the animals were killed by cervical dislocation, and gastrocnemius and quadriceps muscles were removed from both hindlimbs.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Lemmings alter their body mass and composition in response to changes in photoperiod. The molecular mechanisms whereby these changes occur may provide important insights for understanding the regulation of energy expenditure and obesity. In this study, we sought to understand how Ucp3 mRNA expression is affected by situations of known altered energy expenditure (i.e., photoperiod-induced weight gain and loss, cold exposure, and fasting). These conditions either increase or decrease the energy expenditure requirements of collared lemmings. If UCP3 is involved in modulating energy expenditure, changes in Ucp3 mRNA expression should parallel the increase or decrease in energy expenditure induced during these states.

Ucp3 mRNA expression was affected by photoperiod in lemmings. These results suggest that, because the Ucp3 mRNA expression is altered in parallel with changes in REE, it is possible that Ucp3 could be a thermoregulatory gene involved in these changes. The mechanism underlying the change in Ucp3 mRNA expression by photoperiod is not known. It is possible that the changes in Ucp3 mRNA expression are a result of photoperiod-induced changes in thyroid hormone. It has been shown that thyroid hormone concentration is significantly higher in LD lemmings compared with SD lemmings (9,15). Several studies have shown that Ucp3 mRNA expression is increased by treatment with thyroid hormone (2,16,17). Lanni et al. have shown that, in rats transitioned from hypothyroid to euthyroid to hyperthyroid states, Ucp3 mRNA expression is increased (18). It is possible that the photoperiod effects on Ucp3 mRNA expression in the collared lemming are mediated through changes in thyroid hormone.

Because the changes in Ucp3 mRNA expression with photoperiod were in accordance with the hypothesis that it may be a thermoregulatory gene, we sought to understand the effects of mild cold exposure on Ucp3 mRNA expression. When animals are housed at temperatures below their thermoneutral zone, there is an increase in energy expenditure to provide the heat necessary for homeothermy (19). Paradoxically, the results showed that Ucp3 mRNA expression was decreased rather than increased by mild cold exposure. These data suggest that Ucp3 does not function as a thermoregulatory gene in collared lemmings. A number of studies have shown the differential effects of cold exposure in muscle over time, with acute exposure increasing Ucp3 mRNA expression and longer exposure either not altering or decreasing Ucp3 mRNA expression (20,21,22). These results are in line with previous findings that chronic cold exposure decreases muscle Ucp3 mRNA expression (20). Some studies have shown that BAT Ucp3 mRNA is increased during cold exposure (21). It is possible that the lack of a decrease in Ucp3 mRNA expression in SD lemmings on cold exposure is caused by the increased thermal insulation provided by the dense winter pelage (i.e., 10 °C is not cold to an SD lemming) (23). It is not clear why there was a decrease in Ucp3 expression with cold exposure in BAT of LD lemmings. Further studies will be necessary to understand this phenomenon.

Another condition during which energetic needs are altered is during fasting. Fasting is known to cause a decrease in energy expenditure (24). If UCP3 functions in the process of energy expenditure, its expression should decrease in concert with other energy-conserving processes. The results showed that fasting lemmings for 12 hours caused a 2-fold increase in Ucp3 mRNA expression. These findings are contrary to what would be expected if UCP3 were involved in energy expenditure. It was hypothesized that the increase in Ucp3 mRNA expression in muscle during fasting is a result of muscle assuming a greater role in heat production to maintain body temperature during the fast (2). Data contrary to this hypothesis have shown that rats fasted at thermoneutrality to prevent the need to expend energy to maintain body temperature still had increased Ucp3 mRNA expression levels (25). Numerous other studies have demonstrated that fasting increases Ucp3 mRNA expression, and the increase is correlated to increased FFA levels (2,22,26). These findings support the link between FFA and Ucp3 mRNA expression.

While understanding changes in Ucp3 mRNA expression is of interest, the truly important question is whether the changes in expression correspond with functional differences in mitochondrial respiration. This study sought to understand whether physiological changes in Ucp3 mRNA expression would result in changes in mitochondrial oxygen consumption rates. The results showed that photoperiod transfer did not affect respiratory control ratio, even though Ucp3 mRNA expression was altered. Although we acknowledge that respiratory control ratio may not be the most sensitive measure of mitochondrial coupling, these data suggest that physiological changes in Ucp3 mRNA expression (induced by photoperiod transfer) do not alter mitochondrial coupling in lemmings. These findings are in line with others that have shown that increasing Ucp3 mRNA expression did not alter state 4 respiration or proton conductance (27,28,29).

In conclusion, the main findings of this paper are that the lemming Ucp3 gene has a significant similarity to other published Ucp3 sequences and that its mRNA expression is limited to BAT, skeletal muscle, and heart; the mRNA expression of Ucp3 is decreased when REE is decreased (by transferring lemmings from LD to SD conditions) and by cold exposure and is increased when REE is increased (by transferring lemmings from SD to LD conditions) and by fasting; and the changes in Ucp3 mRNA expression by photoperiod switch do not alter the coupling of mitochondria as assessed by measuring respiratory control ratio. Because the changes in Ucp3 mRNA expression during cold exposure and fasting did not correspond to the expected energy expenditure changes under these circumstances and because the changes in Ucp3 mRNA expression with photoperiod transfer did not produce a corresponding change in mitochondrial coupling, doubts were raised concerning the role of UCP3 in energy expenditure in the collared lemming.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

We thank Carlos Krumdieck for assistance with the mitochondrial isolation and oxygen consumption procedures. Funding for this project was provided by NIH Grants DK54918, DK56336, and CA47888.

Footnotes
  • 1

    Nonstandard abbreviations: UCP1, uncoupling protein 1; UCP3, uncoupling protein 3; LD, long day; SD, short day; REE, resting energy expenditure; BAT, brown adipose tissue; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FFA, free fatty acids; β-BHA, β-hydroxybutyric acid.

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  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References
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