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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 5, 2001 as doi:10.1096/fj.00-0517fje. |
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Nutrition and Toxicology Research Institute NUTRIM
* Department of Movement Sciences;
Department of Human Biology, Maastricht University, 6200 MD Maastricht, The Netherlands; and
Eli Lilly & Company, Lilly Corporate Center, Indianapolis, Indiana, USA
2Correspondence: Department of Movement Sciences, Maastricht University, P.O. Box 616, 6200 MD, Maastricht, The Netherlands. E-mail: matthijs.hesselink{at}bw.unimaas.nl
SPECIFIC AIMS
HumanUCP3, predominantly expressed in skeletal muscle, plays an important role in the dissipation of energy in human energy metabolism and is therefore linked to obesity and type 2 diabetes. It has been deduced from rodent studies that the extent and direction in which, for example, fatty acid levels, GLUT4 content and physical training affect UCP3 mRNA expression is muscle fiber-type specific. To explore the role of UCP3 in human energy metabolism further, we wanted to examine the abundance of protein expression of UCP3 relative to muscle fiber typology in a direct manner and at the protein level in m. vastus lateralis muscle of healthy controls and type 2 diabetics.
PRINCIPAL FINDINGS
1. The antibody used here specifically recognizes UCP3 in
cryo-sections of human m. vastus lateralis sections as
well as in whole muscle homogenates
The UCP3 antibody was raised against a 20 amino acid (147–166 of
human UCP3). Affinity purified serum was tested for specificity in
immunofluorescence of H9C2 cells transfected with hUCP3
(Fig. 1a
) and adult vastus lateralis muscle (Fig. 1b)
.
Immunoblots were run on homogenates of human kidney and vastus
lateralis muscle (Fig. 1c
, 1d
). Preincubation
of the primary antibody with the peptide and omission of the primary
antibody yielded no detectable labeling. The signal raised by the UCP3
antibody was consistent with mitochondria (Fig 1b
), it
showed no cross reactivity with proteins of a distinct molecular mass
(Fig. 1c
), and it was absent in human kidney (Fig. 1d
). A single band was detected in muscle homogenates at 34
kDa, representing UCP3L, which possibly indicates that UCP3S is not
expressed at the protein level in human skeletal muscle (for in-depth
discussion, refer to the full paper).
|
2. Expression of UCP3 at the protein level is fiber-type specific:
2b >2a >1
Myosin ATP-ase staining at pH 4.3 (Fig. 2a
, c
) yielded the chessboard pattern of two
distinguishable fiber types (type 1 and type 2). Immunofluorescence
against UCP3 showed an abundant signal in fibers classified as type 2
and was only moderately expressed in type 1 fibers (Fig. 2b
for controls and Fig. 2d
for diabetics). Subclassification
of type 2 fibers in 2a and 2b after staining at pH 4.5 (Fig. 2e
) revealed that UCP3 was most abundantly expressed in type
2b compared with type 2a fibers (Fig. 2f
). These
findings were consistent in all sections examined. On all sections
examined, at least four corresponding fields, comprising at least 150
different fibers, were matched offline and studied by visual
inspection.
|
3. The pattern of fiber-type specific expression of UCP3 does not
deviate between healthy controls and type 2 diabetic subjects
Ten middle-aged male, type 2 diabetics were included along with
three healthy male controls. Subjects were instructed to fast overnight
and refrain from any vigorous exercise for 24 h prior to the
biopsy. No differences were observed between healthy controls and type
2 diabetic with regard to their relative expression of UCP3 in type 1
and type 2 fibers (Fig. 2b
compared with Fig. 2d
). Similarly, the pattern of relative expression of UCP3
in type 2a and type 2b fibers was comparable between healthy controls
and type 2 diabetics.
CONCLUSIONS AND SIGNIFICANCE
Skeletal muscle plays a major role in energy expenditure, under resting conditions as well as during exercise. UCP3 is considered an important mediator of the metabolic rate and therefore has a putative role in the pathophysiology of obesity and type 2 diabetes. However, studies on the functional role of UCP3 in human energy and substrate metabolism are all at the mRNA level, and convincing reports on protein expression of UCP3 are scarce due to the lack of specific antibodies. Here, we present an antibody that successfully passed all specificity checks and has proven to recognize UCP3L in human skeletal muscle by Western blotting as well as by immunofluorescence. Therefore, we conclude that the antibody presented here does not cross-react with any of the other mitochondrial proton carriers presently known in skeletal muscle and is therefore the first antibody that specifically recognizes UCP3 in human skeletal muscle. In human skeletal muscle, UCP3 mRNA is expressed as UCP3S and UCP3L. Remarkably, the antibody raised one single band at 34 kDa, while the peptide used to raise the antibody comprises amino acids 147–166, which are common to UCP3S and UCP3L. This finding raises the possibility that UCP3S is not expressed at the protein level in human skeletal muscle, but because UCP3S lacks the sixth membrane-spanning domain, it could also be argued that UCP3S is not, or is only loosely, inserted into mitochondria and may thus be lost during homogenization. In liver mitochondria, however, UCP3S and UCP3L are both imported and inserted into the mitochondrial inner membrane; whether this also holds for human skeletal muscle mitochondria is presently unknown.
We consistently observed that fibers classified as type 1 by conventional ATP-ase staining showed the lowest expression of UCP3, whereas UCP3 was more abundantly expressed in human type 2 fibers. Upon subclassification of type 2 fibers into type 2a and 2b, we found that type 2b fibers exhibited the highest expression of UCP3. This finding is in line with observations on the mRNA level in whole muscle homogenates of selected rat muscles. The relation between muscle fiber typology and UCP3 expression in sections from type 2 diabetics did not deviate from the relationship reported in healthy controls. Type 2 diabetics have more type 2b fibers compared with healthy controls. Thus, the observation that UCP3 is expressed most abundantly in type 2b fibers agrees with reports on increased UCP3 mRNA in type 2 diabetics. We are aware of two studies reporting higher UCP3 mRNA expression in type 2 diabetics and one report that shows a decrease in UCP3 mRNA expression. Transgenic mice overexpressing GLUT4 also up-regulate UCP3 simultaneously and show increased glucose uptake in gastrocnemius muscle (type 2 fibers). Conversely, transgenic mice overexpressing UCP3 have lower fasting glucose levels and improved glucose tolerance. This finding might imply that UCP3 is involved directly in glucose uptake in glycolytic (type 2) fibers. However, a plausible alternative explanation is that increased glucose uptake is secondary to a declined ATP/ADP ratio because of the uncoupling nature of UCP3. Indeed, it has been shown before that adding a chemical uncoupler to L6 muscle cells results in an increased uptake of glucose. The abundant expression of UCP3 in glycolytic type 2 fibers compared with type 1 fibers matches with a role for UCP3 in glucose uptake in the glycolytic fibers. However, it has been shown that contraction- and insulin-induced glucose uptake is highest in oxidative (type 1) muscles.
In people with high free fatty acid levels (starvation and high-fat feeding) UCP3 mRNA is most prominently up-regulated in glycolytic muscles (with a preponderance of type 2 fibers), which is unexpected considering the fasting-induced decline in energy expenditure. However, it has been hypothesized that UCP3, a mitochondrial anion carrier, is involved in cycling of fatty acid anions across the mitochondrial membrane. Thus a high expression in type 2a fibers during fasting may offer an alternative route to force fatty acid anions into the mitochondria and to use fatty acids as a substrate. However, type 2b fibers express the highest levels of UCP3 while having the lowest capacity to oxidize fatty acids. In contrast with type 2a fibers, which can switch rapidly from glucose to fatty acid oxidation, the abundant expression of UCP3 in type 2b fibers does not fit with a role for UCP3 in fatty acid handling.
Recently, it was shown that after ablation of the UCP3 gene more reactive oxygen species (ROS) were produced due to an altered mitochondrial membrane potential, which is related to UCP3 expression. This finding has led to the conclusion that UCP3 has a role in minimizing the production of ROS, thereby preventing excessive oxidative stress. Clearly, oxidative stress is highest in type 2b fibers and lowest in type 1 fibers (for in-depth discussion, refer to the full paper). Hence, the abundant expression of UCP3 in type 2b fibers is compatible with a role of UCP3 in minimizing oxidative stress.
With regard to the effect of endurance training on UCP3 expression, reports exist showing declined expression, no effect, and increased expression. Based on reports showing an inverse relationship between UCP3 mRNA and gross mechanical efficiency and maximal oxygen uptake (19), endurance training would be expected to lower UCP3 expression. This finding is consistent with the low expression of UCP3 in type 1 fibers reported here, because endurance-trained athletes have a preponderance of type 1 fibers. During exercise of high intensity, however, type 2b fibers are progressively recruited. The abundance of UCP3 in these fibers would indicate that ATP formation becomes less efficient with increasing exercise intensity, which could be considered remarkable.
In summary, we present a specific UCP3 antibody. Moreover, the present study is the first to show, in a direct manner and at the protein level, that UCP3 is most abundantly expressed in type 2b (fast glycolytic) fibers, to a lesser extent in type 2a (fast oxidative glycolytic) fibers, and is only moderately expressed in type 1 (slow oxidative) fibers of human vastus lateralis muscle. We observed no differences in this relationship between healthy controls and type 2 diabetics.
Of the postulated roles of UCP3, only a role of UCP3 in preventing oxidative stress appears to be fully compatible with the fiber-type specific expression of UCP3. It could therefore be hypothesized that UCP3-induced dissipation of energy serves multiple muscle fiber type- specific goals.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0517fje ; to cite this
article, use (February 5, 2001) FASEB J. 10.1096/fj.00-0517fje 
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15 mg of protein was loaded. No cross-reaction
was observed in the 5 to 60 kDa region. d) Autoradiograph
after Western blotting using the antibody described here, in human
skeletal muscle (left lane) and human kidney (right lane). The lack of
signal in the human kidney sample, known to express UCP2 but not UCP3,
shows that the present antibody does not cross-react with
UCP2.
