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Original Studies |
Pennington Biomedical Research Center, Louisiana State University (J.H.P., G.A.B.), Baton Rouge, Louisiana 70808-4124; Department of Medicine, University of Bristol, Bristol Royal Infirmary (J.H.P.), Bristol, United Kingdom BS2 8HW; Faculty of Medicine, Medical Biochemistry, University of Geneva (O.B.), 1211 Geneva 4, Switzerland; and Department of Medicine, University College London Medical School, Whittington Hospital (K.B., S.W.C., V.M.-A.), London, United Kingdom N19 3UA
Address all correspondence and requests for reprints to: Dr. Jonathan H. Pinkney, University of Bristol, Department of Medicine, Research Centre for Neuroendocrinology, Bristol Royal Infirmary, Marlborough Street, Bristol BS2 8HW, United Kingdom. E-mail: j.pinkney{at}bris.ac.uk
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In contrast, less information is available regarding UCP-2 in human adipose tissue. Studies in humans have suggested a correlation between UCP-2 gene expression in WAT and body mass index (BMI), and increasing levels of expression with fasting (13). However, these investigators found no effect on UCP-2 expression with less severe, but more prolonged caloric restriction (14). In contrast, reduced UCP-2 gene expression was observed in ip, but not extraperitoneal, adipose tissue of obese humans, with no change in expression with weight loss (15). Thus, the animal and human studies do not yet afford a clear view of the role of UCP-2 in human adipose tissue. In particular, doubts have been cast about UCP-2 as a prime regulator of thermogenesis because of its induction by starvation, its inconsistent response to adrenergic agonists, and the belief that WAT is not a major thermogenic tissue in humans. This idea would be in keeping with the hypothesis that the skeletal muscle homolog UCP-3 (16, 17) could play a role in lipid metabolism (18, 19, 20, 21).
The present study was set up to examine quantitatively levels of UCP-2 gene expression in human adipose tissue and to relate this measure to major physiological variables, including local concentrations in adipose tissue of free fatty acids (FFA) and leptin, levels of obesity, and nutritional status, as indicated by responses to food intake. To facilitate these analyses, adipose tissue leptin and FFA production were measured directly in vivo using the arterio-venous balance technique, with adjustment for blood flow measured from 133Xe washout, and we established a sensitive nuclease protection assay for UCP-2 to examine, quantitatively, levels of gene expression in abdominal sc adipose tissue.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The studies were approved by the institutional ethical committee, and subjects gave informed written consent. Human volunteers (15 women and 10 men) with a wide range of levels of adiposity were recruited [median BMI, 32.9 kg/m2 (range, 18.0–77.1); percent body fat, 41.9% (range, 27.4–50.8%)] from an obesity clinic and hospital staff. The mean ± SD age was 40.5 ± 13.8 yr. Subjects were all nondiabetic and euthyroid, and obese subjects had simple obesity.
Study design
Subjects were instructed by a nutritionist on how to maintain a high carbohydrate diet for 2 days before the day of study and completed 3-day food diaries, which were analyzed using Dietplan 5 (Forestfield Software Ltd., West Sussex, UK).
Subjects were studied in the postabsorptive state (at least 15 h of fasting) and for 5 h after a high carbohydrate meal (energy content adjusted to represent 50% of the basal metabolic rate, 70% energy from carbohydrate, 20% from fat, and 10% from protein). Blood was sampled at baseline (in duplicate) after a 15-h fast and at 2 and 4 h after the meal.
Anthropometric determinations
Subjects were weighed, height was recorded, and BMI was calculated (kilograms per m2). Body composition was measured by electrical bioimpedance (Bodystat, Douglas, Isle of Man).
Adipose tissue biopsy
An open biopsy of sc anterior abdominal adipose tissue was performed, with the removal of 2–5 g adipose tissue. Biopsies were carried out in the fasting state and in a subset at 2 h (n = 16) and 4 h postprandially (n = 11).
Blood sampling
Cannulas were inserted, using local anesthesia, into a radial artery and a superficial epigastric vein draining the sc abdominal adipose tissue. All lines were kept patent by a slow infusion of isotonic saline. Blood samples were taken simultaneously from the two sites. Previous work has shown that venous blood from superficial epigastric veins approximates the effluent from an adipose tissue bed, and arterio-venous differences across the abdominal adipose tissue yield results in good agreement with microdialysis studies (22).
Blood flow measurements and assays
Adipose tissue blood flow was determined with 133Xe washout (23) during the baseline sampling and after food intake. A tissue/blood coefficient of 10 was assumed for all subjects, as previously advocated (23, 24). Plasma flow was calculated from blood flow and hematocrit. The local leptin production by adipose tissue was calculated as the product of the arterio-venous difference and local plasma flow (25).
Leptin was measured in plasma with an in-house RIA, using an antihuman leptin rabbit polyclonal antibody and 125I-labeled leptin. This assay shows good correlation with the Linco leptin RIA (r = 0.92; Linco Research, Inc., St. Charles, MO). The assay was sensitive to 0.1 ng/mL, with inter- and intraassay coefficients of variation less than 10% (26). FFA were measured by an enzymatic colorimetric assay (Roche Molecular Biochemicals, Lewes, UK). Insulin concentrations were determined with a specific immunoenzymometric assay (DAKO Corp., Cambs, UK), with a detection limit of 3 pmol/L. Plasma glucose was measured by the glucose oxidase method (Beckman Coulter, Inc., Brea, CA).
UCP-2 mRNA expression
Total RNA was extracted from adipose tissue with Tri-Reagent
(Sigma, Poole, UK). The UCP-2 riboprobe was made from
human UCP-2 complete coding sequence (GenBank U82819) (16) cloned in
pBluescript SK-. Purified plasmid DNA was
linearized with the enzyme RsaI (New England Biolabs, Inc., Beverley, MA). A high specific activity riboprobe was
synthesized by in vitro transcription with T3 polymerase
(Maxiscript-T3, Ambion, Inc., Austin, TX) and randomly
labeled by incorporation of [
-32P]UTP (New
England Nuclear Corp., Boston, MA) in the absence of limiting
nucleotide. A 300-nucleotide UCP-2 riboprobe was produced, protecting a
270-nucleotide fragment of UCP-2 mRNA. For the internal control, a
ß-actin riboprobe protecting a 125-nucleotide sequence of ß-actin
mRNA was transcribed from p-TRI-Actin-125-human (Ambion, Inc.) with T3 polymerase, and labeled by random incorporation of
[-32P]UTP. Riboprobes were purified by spin
column (CLONTECH Laboratories, Inc., Palo Alto, CA), and
isolated by 10% urea/formaldehyde gel electrophoresis. RNA Century
Marker Template (Ambion, Inc.) was used as the RNA marker.
Reagents for the nuclease protection assay (RPA-III kit) were obtained
from Ambion, Inc. Hybridization reactions were set up with
10 µg RNA/reaction and incubated with both riboprobes for 24 h.
Single stranded RNA was digested with ribonuclease A/ribonuclease T1
(1:100). Hybridized fragments were resolved by urea-formaldehyde gel
electrophoresis, with appropriate yeast RNA controls. Gels were exposed
in a PhosphorImager cassette (Molecular Dynamics, Inc.,
Sunnyvale, CA) and quantified with ImageQuant. No cross-reactivity was
apparent between the UCP-2 riboprobe and additional homologous mRNAs,
suggesting very low levels of expression of UCP-1 and UCP-3 messages.
Thus, in pilot studies UCP-2 and ß-actin were readily detectable in
as little as 1 µg total adipose tissue RNA, whereas, using similar
in vitro transcription and nuclease protection assay
protocols for human UCP-1 and UCP-3 (GenBank U84763) (16), neither of
these messages was detectable in 20 µg sc adipose tissue RNA.
Statistical analyses
Statistical analyses were performed with the program SPSS, Inc. (Statistical Package for the Social Sciences), version 6.0. UCP-2 gene expression was expressed as a ratio to ß-actin expression. Data were analyzed using parametric methods. Cross-sectional between-group comparisons were made with unpaired t test, and differences between variables at baseline and during a high carbohydrate diet were made using paired t test. Relationships between continuous variables were determined with linear regression analysis. Two-tailed P values were determined, and statistical significance was defined as P < 0.05.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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is a representative gel
showing expression of UCP-2, but neither UCP-1 nor UCP-3, in human
abdominal sc white adipose tissue. UCP-2 was detected in all samples
measured, whereas UCP-1 and UCP-3 were not detected. The faint bands
noted around 230 nucleotides in the UCP-1 assay were present in human
and yeast control lanes alike, excluding the possibility of significant
binding to human UCP-2. However, the faint additional band around 180
nucleotides in the UCP-2 lane could represent a signal from a
homologous sequence. The absence of significant UCP-3 expression
reduces the likelihood that this signal represents a UCP-3 fragment
bound by the UCP-2 riboprobe. It seems probable that this band
represents binding to either homologous sequences of UCP-1 or other
homologous RNAs.
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All subjects were studied in the fasting state. The expected
positive correlations were observed for both arterial and venous
insulin and FFA with BMI (r = 0.44–0.66; 0.05 <
P < 0.001) and for percent body fat (r =
0.40–0.55; 0.06 < P < 0.007). However, the
UCP-2/ß-actin ratio correlated inversely with both indexes of
adiposity, BMI (r = -0.45; P = 0.026) and percent
body fat (r = -0.41; P = 0.05; Fig. 2, A and B). UCP-2/ß-actin gene
expression ratios of biopsies obtained in the fasting state were then
examined in relation to biochemical variables in both radial arterial
and epigastric venous plasma taken concurrently with the biopsy. The
UCP-2/ß-actin ratio correlated inversely with arterial plasma insulin
(r = -0.47; P = 0.02) and epigastric venous FFA
levels (r = -0.45; P = 0.04; Fig. 3, A and B). Both arterial and epigastric
venous plasma leptin concentrations correlated inversely with the
UCP-2/ß-actin ratio (r = -0.51; P = 0.014 and
r = -0.50; P = 0.018; Fig. 3, C and D). In view
of the inverse correlations between the UCP-2/ß-actin ratio and
measures of adiposity as well as biochemical measures, this analysis
was repeated with biochemical indexes expressed as a ratio to percent
body fat to determine whether the relationships were independent of
body fat. The UCP-2/ß-actin ratio no longer correlated with arterial
or venous insulin or FFA (r = -0.002–0.29; 0.23 <
P < 0.99), but was still significantly related to
arterial and venous leptin concentrations (r = -0.44 and -0.45;
both P = 0.04). However, neither the arterio-venous
difference in leptin concentrations nor the adipose tissue leptin
production rate was significantly related to the UCP-2/ß-actin ratio
(r = -0.31; P = 0.16 and r = -0.21;
P = 0.37). Arterial triglycerides (r = -0.39;
P = 0.07) also tended to correlate inversely with the
UCP-2/ß-actin ratio, although this correlation did not reach
statistical significance.
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In the postprandial study both FFAs and insulin concentrations changed significantly, with a reduction in epigastric venous FFA (P = 0.0007) and an increase in arterial insulin concentrations (P = 0.0001). However, no significant changes in gene expression were observed among these three time points. No significant relationship was observed between plasma FFA and the UCP-2/ß-actin ratio in the postprandial studies, either singly or combining the 2 and 4 h study groups.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In the present studies UCP-2 gene expression was measured by nuclease protection assay. This approach was adopted by Oberkofler et al. (15), although these investigators observed reduced UCP-2 expression in visceral adipose tissue, but not sc abdominal adipose tissue of obese patients compared to lean subjects. Using this approach together with adipose tissue obtained at open biopsy may offer more accurate measurement of gene expression than PCR-based methods on smaller samples, where amplification introduces further error. In agreement with the results of previous studies, neither UCP-1 nor UCP-3 (16) was expressed to a significant degree in human sc adipose tissue, but the present studies demonstrate this point well in the same assay. The principal conclusion of the present study was that UCP-2 gene expression was reduced with increasing levels of adiposity. This conclusion was supported by concordant biochemical data, including inverse relationships with circulating plasma insulin, leptin, and fatty acid concentrations.
In the fasting state epigastric venous FFA concentrations were inversely related to levels of UCP-2 gene expression. The use of epigastric vein sampling, as opposed to measurements of mixed venous blood from the forearm, is a more sensitive way to examine concentrations of substrates and hormones emitted from same local WAT depot. The increase in epigastric venous FFA during fasting is a convincing index of lipolysis, implying mobilization and release of adipocyte lipid stores. The elevated venous FFA levels make it unlikely that intracellular FFA concentrations are lower in obesity. Therefore, in obese subjects and in the fasting state, we anticipated that raised intracellular FFA might lead to increased UCP-2 gene expression, as suggested by Aubert et al. (10). However, to the contrary, UCP-2 gene expression was lower in more obese subjects and was not further increased in the fasting state. In the fed state, no differences were noted in UCP-2 gene expression, suggesting that individual meals do not affect UCP-2 gene expression in human adipose tissue.
Regarding insulin, measurements were made on arterial plasma, in view of the previous demonstration by our group that adipose tissue contributes significantly to the extraction of insulin from plasma (27). There are relatively few data regarding the effects of insulin on the expression of any member of the UCP family. Although insulin has been shown to increase UCP-1 protein (28), euglycemic clamps affected neither UCP-2 mRNA expression in human sc adipose tissue and skeletal muscle nor UCP-3 mRNA expression in skeletal muscle (13, 18). In our study, in which obesity was linked to hyperinsulinemia, we cannot distinguish independent effects of adiposity and insulin on UCP-2 expression.
Both arterial and epigastric venous leptin concentrations correlated inversely with UCP-2 expression in the fasting state, after adjusting for adiposity, suggesting an independent relationship between local leptin concentrations and UCP-2 gene expression. In previous studies in rodents leptin was suggested to increase UCP-3 mRNA expression in skeletal muscle (7, 29, 30, 31) and increase UCP-2 mRNA expression in WAT (29, 31), whereas some researchers reported a lack of effect of leptin on UCP-2 mRNA levels in certain WAT depots (31, 32). However, UCP regulation increasingly appears to be dependent on the UCP subtype, the tissue in question, the location of the WAT depot, and the time course studied (9, 31, 33, 34).
In the postprandial studies no significant relationship was observed between UCP-2 expression and the metabolic variables addressed. This may in part be on account of the smaller size of the postprandial study groups together with the postprandial time points being relatively early to observe significant effects on levels of gene expression. These data suggest that there is no acute effect of food intake on UCP-2 gene expression in WAT, and therefore that changes in UCP-2 gene expression are likely to occur in relation to more long-term adaptive changes in adipose tissue.
From the present work and previous literature on UCP-2 mRNA expression, it is difficult to draw firm conclusions about the functional significance of observed differences in levels of UCP-2 mRNA expression. The present results could be consistent with an impairment of thermogenesis in the sc adipose tissue of obese human subjects. However, it is unclear whether this stands in a primary or a secondary relationship to obesity. Reduced UCP-2 expression also might be predicted to enhance the mitochondrial electrochemical gradient and to promote adipocyte ATP synthesis, which might inappropriately facilitate biosynthetic processes, including fatty acid reesterification as well as de novo lipogenesis.
In conclusion, this study, by bringing together in vivo physiological techniques with molecular methods in humans, affords new insights into physiological relationships of gene expression that may not necessarily be apparent from work in animals or in vitro. UCP-2 gene expression in sc abdominal adipose tissue is inversely related to adiposity and its biochemical correlates. Furthermore, it is inversely related to plasma leptin levels independent of body fat, supporting an inverse relationship between leptin and UCP-2 gene expression in vivo. Reduced UCP-2 expression in obese individuals may be a maladaptive phenomenon and could offer a target of therapeutic potential in the treatment of obesity.
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Acknowledgments |
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Footnotes |
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Received June 11, 1999.
Revised February 9, 2000.
Accepted February 27, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, tumor necrosis factor, and
uncoupling protein-2 expression in adipose tissues. Biochem Biophys Res
Commun. 246:660–667.[CrossRef][Medline]
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