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Endocrinology |
Diabetes and Endocrinology Research Group, Department of Medicine, University of Liverpool, Liverpool L69 3GA [C. B., M. B., P. K., P. C., G. W.], and Pharmaceutical Sciences Research Institute, Aston University, Birmingham B4 7ET [M. J. T.], United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MAC16 mice showed significantly higher UCP1 mRNA levels in brown adipose tissue (BAT) than in controls (+63%, P < 0.01), and pair-feeding had no effect. UCP2 and -3 expression in BAT did not differ significantly between groups. By contrast, UCP2 mRNA levels in skeletal muscle were comparably increased in both MAC16 and pair-fed groups (respectively, 183 and 163% above controls; both, P < 0.05), with no significant difference between these two groups. Similarly, UCP3 mRNA was significantly higher than controls in both MAC16 (+163%, P < 0.05) and pair-fed (+253%, P < 0.01) groups, with no significant difference between the two experimental groups.
Overexpression of UCP1 in BAT in MAC16-bearing mice may be an adaptive response to hypothermia, which is apparently induced by tumor products; increased thermogenesis in BAT could increase total energy expenditure and, thus, contribute to tissue wasting. Increased UCP2 and -3 expression in muscle are both attributable to reduced food intake and may be involved in lipid utilization during lipolysis in MAC16-induced cachexia.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Weight loss is not simply caused by increased nutrient consumption by the tumor itself, because the tumor burden is usually trivial (<5% of total body weight), but rather by concerted changes in host metabolism induced by the tumor or the host’s response to it (4) . These changes include hypophagia, increased energy expenditure, and depletion of fat and muscle (4–7) . The failure to maintain or increase energy intake to compensate for energy loss is a major factor in causing cachexia (8, 9) . Various tumor products that inhibit feeding and/or stimulate energy expenditure have been identified (1) , but the specific molecular mechanisms responsible are not known.
In this study, we focused on the possible roles of the UCPs3 (1, 2, and 3) , which are postulated to be involved in the control of energy metabolism. UCP1, a mitochondrial protein expressed exclusively in BAT, is responsible for dissipating energy as heat instead of generating ATP from the oxidation of FFA (10) ; it determines the thermogenic capacity of BAT, a major heat-producing tissue in rodents and human neonates (11) . Gene expression of UCPl is stimulated by the sympathetic nervous system, mediated by the ß3 adrenoceptor (12–15) .
Recently, two structurally similar proteins with high homology to UCP1 have been identified and designated UCP2 and UCP3 (16, 17) . UCP2 is widely expressed in most tissues including white fat, BAT, muscle, heart, and liver, whereas UCP3 is mainly confined to thermogenic tissues such as skeletal muscle and brown fat in rodents; in humans, it is predominantly expressed in muscle (16–18) . It has been suggested that these novel proteins also uncouple mitochondrial oxidative phosphorylation and contribute to thermogenesis in vivo (19, 20) . This could be important for energy expenditure in humans, who virtually have no BAT after the neonatal period (12) . However, UCP3 expression in muscle is up-regulated by fasting, a condition in which there are substantial decreases in BAT thermogenic activity (12, 21) . Other possible physiological functions of UCP2 and UCP3 have been suggested, for example, involvement in the use of FFA (22) ; high levels of FFA are postulated to be responsible for the up-regulation of muscle UCP3 (23) .
The possible roles of the UCPs in the increased lipid catabolism and/or the enhanced thermogenic activity found in cancer cachexia have not been systematically investigated and may vary among different cancers and species. Little is known about the changes of BAT UCP1 mRNA in cancer cachexia, although increased skeletal muscle UCP2 and -3 mRNA has recently been reported in a rat cancer cachexia model that displays increased energy expenditure (24) .
Here, we studied the murine colonic adenocarcinoma, MAC16, a model for many human gastrointestinal and pancreatic cancers (25) . The MAC16 induces profound weight loss (up to 30%) with a minimal tumor burden (<3%) and prevents the compensatory hyperphagia that normally follows weight loss (25, 4) . Changes in energy expenditure or thermogenic capacity have not been characterized in MAC16-bearing mice. Recently, circulating catabolic factors PIF and LMF that, respectively, cause proteolysis (26) and lipolysis (27) have been isolated from MAC16-tumor tissue (26) . In vitro, LMF enhances FFA release from adipose tissue (4) , and, in vivo, it specifically depletes carcass lipid, increases serum glycerol levels, and stimulates oxygen uptake by BAT (28) . Both PIF and LMF are also found in the urine of patients with cancer cachexia.
The aim of this study was to determine whether the expression of UCP1,
-2, and -3 was altered in mice bearing the MAC-16 tumor and to relate
any such alterations to changes in food intake, body weight, and
composition. UCP1, -2, and -3 mRNA levels were measured in BAT, and
UCP2 and -3 mRNA in skeletal muscle of MAC16-tumor-bearing mice, and
the effects of hypophagia per se were investigated by
comparisons with both freely fed and food-restricted non-tumor-bearing
animals. To clarify peripheral signals that might mediate cachexia
induced by the MAC16 tumor, we also measured circulating FFA, leptin,
and the cytokines, TNF-
, IL-1ß, and IL-6. Leptin, the
ob gene product, is secreted by adipocytes and inhibits
feeding, stimulates thermogenesis, and mobilizes triglyceride
(29)
; its involvement has not been studied in this model.
Moreover, each of these factors affects the UCPs.
Intracerebroventricular leptin administration enhances the expression
of UCP1, UCP2, and UCP3 in rats (30)
, whereas it is
suggested that TNF-, produced by macrophages in response to
malignancy and inflammation, stimulates gene expression of UCP2 and
UCP3 in rats (31, 32)
.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fragments of the MAC16 tumor maintained in mice within the colony were
implanted s.c. in the flank of one group of mice
(n = 16) using a trocar (4)
.
Sham operations were performed for the other two non-tumor-bearing
groups. In one of the latter, each animal was pair-fed to match the
food intake of an individual MAC16-bearing mouse, whereas the other was
fed freely. After 18 days, weight loss had reached 
20% in the
tumor-bearing mice, and all of the mice were killed by
CO2 inhalation. Blood was removed by cardiac
puncture, and plasma was separated and stored at -40°C until assay.
The gastrocnemius muscle, interscapular BAT, and gonadal fat pads were
dissected, snap-frozen in liquid nitrogen, and stored at -80°C until
extraction of RNA. The whole tumor was also dissected out and weighed.
In a separate study, body temperature was measured by the insertion of a plastic-coated thermocouple into the rectum for each MAC16-bearing mouse (n = 8) at day 18 after tumor transplantation and for freely fed (n = 8) and pair-fed (n = 8) controls. In this study, weight and food-intake changes were similar to those in the first experiment.
Assays.
Plasma leptin concentrations were determined by using a ELISA kit
(Crystal Chem; Chicago, IL). Plasma FFA levels were measured using an
enzymatic colorimetric assay kit (Boehringer Mannheim; Mannheim,
Germany). Plasma TNF, IL-1ß, and IL-6 were all analyzed with ELISA
kits (Peninsula Laboratories Europe, St Helen’s, United Kingdom).
Expression of UCP1, -2, and -3 mRNA.
Total RNA was extracted from BAT and gastrocnemius muscle using
Tri-reagent (Sigma; Poole, United Kingdom) and RNA concentration
determined from the absorbance at 260 nm. Twenty µg of total RNA per
sample was applied to a 1% agarose-formaldehyde gel and separated by
electrophoresis. RNA was transferred overnight to a charged membrane by
capillary blotting and then cross-linked under UV light.
UCP1, -2, and -3 mRNAs were detected by Northern blotting in conjunction with the chemiluminescence method. The membranes were prehybridized in Easyhyb solution (Boehringer Mannheim) at 42°C for 1 h and were hybridized in the same solution with a digoxigenin-labeled 32-mer antisense oligonucleotide probe for mouse UCP1 (33) , or digoxigenin-labeled 30-mer oligo probes for mouse UCP2 (5'-ACTGTTTGACAGAGTCG TAGAGGCCAATGC-3'; GenBank accession number: U69135) and UCP3 (5'-CGTAGG TCACCATCTCAGCACAGTTGACAA-3'; GenBank accession number: AB008216), respectively. Each blot was stripped and reprobed for 18S rRNA with a 31-mer digoxigenin-labeled oligonucleotide, as described previously (34) . The amount of mRNA was expressed as the ratio of UCP mRNA:18S rRNA signals.
Statistical Analyses.
Data are expressed as mean ± SE. Differences in food
intake and body weight between groups were compared using two-way ANOVA
coupled to a Bonferroni t test. Tissue weights, plasma
levels of FFA and cytokines, and mRNA levels of the UCPs were compared
by one-way ANOVA. ARCUS statistical software (Medical Computing,
Aughton, United Kingdom) was used throughout. A P of 0.05 or
less was considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Total food intake between days 1 and 18
was 14% less in the MAC16 group than in controls
(P < 0.01; Table 1
).
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). Thereafter, non-tumor-bearing freely fed controls continued to gain
weight, whereas tumor-bearing mice weighed progressively less; on day
18, the final body weight was 24% (P < 0.01) lower than that of freely fed animals (Table 1)
. Tumor weight was
0.38 ± 0.03 g, averaging 1.8% of final body
weight, and was inversely correlated with final body weight
(r, -0.56; P < 0.02). Pair-fed
animals also lost weight after day 13 but to a lesser degree than the
MAC16 mice (Fig. 2)
; final body weight was 10% (P < 0.01) lower than in the freely fed group and 16% higher than
in the tumor-bearing group (P < 0.01; Table 1
).
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. Interscapular BAT also weighed less
in both MAC16 and pair-fed groups (-36% and -24%, respectively,
both P < 0.01 versus freely fed
controls). Gastrocnemius muscle weight showed a similar
relationship, with decreases below controls of 20%
(P < 0.01) in MAC16 and 7%
(P < 0.05) in pair-fed groups (Table 1)
.
As shown in Table 2
, plasma leptin levels fell significantly in tumor-bearing mice (-87%,
P < 0.01) and to a lesser degree in the
pair-fed group (-70%; P < 0.01). Plasma
leptin positively correlated with both body weight (r, 0.80;
P < 0.001; Fig. 3
) and gonadal fat mass (r, 0.76; P < 0.001), across all of the three experimental groups. Plasma FFA
concentrations were significantly higher in both MAC16 (+79%;
P < 0.05) and pair-fed (+99%;
P < 0.01) groups than in freely fed
controls, with no significant difference between the two experimental
groups (Table 2)
. Circulating concentrations of TNF, IL-1ß, and
IL-6 did not differ significantly between any of the three groups
(Table 2)
.
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). Neither UCP2 nor UCP3 expression in BAT was altered in
MAC16-tumor-bearing or in pair-fed mice, relative to controls (Table 1)
.
|
). UCP3 mRNA levels in muscle showed significant increases above
controls in MAC16 mice (+163%; P < 0.05)
and in pair-fed animals (+253%; P < 0.01),
again with no significant difference between these two groups (Fig. 6
). There was a highly significant correlation between muscle UCP3 mRNA
and plasma FFA levels (r, 0.70; P < 0.001; Fig. 7
) but not between UCP2 mRNA and plasma FFA (r, 0.38;
P > 0.05). Muscle UCP3 mRNA was also
inversely correlated with gonadal fat mass (r, -0.43;
P < 0.05), whereas UCP2 was not
(r, -0.28; P > 0.05).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In pair-fed animals that consumed the same amount of food as the
tumor-bearing animals, body weight was also reduced below controls but
to a lesser degree, as were white and BAT weights; however,
gastrocnemius muscle mass was not significantly affected. The
significantly greater reductions in body fat and body weight in
tumor-bearing than in pair-fed animals may suggest the importance of
the lipolytic factor LMF in vivo (26)
. Our
observations further highlight the complexity of cachexia in this
model: it is not caused solely by reduced energy intake, and tissue
breakdown and/or increased energy expenditure must contribute (see Fig. 2
). Increased energy expenditure has been reported both in humans and
in experimental animals with cancer cachexia (9, 35, 36, ) , but the underlying molecular mechanisms are not fully
identified.
Here, BAT UCP1 mRNA was increased in tumor-bearing mice, which indicated activation of non-shivering thermogenesis. This was a highly selective change, because UCP2 and -3 mRNA in the same tissue were unaltered. By contrast, the pair-fed animals showed no elevation in UCP1 mRNA, which suggested that the additional weight loss in tumor-bearing animals is partly due to inappropriately increased energy expenditure by BAT and/or to excessive catabolism, both presumably driven by tumor products. Non-shivering thermogenesis in BAT contributes substantially to resting energy expenditure in rodents (37, 38) . Increased thermogenic activity of BAT as assessed by GDP binding has also been reported in several rat models of malignancy, such as leukemia and the Yoshida sarcoma (9, 39) , and is likely to be sympathetically mediated stimulation as it is attenuated by propranolol treatment (9) . Moreover, BAT oxygen uptake in vivo is stimulated by a LMF, isolated from cancer patients, which showed similar lipolytic effects to the MAC16-derived LMF (28) . In our study, tumor-bearing mice had significantly lower body temperature at day 18, whereas pair-feeding did not alter body temperature, which suggested that MAC16-tumor products may independently induce hypothermia that can override additional heat produced by BAT activation. Separate studies from our laboratories have shown that PIF given peripherally can lower body temperature in normal mice.4 The mechanism of this effect is not known but may be attributable to an action on thermoregulatory centers in the brain or to loss of skeletal muscle protein. In addition, body temperature can decrease at a late stage of cachexia in leukemic rats (9) and in terminal cancer patients,4 which might be the results of emaciation. Structural alterations have been reported in BAT mitochondria from cancer cachectic mice, alterations that are similar to those cold-adapted animals and that are not seen in pair-fed animals (36) . However, it is not clear whether the increased BAT thermogenic activity in MAC16 mice (a) reflects a compensatory mechanism in response to hypothermia induced by tumor product as occurs in cold-exposure (12, 40) ; or (b) results from a direct or indirect stimulatory effect of LMF on BAT.
The newly described UCP2 and UCP3 have been implicated in energy metabolism in skeletal muscle, an important site for thermogenesis, particularly in humans (16, 19) . Their roles in cachectic states such as malignancy or infection remain unclear, although increased UCP2 and UCP3 expression in skeletal muscle was reported in rats bearing the Yoshida AH-130 ascites hepatoma (24) , while our studies were in progress. In MAC16-tumor-bearing mice, gastrocnemius muscle UCP2 and-3 expression were markedly up-regulated, which is in agreement with the study by Sanchis et al. (24) . Because skeletal muscle contributes to thermogenic response to cold (38) , up-regulation of UCP homologous by the tumor could point to enhanced energy production that could exacerbate tissue losses. However, this notion is challenged by the fact that pair-feeding induced similar increases in muscle UCP2 and -3 expression. Muscle UCP2 and-3 may, therefore, serve other nonthermogenic functions, perhaps in lipid utilization (22, 23) .
FFA are raised in some patients with cancer cachexia (41, 42) , and, in our study, plasma FFA levels were comparably increased in both pair-fed and MAC16-tumor-bearing animals. Interestingly, we found a highly significant positive correlation between plasma FFA and muscle UCP3 mRNA, consistent with suggestions that FFA induce muscle UCP3 expression both in vivo (22, 43) and in vitro (23) . Several studies demonstrate that skeletal muscle UCP3 mRNA levels are increased dramatically during fasting, which also raises circulating FFA levels (21, 22, 44 ) . Because fasting reduces energy expenditure, increased muscle UCP3 expression is unlikely to regulate thermogenesis in this state but may instead play a role in the utilization of FFA or perhaps the removal of toxic free radicals resulting from enhanced ß-oxidation of FFA. The inverse correlation between muscle UCP3 and fat mass observed in our study further supports the possible involvement of this protein in lipid mobilization and metabolism. Any direct association of muscle UCP2 to MAC16 was not established in this study.
Leptin induces weight loss by suppressing appetite and stimulating heat
production, mobilizing fat with little loss of lean body mass
(45)
. Intracerebroventricular leptin infusion increases
BAT UCP1 mRNA, and UCP2 and UCP3 mRNA in brown and white adipose
tissues as well as in skeletal muscle (30)
. In
MAC16-tumor-bearing mice, circulating leptin fell markedly (-87%) in
proportion to fat and weight loss, which suggests that leptin
production has been severely suppressed in this model; this indicates
that leptin is unlikely to mediate hypophagia, wasting, or enhanced UCP
expression in MAC16-induced cancer cachexia. Indeed, low leptin levels
would be predicted to stimulate hunger and feeding, which highlights
the powerful appetite-suppressing effect of tumor products. Low or
undetectable circulating leptin concentrations have also been reported
in patients with lung cancer and weight loss (46)
and
gastrointestinal cancer (47)
. The unchanged levels of
circulating TNF, IL-1ß, and IL-6 levels in MAC16-bearing mice make
it unlikely that wasting or UCP expression are mediated by these
cytokines. We are currently investigating whether UCP expression is
affected by the MAC16 products, PIF and LMF.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by North West Cancer Research
Fund. 
2 To whom requests for reprints should be
addressed, at Diabetes and Endocrinology Research Group, Department of
Medicine, University of Liverpool, Duncan Building, Daulby Street,
Liverpool L69 3GA, United Kingdom.
3 The abbreviations used are: UCP, uncoupling
protein; BAT, brown adipose tissue; LMF, lipid-mobilizing factor; PIF,
proteolysis-inducing factor; FFA, free fatty acid(s); TNF, tumor
necrosis factor; IL, interleukin; GDP, guanosine diphosphate.
Received 10/18/99. Accepted 3/ 3/00.
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