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Department of Internal Medicine, Divisions of Endocrinology and Metabolism, University of Iowa and Iowa City Veterans Affairs Medical Center, Iowa City, Iowa 52246
Address all correspondence and requests for reprints to: Dr. William Sivitz, Department of Internal Medicine, University of Iowa Hospitals and Clinics, 3E-17 VA, Iowa City, Iowa 52246. E-mail: william-sivitz{at}uiowa.edu
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Abstract |
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Within certain limitations, which are discussed, these data are the first to demonstrate increased respiration and impaired coupling of oxidative phosphorylation as a result of UCP homolog expression in isolated mammalian mitochondria. Our results also suggest an important role for UCP in lipid metabolism and, possibly, insulin secretion.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is evidence that UCP2 and UCP3, like UCP1, function to dissipate the proton electrochemical gradient; however, this issue is controversial (11, 12). Major support for a role of UCP2 and/or UCP3 in catalysis of the proton leak includes sequence homology to UCP1 (4, 5), increased respiration in yeast expressing UCP2 and UCP3 (4, 13, 14, 15), and decreased ability of yeast expressing UCP2, UCP3L, and UCP3S to accumulate membrane-sensitive probes (4, 5, 13, 14, 15). On the other hand, fasting, which increases skeletal muscle UCP3 expression, was not associated with altered proton conductance (16), and enhanced UCP2 expression in UCP1 knockout mice is not associated with increased proton conductance (17). Further, proton conductance can be readily demonstrated in normal hepatocytes even though UCP homologs have not been detected in these cells under normal physiological circumstances (11).
Aside from the issue of whether UCPs uncouple the proton electrochemical gradient, the metabolic roles of these proteins still need definition. Various proposed functions include regulation of lipid metabolism, thermogenesis, modulation of reactive oxygen species, fatty acid transport, and regulation of ATP synthesis (2, 11). In pancreatic islets, a role of UCP in regulating insulin release has been suggested (18, 19).
The direct effects of expressed UCP homologs in mammalian mitochondria has not been reported to date. In the current work we subjected an insulin-secreting pancreatic insulinoma cell line (INS-1 cells) to adenoviral expression of UCP2 and UCP3 in mitochondria. Using this approach, we were able to obtain a high level of expression of both proteins in adequate numbers of cells for metabolic studies of isolated mitochondria. Hence, we were able to quantify mitochondrial oxygen use in the presence (state 3 respiration) or absence of ADP and under conditions of ATP synthesis inhibition by oligomycin (state 4 respiration). In addition, at the whole cell level, we examined the effects of UCP2 and UCP3 overexpression on lipid and glucose oxidation, insulin release, and ATP and ADP contents in this cell line.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adenovirus generation
The complementary DNAs encoding the full-length rat UCP2 and
UCP3 proteins were amplified from isolated spleen (UCP2) and
gastrocnemius muscle (UCP3) total RNA by RT-PCR according to standard
methodology (20). We used the sense and antisense primers:
5'-AGACGCGGTACCGGAAATCAAGGGGATCAG-3' and
5'-AGACGCATCGATAAGGAAAAGACAGGGCAG-3' for UCP2 (positions 137–154 and
1313–1330, respectively; GenBank accession no. AB010743), and
5'-AGACGCCTCGAGTCACAGGCAGCAAAGGAAC-3' and
5'-AGACGCATCGATTGAGGAAAGTACCAAGCGG-3' for UCP3 (positions 98–116 and
1077–1095, respectively; GenBank accession no. U92069). The amplified
complementary DNA products were ligated as
KpnI/ClaI (UCP2) and
XhoI/ClaI (UCP3) fragments into a similarly
restricted shuttle plasmid pAd5CMVK-NpA (provided by the Gene Transfer
Vector Core of our institution) between the cytomegalovirus promoter
and simian virus 40 polyadenylation signal site. Final recombinant
plasmids containing the insert were verified by restriction
endonuclease digestion and automated fluorescent DNA sequencing by the
DNA core of our Diabetes and Endocrine Research Center and used to
prepare the recombinant adenoviruses, Ad5CMV-UCP2 and Ad5CMV-UCP3.
Plasmids containing insert DNA were transfected into human embryonic
kidney 293 (HEK-293) cells with near full-length adenoviral DNA
previously restricted to remove the E1 region, rendering the virus
replication deficient. After transfection, individual viral plaques
were isolated, and plaques containing the insert DNA were identified by
PCR and restriction enzyme digestion. Then, the recombinants were
amplified in HEK-293 cells and purified by CsCl gradient. The
preparations were collected and desalted, and titers were determined.
Identical adenoviral constructs, Ad5CMVlacZ and Ad5CMV-MnSOD,
expressing bacterial ß-galactosidase (ß-gal) or the mitochondrial
protein, MnSOD, rather than UCP2 or UCP3 were available and obtained
from our Gene Transfer Vector Core for use as controls. The adenoviral
human MnSOD was engineered by the core in conjunction with the
laboratory of Dr. John Engelhardt at our institution
(21).
Cell culture adenoviral infection
INS-1 cells were provided by Dr. Claes Wolheim (Geneva,
Switzerland). Cells were seeded into 100-mm dishes for mitochondrial
isolation and 12-well plates for lipid and glucose oxidation. Cells
were grown in RPMI with 10% FBS, 1 mM sodium pyruvate, 4
mM L-glutamine, and 50 µM
2-mercaptoethanol at 37 C under 5% CO2. Cells
were allowed to reach near confluence. Then, cells were infected with
the recombinant UCP or control viral stocks of 1 x
1010 plaque-forming units (pfu)/ml. The efficacy
of infection for varying viral loads was determined by staining for
ß-gal (Fig. 1
). For studies of
respiration and metabolism, virus was applied to cells at 3.1 x
106 pfu/cm2 for 4 h in
the absence of serum. The cells were then washed of virus, 10%
serum-containing medium was added, and the cells were allowed to grow
for 18 h.
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Polarography
Mitochondrial respiration was measured using a Clark miniature
oxygen electrode and small (0.6-ml) volume chamber with stir bar
(Instech Laboratories, Inc., Plymouth Meeting, PA) at 37 C in
respiratory medium (220 mM mannitol, 70 mM
sucrose, 2.5 mM
KH2PO4, 2 mM
MgCl2, 1 mM EDTA, and 2
mM HEPES, pH 7.4) with 0.1% fatty acid-free BSA. Isolated
mitochondria (0.5 mg protein/ml) were incubated in the respiratory
medium, and oxygen consumption was quantified. To determine state 3
respiration, oxygen consumption was recorded with sequential additions
of 5 mM succinate, 0.2 mM ADP, and finally 0.2
µM carbonyl cyanide
p-[trifluoromethoxy]-phenyl-hydrazone to induce maximal
chemical uncoupling. To determine state 4 respiration, 2
µM oligomycin was added to inhibit ATP synthase
before the above sequential additions.
Immunoblotting
Four micrograms of protein per lane were separated on 12.5%
polyacrylamide gels and electroblotted to nitrocellulose membranes
(Millipore Corp., Bedford, MA). Blots were blocked with
5% BSA in Tris-buffered saline with 0.1% Tween-20 (T-TBS) for 40 min
and incubated overnight at 4 C with affinity-purified antibody to UCP2
(1 µg/ml goat anti-UCP2), UCP3 (0.5 µg/ml rabbit anti-UCP3), or
UCP1 (0.5 µg/ml rabbit anti-UCP1) or with unpurified MnSOD (1:4000
dilution of rabbit serum). Blots were washed with T-TBS and exposed to
antigoat or antirabbit horseradish peroxidase-conjugated secondary
antibody at a 1:10,000 dilution in T-TBS for 40 min at room
temperature. Blots were washed again and developed by enhanced
chemiluminescence using a standard kit (ECL, Amersham Pharmacia Biotech, Piscataway, NJ).
The specificity of the antibodies to UCP1 and UCP3 has been
previously documented (22) with competition by
specific (but not by nonspecific) peptide to which these antibodies
were raised, by demonstration of the appropriate tissue
distribution of UCP1 or UCP3 immunoreactivity based on the reported
messenger RNA (mRNA) distribution, and by demonstration of
enhanced immunoreactivity in mitochondrial compared with whole cell
extracts and lack of cytoplasmic immunoreactivity. We also found that
the UCP2 antibody used in the current studies meets the same criteria.
In addition, our current data (Fig. 2)
demonstrate that the UCP2 and UCP3 antibodies show the expected
immunoreactivity with the expressed proteins, albeit with minor
cross-reactivity separable by migration on gel. The specificity of the
rabbit anti-MnSOD antibody has been previously documented
(23).
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Glucose oxidation
Cells were washed and preincubated in RPMI 1640 containing 1.0
mM glucose for 45 min in 12-well plates as described for
the lipid oxidation experiments. Then, 0.6 µCi
D-[U-14C]glucose (SA, 261 mCi/mmol)
and 5 mM glucose were added to each well, and incubation
was continued for 1 h. After incubation,
[14C]CO2 was quantified
by filter trapping as described for the lipid oxidation experiments.
The contents of the wells were neutralized with NaOH, and protein was
determined.
Insulin secretion
Cell cultures were preincubated at 5 mM glucose for
18 h, washed, and incubated for 30 min at 37 C in 1.0 ml modified
Krebs-Ringer bicarbonate buffer (119 mM NaCl, 4.6
mM KCl, 2 mM CaCl2, 1
mM
MgSO4·7H2O, 0.4
mM KH2PO4,
0.05% fatty acid-free BSA, and 20 mM HEPES, pH 7.4) with
0.1 mM glucose. The cells were then incubated with 1.0 ml
modified Krebs-Ringer bicarbonate buffer containing 15 mM
glucose for 30 min at 37 C. The medium was removed and cleared of
nonadherent cells. Insulin content in the supernatant was determined by
RIA for rat insulin (Linco Research, Inc., St. Charles,
MO). Total cell protein was determined for each well.
ATP and ADP concentrations
INS-1 cells were incubated exactly as described above for
assessment of insulin secretion. To prepare cell extracts, cells were
scraped in PBS, suspended in 100 mM Tris with 0.4
mM EDTA (pH 7.75), lysed at 100 C for 3 min, and
centrifuged at 10,000 x g for 60 sec. Half of the
extract (supernatant) was treated with phosphoenolpyruvate and pyruvate
kinase as previously described (24) to convert ADP to ATP.
The ATP content was determined on the treated and untreated
half-extracts by bioluminescence using a kit (ATP determination kit,
A-6608) purchased from Molecular Probes, Inc. (Eugene,
OR). ADP content was calculated as the difference between the two
half-portions.
Statistics
Data were analyzed by ANOVA using Dunnett’s test for multiple
comparisons with a control group.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). At the concentration
used for subsequent expression and metabolic studies of the UCP
constructs (3.1 x 106
pfu/cm2), over 95% of the cells expressed
ß-gal, as evidenced by light microscopy. Infection of INS-1 cells by
the recombinant adenoviruses, Ad5CMV-UCP2 and Ad5CMV-UCP3, enhanced the
mitochondrial content (specific immunoreactivity) of the corresponding
proteins severalfold compared with that in control cells infected with
the same adenovirus containing the bacterial ß-gal gene (Fig. 2).
There was some cross-reactivity of the antibodies against UCP2 and
UCP3, as described in Fig. 2, although this appeared minor in extent.
From the relative migratory positions of UCP2 immunoreactivity in Fig. 2, A and B, and UCP3 immunoreactivity in Fig. 2C, it appears that any
cross-reactive UCP3 protein migrates slightly slower on electrophoretic
gel than UCP2. This is evident comparing UCP2 immunoreactivity between
cells infected with UCP2 and UCP3 in Fig. 2, A and B, and also by
comparing UCP3 immunoreactivity between cells infected with UCP2 and
BAT extract (known to express UCP3 and UCP2 as well as UCP1) or cells
infected with UCP3 in Fig. 2C. Figure 2D shows that infection of INS-1
cells with Ad5CMV-MnSOD results in marked mitochondrial overexpression
of MnSOD compared with expression of the native protein in control
cells infected with Ad5CMVlacZ or Ad5CMV-UCP2. Although BAT UCP3
immunoreactivity (Fig. 2C) and a weak BAT UCP2 immunoreactive signal
(not shown) comigrate exactly with the expressed proteins, there is
still the possibility of cross-reactivity of UCP2 and UCP3 antibodies
with UCP1. However, the issue appears irrelevant in INS-1 cells,
because, using a previously characterized (22) UCP1
antibody, we detected no immunoreactivity in INS-1 mitochondrial
extracts despite strong signals from BAT as a positive control (not
shown).
Adenoviral expression of UCP2 decreased coupling of mitochondrial
oxidation and phosphorylation, as evidenced by a decrease in the ADP/O
ratio (more oxygen consumed per amount of ADP converted to ATP) in
mitochondria from these cells compared with those infected with
Ad5CMVlacZ (Figs. 3 and 4A). State 4
respiration, as manifest by oxygen consumption in the presence of
oligomycin to inhibit ATP synthase, was also significantly greater in
mitochondria isolated from INS-1 cells infected with Ad5CMV-UCP2 (Figs. 3 and 4B). Very similar effects of UCP2 were noted in a completely
separate series of experiments (Fig. 4, C
and D) comparing overexpression of UCP2 to MnSOD, which, unlike
ß-gal, is a mitochondrial inner membrane and matrix protein
(25, 26, 27). Adenoviral UCP3 expression also decreased the
ADP/O ratio and increased state 4 respiration, but these differences
were significant only in comparison to control cells expressing MnSOD
(Fig. 4).
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). As
[14C]oleic acid was labeled at the 1 position,
in molar terms 18 molecules of CO2 generated
represent 1 molecule of completely metabolized oleate. In contrast to
these effects on lipid metabolism, no change was detected in glucose
oxidation after expression of either adenoviral UCP2 or UCP3 (Fig. 6). As
D-[U-14C]glucose was used, the
labeled carbon is variably positioned, and the exact amount oxidized to
CO2 is not known. Hence, the reported molar
amounts of CO2 produced assume complete
metabolism of each molecule of labeled glucose. However, even if this
is not the case, there is no reason to believe that the relative
amounts of CO2 produced between the conditions
studied would be affected.
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are expressed per well.
However, we determined protein in cells exposed to similar conditions
until initiation of the procedures for lipid oxidation. The protein
content of the cells expressing UCP2 and UCP3 was actually less than
that of the cells expressing ß-gal (212 ± 7, 208 ± 7, and
246 ± 6 µg/well for UCP2, UCP3, and ß-gal, respectively;
P < 0.01 for UCP2 and UCP3 compared with ß-gal;
n = 8/group). Thus, it is very unlikely that differences in cell
mass (as reflected by protein) could explain the increase in oleate
oxidation observed for the cells expressing UCP2 and UCP3 compared with
ß-gal.
Insulin release by INS-1 cells expressing ß-gal, UCP2, or UCP3 was
determined in the presence of 15 mM glucose. Adenoviral
UCP2 infection (compared with ß-gal) significantly inhibited insulin
secretion (Fig. 7). Insulin release by
cells expressing UCP3 did not differ from the control value. ATP and
ADP were measured in cells exposed to identical conditions. UCP2
overexpression significantly reduced ATP content (Table 1), whereas UCP3 had a lesser and
nonsignificant effect. As ADP content was also reduced by UCP
overexpression, there were no significant changes in the ATP/ADP ratio
(Table 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the current work we used adenoviral infection to enhance the
expression of rat UCP2 and UCP3 within a rat insulinoma cell line. As
evident in Figs. 1 and 2, high levels of expression could be achieved
in this fashion. Regarding Fig. 2, the issue of specific
immunoreactivity detected by currently available UCP antibodies has
been a matter of some debate. We have documented that our UCP3 antibody
(22) and the UCP2 antibody used here recognize proteins of
expected size based on gel migration; that immunoreactivity was
inhibited by specific competing peptide, but not by nonspecific
peptide; that the immunoreactivity conformed to the appropriate tissue
distribution based upon mRNA distribution; and that immunoreactivity
was increased in mitochondrial fractions compared with whole cell
fractions. Based on the immunoreactivity of the currently expressed
proteins, this issue has now been addressed further. In fact, the
current data do show some cross-reactivity between the UCP2 and UCP3
antibodies. However, the relative levels of immunodetection between the
exogenously expressed and nonexpressed proteins as well as slight, but
clearly evident, differences in UCP2 and UCP3 gel migration enable
resolution of specific UCP2 or UCP3 immunoreactivity.
The major finding reported here is that rat UCP2 overexpression enhanced respiration in rat mitochondria isolated from an insulinoma cell line. Overexpressed UCP2 significantly increased state 4 respiration and decreased the ADP/O ratio or, in other words, increased the amount of oxygen required to consume a given amount of ADP. To our knowledge, this is the first direct demonstration of a role for UCP2 in respiration by isolated mammalian mitochondria from any cell type. Similar effects were observed for UCP3, however, the increase in state 4 respiration and the decrease in ADP/O ratio after UCP3 infection were less in magnitude and only significant in comparison to MnSOD.
Moreover, the changes in mitochondrial respiration resulting from overexpression of UCP2 were associated with significantly increased lipid oxidation to CO2. Thus, our data are consistent with findings by Wang et al. (18) that adenoviral expression of UCP2 in isolated pancreatic islets from Zucker diabetic fatty (ZDF) rats increased palmitate oxidation. Our current studies also showed that UCP3 overexpression as well as UCP2 significantly increased oleate metabolism to CO2, suggesting a role for both UCP homologs in fat oxidation.
Several physiological studies support a role for both UCP3 and UCP3 in
fat metabolism. Conditions in which circulating FFA concentrations are
elevated, including fasting (22, 29, 30) and lipid
infusion (31), are associated with increased UCP3
expression in skeletal muscle. Further, provision of fat by suckling to
newborn mice enhances muscle UCP3 mRNA expression (32)
and, very recently, Clapham et al. (33)
reported that transgenic mice overexpressing UCP3 in skeletal muscle
show a marked reduction in adipose tissue mass. In addition, white
adipose tissue UCP2 mRNA is present at higher levels in obese
ob/ob and db/db mice (5) and can be
enhanced in obesity-resistant, but not obesity-prone, mice by high fat
feeding (34). Also, UCP2 is expressed at higher levels in
brown fat of UCP1 knockout mice with increased fat stores, perhaps to
compensate for the loss of UCP1. Thus, our current findings along with
the above studies support the emerging concept that uncoupling proteins
in some way are important in lipid metabolism. Also, consistent with
this concept are observations that activators of peroxisome
proliferator-activated receptor-
, a known regulator of multiple
genes involved in lipid oxidation, induced the expression of UCP3 gene
in skeletal muscle (35).
Although uncoupling protein homologs may be important to lipid oxidation per se, other proposed functions include thermogenesis and protection against free radical formation (2). Although not ruling out these latter possibilities, our current finding that expressed UCP2 and UCP3 increase lipid oxidation supports a role for these proteins in the former capacity. Whether this occurs as a result of increased oxidation alone or somehow involves enhanced fatty acid transport into mitochondria remains to be elucidated.
In contrast to fat oxidation, we were not able to show a significant effect of UCP overexpression on glucose oxidation, suggesting that UCP2 and UCP3 may be less important in this regard. This seems consistent with observations by Brun et al. (32) that a high fat diet maintains UCP3 mRNA in skeletal muscle of newborn mice, whereas UCP3 message expression decreases if the mice are fed a high carbohydrate diet. On the other hand, Krook et al. (36) noted a correlation between UCP3 mRNA levels in human skeletal muscle and whole body insulin-stimulated glucose uptake in subjects with type 2 diabetes, and Tsuboyama-Kasaoka et al. (37) showed a positive association between GLUT-4 and UCP3 gene expression in gastrocnemius muscle. Thus, more work is needed to clarify the roles of UCPs in relation to carbohydrate metabolism.
We also noted a reduction in insulin released into the culture medium in INS-1 cells exposed to 15 mM glucose. Although we must acknowledge that insulin secretion is not regulated in the same way in insulinoma cell lines as in normal islet ß-cells, our data are consistent with the results of Chan et al. (19), who found that adenoviral expression of UCP2 in islets of normal rats decreased glucose-stimulated insulin release. In contrast, Wang et al. (18) found that adenoviral expression of UCP2 in islets isolated from ZDF rats increased proinsulin and improved glucose-induced insulin secretion. However, this discrepancy may be explained by the effect of UCP on fat oxidation in islets from these rats, as ZDF islets are known to contain large amounts of fat, inducing a lipotoxic state. In fact, in vivo depletion of islet fat as a result of troglitazone treatment of these animals induces UCP2 expression, reduces islet fat, and improves insulin secretion (3, 38).
UCP2 overexpression significantly reduced cell ATP content, but did not change the ATP/ADP ratio consequent to concurrent reduction in ADP. Although presumptive, the decrease in ATP may have resulted from impaired oxidative phosphorylation with inability of increased lipid oxidation to adequately compensate. It is more difficult to explain the reduction in ADP. However, we point out that a similar reduction in ADP was noted by Wang et al. (18) in their above-cited studies of UCP2 overexpression in isolated islets.
In any case, the reduction in ATP may explain the observed decrease in insulin secretion as ATP and/or the ATP/ADP ratio appear important in closing ATP-sensitive K+ channels, a proximal event toward calcium influx, depolarization, and insulin release. Although the ATP/ADP ratio may be of major importance in K+ channel closure (39, 40), it seems plausible that in the presence of a substantial reduction in both ATP and ADP, reduced ATP per se may be important. Alternative to any effect on ATP-sensitive K+, channels, reduced ATP may have decreased insulin release through impairment in energy-dependent insulin granular movement or nonspecific impairment of phosphorylation of critical cell targets.
There are some important limitations to the current studies that also apply to the above-cited previous studies (18, 19) of adenoviral UCP2 expression in pancreatic islets. First, although both adenoviral UCP2 and UCP3 as well the control adenoviral proteins ß-gal and MnSOD are highly expressed, we have not quantified their expression in molar terms. Thus, we are limited to comparing expression of UCP-2 and UCP3 to control adenoviral proteins expressed at the same viral loads. Further, we cannot quantify any of the measured parameters per unit UCP2 or UCP3 expression, and thus, we cannot quantitatively compare the effects of UCP2 and UCP3 to each other. In addition, there are limitations related to the nature of the control adenoviral proteins. We used adenoviral ß-gal and the mitochondrial matrix and inner membrane protein MnSOD as controls. MnSOD was expressed at levels severalfold above basal levels, so our findings are not likely to have simply resulted from excess mitochondrial protein. However, the ideal control would be expressed in the inner membrane in quantitative proportion and in the same structural manner as UCP2 and UCP3. This potentially could be addressed by expressing inactive UCP2 and UCP3 mutants targeted to mitochondria in the same way. However, this will require future efforts toward engineering and characterizing these proteins.
Although we must be cautious about quantitative comparisons, our data suggest that there may be some differences between the overall effects of overexpressed UCP2 and UCP3. UCP2 significantly altered respiration, insulin release, and ATP content. UCP3 had less effect on respiration (only significant in comparison to MnSOD) and had similar, but nonsignificant, directional effects on insulin release and ATP content, whereas both proteins increased lipid oxidation. Thus, the effect of UCP2 may be more prominent in these cells. Although speculative, the reason for this may be that UCP2 is the native UCP in islet ß-cells (41) and was the only endogenous UCP protein detected in our control INS-1 cells by immunoblotting. Hence, overexpressed UCP2 may be more active than UCP3 in these cells, as the transport, insertion, and activity in the mitochondrial membrane of the native protein may be more effective.
In summary, within the limitations discussed, our results show that overexpression of rat UCP2 increases state 4 respiration and reduces the ADP/O ratio in isolated mitochondria from a rat insulinoma cell line. Thus, our findings support a role for this UCP homolog as a mediator of uncoupling in this mammalian cell line. Our data further support the concept that UCP2 and UCP3 are important in fat metabolism and are consistent with the concept that UCP2 may be a factor in regulating ß-cell insulin release.
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Footnotes |
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Received June 22, 2000.
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B
activation. Nat Med 4:698–704[CrossRef][Medline]
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