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Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward, C1A 4P3 Canada
1 Departments of Physiology and Medicine, University of Toronto, Toronto, Ontario, M5S 1A8 Canada
(Requests for offprints should be addressed to C B Chan; Email: cchan{at}upei.ca)
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Abstract |
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Introduction |
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and 1 (Wang et al. 2000a, 2000b), peroxisome-proliferator activated receptors (PPAR)- and -
(Tordjman et al. 2002, Ito et al. 2004), and sterol response element-binding protein-1c (Yamashita et al. 2004) leads to the induction of UCP2 and suppression of GSIS. Conversely, the induction of Sirt1 in ß-cells improves GSIS coincident with the downregulation of UCP2 (Moynihan et al. 2005). However, an important unanswered question is whether the upregulation of endogenous UCP2 coincides developmentally with loss of glucose homeostasis in diabetic models. In several obese-diabetic rodent models examined in adulthood (Kassis et al. 2000, Laybutt et al. 2002), including the ob/ob mouse (Zhang et al. 2001), islet UCP2 mRNA or protein expression is elevated. Insulin secretion in ob/ob mice is elevated but nonetheless insufficient to maintain euglycemia (Zhang et al. 2001). Although increased UCP2 is predicted to contribute to the reduction in GSIS, there is little direct evidence to support this hypothesis. However, when ucp2 is eliminated in ob/ob mice by crossing them with ucp2 knockout mice, first phase insulin secretion is increased and hyperglycemia is reduced (Zhang et al. 2001). No longitudinal studies examining concurrent changes in GSIS with UCP2 protein expression have yet been reported. It is also unclear how short-term reduction of UCP2 affects insulin secretion.
The ob/ob mouse develops mild type 2 diabetes by 8–9 weeks of age, but then demonstrates a phenotypic improvement by 4–5 months of age (Garthwaite et al. 1980, Menahan 1983, Tassava et al. 1992). Leptin induces UCP2 expression in islets (Shimabukuro et al. 1997), yet in the ob/ob mouse the upregulation of UCP2 (Zhang et al. 2001) is clearly independent of leptin. Other factors, such as non-esterified fatty acid (NEFA) may be important regulators of UCP2 transcription and activity in the ß-cell (Lameloise et al. 2001, Medvedev et al. 2002, Koshkin et al. 2003). We hypothesized that the developmental loss of insulin secretion capacity would coincide with changes in UCP2 expression in ob/ob mice. Thus, UCP2 mRNA and protein expression were correlated with metabolic indices and GSIS in 5-, 8- and 16-week-old ob/ob and lean mice. In addition, the effect of acutely reducing UCP2 expression by ~50% on GSIS was examined.
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Materials and Methods |
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Control (C57Bl/6) and obese (ob/ob) female mice obtained from Jackson Laboratories (Bar Harbor, ME, USA) were fed standard rodent chow (Purina 5001; St Louis, MO, USA) and water ad libitum and were housed under standard temperature and light-controlled conditions. All protocols were approved by the University of Prince Edward Island Animal Care Committee, following the Guidelines of the Canadian Council on Animal Care.
Body weights were measured at 5, 8 and 16 weeks of age. Insulin tolerance tests were performed after a 4 h fast, as described (Zhang et al. 2001) in ob/ob mice from all age groups. Lean mice from the 16-week age group served as controls. In a second group of overnight-fasted mice, glucose tolerance and in vivo insulin secretion were assessed following i.p. injection of glucose (Joseph et al. 2002).
Prior to islet isolation, mice were fasted for 4 h, then anesthetized with pentobarbital (60 mg/kg i.p.). A ~0.5 ml blood sample was obtained by cardiac puncture for measurement of serum NEFA (Roche Diagnostics, Laval, QC, Canada), triglyceride (TG; Sigma), and insulin (Crystal Chem, Chicago, IL, USA).
Islet isolation, culture, and insulin secretion and ATP studies
Islets were isolated as described (Zhang et al. 2001) and cultured overnight in Dulbecco’s modified Eagle medium (DMEM) containing 8.3 mmol/l glucose (Kibenge & Chan 1995), unless otherwise indicated. For measurement of insulin secretion under static conditions, the culture medium was replaced with 1.0 ml DMEM plus 0.1% gelatin and glucose concentrations as indicated in the figures. Insulin release and islet insulin content were measured by RIA, using human insulin as standard. For ATP measurement, islets were transferred to fresh DMEM containing 2.8 mmol/l glucose and 0.1% gelatin for 30 min at 37 °C, centrifuged and the medium aspirated. Islets were then incubated in DMEM containing 2.8 or 16.5 mmol/l glucose for 60 min at 37 °C. Following centrifugation, the islet pellets were lysed with 100 µl ATP lysis buffer (ATP-Lite, Perkin–Elmer, Boston, MA, USA) and frozen at –80 °C until assayed for ATP content using the ATP-Lite kit. Protein content of each sample was determined using the Lowry method (Sigma).
Knockdown of ucp2 gene expression
Small interfering RNA (siRNA) against ucp2 mRNA (siUCP2) and control constructs (siControl) were purchased from Ambion (Austin, TX, USA). In a pilot study, the optimal dose of siUCP2 resulting in ~50% knockdown of UCP2 protein expression was determined (not shown). The siRNAs were transfected into intact islets from 8-week-old rats at a dose of 25 pmol per 100 islets with the transfection agent N-(2,3-dioleoyloxy-1-propyl)trimethylammonium methyl sulfate (DOTAP; Sigma) following the suggested protocol (Lakey et al. 2001). The islets were harvested 48 h following transfection for insulin secretion experiments or measurement of UCP2 expression.
Determination of islet UCP2 and PPAR expression
After overnight culture, total RNAwas extracted from 100 islets per mouse using Trizol (Invitrogen), and cDNA was created using oligo-dT primers and the Cloned AMV First Strand cDNA Synthesis Kit (Invitrogen). The ucp2-specific primer sequences for quantitative PCR (qPCR) were reported elsewhere (Joseph et al. 2002). For normalization, ß-actin primers were utilized. Gene expression was quantified using SYBR Green ready-mix for qPCR (Invitrogen). Standards and samples were heated at 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s, annealing at 57 °C for 30 s, and extension at 72 °C for 20 s using a Rotor-Gene qPCR thermocycler (Corbett Research, Sydney, Australia). Relative quantities of qPCR product were determined by comparison to standard curves constructed from a dilution series of a cDNA sample mixture. Melt curve analysis for each primer set revealed only one peak for each product. The size of the qPCR products was confirmed by comparing the size of product with a commercial ladder after agarose gel electrophoresis.
For UCP2 protein expression, 100 islets per sample (cultured overnight) were lysed in LIPA buffer (25 mM Tris-HCl, pH 7.4, 0.1% SDS, 0.1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 1 mM, EDTA, 1 mM Na3VO4, 1 mM PMSF, 10 µg/mL aprotinin and 5 µg/mL leupeptin Kim et al. 2004). Lysate proteins were separated by 10% PAGE and transferred to nitrocellulose membranes (Bio-Rad). Primary antibodies for UCP2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:2500), PPAR (Affinity Bio Reagents/Cedarlane, Hornby, ON, Canada; 1:500), and ß-actin (Sigma; 1:10 000) were applied for 24 h at 4 °C. Membranes were then exposed to appropriate peroxidase conjugated secondary antibodies (1:5000) for 2 h at room temperature. Proteins of interest were detected using enhanced chemiluminescence (Amersham) and imaged on a Kodak Image Station.
Statistical analysis
All data are expressed as means ± S.E.M. Significant differences at P<0.05 were determined using unpaired t-test or two-way ANOVA followed by Bonferroni post hoc tests, where appropriate.
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Results |
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Metabolic profile summaries are provided in Table 1
. Obese mice weighed 0.5- and 2.5-fold more than lean mice at age 5 and 16 weeks of age respectively (P<0.0001 for phenotype; P<0.0001 for age; P<0.0001 for phenotype x age). Blood glucose concentrations were elevated (P<0.05) in the 8-week-old ob/ob mice, but not the 5- or 16-week-old ob/ob mice compared with lean controls. At 5 weeks, plasma insulin was ninefold higher in ob/ob than lean mice. At 8 weeks, insulin secretion was increased by 30-fold compared with lean mice (P<0.001). The insulin:glucose (ins:gluc) ratio is an approximate measure of insulin resistance; the higher the ratio, the more insulin is required to maintain appropriate glucose concentrations. Ins:gluc was constant in lean mice but decreased significantly by 32% with increasing age in ob/ob mice (P<0.05). Serum NEFA concentrations were elevated in 8- and 16-week-old ob/ob mice (P<0.05). A significant difference in serum TG between phenotypes was detected only at 16 weeks of age (P<0.05).
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). Fasting glucose concentrations exceeded 13 mmol/l in ob/ob mice, compared with ~5.5 mmol/l in lean mice (P<0.01). Circulating glucose concentrations in ob/ob mice peaked at ~34 mmol/l 30 min after glucose administration, then declined to fasting concentrations by 2 h. Peak glucose concentrations of ~12 mmol/l occurred 15 min after administration in lean mice. Fasting plasma insulin concentrations were 15-fold higher in ob/ob than lean mice (P< 0.0001, Fig. 1B). The ob/ob mice had profoundly impaired GSIS in vivo, with no significant differences in concentration observed over the 2-h period. Lean mice exhibited a rapid 2.2-fold increase in insulin secretion within 5 min of glucose administration, returning to baseline by 30 min (inset of Fig. 1B).
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) of ob/ob mice were related to changes in tissue insulin sensitivity. The 16-week-old lean mice were more sensitive to insulin than ob/ob mice in any age grouping (Fig. 1C, P<0.001). There was no effect of age on the insulin tolerance of ob/ob mice. Islet and insulin secretion characteristics
Islet enlargement, as indicated by islet insulin content, was not present at 5 weeks of age. At both 8 and 16 weeks of age, the insulin content of ob/ob islets was approximately twofold greater than lean islets (P<0.05, Table 1).
When expressed as percentage of islet content, basal insulin secretion (at 2.8 mmol/l glucose) was significantly elevated in isolated islets from 8- and 16-week-old ob/ob mice compared with lean animals (Fig. 2B and C; P<0.05) but not in 5-week-old mice (Fig. 4A). Absolute insulin secretion was elevated approximately four- to fivefold in the older groups (insets of Fig. 2B and C). Lean islets incubated with 11–22 mmol/l glucose had insulin secretion elevated by four- to sevenfold over basal in all age groups. In contrast, GSIS was observed only at 5 weeks of age in the ob/ob mice (Fig. 2A). In vivo fasting glucose concentrations were also considered in the analysis of in vitro data. Data from the glucose tolerance tests showed that fasting glucose in lean mice was ~5.5 mmol/l compared with >13 mmol/l in ob/ob mice (indicated on Fig. 2B by arrows). In lean islets, raising glucose from 5 to 22 mmol/l increased insulin secretion from 3 to 7.5% of total content. In comparison, when glucose increased from 11 to 22 mmol/l in ob/ob mouse islet incubations, the insulin secretion was nearly constant (from 4 to 4.5% of total content).
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ATP content of isolated, overnight-cultured islets was measured in 8-week-old mice (Fig. 3), concurrent with the onset of impaired glucose homeostasis. Lean islets had a fivefold increase in ATP content in high compared with low glucose (P<0.05). In contrast, ob/ob islets had elevated ATP at low glucose (P<0.05 compared with lean islets) but failed to exhibit a significant increase when exposed to higher glucose.
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Ucp2 mRNA expression was measured by qPCR at 5, 8 and 16 weeks of age. Significant effects of age (P=0.0014), genotype (P=0.0028), and an age x genotype interaction were detected (P=0.0009). The amount of ucp2 mRNA, relative to ß-actin, declined by 60% from 5 to 16 weeks of age in lean islets (Fig. 4A). At 5 weeks of age, ob/ob and lean islet UCP2 expressions were similar, but ob/ob mice aged 8 or 16 weeks had approximately twofold higher islet ucp2 mRNA expression than lean controls (P<0.05).
UCP2 (Fig. 4B) protein expression was quantified by Western blotting. UCP2 expression in ob/ob islets was increased relative to lean islets at age 5 (P=0.004) and 8 weeks (P<0.01) but not at 16 weeks of age. Because the activation of PPAR is reported to induce UCP2 expression, Western blotting for PPAR was also conducted (Fig. 4C). Expression of PPAR protein was similar in ob/ob and lean islets at 8 weeks of age.
Effects of UCP2 knockdown on insulin secretion
Following transfection of siUCP2 or siControl vectors for 48 h, protein expression of UCP2 was quantified by Western blotting for lean (Fig. 5A) and ob/ob islets (Fig. 5B). In both cases, siControl had no effect on UCP2 expression, whereas siUCP2 transfection reduced UCP2 expression by 40–50% (P<0.05).
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, P<0.01). In contrast, siUCP2 transfection had no significant effect on GSIS from ob/ob islets (Fig. 5D). Neither siUCP2 nor siControl vector transfection had anyeffect on islet insulin content (Fig. 5E and F). |
Discussion |
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The long-term absence of leptin, as experienced by the ob/ob mouse, would be expected to elevate insulin secretion (Ceddia et al. 2002). Leptin regulates expression of genes involved in ß-cell fatty acid metabolism and also induces ucp2 transcription in rat islets (Zhou et al. 1997). However, in the ob/ob mouse, the regulation of UCP2 must be independent of leptin. Other factors, such as NEFA, may be important regulators of UCP2 expression and activity in the ß-cell (Lameloise et al. 2001, Koshkin et al. 2003). Since UCP2 induction by overexpression strategies inhibits GSIS, it is important to determine whether impaired insulin response of islets and glucose intolerance in diabetes is associated with an increase in endogenous UCP2.
The present study reiterates the classical pattern of development of insulin resistance and diabetes in ob/ob mice (Garthwaite et al. 1980, Menahan 1983, Tassava et al. 1992). The earliest phenotypical changes were weight gain, insulin resistance and hyperinsulinemia at 5 weeks of age. ß-cell function and insulin content were normal, as was islet ucp2 mRNA expression. However, by 8 weeks of age the ob/ob mice exhibited glucose intolerance with marked fasting hyperinsulinemia and loss of GSIS in vivo and in vitro. Fasting hyperinsulinemia may be partially attributed to islet hyperplasia and hypertrophy, given the 2.5-fold increase in insulin content per islet but is also consistent with the toxic effects of NEFA (Carlsson et al. 1999). We observed increased islet ATP content (normalized to total protein) at low glucose, also seen after NEFA exposure of islets (Carlsson et al. 1999), which is consistent with increased glucokinase activity (Chan 1995) and KATP channel closure at lower glucose concentrations (Chan & MacPhail 1996) found in other obese/diabetic rodents.
Fasting hyperinsulinemia with loss of GSIS is consistent with glucolipotoxicity, the term describing concurrent hyperglycemia and hyperlipidemia, which together impair ß-cell function (Poitout & Robertson 2002). Notably, 8-week-old ob/ob mice had elevated circulating NEFA in addition to hyperglycemia, thereby creating a glucolipotoxic condition. Exposing ß-cells to NEFA with glucose >5 mmol/l causes altered transcription of hundreds of genes, many associated with fatty acid metabolism (Wang et al. 2004). One such gene receiving considerable attention recently because of its putative involvement in the regulation of GSIS is ucp2 (Lameloise et al. 2001, Joseph et al. 2002, 2004, Patane et al. 2002). Our results showed that an increase in UCP2 protein was detectable at 5 and 8 weeks, whereas ucp2 mRNA expression was elevated at 8 and 16 weeks. Therefore, the induction of UCP2 occurs prior to development of impaired GSIS and hyperglycemia, suggesting it may be a causal factor. The findings are also consistent with our previous work showing that knockout of ucp2 in ob/ob mice from conception partially corrects the diabetic phenotype.
The factors that induce UCP2 expression at this time are unknown; however, given the rapid growth of the ob/ob mice during this timeframe, a generalized metabolic stress may contribute. Indeed, insulin resistance and hyperinsulinemia were already present at 5 weeks, indicative of a ß-cell response to metabolic demand. At 8 weeks, the upregulation of ucp2 mRNA in ob/ob mouse islets was not associated with increased PPAR expression. The discordance between mRNA and protein expression of UCP2 suggest that post-transcriptional regulation of mRNA activity or turnover strongly influences protein induction. Other researchers (Pecqueur et al. 2001) previously showed that UCP2 protein is less ubiquitously expressed in tissues than its mRNA. These data confirm that UCP2 protein translation is tightly regulated and may not parallel changes in mRNA transcription as was found in the brain (Ho et al. 2005). Interestingly, in the db/db mouse, the reduction of plasma glucose concentrations normalized GSIS but ucp2 mRNA expression remained elevated (Kjorholt et al. 2005), another example of dissociation of UCP2 mRNA expression and insulin secretion. It has been wondered why UCP2 is expressed in ß-cells when its presence can be detrimental to insulin secretion, but it appears that a strong stimulus is required to elicit upregulation of UCP2 protein and that temporal regulation may occur. For example, the overexpression of hormone-sensitive lipase by ~50-fold in transgenic mice caused a marginal increase in UCP2 protein expression (not quantified) vs a 1.5-fold induction of ucp2 mRNA (Sorhede Winzell et al. 2003).
Lack of induction of UCP2 at later ages does not eliminate it as a potential cause of impaired GSIS in ob/ob islets. In addition to regulating transcription (Medvedev et al. 2002), NEFA strongly activate the uncoupling action of UCP2 in ß-cells by increasing ROS production (Koshkin et al. 2003). Notably, glucose-stimulated ob/ob ß-cells produce significantly more ROS than lean ß-cells (Krauss et al. 2003). Thus, the glucolipotoxic environment is predicted to strongly induce uncoupling in ob/ob ß-cells even if UCP2 expression is normal. The observation that glucose failed to increase ATP concentration in the ob/ob islets is consistent with the uncoupling of respiration. In ob/ob hepatocytes, which express ucp2 (Rashid et al. 1999), proton leak is elevated compared with lean hepatocytes (Chavin et al. 1999, Porter et al. 1999), which do not express ucp2 (Rashid et al. 1999). In islets, reduced ATP production leads to impaired closure of KATP channels, thereby suppressing GSIS (Chan et al. 2001). Interestingly, muscle cells from insulin-resistant but non-diabetic human subjects exhibit reduced insulin-stimulated ATP synthesis (Petersen et al. 2005), pointing to the importance of adequate cellular energy to maintain function in a variety of cell types implicated in diabetes.
The present findings are at odds with our previous demonstration of elevated UCP2 in ob/ob islets at 4 months of age (Zhang et al. 2001). The earlier experiments were performed in male mice. Effects of sex steroids on ucp2 are apparently small in most tissues (Pedersen et al. 2001), but have not been studied in islets. Interestingly, our data indicate that ucp2 mRNA expression declines with increasing age in lean but not ob/ob mice. The elevation of ucp2 mRNA seen in all post-weaning mice may be an adaptation to the high fat diet preweaning, similar to the fatty acid-dependent post-natal increase in UCP3 transcription in skeletal muscle (Brun et al. 1999). Hyperlipidemia, caused byelevated fatty acid synthesis by liver and adipose by 8 weeks in ob/ob mice (Kaplan & Leveille 1981), may prevent further regression of ucp2 mRNA.
Eliminating ucp2 from the ob/ob mouse genome (by crossing lep–/– mice onto the ucp2–/– background) partially ameliorated in vivo glucose intolerance because of enhanced insulin secretion (Zhang et al. 2001). Moreover, a gene-dose effect of UCP2 was apparent, with mice heterozygous for UCP2 (ucp2+/–) secreting more insulin and having lower blood glucose than ucp2+/+ mice (Zhang et al. 2001). Knockdown of UCP2 protein by ~50% using a siRNA approach improved in vitro GSIS in lean islets in the present study, as anticipated. However, acute knockdown of UCP2 by a similar amount in the ob/ob islets failed to improve insulin secretion, suggesting that short-term reduction of UCP2, unless coupled with a return to normal of other genes, whose expression is altered in ob/ob islets, inadequately regulates insulin secretion. To our knowledge, genetic profiling has not been done in islet tissue of ob/ob vs lean mice. However, in one study of ob/ob liver, 3% of >5000 unique cDNAs examined were consistently up- or downregulated (Ferrante et al. 2001). When insulin-secreting cells were treated with NEFA in culture, an average 5% of genes and expressed sequence tags were altered (Wang et al. 2004). Thus, it is not surprising that short-term reduction of a single gene failed to normalize GSIS, given that ob/ob mice have existed without a functional leptin gene since conception, perhaps resulting in altered expression of 3–5% of ß-cell genes.
These studies demonstrated a developmental relationship between UCP2 protein expression and impaired glucose homeostasis in ob/ob mice, with the induction of UCP2 preceding impairment of GSIS. Short-term knockdown of UCP2 did not elicit any improvement in GSIS in ob/ob islets, although GSIS in lean islets was potentiated. These studies provide additional evidence that UCP2 modulates insulin secretion under physiological and pathophysiological conditions.
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Funding |
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References |
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Received 22 December 2005
Received in final form 25 April 2006
Accepted 26 May 2006
Made available online as an Accepted Preprint 16 June 2006
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, n=7) to determine the incremental change in (A) blood glucose and (B) plasma insulin. Animals were overnight fasted prior to administration of 1 g/kg glucose, i.p. Blood glucose concentrations were significantly higher in ob/ob than lean mice at all time points (*P<0.05, 
P<0.01, 
P<0.001). Plasma insulin concentrations were higher in ob/ob mice than lean mice at all time points (P<0.001 for all). The inset in B shows the pattern of insulin responsiveness to glucose for the lean mice, with S.E. of the measurements indicated. (C) Insulin tolerance test results in 16-week-old lean(
) and 16-week old ob/ob mice (
).Blood sampleswere obtained from the tail vein of 4-h fasted mice. Mice were injected with 1 U/kg human insulin at time=0. n=7–8. There was no significant effect of age on the insulin tolerance of ob/ob mice.
