Uncoupling protein 2 (UCP2) may act as an important regulator of insulin secretion. In this study, β-cell function in UCP2-deficient mice was examined after a 45% high-fat diet (HFD) to assess its role during the development of diet-induced type 2 diabetes. HFD-fed UCP2 (−/−) mice have lower fasting blood glucose and elevated insulin levels when compared with wild-type (WT) mice. UCP2 (−/−) mice also have enhanced β-cell glucose sensitivity compared with WT mice after HFD, a result that is due in part to the deterioration of glucose responsiveness in WT mice. HFD-fed UCP2 (−/−) mice have increased insulin secretory capacity as a result of increased pancreatic β-cell mass and insulin content per islet. Islets from WT mice exposed to 0.5 mmol/l palmitate for 48 h have significantly reduced mitochondrial membrane potential, ATP concentrations, and glucose responsiveness compared with UCP2 (−/−) islets, suggesting that elevated UCP2 in WT mice increases proton leak and decreases mitochondrial ATP production. Highly increased carnitine palmitoyl transferase-1 gene expression in UCP2 (−/−) mice is suggestive of enhanced fatty acid oxidizing capacity, particularly after HFD stress. These results further establish UCP2 as a component in glucose sensing and suggest a possible new aspect of UCP2 function during the progression of type 2 diabetes.

Oxidative metabolism in the β-cell produces NADH and FADH2, which donate electrons to the electron transport chain, leading to the generation of a proton-motive force that drives ATP production catalyzed by ATP synthase. The production of mitochondrial ATP is critical for glucose-stimulated insulin secretion (GSIS) (1,2). Given the importance of oxidative metabolism in β-cell glucose sensing and insulin secretion, it is important to identify the key proteins that regulate mitochondrial ATP production. Uncoupling proteins (UCPs) (3,4), of which UCP2 is the only member found in islets thus far (5), are thought to fulfill such a role. These proteins are localized to the inner mitochondrial membrane and have a high degree of homology (3,4).

The functions of UCP2 in any tissue, including islets, have not been established. Data from several laboratories support a role for UCP2 as a “typical” uncoupler (4,6,7) that modulates the efficiency of ATP production (3,4). We hypothesized that by regulating β-cell energy levels, insulin secretion would be enhanced in the absence of UCP2 (8), a concept verified by the demonstration of enhanced insulin secretion in UCP2 knockout (−/−) mice. The induction of UCP2 by high-fat diet (HFD) (9) or free fatty acid (FFA) exposure (10) may contribute to so-called lipotoxicity, wherein GSIS is suppressed. Alternatively, induction of UCP2 may be part of a cellular lipid detoxification effort (11), along with fat-metabolizing enzymes such as carnitine palmitoyl transferase-1 (CPT-1). A third possibility is that UCP2, by dissipating the mitochondrial proton-motive force, decreases reactive oxygen species (ROS), thereby promoting cell survival under a stress such as HFD. The latter is supported by data from the UCP2 knockout mouse, which has elevated ROS formation when exposed to a pathogen (12). Although each outcome is theoretically plausible, the actual contribution of UCP2 to islet dysfunction after HFD is unknown. The UCP2 (−/−) mouse provides a unique model for investigating these possibilities. The present data demonstrate that HFD UCP2 (−/−) mice maintain superior pancreatic glucose responsiveness over wild-type (WT) mice in vivo and in situ at least partly caused by an expanded β-cell secretory capacity.

Animals.

Male and female UCP2 (WT) or (−/−) mice were used in the present study and were bred from lines generated previously, as described by us (8). At 4 months of age, half of the mice were placed on either a control diet (Rodent Diet #8664; Harlan Teklad, Madison, WI) or an HFD (#D12451; Research Diets, New Brunswick, NJ). The fat source was lard and composed 45% of the total calories. The mice remained on the HFD or control diet for 4.5 months.

Blood and plasma measurements.

Blood glucose levels were measured using a Lifescan Elite glucose meter (Lifescan, Toronto, ON, Canada). Plasma insulin levels were determined using a rat insulin enzyme-linked immunosorbent assay kit (Crystal Chem, Chicago, IL). Plasma glucagon levels were measured by radioimmunoassay (Linco, St. Charles, MO). Plasma FFAs were measured using a NEFA C FFA kit (Wako Chemicals USA, Richmond, VA). Plasma triglycerides were measured using a BioScanner 2000 Triglyceride system (Polymer Technologies Systems, Indianapolis, IN). Intraperitoneal glucose tolerance tests (IPGTTs) and insulin tolerance tests (ITTs) were performed as previously described (8). The area under the curve (AUC) analysis represents the AUC independent of basal values.

Pancreatic islet measurements.

Pancreatic islets were isolated as previously described (8). RNA was extracted from cultured islets using TRIzol reagent (Gibco BRL) following the manufacturer’s instructions. Pancreatic insulin and glucagon contents were determined as previously described (13,14).

Immunocytochemistry.

Pancreatic tissues were fixed in Bouin’s fixative (pH 7.2) for 45 min and embedded in paraffin. Sections were prepared at three levels of the pancreas separated by 100 μm. For insulin staining, the primary antibody was guinea pig anti-insulin (1:1,000; RA Pederson, Vancouver, BC, Canada) and the secondary was rabbit anti-guinea pig peroxidase (1:100; Dako, Mississauga, ON). Slides were labeled with Signet USA Level 2 labeling reagent (Ultra Streptavidin-Horseradish Peroxidase Complex) and developed with Nova Red (Vector Labs, Burlington, ON, Canada). For β-cell replication, 100 mg/kg 5-bromo-2′deoxyuridine (BrdU; Sigma) was injected intraperitoneally 6 h before pancreas removal. After staining for insulin, sections were stained for BrdU with a monoclonal BrdU antibody (Clone IU-4; Caltag Labs, Burlingame, CA). The secondary antibody was biotinylated horse anti-mouse linking antibody (Vector Labs). The labeling reagent was Signet Ultra Streptavidin detection system-Alkaline phosphatase (Signet, Denham, MA). The substrate was BCIP/NBT (Dako). The slides were then counterstained with hematoxylin. For β-cell apoptosis, sections were stained using ApopTag Peroxidase In Situ Apoptosis Detection Kit (Intergen, Purchase, NY), with addition of a 15-min microwave step (medium power) before the proteinase K step. Data were expressed as percentage of positive β-cells for either BrdU or apoptosis. At least 5,000 insulin-positive cells were counted for each treatment. Apoptotic nuclei were identified by both presence of brown nuclei and morphological analysis (15). The percentage of β-cell area in the pancreas was calculated by dividing the area of all insulin-positive cells in one section by the total area of this section and then multiplying this ratio by 100.

Perfused pancreas.

Mice were fasted overnight (15–18 h) and anesthetized with 80 mg/kg sodium pentobarbital intraperitoneally. The surgical procedure for the perfusion of the pancreas was modified from that described previously (16,17). In brief, PE 50 tubing (Intramedic, Parsippany, NJ) was used to cannulate the dorsal aorta and the hepatic portal vein. The perfusate was a Krebs-Ringer 2% BSA glucose 3% Dextran solution (17). The solution was gassed with 95% O2/5% CO2 to achieve a pH of 7.4 and heated to 37°C. The glucose concentration was delivered as a linear gradient between 1.4 and 20 mmol/l over a 55-min period. Samples were stored at −20°C until assayed for insulin, as described (18).

Islet studies with palmitic acid.

Medium containing palmitic acid (Sigma) was prepared as a stock solution in 5% fatty acid free BSA (Sigma) and diluted to 0.5 mmol/l on the day of the experiment. Control medium was prepared from a stock solution that did not contain palmitic acid. The FFA concentration of the stock was confirmed by analysis with a NEFA C kit. Islets were incubated for 48 h with or without palmitic acid in the presence of 8.3 mmol/l glucose, after which insulin secretion or ATP concentrations were determined. ATP was assayed as previously described (19). Briefly, islets were extracted in groups of 50 islets with 0.1 mol/l NaOH/0.5 mmol/l EDTA. ATP measurements were performed using a bioluminescent assay kit (Sigma). Mitochondrial membrane potential was measured in isolated islets using JC-1 (Molecular Probes, Eugene, OR) (20). Islets from WT or UCP2 (−/−) mice were loaded with 10 μg/ml JC-1 for 20 min at room temperature in a 96-well fluorescent plate reader (excitation 485, emission 590). Results were normalized by DNA content that was estimated with SYBR Green I dye (Sigma) in islets extracted as according to Laird et al. (21).

Quantification of gene expression.

Probes for UCP2, CPT-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to quantify the level of gene expression in isolated WT and UCP2 (−/−) mouse islets (Table 1). UCP2- and GAPDH-specific fluorescent probes were obtained from Biosearch Technologies (Novato, CA). UCP2 and GAPDH expression levels were measured simultaneously using a Taqman Universal PCR master mix and an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Branchburg, NJ). All mRNA levels were quantified using a standard curve of a known amount of mouse cDNA (cDNAs were created from forward and reverse primers; Table 1). All cDNA PCR products that were used in the standard curve were purified using a MinElute PCR Purification Kit (Qiagen, Mississauga, ON, Canada). Quantification of CPT-1 gene expression levels was performed using a SYBR green detection system (SYBR Green PCR Master Mix, Applied Biosystems). The real-time PCR conditions were 2 min at 50°C, 10 min at 95°C, and then 40 cycles for 15 s at 95°C and 1 min at 53°C. Melt curve analysis demonstrated that each primer set described in Table 1 amplified a single predominant product with a distinct melting temperature (Tm), and the size of the PCR products was confirmed by running the PCR products on a 1.2% agarose gel. UCP2 and CPT-1 gene levels were corrected for GAPDH levels.

Statistics.

Statistical significance was assessed by using either Student’s t test or one-way or two-way ANOVA for repeated measures followed by multiple Bonferroni comparisons using a Statistical Analysis System program (SAS). All data are expressed as means ± SE.

In vivo measurements of body weight, blood glucose, insulin, glucagon, FFAs, and triglycerides.

All WT and UCP2 (−/−) mice on HFD gained a significant amount of weight during the treatment (Table 2). The net weight gain of UCP2 (−/−) mice was greater than that of WT mice (P < 0.05; Table 2). There were no differences in fasting blood glucose concentrations for control diet WT and UCP2 (−/−) mice (Fig. 1A). HFD WT mice significantly increased their fasting blood glucose compared with control diet WT mice, but this increase did not occur in UCP2 (−/−) mice (Fig. 1A). UCP2 (−/−) mice also had significantly lower fed blood glucose concentrations compared with WT mice fed the control diet (P < 0.01) and fed the HFD (P < 0.001; Fig. 1B). Fasting insulin concentrations in the control diet UCP2 (−/−) mice were significantly (P < 0.01) higher than the control diet WT mice as previously reported (8) (Fig. 1C). HFD fasting plasma insulin concentrations rose significantly in both WT (P < 0.001) and UCP2 (−/−) mice (P < 0.01) compared with the control diet fed animals. However, the increase in insulin secretion was significantly (P < 0.01) higher in HFD UCP2 (−/−) mice compared with HFD WT mice (Fig. 1C). Similar results were found with fed insulin values (Fig. 1D).

Control diet fasting plasma FFAs were not different between UCP2 (−/−) mice and WT mice. HFD WT mice had significantly (P < 0.05) higher fasting FFA concentrations than their control diet counterparts; however, UCP2 (−/−) mice did not (Table 2). Fed plasma FFA concentrations of control diet fed mice were not different; however, HFD-fed plasma FFAs were significantly elevated in both groups. Plasma triglycerides were not significantly different from each other in control diet fed mice. However, HFD plasma triglycerides were only significantly elevated in HFD WT mice compared with control diet mice (Table 2). Immunoreactive plasma glucagon levels in the control diet and HFD mice were not different between the two groups. HFD WT and UCP2 (−/−) mice had significantly increased plasma glucagon concentrations (P < 0.05; Table 2).

Blood glucose and insulin concentrations after an intraperitoneal glucose load.

Mice were assessed for glucose tolerance after an IPGTT. UCP2 (−/−) mice had significantly (P < 0.05 to P < 0.001) lower blood glucose concentrations at 10, 20, and 30 min after glucose load than WT mice on HFD (Fig. 2A). The AUC for the blood glucose responses of control diet mice was significantly lower for UCP2 (−/−) mice than WT mice (277.8 ± 15.8 vs. 406.1 ± 13.5 mmol · l−1 · 120 min−1, respectively; P < 0.001). Of importance, for HFD mice, all time points from 10 to 120 min after the glucose load were significantly (P < 0.01) lower for UCP2 (−/−) mice compared with WT mice (Fig. 2B). HFD UCP2 (−/−) mice had a 32% lower AUC than WT mice (526.1 ± 53.6 vs. 789.1 ± 55.6 mmol · l−1 · 120 min−1, respectively; P < 0.05).

The plasma insulin response was also assessed during the IPGTT. Insulin concentrations were measured at 0, 10, 30, and 120 min after glucose challenge. Control diet UCP2 (−/−) insulin responses were significantly elevated at 0, 10, and 30 min compared with control diet WT mice (Fig. 2C). Control diet UCP2 (−/−) mice had significantly higher AUC for 0–30 min than WT mice (24.3 ± 1.6 vs. 14.5 ± 1.2 ng insulin/30 min, respectively; P < 0.05). A similar trend was found in the HFD animals, but the peak insulin response was further exaggerated in the UCP2 (−/−) mice (Fig. 2D). For insulin, UCP2 (−/−) mice had a twofold higher AUC for 0–30 min than WT mice (42.2 ± 6.0 vs. 21.4 ± 2.3 insulin/30 min, respectively; P < 0.05).

ITT.

Given that UCP2 (−/−) mice had markedly elevated plasma insulin levels, one might expect that they could develop insulin resistance, particularly when fed an HFD. To test this possibility and whether changes in the glucose response to the IPGTT were due to changes in insulin sensitivity, we performed an ITT. Control diet mice showed no difference in their ITT responses (Fig. 2E). However, HFD WT mice were significantly more insulin-resistant than UCP2 (−/−) mice (Fig. 2F).

In situ pancreatic insulin secretion.

For assessing β-cell glucose responsiveness in control diet and HFD animals, glucose was presented to the isolated pancreas as a gradient (1.4–17.4 mmol/l; Fig. 3). The control diet UCP2 (−/−) mice showed a significant increase in their glucose sensitivity when compared with control mice (Fig. 3A). The HFD-fed WT mice had a rightward shift in their glucose sensitivity as compared with control diet WT mice (Fig. 3A and B); however, this did not occur in the UCP2 (−/−) mice.

Islet morphology.

On control diet, pancreatic insulin content was not different between WT and UCP2 (−/−) mice (Table 2). On the HFD, UCP2 (−/−) mice had significantly higher pancreatic insulin content (P < 0.001; Table 2) and islet insulin content corrected for cell number (P < 0.01) than WT mice (Fig. 4A). The glucagon content of the pancreas was not different for control diet or HFD mice (Table 2). To examine the possible mechanism of the increased insulin content, we performed β-cell apoptosis, replication, and islet morphometric analyses. The relative β-cell area of the pancreas was significantly higher in both control diet and HFD-fed UCP2 (−/−) mice (Figs. 4B and 5A and B). HFD UCP2 (−/−) mice had a 2.3-fold higher relative β-cell area compared with HFD control diet mice. There was also an increase in islet number (Fig. 4C) as well as a significant increase in average size of the islets in HFD UCP2 (−/−) mice (Fig. 4D). Apoptosis was two times higher in control diet UCP2 (−/−) mice (P < 0.05; Figs. 4E and 5C and D). After the HFD, apoptosis significantly increased in both WT and UCP2 (−/−) mice (P < 0.001 and P < 0.05, respectively). The percentage of BrdU-positive β-cells was significantly elevated in control diet UCP2 (−/−) mice (Figs. 4F and 5E and F). HFD significantly increased the percentage of BrdU-positive cells in UCP2 (−/−) mice as compared with control diet UCP2 (−/−) mice as well as HFD WT mice.

Short-term effects of palmitic acid on islet function.

To examine further the effects of the HFD in mice, we implemented a 48-h in vitro 0.5 mmol/l palmitic acid incubation of islets from either WT or UCP2 (−/−) mice. Before and after FFA treatment, there was no significant difference in islet insulin content among the groups. The WT mice showed elevated basal but attenuated GSIS, whereas UCP2 (−/−) mouse islets showed elevated basal and enhanced GSIS (Fig. 6A and B following palmitate treatment). Islet ATP levels and mitochondrial membrane potential were significantly (P < 0.05) reduced in palmitate-treated WT mouse islets as compared with control islets, whereas UCP2 (−/−) mouse islets showed no significant reduction in ATP levels (Fig. 6C) or mitochondrial membrane potential (Fig. 6D).

Quantification of gene expression.

To assess the effect of HFD on UCP2 and CPT-1 gene expression in islet RNA, we performed real-time PCR. No detectable UCP2 expression was found in UCP2 (−/−) mice. HFD WT mice, however, had a 6.8-fold increase in UCP2 mRNA compared with their control diet WT counterparts (Table 3). CPT-1 is a key enzyme involved in transporting fatty acids into the mitochondria for β-oxidation and can act, in some situations, to remove lipid accumulation in cells. CPT-1 gene expression was also significantly increased in both WT (P < 0.05) and UCP2 (−/−) mice (P < 0.01) after HFD, but the level of mRNA expression was higher in the UCP2 (−/−) mice (P < 0.05; Table 3).

The detrimental effects of a long-term HFD on β-cell function and insulin sensitivity in mice have been clearly demonstrated and can lead to type 2 diabetes (22,23). The present study focused on comparing the effects of a 4.5-month HFD on glucose homeostasis and pancreatic islet function in WT and UCP2 (−/−) mice. Previous studies have shown that the loss of expression of UCP2 improved glucose tolerance in ob/ob mice; therefore, we hypothesized that UCP2 (−/−) mice would likewise tolerate HFD better than WT mice. We have shown that giving mice an HFD led to elevated plasma triglyceride and fasting FFA concentrations in WT mice, as has been shown in other models of rodent fed diets with elevated fat content (2426).

As expected, administration of HFD to WT mice was associated with a number of metabolic perturbations, notably insulin resistance and glucose intolerance (Fig. 2). HFD WT mice were more insulin-resistant than HFD UCP2 (−/−) mice. The lack of insulin resistance seen in HFD UCP2 (−/−) mice could be secondary to improved control of diabetes. Although plasma insulin concentrations of HFD WT mice increased in both fasting and postglucose states, the additional secretion was insufficient to maintain a normal glucose profile after an IPGTT. Despite this, HFD UCP2 (−/−) mice maintained lower blood glucose levels than HFD WT mice in both fasting and fed states. It seems that UCP2 (−/−) mice achieved better glucose homeostasis at least partly by a compensatory increase in insulin secretion (Fig. 2D). Therefore, the present data are consistent with a protective effect of low UCP2 on insulin secretion capacity, even when stressed by an HFD. The enhancement seems to be due, in part, to an effect on first-phase insulin secretion in UCP2 (−/−) mice (Fig. 2D). This is further supported in the perfused pancreas model, which shows the maintenance or a small enhancement of glucose sensitivity (Fig. 3).

One mechanism by which pancreatic islet function was preserved in the UCP2 (−/−) mice on HFD was an increase in their insulin secretory capacity. Islet insulin content was significantly higher in HFD UCP2 (−/−) mice than in HFD WT mice. Thus, despite overall higher secretion in vivo, the UCP2 (−/−) mice are able to maintain a sufficient insulin-secretory response when subjected to a long-term HFD. Saturated FFA (the major FFA in lard) inhibits insulin biosynthesis in vitro (2731).

The total β-cell mass of HFD UCP2 (−/−) mice was also increased; this likely allowed HFD UCP2 (−/−) mice to maintain glucose responsiveness, whereas this did not occur in WT mice. Glucose has been proposed to be one stimulus for increasing pancreatic β-cell mass. Short-term hyperglycemia can stimulate β-cell proliferation, decrease apoptosis, and promote β-cell neogenesis (3235). The number of functionally intact β-cells in the islet is important for the development, course, and outcome of diabetes. The total β-cell mass reflects the balance between the renewal and loss of these cells (36). Several studies in vitro have so far confirmed the mitogenic action of a high extracellular glucose concentration (3235,37). However, there is no explanation of how glucose stimulates these changes or how β-cells lose this ability during chronic hyperglycemia. The current data suggest that hyperglycemia per se is not essential for stimulating β-cell replication or proliferation. Instead, enhanced glucose metabolism permitted by higher levels of coupled oxidative phosphorylation may be the stimulus for β-cell proliferation and neogenesis. Thus, the expression of UCP2 may limit the ability of the β-cell to compensate for increasing insulin resistance by increasing β-cell mass. The mechanism by which UCP2 may perform this task is to limit the production of a metabolic stimulus, such as ATP, for increasing β-cell mass. ATP, for example, could be working through the mammalian target of rapamycin to stimulate β-cell proliferation (38). Work is currently under way in our laboratory to explore further this potential relationship.

CPT-1 activity is critical to promoting FFA acid oxidation in cells; moreover, its expression is induced in β-cells by FFA exposure (39). Preventing transport of FFAs to the mitochondrial matrix by use of CPT-1 inhibitors prevents the inhibition in GSIS observed after exposure of islets to long-chain FFAs (28). Although gene expression levels do not necessarily equate to functional activity, it is of interest to point out that UCP2 (−/−) mice fed control diet had elevated CPT-1 mRNA expression compared with WT islets. After HFD, the difference increased. The implication of this finding is that islets from UCP2 (−/−) mice may be able to oxidize greater amounts of FFA compared with WT mice. This would prevent accumulation of islet triglyceride, which is a key marker of lipotoxicity (40). CPT-1 induction could be the dominant mechanism by which islets from UCP2 (−/−) mice maintain their high level of functionality under stress. Alternatively, it could be a complementary mechanism to our primary hypothesis: that maintenance of higher levels of ATP (whether via glucose or fat metabolism) is key to enhanced β-cell glucose responsiveness and proliferative capacity. Additional studies, such as the measurement of the ATP/ADP ratio and a comprehensive assessment of glucose and FFA metabolism, are required to quantify the relative contributions to islet health from these two possible outcomes in UCP2 (−/−) mice. It is also important to note that UCP2 and CPT-1 are coordinately upregulated by FFA in WT mice, yet the upregulation of CPT-1 alone does not prevent the loss of β-cell function. Moreover, UCP2 is downregulated in β-cells exposed to high glucose, whereas CPT-1 expression remains unchanged (41); therefore, simply examining the ratio between the two entities does not predict the outcome on β-cell metabolism.

The concept of lipotoxicity in β-cells is attractive as an explanation for the loss of GSIS in obesity-induced diabetes. However, evidence is accumulating that fats exert most of their deleterious effects on islets only when glucose is also elevated; this concept is termed “glucolipotoxicity” (42). Thus, only in hyperglycemic rats did HFD cause a loss of GSIS concomitant with induction of UCP2 (43). Our current results are similar because UCP2 (−/−) mice that maintained fasting euglycemia were also resistant to HFD. It is interesting that in the Briaud et al. study (43), HFD did not increase islet triglyceride, suggesting that fat accumulation is not a necessary consequence of exposure of islets to high fat.

As a consequence of its ability to dissipate the proton-motive force, UCP2 is predicted to reduce the generation of ROS (44). This hypothesis is supported by data showing that macrophages from UCP2 (−/−) mice produce more ROS when challenged with infection (12) and that UCP2 is activated by ROS in mitochondria from MIN6 cells (7). An increase in ROS can lead to β-cell apoptosis (45); therefore, the presence of elevated UCP2 may reduce ROS production by dissipating the excess energy. This excessive energy metabolism, as seen with lipid exposure, may induce multiple mechanisms that lead to cell damage, such as ceramide formation (46) and apoptosis (47) in islets. At the outset, it was unclear whether the positive effects of the lack of UCP2 on insulin secretion would be negated by the putative greater increase in apoptosis in HFD UCP2 (−/−) mice. However, this was not the case, at least in the time frame studied here. These results also suggest that inhibition and not activation of UCP2 could be an effective means of improving β-cell function in individuals with type 2 diabetes. Physiologically, an advantage of increasing the expression of UCP2 may be to prevent long-term effects of excessive fat expression often associated with increasing ROS production; however, as we have shown, UCP2 negatively impacts insulin secretion and the compensatory increase in pancreatic β-cell mass.

In conclusion, UCP2 (−/−) mice have enhanced β-cell responsiveness to a glucose challenge in vivo after an HFD. These mice also have preserved islet sensitivity to glucose, enhanced insulin content, and increased β-cell mass after an HFD. These factors seem to contribute to an increased insulin response to glucose in vivo that produces improvements in glucose tolerance in the face of insulin resistance.

FIG. 1.

Fasting and fed glucose and insulin values for control diet and HFD WT and UCP2 (−/−) mice (n ≥ 30). A: Fasting blood glucose. B: Fed blood glucose. C: Fasting plasma insulin values. D: Fed plasma insulin values. [cjs2108], WT mice; [cjs2112], UCP2 (−/−) mice.

FIG. 1.

Fasting and fed glucose and insulin values for control diet and HFD WT and UCP2 (−/−) mice (n ≥ 30). A: Fasting blood glucose. B: Fed blood glucose. C: Fasting plasma insulin values. D: Fed plasma insulin values. [cjs2108], WT mice; [cjs2112], UCP2 (−/−) mice.

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FIG. 2.

Blood glucose and plasma insulin values during an IPGTT and ITT of WT and UCP2 (−/−) mice under control diet and HFD conditions. A and B: IPGTT blood glucose responses in control diet (A) and HFD (B) (n = 30). C and D: IPGTT plasma insulin values in control diet (C) and HFD (D) (n = 20). E: ITT in control diet mice (n = 20). F: ITT in HFD mice (n = 20). ▪, WT mice; ▴, UCP2 (−/−) mice. *P < 0.05; **P < 0.01; ***P < 0.001.

FIG. 2.

Blood glucose and plasma insulin values during an IPGTT and ITT of WT and UCP2 (−/−) mice under control diet and HFD conditions. A and B: IPGTT blood glucose responses in control diet (A) and HFD (B) (n = 30). C and D: IPGTT plasma insulin values in control diet (C) and HFD (D) (n = 20). E: ITT in control diet mice (n = 20). F: ITT in HFD mice (n = 20). ▪, WT mice; ▴, UCP2 (−/−) mice. *P < 0.05; **P < 0.01; ***P < 0.001.

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FIG. 3.

Perfused pancreas data of WT and UCP2 (−/−) mice on control diet or HFD. Mice were perfused with increasing concentrations of glucose starting at 1.4 mmol/l glucose and ending at 20 mmol/l (n = 10 each). The rate of glucose increase was 0.3 mmol · l−1 · min−1, and the pancreas was perfused over a 55-min period. A: Control diet mice. B: HFD mice. ▪, WT mice; ▴, UCP2 (−/−) mice.

FIG. 3.

Perfused pancreas data of WT and UCP2 (−/−) mice on control diet or HFD. Mice were perfused with increasing concentrations of glucose starting at 1.4 mmol/l glucose and ending at 20 mmol/l (n = 10 each). The rate of glucose increase was 0.3 mmol · l−1 · min−1, and the pancreas was perfused over a 55-min period. A: Control diet mice. B: HFD mice. ▪, WT mice; ▴, UCP2 (−/−) mice.

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FIG. 4.

Islet morphological analysis. A: Islet insulin content corrected for DNA content. Insulin was extracted using acid/ethanol from groups of 20 islets (n = 10). B: Relative β-cell area (n = 6). C: Number of islets per mm2 pancreas (n = 6). D: Average islet size (mm2; n = 6). E: Apoptosis in β-cells only (percentage β-cells; n = 6). F: BrdU-positive cells in β-cells only (percentage β-cells; n = 6). [cjs2108], WT mice; [cjs2112], UCP2 (−/−) mice.

FIG. 4.

Islet morphological analysis. A: Islet insulin content corrected for DNA content. Insulin was extracted using acid/ethanol from groups of 20 islets (n = 10). B: Relative β-cell area (n = 6). C: Number of islets per mm2 pancreas (n = 6). D: Average islet size (mm2; n = 6). E: Apoptosis in β-cells only (percentage β-cells; n = 6). F: BrdU-positive cells in β-cells only (percentage β-cells; n = 6). [cjs2108], WT mice; [cjs2112], UCP2 (−/−) mice.

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FIG. 5.

Islet morphological analysis. A and B: Representative images for relative β-cell area (HFD WT mice [A], HFD UCP2 [−/−] mice [B]). Magnification ×4. C and D: Representative images for apoptosis (HFD WT mice [C], HFD UCP2 [−/−] mice [D]). Magnification ×40. Arrows indicate apoptotic positive cells. E and F: Representative images for BrdU-positive cells (HFD WT mice [E], HFD UCP2 [−/−] mice [F]). Magnification ×40. Arrows indicate BrdU-positive cells.

FIG. 5.

Islet morphological analysis. A and B: Representative images for relative β-cell area (HFD WT mice [A], HFD UCP2 [−/−] mice [B]). Magnification ×4. C and D: Representative images for apoptosis (HFD WT mice [C], HFD UCP2 [−/−] mice [D]). Magnification ×40. Arrows indicate apoptotic positive cells. E and F: Representative images for BrdU-positive cells (HFD WT mice [E], HFD UCP2 [−/−] mice [F]). Magnification ×40. Arrows indicate BrdU-positive cells.

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FIG. 6.

Insulin, ATP, and mitochondrial membrane potential responses after incubating WT or UCP2 (−/−) mouse islets with or without 0.5 mmol/l palmitic acid for 48 h. A: Islets insulin responses to glucose for WT islets (n = 5 groups of 10 islets). B: Islets insulin responses to glucose for UCP2 (−/−) islets (n = 5 groups of 10 islets). □, media without palmitic acid; ▪, islets incubated with 0.5 mmol/l palmitic acid. C: ATP levels in control media and palmitic acid treated islets. [cjs2108], WT mice; [cjs2112], UCP2 (−/−) mice (n = 4 groups of 50 islets). D: Mitochondrial membrane potential in control media and palmitic acid treated islets. [cjs2108], WT mice; [cjs2112], UCP2 (−/−) mice (n = 4 groups of 20 islets).

FIG. 6.

Insulin, ATP, and mitochondrial membrane potential responses after incubating WT or UCP2 (−/−) mouse islets with or without 0.5 mmol/l palmitic acid for 48 h. A: Islets insulin responses to glucose for WT islets (n = 5 groups of 10 islets). B: Islets insulin responses to glucose for UCP2 (−/−) islets (n = 5 groups of 10 islets). □, media without palmitic acid; ▪, islets incubated with 0.5 mmol/l palmitic acid. C: ATP levels in control media and palmitic acid treated islets. [cjs2108], WT mice; [cjs2112], UCP2 (−/−) mice (n = 4 groups of 50 islets). D: Mitochondrial membrane potential in control media and palmitic acid treated islets. [cjs2108], WT mice; [cjs2112], UCP2 (−/−) mice (n = 4 groups of 20 islets).

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TABLE 1

Primer sets for real-time PCR analysis

Primer NameSequenceProduct size
RT-UCP2 primer forward CAG CCA GCG CCC AGT ACC 127 bp 
RT-UCP2 primer reverse CAA TGC GGA CGG AGG CAA AGC 127 bp 
UCP2 specific fluorescent probe 5′ 6-FAM TGA GGG TCC ACG CAG CCT CTA — 
 CAA TG BHQ-13′  
RT-GAPDH primer forward GTG GCA GTG ATG GCA TGG AC 193 bp 
RT-GAPDH primer reverse CAG CAC CAG TGG ATG CAG GG  
GAPDH specific fluorescent probe 5′ HEX TTG GCA TTG TGG AAG GGC TCA — 
 TGA CCA CAG BHQ-13′  
RT-CPT1 primer forward CAG GAT TTT GCT GTC AAC CTC 161 bp 
RT-CPT1 primer reverse GAG CAT CTC CAT GGC GTA G  
UCP2 cDNA forward GAG GTA GCA GGA AAT CAG AAT C 1176 bp 
UCP2 cDNA forward ATG GAG AGG CTC AGA AAG G  
GAPDH cDNA forward CAA CTA CAT GGT CTA CAT GTT CC 724 bp 
GAPDH cDNA reverse CAA CCT GGT CCT CAG TGT AG  
CPT1 cDNA primer forward GCA TCA TCA CTG GTG TGT TC 671 bp 
CPT1 cDNA primer reverse GTG TTG CCA GCT CTC GCT G  
Primer NameSequenceProduct size
RT-UCP2 primer forward CAG CCA GCG CCC AGT ACC 127 bp 
RT-UCP2 primer reverse CAA TGC GGA CGG AGG CAA AGC 127 bp 
UCP2 specific fluorescent probe 5′ 6-FAM TGA GGG TCC ACG CAG CCT CTA — 
 CAA TG BHQ-13′  
RT-GAPDH primer forward GTG GCA GTG ATG GCA TGG AC 193 bp 
RT-GAPDH primer reverse CAG CAC CAG TGG ATG CAG GG  
GAPDH specific fluorescent probe 5′ HEX TTG GCA TTG TGG AAG GGC TCA — 
 TGA CCA CAG BHQ-13′  
RT-CPT1 primer forward CAG GAT TTT GCT GTC AAC CTC 161 bp 
RT-CPT1 primer reverse GAG CAT CTC CAT GGC GTA G  
UCP2 cDNA forward GAG GTA GCA GGA AAT CAG AAT C 1176 bp 
UCP2 cDNA forward ATG GAG AGG CTC AGA AAG G  
GAPDH cDNA forward CAA CTA CAT GGT CTA CAT GTT CC 724 bp 
GAPDH cDNA reverse CAA CCT GGT CCT CAG TGT AG  
CPT1 cDNA primer forward GCA TCA TCA CTG GTG TGT TC 671 bp 
CPT1 cDNA primer reverse GTG TTG CCA GCT CTC GCT G  

BHQ, black hole quencher; RT, real-time PCR primer. FAM and HEX are fluorescent dyes.

TABLE 2

Body weights and FFA, triglyceride, and glucagon levels of control diet and HFD mice

TreatmentWTUCP2 (−/−)Significance
Female body weight (g) (n = 25)    
Control diet 20.8 ± 0.4 19.9 ± 0.4 NS 
HFD 23.0 ± 0.5* 23.5 ± 0.6* NS 
Female net weight gain (g)    
Control to HFD 2.5 ± 0.5 3.7 ± 0.6 P < 0.05 
Male body weight (g) (n = 25)    
Control diet 24.7 ± 0.6 25.8 ± 0.6 NS 
HFD 34.7 ± 1.4 38.5 ± 1.6 NS 
Male net weight gain (g)    
Control to HFD 9.8 ± 1.1 12.7 ± 1.5 P < 0.05 
Fasting plasma FFA (mEq/l) (n = 20)    
Control diet 1.80 ± 0.19 1.74 ± 0.20 NS 
HFD 2.15 ± 0.17* 1.73 ± 0.11 P < 0.05 
Fed plasma FFA (mEq/l) (n = 20)    
Control diet 0.82 ± 0.05 0.88 ± 0.04 NS 
HFD 0.99 ± 0.09* 1.08 ± 0.1* NS 
Fed plasma triglyceride (mg/dl) (n = 20)    
Control diet 72.2 ± 4.5 75.2 ± 3.9 NS 
HFD 167.8 ± 17.7 89.5 ± 6.1 P < 0.001 
Fed plasma glucagon (pg/ml) (n = 20)    
Control diet 34.2 ± 3.3 38.5 ± 4.5 NS 
HFD 48.7 ± 3.4 46.9 ± 4.3* NS 
Pancreatic insulin content (ng insulin/mg protein) (n = 10)    
Control diet 14.5 ± 2.8 14.0 ± 2.2 NS 
HFD 12.2 ± 1.3 55.6 ± 8.9 P < 0.001 
Pancreatic glucagon content (ng glucagon/mg protein) (n = 10)    
Control diet 3.1 ± 0.4 3.3 ± 0.5 NS 
HFD 3.3 ± 0.6 3.1 ± 0.4 NS 
TreatmentWTUCP2 (−/−)Significance
Female body weight (g) (n = 25)    
Control diet 20.8 ± 0.4 19.9 ± 0.4 NS 
HFD 23.0 ± 0.5* 23.5 ± 0.6* NS 
Female net weight gain (g)    
Control to HFD 2.5 ± 0.5 3.7 ± 0.6 P < 0.05 
Male body weight (g) (n = 25)    
Control diet 24.7 ± 0.6 25.8 ± 0.6 NS 
HFD 34.7 ± 1.4 38.5 ± 1.6 NS 
Male net weight gain (g)    
Control to HFD 9.8 ± 1.1 12.7 ± 1.5 P < 0.05 
Fasting plasma FFA (mEq/l) (n = 20)    
Control diet 1.80 ± 0.19 1.74 ± 0.20 NS 
HFD 2.15 ± 0.17* 1.73 ± 0.11 P < 0.05 
Fed plasma FFA (mEq/l) (n = 20)    
Control diet 0.82 ± 0.05 0.88 ± 0.04 NS 
HFD 0.99 ± 0.09* 1.08 ± 0.1* NS 
Fed plasma triglyceride (mg/dl) (n = 20)    
Control diet 72.2 ± 4.5 75.2 ± 3.9 NS 
HFD 167.8 ± 17.7 89.5 ± 6.1 P < 0.001 
Fed plasma glucagon (pg/ml) (n = 20)    
Control diet 34.2 ± 3.3 38.5 ± 4.5 NS 
HFD 48.7 ± 3.4 46.9 ± 4.3* NS 
Pancreatic insulin content (ng insulin/mg protein) (n = 10)    
Control diet 14.5 ± 2.8 14.0 ± 2.2 NS 
HFD 12.2 ± 1.3 55.6 ± 8.9 P < 0.001 
Pancreatic glucagon content (ng glucagon/mg protein) (n = 10)    
Control diet 3.1 ± 0.4 3.3 ± 0.5 NS 
HFD 3.3 ± 0.6 3.1 ± 0.4 NS 
*

P < 0.05,

P < 0.001,

P < 0.01 comparing control diet with HFD weight and significance column comparing HFD WT with HFD UCP2 (−/−) mice. NS, not significant.

TABLE 3

Pancreatic islet mRNA expression

TreatmentWTUCP2 (−/−)Significance
UCP2/GAPDH ratio (n = 3)    
Control diet 0.27 ± 0.13 ND NS 
HFD 1.84 ± 0.16* ND NS 
CPT1/GAPDH ratio (n = 3)    
Control diet 0.75 ± 0.10 1.59 ± 0.28 P < 0.05 
HFD 3.70 ± 0.65 12.67 ± 1.66 P < 0.05 
TreatmentWTUCP2 (−/−)Significance
UCP2/GAPDH ratio (n = 3)    
Control diet 0.27 ± 0.13 ND NS 
HFD 1.84 ± 0.16* ND NS 
CPT1/GAPDH ratio (n = 3)    
Control diet 0.75 ± 0.10 1.59 ± 0.28 P < 0.05 
HFD 3.70 ± 0.65 12.67 ± 1.66 P < 0.05 
*

P < 0.001,

P < 0.05,

P < 0.01 comparing control diet with HFD and significance column comparing HFD WT with HFD UCP2 (−/−) mice. ND, not detectable; NS, not significant.

This work was supported by operating grants from the Canadian Institutes of Health Research (CIHR) to M.B.W. and C.B.C. (MOP 12898 and MOP 85464). J.W.J. was supported by the Banting Best Diabetes Center studentship award and by a CIHR doctoral award. M.B.W. is supported by a CIHR investigator award. C.-Y.Z. and B.B.L. are supported by research grants from the National Institutes of Health and the American Diabetes Association.

We thank Dr. Sandy Der for assistance with real-time PCR studies.

1.
Erecinska M, Bryla J, Michalik M, Meglasson MD, Nelson D: Energy metabolism in islets of Langerhans.
Biochim Biophys Acta
1101
:
273
–295,
1992
2.
Ashcroft SJ, Ashcroft FM: Properties and functions of ATP-sensitive K-channels.
Cell Signal
2
:
197
–214,
1990
3.
Klingenberg M, Huang SG: Structure and function of the uncoupling protein from brown adipose tissue.
Biochim Biophys Acta
1415
:
271
–296,
1999
4.
Echtay KS, Winkler E, Frischmuth K, Klingenberg M: Uncoupling proteins 2 and 3 are highly active H(+) transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone).
Proc Natl Acad Sci U S A
98
:
1416
–1421,
2001
5.
Chan CB, MacDonald PE, Saleh MC, Johns DC, Marban E, Wheeler MB: Overexpression of uncoupling protein 2 inhibits glucose-stimulated insulin secretion from rat islets.
Diabetes
48
:
1482
–1486,
1999
6.
Echtay KS, Winkler E, Klingenberg M: Coenzyme Q is an obligatory cofactor for uncoupling protein function.
Nature
408
:
609
–613,
2000
7.
Echtay KS, Roussel D, St Pierre J, Jekabsons MB, Cadenas S, Stuart JA, Harper JA, Roebuck SJ, Morrison A, Pickering S, Clapham JC, Brand MD: Superoxide activates mitochondrial uncoupling proteins.
Nature
415
:
96
–99,
2002
8.
Zhang CY, Baffy G, Perret P, Krauss S, Peroni O, Grujic D, Hagen T, Vidal-Puig AJ, Boss O, Kim YB, Zheng XX, Wheeler MB, Shulman GI, Chan CB, Lowell BB: Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes.
Cell
105
:
745
–755,
2001
9.
Chan CB, De Leo D, Joseph JW, McQuaid TS, Ha XF, Xu F, Tsushima RG, Pennefather PS, Salapatek AM, Wheeler MB: Increased uncoupling protein-2 levels in β-cells are associated with impaired glucose-stimulated insulin secretion: mechanism of action.
Diabetes
50
:
1302
–1310,
2001
10.
Lameloise N, Muzzin P, Prentki M, Assimacopoulos-Jeannet F: Uncoupling protein 2: a possible link between fatty acid excess and impaired glucose-induced insulin secretion?
Diabetes
50
:
803
–809,
2001
11.
Wang MY, Shimabukuro M, Lee Y, Trinh KY, Chen JL, Newgard CB, Unger RH: Adenovirus-mediated overexpression of uncoupling protein-2 in pancreatic islets of Zucker diabetic rats increases oxidative activity and improves β-cell function.
Diabetes
48
:
1020
–1025,
1999
12.
Arsenijevic D, Onuma H, Pecqueur C, Raimbault S, Manning BS, Miroux B, Couplan E, Alves-Guerra MC, Goubern M, Surwit R, Bouillaud F, Richard D, Collins S, Ricquier D: Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production.
Nat Genet
26
:
435
–439,
2000
13.
Brubaker PL, Lee YC, Drucker DJ: Alterations in proglucagon processing and inhibition of proglucagon gene expression in transgenic mice which contain a chimeric proglucagon-SV40 T antigen gene.
J Biol Chem
267
:
20728
–20733,
1992
14.
Brubaker PL, So DCY, Drucker DJ: Tissue-specific differences in the levels of proglucagon-derived peptides in streptozotocin-induced diabetes.
Endocrinology
124
:
3003
–3009,
1989
15.
Wyllie AH, Kerr JF, Currie AR: Cell death: the significance of apoptosis.
Int Rev Cytol
68
:
251
–306,
1980
16.
Pederson RA, Satkunarajah M, McIntosh CHS, Scrocchi LA, Flamez D, Schuit F, Drucker DJ, Wheeler MB: Enhanced glucose-dependent insulinotropic polypeptide secretion and insulinotropic action in glucagon-like peptide 1 receptor (−/−) mice.
Diabetes
47
:
1046
–1052,
1998
17.
Grodsky GM, Batts AA, Bennett LL, Vcella C, McWilliams NB, Smith DF: Effects of carbohydrates on secretion of insulin from isolated rat pancreas.
Am J Physiol
205
:
638
–644,
1963
18.
Drucker DJ, Campos R, Reynolds R, Stobie K, Brubaker PL: The rat glucagon gene is regulated by a protein kinase A-dependent pathway in pancreatic islet cells.
Endocrinology
128
:
394
–400,
1991
19.
Tillmar L, Carlsson C, Welsh N: Control of insulin mRNA stability in rat pancreatic islets: regulatory role of a 3′-untranslated region pyrimidine-rich sequence.
J Biol Chem
277
:
1099
–1106,
2002
20.
Carlsson C, Borg LA, Welsh N: Sodium palmitate induces partial mitochondrial uncoupling and reactive oxygen species in rat pancreatic islets in vitro.
Endocrinology
140
:
3422
–3428,
1999
21.
Laird PW, Zijderveld A, Linders K, Rudnicki MA, Jaenisch R, Berns A: Simplified mammalian DNA isolation procedure.
Nucleic Acids Res
19
:
4293
,
1991
22.
Saltiel AR: New perspectives into the molecular pathogenesis and treatment of type 2 diabetes.
Cell
104
:
517
–529,
2001
23.
Kahn CR: Insulin action, diabetogenes, and the cause of type II diabetes.
Diabetes
43
:
1066
–1084,
1994
24.
Ahren B, Simonsson E, Scheurink AJ, Mulder H, Myrsen U, Sundler F: Dissociated insulinotropic sensitivity to glucose and carbachol in high-fat diet-induced insulin resistance in C57BL/6J mice.
Metabolism
46
:
97
–106,
1997
25.
Akiyama T, Tachibana I, Shirohara H, Watanabe N, Otsuki M: High-fat hypercaloric diet induces obesity, glucose intolerance and hyperlipidemia in normal adult male Wistar rat.
Diabetes Res Clin Pract
31
:
27
–35,
1996
26.
Ahren B, Scheurink AJ: Marked hyperleptinemia after high-fat diet associated with severe glucose intolerance in mice.
Eur J Endocrinol
139
:
461
–467,
1998
27.
Ritz-Laser B, Meda P, Constant I, Klages N, Charollais A, Morales A, Magnan C, Ktorza A, Philippe J: Glucose-induced preproinsulin gene expression is inhibited by the free fatty acid palmitate.
Endocrinology
140
:
4005
–4014,
1999
28.
Zhou YP, Grill V: Long term exposure to fatty acids and ketones inhibits B-cell functions in human pancreatic islets of Langerhans.
J Clin Endocrinol Metab
80
:
1584
–1590,
1995
29.
Bollheimer LC, Skelly RH, Chester MW, McGarry JD, Rhodes CJ: Chronic exposure to free fatty acid reduces pancreatic beta cell insulin content by increasing basal insulin secretion that is not compensated for by a corresponding increase in proinsulin biosynthesis translation.
J Clin Invest
101
:
1094
–1101,
1998
30.
Skelly RH, Bollheimer LC, Wicksteed BL, Corkey BE, Rhodes CJ: A distinct difference in the metabolic stimulus-response coupling pathways for regulating proinsulin biosynthesis and insulin secretion that lies at the level of a requirement for fatty acyl moieties.
Biochem J
331
:
553
–561,
1998
31.
Katahira H, Nagamatsu S, Ozawa S, Nakamichi Y, Yamaguchi S, Furukawa H, Takizawa M, Yoshimoto K, Itagaki E, Ishida H: Acute inhibition of proinsulin biosynthesis at the translational level by palmitic acid.
Biochem Biophys Res Commun
282
:
507
–510,
2001
32.
Bonner-Weir S, Deery D, Leahy JL, Weir GC: Compensatory growth of pancreatic β-cells in adult rats after short-term glucose infusion.
Diabetes
38
:
49
–53,
1989
33.
Bonner-Weir S, Smith FE: Islet growth and the growth factors involved.
Trends Endocrinol Metab
5
:
60
–64,
1994
34.
Swenne I, Andersson A: Effect of genetic background on the capacity for islet cell replication in mice.
Diabetologia
27
:
464
–467,
1984
35.
Chick WL, Lauris V, Flewelling JH, Andrews KA, Woodruff JM: Effects of glucose on beta cells in pancreatic monolayer cultures.
Endocrinology
92
:
212
–218,
1973
36.
Bonner-Weir S: Islet growth and development in the adult.
J Mol Endocrinol
24
:
297
–302,
2000
37.
Chick WL: Beta cell replication in rat pancreatic monolayer cultures: effects of glucose, tolbutamide, glucocorticoid, growth hormone and glucagon.
Diabetes
22
:
687
–693,
1973
38.
Dennis PB, Jaeschke A, Saitoh M, Fowler B, Kozma SC, Thomas G: Mammalian TOR: a homeostatic ATP sensor.
Science
294
:
1102
–1105,
2001
39.
Assimacopoulos-Jeannet F, Thumelin S, Roche E, Esser V, McGarry JD, Prentki M: Fatty acids rapidly induce the carnitine palmitoyltransferase I gene in the pancreatic beta-cell line INS-1.
J Biol Chem
272
:
1659
–1664,
1997
40.
Unger RH: Lipotoxicity in the pathogenesis of obesity-dependent NIDDM: genetic and clinical implications.
Diabetes
44
:
863
–870,
1995
41.
Roduit R, Morin J, Masse F, Segall L, Roche E, Newgard CB, Assimacopoulos-Jeannet F, Prentki M: Glucose down-regulates the expression of the peroxisome proliferator-activated receptor-alpha gene in the pancreatic beta-cell.
J Biol Chem
275
:
35799
–35806,
2000
42.
Prentki M, Corkey BE: Are the β-cell signaling molecules malonyl-CoA and cystolic long-chain acyl-CoA implicated in multiple tissue defects of obesity and NIDDM?
Diabetes
45
:
273
–283,
1996
43.
Briaud I, Kelpe CL, Johnson LM, Tran PO, Poitout V: Differential effects of hyperlipidemia on insulin secretion in islets of Langerhans from hyperglycemic versus normoglycemic rats.
Diabetes
51
:
662
–668,
2002
44.
Boss O, Hagen T, Lowell BB: Uncoupling proteins 2 and 3. Potential regulators of mitochondrial energy metabolism.
Diabetes
49
:
143
–156,
2000
45.
Chandra J, Zhivotovsky B, Zaitsev S, Juntti-Berggren L, Berggren PO, Orrenius S: Role of apoptosis in pancreatic beta-cell death in diabetes.
Diabetes
50 (Suppl. 1)
:
S44
–S47,
2001
46.
Unger RH, Zhou YT: Lipotoxicity of β-cells in obesity and in other causes of fatty acid spillover.
Diabetes
50 (Suppl. 1)
:
S118
–S121,
2001
47.
Shimabukuro M, Zhou YT, Levi M, Unger RH: Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes.
Proc Natl Acad Sci U S A
95
:
2498
–2502,
1998

Address correspondence and reprint requests to Michael B. Wheeler, Department of Physiology, University of Toronto, 1 King’s College Circle, Room 3352, Toronto, ON, M5S 1A8, Canada. E-mail: michael.wheeler@utoronto.ca.

Received for publication 15 November 2001 and accepted in revised form 4 July 2002.

AUC, area under the curve; BrdU, 5-bromo-2′deoxyuridine; CPT-1, carnitine palmitoyltransferase 1; FFA, free fatty acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSIS, glucose-stimulated insulin secretion; HFD, high-fat diet; IPGTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test; ROS, reactive oxygen species; UCP, uncoupling protein.