Evidence for a Role of Superoxide Generation in Glucose-Induced β-Cell Dysfunction In Vivo

  1. Christine Tang1,
  2. Ping Han1,
  3. Andrei I. Oprescu2,
  4. Simon C. Lee1,
  5. Armen V. Gyulkhandanyan1,
  6. Gary N.Y. Chan1,
  7. Michael B. Wheeler1 and
  8. Adria Giacca1,2,3
  1. 1Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
  2. 2Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
  3. 3Department of Medicine, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
  1. Address correspondence and reprint requests to Adria Giacca, 1 King's College Circle, Medical Sciences Building, Room 3336, Toronto ON, Canada. E-mail: adria.giacca{at}utoronto.ca

Abstract

OBJECTIVE— Prolonged elevation of glucose can adversely affect β-cell function. In vitro studies have linked glucose-induced β-cell dysfunction to oxidative stress; however, whether oxidative stress plays a role in vivo is unclear. Therefore, our objective was to investigate the role of oxidative stress in an in vivo model of glucose-induced β-cell dysfunction.

RESEARCH DESIGN AND METHODS— Wistar rats were infused intravenously with glucose for 48 h to achieve 20 mmol/l hyperglycemia with/without co-infusion of one of the following antioxidants: taurine (2-amino ethanesulfonic acid) (TAU), an aldehyde scavenger; N-acetylcysteine (NAC), a precursor of glutathione; or tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) (TPO), a superoxide dismutase mimetic. This was followed by islet isolation or hyperglycemic clamp.

RESULTS— A 48-h glucose infusion decreased glucose-stimulated insulin secretion (GSIS) and elevated reactive oxygen species (ROS), total superoxide, and mitochondrial superoxide in freshly isolated islets. TPO prevented the increase in total and mitochondrial superoxide and the β-cell dysfunction induced by high glucose. However, TAU and NAC, despite completely normalizing H2DCF-DA (dihydro-dichlorofluorescein diacetate)-measured ROS, did not prevent the increase in superoxide and the decrease in β-cell function induced by high glucose. TPO but not TAU also prevented β-cell dysfunction induced by less extreme hyperglycemia (15 mmol/l) for a longer period of time (96 h). To further investigate whether TPO is effective in vivo, a hyperglycemic clamp was performed. Similar to the findings in isolated islets, prolonged glucose elevation (20 mmol/l for 48 h) decreased β-cell function as assessed by the disposition index (insulin secretion adjusted for insulin sensitivity), and co-infusion of TPO with glucose completely restored β-cell function.

CONCLUSIONS— These findings implicate superoxide generation in β-cell dysfunction induced by prolonged hyperglycemia.

Type 2 diabetes is characterized by insulin resistance and defective insulin secretion. The progressive failure of pancreatic β-cells to secrete sufficient insulin to compensate for insulin resistance leads to hyperglycemia, which can in turn exert deleterious effects on β-cells (1).

Previous in vitro studies have shown that oxidative stress mediates the impairment in β-cell function induced by chronic exposure of cultured islets or β-cell lines to high glucose (26). However, the association between glucose-induced β-cell dysfunction and oxidative stress has not been shown in all studies (7,8).

In vivo evidence demonstrating a link between oxidative stress and glucose-induced β-cell dysfunction is derived from studies performed in animal models of type 2 diabetes. It has been demonstrated that the antioxidant N-acetylcysteine (NAC) can prevent diabetes in the ZDF rat and db/db mouse (9,10), an effect due to amelioration of defective glucose-stimulated insulin secretion (GSIS). To date, however, no studies have been performed to investigate the role of oxidative stress in a selective model of glucose-induced β-cell dysfunction (i.e., in the absence of other metabolite/hormone changes related to diabetes).

To do this, we examined the effects of 48 h of in vivo exposure to elevated glucose with or without antioxidants on β-cell function in rats. Three different antioxidants, taurine (2-amino ethanesulfonic acid; TAU), NAC, and tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl; TPO), were tested. TAU is an effective aldehyde scavenger due to its ability to form a Schiff base with the amino group at its β position (11). (Toxic aldehydes are byproducts of lipid peroxidation.) NAC is a scavenger of free radicals and aldehydes and can provide cysteine for the synthesis of glutathione, an integral part of the intracellular antioxidant defense system. TPO is a membrane-permeable and metal-independent superoxide dismutase mimetic that has been shown to prevent endothelial dysfunction in streptozotocin-induced diabetic rats (12). Similar to TAU and NAC, TPO has been used in vivo without adverse effects (13).

Following a 48-h infusion of glucose/saline with/without antioxidants, β-cell function was assessed in isolated islets and reactive oxygen species (ROS) and total and mitochondrial superoxide levels measured using the fluorescent dye dihydro-dichlorofluorescein diacetate (H2DCF-DA), hydroethidine, and MitoSOX, respectively. The effect of TPO was also evaluated in vivo using a hyperglycemic clamp.

RESEARCH DESIGN AND METHODS

Female Wistar rats (Charles River), weighing 250–300 g, were cannulated as previously described (14). The jugular catheter served for infusion and the carotid catheter for blood sampling. Rats were allowed at least 2–3 days of postsurgery recovery before infusions. All procedures were approved by the University of Toronto Animal Care Committee.

Ex vivo studies

Forty-eight–hour infusions.

Rats were randomized and infused for 48 h with one of the following: 1) saline, 2) a variable infusion of 37.5% glucose to achieve and maintain plasma glucose at 20–22 mmol/l (HG), 3) HG + low-dose TAU (l-TAU), 4) HG + medium-dose TAU (m-TAU), 5) HG + high-dose TAU (h-TAU), 6) HG + NAC, 7) HG + TPO, or 8) TAU, NAC, or TPO without HG infusion. TAU was given at three doses: 2.14 μmol · kg−1 · min−1, which reversed insulin resistance induced by high glucose (15); 2.76 μmol · kg−1 · min−1, which prevented β-cell dysfunction induced by 48-h fat infusion (16); and 5.52 μmol · kg−1 · min−1. NAC was given at 2.76 μmol · kg−1 · min−1 (equimolar to m-TAU), which also prevented fat-induced β-cell dysfunction (16). TPO was given at 2.41 μmol · kg−1 · min−1, which protected against ROS toxicity in experimental pancreatitis (17). All antioxidants (Sigma, St. Louis, MO) were dissolved in saline at pH = 7.4.

Pancreatic islets were isolated in overnight-fasted rats, after the 48-h infusions, using the Ficoll/Histopaque method (18).

The freshly isolated islets were preincubated for 1 h at 37°C in Krebs Ringer buffer containing 10 mmol/l HEPES (KRBH) and 2.8 mmol/l glucose and incubated in triplicate for 2 h at 37°C in KRBH at the following glucose concentrations: 2.8 mmol/l, to evaluate non-GSIS; 6.5 mmol/l, which is basal glucose in rats; 13 mmol/l, the upper physiological glucose level in rats; and 22 mmol/l, which is a maximum stimulatory concentration. Insulin was measured in the supernatant with a Linco radioimmunoassay kit. The islet pellets were subjected to acid-ethanol extraction for measurement of insulin content using the kit from Linco (19).

Islet ROS was measured with H2DCF-DA (18), which detects most ROS including hydrogen peroxide, peroxyl radical, and peroxynitrite anion (20). H2DCF-DA does not detect superoxide directly; however, superoxide can be detected indirectly via its ROS metabolites (21). Total superoxide was measured by hydroethidine (22), and mitochondrial superoxide was measured by MitoSOX (23).

Islet apoptosis was measured in dispersed islet cells using fluorescein isothiocyante–conjugated Annexin-5 (Sigma) and propidium iodide (Sigma). Annexin-5 detects membrane phosphotidylserine flip-flop once viability is compromised (24). Propidium iodide stains dead and necrotic cells. Cells stained with annexin-5 only were identified as apoptotic. After islet dissociation, performed as previously described (25), islet cells were loaded onto coverslips and incubated at 37°C in RPMI-1640 containing 2.8 mmol/l glucose for 2 h. Cells were then washed with KRBH buffer and incubated with annexin-5 (0.15 μg/ml) and propidium iodide (5 μmol/l) in KRBH for 20 min at 25°C. After washing, fluorescence was measured at 480/545 nm excitation and 510/610 nm emission for annexin and propidium iodide, respectively.

Ninty-six–hour infusions.

A set of rats was randomized into the following groups and infused for 96 h with one of the following: 1) saline, 2) a variable infusion of 37.5% glucose to achieve and maintain plasma glucose at 15 mmol/l (HG), 3) HG + m-TAU, or 4) HG + TPO. The m-TAU and TPO doses used were the same as those in the 48-h infusion study. Following the 96-h infusion, islets were isolated and ex vivo evaluation of GSIS performed as described above.

In vivo studies.

Rats were randomized and infused for 48 h with one of the following: 1) saline, 2) a variable infusion of 37.5% glucose to achieve and maintain plasma glucose at 20–22 mmol/l (HG), 3) HG + TPO, 4) TPO alone, or 5) mannitol to control for plasma osmolarity as in ref. 26, infused at a rate comparable with that of the glucose infusion (∼45 μl/min) in HG rats. After 48 h of infusion and overnight fasting, all groups were subjected to a two-step hyperglycemic clamp to evaluate GSIS in vivo.

Two-step hyperglycemic clamp.

At the end of 48 h, glucose infusion in the HG alone and HG + TPO groups was decreased to ∼5 μl/min for ∼75 min, which was required to achieve basal glucose without hypoglycemia. Basal insulin and C-peptide were measured at −20 and 0 min. At time = 0, an infusion of 37.5% glucose was started. Plasma glucose was maintained at 13 mmol/l by adjusting the rate of glucose infusion according to frequent (5–10 min) glycemic determinations. At 120 min, the glucose infusion was again raised to 22 mmol/l until the end of the experiment (time = 240 min). Samples for insulin and C-peptide were taken at regular intervals.

Plasma assays.

Plasma glucose was measured using a Beckman Analyzer II (Fullerton, CA). Radioimmunoassay kits (Linco, St. Charles, MO) specific for rat insulin and C-peptide were used.

Calculations.

The M/I index of insulin sensitivity was calculated by dividing the glucose infusion rate (GIR) by the plasma insulin level.

Regarding β-cell function, C-peptide response was taken as an index of absolute insulin secretion, as insulin secretion rate cannot be calculated in rats. This is due to the fact that rat C-peptide kinetics have not been determined because species-specific rat C-peptide is unavailable for injection. Insulin secretion in vivo has to be evaluated in the context of insulin sensitivity, since the normal β-cell compensates for insulin resistance. In normal subjects, the relationship between insulin sensitivity and insulin secretion is hyperbolic (27,28), i.e., the product of insulin sensitivity and insulin secretion is a constant defined as disposition index (DI) and considered a measure of β-cell function. We have shown that the relationship between C-peptide and M/I index is indeed hyperbolic in control rats (29). Therefore, we calculated DI during the last 40 min of each step of the hyperglycemic clamp.

Islet morphology.

At the end of the hyperglycemic clamp, rats were anesthetized with a ketamine:xylazine:acepromazine cocktail (87:1.7:0.4 mg/ml) and pancreases were removed, fixed overnight in 10% formalin, and stored in 70% ethanol. Samples were processed and embedded in paraffin within 5 days of collection.

Three sections (separated by 100 μm) were immunostained for insulin using an antibody from Biomeda (Foster City, CA) coupled with peroxidase detection. Relative β-cell area was determined from the ratio between areas occupied by insulin-positive cells and area occupied by total pancreatic tissue. β-Cell mass was determined by multiplying this ratio by pancreas weight. Individual β-cell area (index of size) was calculated by dividing insulin-positive areas by the number of nuclei within each area.

Sections (three per rat) adjacent to those used for insulin immunostaining were used to detect islet apoptotic nuclei with TUNEL (transferase-mediated dUTP nick-end labeling) staining (30).

Statistics.

Data are presented as means ± SE. One-way nonparametric ANOVA for repeated measurements followed by Tukey's t test was used to compare differences between treatments. Calculations were performed using SAS (Cary, NC).

RESULTS

Forty-eight–hour infusions.

Baseline fed plasma glucose and insulin were not different between groups before the onset of the 48-h infusions. In rats infused for 48 h with HG alone or in combination with antioxidants, glucose levels were elevated to ∼20–22 mmol/l by ∼6 h and were maintained at that level for the remainder of the 48-h period (not shown).

Figure 1A shows that at 48 h, co-infusion of TAU (all doses) or NAC tended to increase and TPO to significantly increase GIR compared with HG alone (P < 0.001). HG + TPO also increased GIR versus HG + l-TAU and HG + h-TAU (P < 0.05). Plasma insulin tended to decrease compared with HG with all antioxidants (Fig. 1B). The HG + TAU (all doses) and HG + NAC groups had significantly lower C-peptide levels than the group treated with HG alone at 48 h (Fig. 1C). Although HG + TPO tended to increase the ratio of C-peptide to insulin (index of insulin clearance) compared with HG alone, this was not significantly different (not shown). Co-infusion of all antioxidants with HG improved insulin sensitivity (Fig. 1D). HG + TPO increased the DI compared with HG alone. However, TAU (all doses) and NAC did not have any effect on DI (Fig. 1E). Non–glucose-infused groups are not shown in the graph because they had GIR = 0, and thus insulin sensitivity index (SI) and DI could not be calculated.

FIG. 1.

Effects of TAU, NAC, and TPO on glucose infusion rate (GIR) (A), insulin (B), C-peptide (C), insulin sensitivity index (D), and disposition index (DI) (E) at the end of the 48-h glucose infusion period. Rats were treated for 48 h with 1) saline; 2) glucose, to maintain glycemia at ∼20–22 mmol/l (HG); 3) glucose + low-dose TAU (HG+L-TAU, 2.12 μmol · kg−1 · min−1); 4) glucose + medium-dose TAU (HG+M-TAU, 2.76 μmol · kg−1 · min−1); 5) glucose + high-dose TAU (HG+H-TAU, 5.52 μmol · kg−1 · min−1); 6) glucose + NAC (HG+NAC, 2.76 μmol · kg−1 · min−1); 7) glucose + TPO (HG+TPO, 2.41 μmol · kg−1 · min−1); 8) medium-dose TAU (TAU 2.76 μmol · kg−1 · min−1); 9) NAC (2.76 μmol · kg−1 · min−1); or 10) TPO (2.41 μmol · kg−1 · min−1). The non–glucose-infused groups are not shown in this graph because GIR = 0; glucose, insulin, and C-peptide are basal; and insulin sensitivity index and DI cannot be calculated. At 48 h, co-infusion of TAU (all doses) or NAC tended to increase GIR, and TPO significantly increased GIR compared with HG alone (A). Plasma insulin levels tended to decrease compared with HG with all antioxidants (B). C-peptide was decreased by TAU (all doses) and NAC but not by TPO (C) vs. HG alone. All three antioxidants improved insulin sensitivity (D), but only TPO increased the disposition index (E) vs. HG alone. Data are means ± SE. *P <0.001 vs. HG; ¶P < 0.01 vs. HG; #P < 0.05 vs. HG. AUnit of M/I index = μmol · kg−1 · min−1 glucose infusion per pmol/l insulin.

GSIS in freshly isolated islets.

Insulin secretion at 2.8 mmol/l glucose did not differ between groups. Forty-eight–hour glucose infusion increased basal insulin secretion at 6.5 mmol/l (P < 0.001 HG vs. saline) but impaired GSIS at 13 and 22 mmol/l glucose (P < 0.001). All three doses of TAU did not prevent the increase in basal insulin secretion induced by glucose or the impairment in GSIS at 13 and 22 mmol/l glucose. In fact, h-TAU tended to accentuate the impairment in GSIS at 22 mmol/l glucose (Fig. 2A). Co-infusion of NAC, similar to TAU, did not prevent the effect of high glucose on either basal secretion or GSIS (Fig. 2B). Co-infusion of TPO did not affect insulin secretion at 6.5 mmol/l glucose but completely restored GSIS at both 13 and 22 mmol/l glucose (Fig. 2C).

FIG. 2.

Effects of hyperglycemia and TAU (A), NAC (B), and TPO (C) on insulin secretion in freshly isolated islets. D: Effect of hyperglycemia and TPO on islet insulin content. Groups are described in the legend of Fig. 1. Following 48-h infusion, islets were isolated and preincubated in KRBH containing 2.8 mmol/l glucose for 1 h at 37°C. To measure insulin secretion, five islets of approximately the same size were transferred in triplicate to vials containing fresh medium plus glucose at the following glucose concentrations: 2.8, 6.5, 13, and 22 mmol/l. Islets were incubated for 2 h at 37°C, and insulin was measured in the supernatant (AC). Islet pellets were subjected to acid-ethanol extraction for measurement of insulin content using radioimmunoassay (D). Data are means ± SE; n represents the number of rats (each studied at all glucose concentrations in triplicate). Typically, islets of four rats (control, glucose, glucose + antioxidant, and antioxidant alone) were studied on the same day. AC show that 48-h glucose infusion increased insulin secretion at 6.5 mmol/l glucose but impaired GSIS at 13 and 22 mmol/l glucose. TPO but not TAU or NAC completely restored GSIS at both 13 and 22 mmol/l glucose. All antioxidants did not affect the increase in insulin secretion at 6.5 mmol/l glucose. Figure 2D shows that insulin content was depleted following 48-h glucose infusion. Addition of TPO doubled the insulin content, but this was still profoundly reduced compared with saline (SAL). *P < 0.001 vs. saline; aP < 0.001 vs. saline; bP < 0.05 vs. HG alone.

Forty-eight–hour hyperglycemia drastically reduced islet insulin content. The addition of TPO doubled the insulin content (P < 0.05 HG + TPO vs. HG alone), but this was still profoundly reduced compared with saline (Fig. 2D).

ROS in islets.

Forty-eight–hour hyperglycemia increased intracellular ROS in islets compared with saline (P < 0.01). Co-infusion of m-TAU or NAC abolished the increase in ROS induced by HG (Figs. 3A and B). Co-infusion of TPO did not have a significant effect (Fig. 3C).

FIG. 3.

Effects of hyperglycemia and TAU (medium dose) (A), NAC (B), and TPO (C) on islet ROS as detected by H2DCF-DA. The groups are described in the legend of Fig. 1. Following 1 h of preincubation at 37°C in KRBH buffer containing 2.8 mmol/l glucose, islets were incubated with 10 μmol/l H2DCF-DA (Sigma) in KRBH containing 2.8 mmol/l glucose for 30 min at 37°C. After washing with KRBH, islet fluorescence was measured at 480 nm excitation and 510 nm emission using an Olympus fluorescent BX51W1 microscope. Approximately 10 islets were measured per each rat (n). Data are expressed as percentage of saline (SAL) ± SE. (A saline control rat was studied on each experiment day.) D: Representative fluorescent images of the islets. The light images are available from the authors upon request. Forty-eight–hour glucose infusion increased H2DCF-DA–detected ROS. Co-infusion of TAU or NAC abolished the increase in ROS induced by high glucose (A and B). TPO did not have a significant effect (C). ¶P < 0.01 vs. saline; #P < 0.05 vs. saline.

Total and mitochondrial superoxide levels in islets.

Forty-eight–hour glucose infusion elevated total islet superoxide (P < 0.05 vs. saline). TAU did not normalize superoxide (Fig. 4A). NAC tended to decrease superoxide, but this was not significant (Fig. 4B). In contrast, TPO prevented the increase in superoxide induced by prolonged glucose infusion (Fig. 4C).

FIG. 4.

Effects of hyperglycemia and TAU (medium dose) (A), NAC (B), and TPO (C) on total islet superoxide levels as detected by hydroethidine. The groups are described in the legend of Fig. 1. Islets isolated from glucose-treated rats were maintained at 20 mmol/l glucose throughout the islet isolation procedure. Following 1 h of preincubation at 37°C in KRBH buffer containing either 2.8 or 20 mmol/l glucose (for glucose-infused groups), islets were incubated with hydroethidine (3 μmol/l) in KRBH buffer containing 2.8 mmol/l glucose for 15 min at 37°C. After washing with KRBH, islet fluorescence was measured at 550 nm excitation and 660 nm emission using an Olympus fluorescent BX51W1 microscope. Approximately 10 islets were measured per rat (n). Data are expressed as percentage of saline (SAL) ± SE. (A saline control rat was studied on each experiment day.) D: Representative fluorescent images of the islets. The light images are available from the authors upon request. Forty-eight–hour glucose infusion increased total islet superoxide level. Co-infusion of TAU and NAC did not normalize superoxide (A and B). TPO abolished the increase induced by 48-h glucose infusion (C). #P < 0.05 vs. saline.

Forty-eight–hour glucose infusion elevated islet mitochondrial superoxide, and co-infusion of TPO completely prevented this increase (Fig. 5).

FIG. 5.

Effects of hyperglycemia, TAU, and TPO on islet mitochondrial superoxide levels as detected by MitoSOX. The groups are described in the legend of Fig. 1. Islets isolated from glucose-treated rats were maintained at 20 mmol/l glucose throughout the islet isolation procedure. Following 1 h of preincubation at 37°C in KRBH buffer containing either 2.8 or 20 mmol/l glucose (for glucose-infused groups), islets were incubated with MitoSOX (5 μmol/l) in KRBH buffer containing 2.8 mmol/l glucose for 15 min at 37°C. After washing with KRBH, islet fluorescence was measured at 545 nm excitation and 610 nm emission using a Zeiss Axiovert 200M microscope. Approximately 10 islets were measured per rat (n). Data are expressed as percentage of saline (SAL) ± SE. (A saline control rat was studied on each experiment day.) B: Representative fluorescent images of the islets. The light images are available from the authors upon request. Forty-eight–hour glucose infusion increased mitochondrial superoxide levels, and co-infusion of TPO completely abolished this increase. *P < 0.001 vs. saline.

β-Cell apoptosis.

The percentage of apoptotic cells was measured in dispersed islet cells from isolated islets using the fluorescent dyes annexin-5 and propidium iodide. Islet cells stained for annexin-5 only were identified as apoptotic. There was no detectable difference in islet cells apoptosis between groups (percentage of apoptotic cells: saline, 1.86 ± 0.08%; HG alone, 1.99 ± 0.17%; HG + TPO, 1.88 ± 0.20%; and TPO, 1.91 ± 0.18%; n = 3–4 per group).

Islet morphology.

Islet morphological studies were performed in the isolated pancreas after 48-h infusion and hyperglycemic clamps. β-Cell mass and individual area (i.e., size) were increased ∼1.5- to 2-fold compared with saline. β-Cell mass and size were similar in HG alone and HG + TPO (Table 1). The number of β-cells per section tended to increase in HG alone and HG + TPO, but these differences were not significant (saline, 3,119 ± 313; HG, 3,431 ± 551; HG + TPO, 3,312 ± 279; and TPO, 3,083 ± 337). Apoptotic cells were very rare without detectable differences between groups (not shown).

TABLE 1

Pancreatic β-cell mass and individual β-cell area following 48-h infusion

Two-step hyperglycemic clamp.

To further investigate whether TPO is effective in preventing glucose-induced β-cell dysfunction in vivo, a two-step hyperglycemic clamp was performed after 48-h infusions. A subset of rats were also infused with mannitol as a control for osmolarity of the glucose infusate, and the results were not different from those of saline.

Basal plasma glucose before the clamp was slightly lower in HG alone and HG + TPO groups (not visible because of graph scale). Plasma glucose was elevated to 13 mmol/l until 120 min and then to 22 mmol/l until 240 min, with no difference between groups (Fig. 6A).

FIG. 6.

Effects of TPO on plasma glucose levels (A), GIR (B), plasma insulin levels (C), plasma C-peptide levels (D), sensitivity index (E), and DI (F) during the two-step hyperglycemic clamp with or without glucose infusion. Rats were infused for 48 h with one of the following: 1) saline (SAL), 2) a variable infusion of 37.5% glucose to achieve and maintain plasma glucose at 20–22 mmol/l (HG), 3) HG + TPO, 4) TPO alone, or 5) mannitol (MAN) to control for plasma osmolarity as in ref. 26, infused at a rate comparable with glucose infusion (∼45 μl/min). Glucose levels were superimposable in all groups during both steps of the clamp (A). HG had a significantly lower GIR than saline at 22 mmol/l, and GIR was completely restored by TPO (B). Absolute insulin and C-peptide levels at basal and 13 mmol/l glucose were significantly increased by glucose infusion. However, the response of insulin and C-peptide to the rise in glucose was blunted in HG compared with saline, suggesting β-cell dysfunction. TPO tended to decrease absolute insulin and C-peptide levels and increase the response of insulin and C-peptide to the rise in glucose (C and D). The insulin sensitivity index was significantly decreased in HG compared with saline at both 13 and 22 mmol/l glucose. This decrease was partially prevented by the co-infusion of TPO at 22 mmol/l glucose (E). The DI was significantly decreased in HG compared with saline at both 13 and 22 mmol/l glucose. TPO completely restored the DI during both steps of the clamp (F). Data are means ± SE. AUnit of M/I index = μmol · kg−1 · min−1. aP < 0.05 HG, HG+TPO vs. SAL at basal glucose; bP < 0.001 HG+TPO vs. HG at 13 mmol/l; cP < 0.05 HG+TPO vs. SAL at 13 mmol/l glucose; dP < 0.001 HG vs. SAL at 22 mmol/l; eP < 0.001 HG, HG+TPO vs. SAL at basal and 13 mmol/l glucose; ¶P < 0.01 vs. SAL; *P <0.001 vs. SAL.

In rats given HG alone, a lower GIR was necessary to clamp glucose at 22 mmol/l, indicating that the circulating insulin was inadequate to compensate for insulin resistance. Co-infusion of TPO completely restored GIR to the saline levels (Fig. 6B).

Basal insulin and C-peptide levels (−20 to 0 min) were higher in rats given HG alone and HG + TPO than in those given saline (P < 0.001). In response to increasing glucose levels, plasma insulin and C-peptide increased as expected. Absolute insulin and C-peptide levels were higher in rats given HG alone than in those given saline (P < 0.001) at 13 mmol/l and tended to be higher at 22 mmol/l. However, the response of insulin and C-peptide to the rise in glucose was blunted in HG compared with saline, suggesting β-cell dysfunction. TPO tended to decrease absolute insulin and C-peptide levels and increased the response of insulin and C-peptide to the rise in glucose (Figs. 6C and D).

The ratio of C-peptide to insulin (index of insulin clearance) was lower in rats given HG than in those given saline at 13 mmol/l glucose (saline 4.43 ± 0.26 vs. HG 3.08 ± 0.25; P < 0.05 vs. saline). This was partially prevented by the co-infusion of TPO (HG + TPO 3.65 ± 0.26; N.S. vs. saline or HG). Similar intergroup differences in C-peptide–to–insulin ratio failed to reach significance at basal and 22 mmol/l glucose.

Insulin sensitivity index (M/I index) was decreased in HG compared with saline at both 13 and 22 mmol/l glucose. Decreased insulin sensitivity was partially prevented by the co-infusion of TPO at 22 mmol/l glucose (Fig. 6E).

Although insulin and C-peptide levels were elevated in HG, the DI, which represents the ability of the β-cell to compensate for insulin resistance, was lower in rats given HG than in those given saline. TPO was able to completely restore DI during both steps of the clamp (Fig. 6F).

Ninty-six–hour infusions.

To investigate the effect of antioxidants in a more chronic, less extreme model of glucose-induced β-cell dysfunction, GSIS in isolated islets was assessed following 96-h hyperglycemia (plasma glucose ∼15 mmol/l) with/without TPO or TAU. Ninty-six–hour hyperglycemia increased insulin secretion at 6.5 mmol/l glucose but impaired GSIS at 22 mmol/l glucose. Insulin secretion at 13 mmol/l glucose was unaffected. Similar to our findings following 48-h glucose infusion, both antioxidants did not prevent the increase in basal insulin secretion induced by hyperglycemia, and only TPO was able to prevent the impairment in GSIS at 22 mmol/l glucose (Fig. 7).

FIG. 7.

Effects of 96-h hyperglycemia and TAU and TPO on insulin secretion in isolated islets. Rats were treated for 96 h with 1) saline (SAL); 2) glucose, to maintain glycemia at ∼15 mmol/l (HG); 3) HG + medium-dose TAU (HG+TAU, 2.76 μmol−1 · kg−1 · min); 4) HG + TPO (2.41 μmol−1 · kg−1 · min)). Ninty-six–hour hyperglycemia (plasma glucose 15 mmol/l) increased insulin secretion at 6.5 mmol/l glucose but impaired insulin secretion at 22 mmol/l glucose. No impairment was observed at 13 mmol/l glucose. Co-infusion of TPO but not TAU completely prevented β-cell dysfunction induced by 96-h hyperglycemia at 22 mmol/l glucose. Both antioxidants did not affect the increase in insulin secretion at 6.5 mmol/l glucose. Data are means ± SE, and n represents the number of rats. The static incubation studies were performed as described in the legend of Fig. 2. ¶P < 0.01 vs. SAL; #P < 0.05 vs. SAL.

DISCUSSION

In this study, the role of oxidative stress in glucose-induced β-cell dysfunction was investigated using ex vivo and in vivo models. Chronic hyperglycemia has been well documented to induce β-cell dysfunction in vitro (3134) and ex vivo (3537). Consistent with previous results, we found that β-cell function was impaired ex vivo in isolated islets and also in vivo, as assessed by the hyperglycemic clamp following 48 h of glucose infusion.

During the hyperglycemic clamp, absolute insulin and C-peptide levels were elevated in rats that received 48-h glucose infusion. However, insulin resistance was also induced in these animals. Insulin secretion in vivo has to be evaluated in the context of insulin sensitivity, since the normal β-cell compensates for insulin resistance by increasing insulin secretion, independent of plasma glucose. In subjects with normal glucose tolerance, insulin secretion and sensitivity are linked through a hyperbolic relationship (27,28), i.e., the product of insulin sensitivity and insulin secretion is a constant. This constant is defined as the DI and is a measure of β-cell function (including β-cell ability to compensate for insulin resistance). DI was decreased in glucose-infused rats, demonstrating that β-cell dysfunction was induced by 48-h hyperglycemia. Aside from a decreased DI, there was also a marked defect in the response of insulin and C-peptide to a rise in plasma glucose, which further demonstrates β-cell dysfunction in glucose-infused rats.

Increased insulin secretion was observed at basal glucose (∼6.5 mmol/l) both in isolated islets and in vivo before the onset of the hyperglycemic clamp. This increase was not observed in islets at 2.8 mmol/l, which suggests that it is likely not due to islet hypertrophy or hyperplasia, but to a shift in the sensitivity of the β-cell to glucose. The mechanisms whereby chronic glucose exposure increases the insulin secretory response to low glucose are unclear but may be linked to the well-described induction of glycolytic genes by glucose (38). From our findings, we postulate that this increase in insulin secretion at low glucose levels is likely not due to oxidative stress, since all three antioxidants did not prevent this effect.

β-Cell mass was increased by short-term glucose infusion as previously reported (3941), mainly because of β-cell hypertrophy (also as reported previously at 48 h [39,40]). There was no detectable change in islet viability or apoptosis. This establishes the 48-h glucose infusion model as a model of β-cell dysfunction rather than loss.

The mechanism whereby prolonged exposure to high glucose impairs β-cell function is not completely understood and is probably due to oxidative stress–dependent and –independent pathways. The studies that have linked oxidative stress to glucose-induced β-cell dysfunction have mainly been performed in vitro (26) or in animal models of type 2 diabetes (9,35), which in addition to hyperglycemia have many other metabolic alterations that can affect β-cell function. Thus, it is important to study the effect of chronic hyperglycemia per se on β-cell function in vivo.

Unexpectedly, the antioxidants TAU and NAC, which we have previously shown to prevent lipid-induced β-cell dysfunction in vivo and in isolated islets (16) (same doses as in this study), were not able to prevent glucose-induced β-cell dysfunction in isolated islets. (The effects of TAU and NAC were not assessed by the hyperglycemic clamp becaue TAU and NAC did not show any effect on the DI at 48 h). One possible explanation may be that a higher dose of TAU and NAC is required to prevent glucose-induced than lipid-induced β-cell dysfunction. However, this is unlikely, since 1) co-infusion of TAU or NAC completely normalized H2DCF-DA–measured ROS and 2) doubling the dose of TAU (compared with the dose infused with lipids) was still ineffective. Another more plausible explanation may be that different types of ROS are important for β-cell dysfunction induced by glucose and lipids. TAU is effective in scavenging toxic aldehydes. NAC contains an −SH group and thus is capable of reducing oxidized proteins. NAC is also a precursor of intracellular glutathione. It is therefore possible that toxic aldehyde generation and glutathione depletion play a lesser role in glucose- than lipid-induced β-cell dysfunction.

In contrast to TAU and NAC, the antioxidant TPO was completely effective in preventing β-cell dysfunction induced by prolonged hyperglycemia in isolated islets and in vivo. Interestingly, TPO did not decrease total ROS (as detected by H2DCF-DA) induced by prolonged glucose infusion. This was not surprising because TPO is a superoxide dismutase mimetic, i.e., it converts superoxide to hydrogen peroxide. Instead, TPO did reduce total superoxide in islets of glucose-treated rats, unlike TAU or NAC. Therefore, our findings show that the effect of antioxidants to prevent glucose-induced β-cell dysfunction parallels their ability to normalize superoxide but not H2DCF-DA–detected ROS, suggesting that superoxide generation is important in our in vivo model of glucose-induced β-cell dysfunction. Our findings are in accordance with an in vitro study by Krauss et al. (42), which showed that overexpression of Mn superoxide dismutase, but not glutathione peroxidase 1 (which decreases H2O2), prevented glucose-induced β-cell dysfunction.

In the β-cell, superoxide can be generated in various ways. One possible source of superoxide is the mitochondrial electron transport chain. During hyperglycemia, superoxide production is increased due to greater electron flow in the electron transport chain. Studies have shown that mitochondrial production of ROS can impair insulin secretion (42,43). Our findings show that glucose-induced β-cell dysfunction in vivo is indeed associated with increased mitochondrial superoxide levels, and this increase was completely prevented by the co-infusion of TPO.

Increased mitochondrial superoxide can affect β-cell function in several ways. Superoxide can decrease mitochondrial glucose oxidation. Superoxide can also activate uncoupling protein 2, resulting in decreased ATP produced by glucose oxidation and thus decreased GSIS (42). The fact that insulin content was only mildly improved by TPO suggests that after 48 h, TPO is mainly acting on the insulin secretory process, presumably by increasing glucose oxidation (44) or decreasing uncoupling (42) via decreased mitochondrial superoxide.

In addition to the mitochondria, the cytosol can also generate superoxide via the activation of NAD(P)H oxidase. β-Cells have been shown to express NAD(P)H oxidase (45), and components of this enzyme are elevated in islets of animal models of type 2 diabetes (46). Furthermore, glucose can activate NAD(P)H oxidase via protein kinase C (47). When NAD(P)H was inhibited in islets of db/db mice, insulin content was partially restored (46), suggesting that cytosolic superoxide may also be important in β-cell dysfunction. Here, we showed that the mitochondria are an important site of superoxide production in a model of glucose-induced β-cell dysfunction. However, further studies need to be performed to assess the role of cytosolic superoxide production.

It is recognized that our glucose infusion model is short-term and extreme in nature and that 48-h glucose infusion does not induce β-cell dysfunction in humans (48). However, there is evidence that glucose-induced β-cell dysfunction does occur in humans following exposure to hyperglycemia (13 mmol/l) for longer than 68 h (48). Thus, we wished to determine the effect of antioxidants in a less extreme, more chronic model of glucose-induced β-cell dysfunction that can be reproduced in humans. We showed that similar to 48-h infusion, TPO but not TAU prevented β-cell dysfunction induced by 96 h of hyperglycemia (plasma glucose ∼15 mmol/l). Further prolongation of in vivo infusion is not feasible, and more chronic glucose exposure requires in vitro studies (49) or studies in hyperglycemic but not hyperlipidemic diabetic models such as GK rats. Future studies in these models may reveal that not only TPO but also other antioxidants are beneficial, perhaps due to their effect on insulin gene transcription (2,10).

Previously, we have shown that TAU and NAC (15) (in doses similar to those in this study) were effective in preventing the decrease in insulin sensitivity induced by 6 h of hyperglycemia. In this study, we showed that following 48-h hyperglycemia, TAU and NAC increased insulin sensitivity in glucose-infused rats. Despite TAU and NAC improving insulin sensitivity, both antioxidants did not increase DI. Oxidative stress has been shown to play a role in both hyperglycemia-induced insulin resistance and hyperglycemia-induced β-cell dysfunction; it is thus unclear why TAU and NAC were effective in the former but not the latter. It is possible that mitochondrial superoxide is important for glucose-induced β-cell dysfunction, at least in the short term, whereas glucose-induced insulin resistance is due to cytosolic production of reactive aldehydes and to cytosolic glutathione depletion.

In vivo, metabolic compensation for insulin resistance involves both increased GSIS and decreased insulin clearance. The C-peptide–to–insulin ratio (index of insulin clearance) was lower at some points after prolonged hyperglycemia, consistent with a reduction in insulin clearance. Addition of TPO tended to reverse the decrease in C-peptide–to–insulin ratio, although not significantly, presumably because this ratio is not a good indicator of insulin clearance. Therefore, it cannot be excluded that a TPO-induced increase in insulin clearance stimulates β-cell compensation for insulin resistance, thus contributing to improved β-cell function in vivo. Nevertheless, data from isolated islets clearly show a direct β-cell effect of TPO.

In conclusion, our study demonstrates that β-cell dysfunction induced by 48- and 96-h glucose elevation in vivo is due to superoxide generation. Furthermore, this study suggests that superoxide dismutase mimetics are of potential interest to preserve β-cell function in type 2 diabetes.

Acknowledgments

This work was supported by Canadian Institutes of Health Research Grants MOP-69018 and NET-54012 to A.G. and MOP-12898 to M.B.W. C.T. was supported by a Banting and Best Diabetes Centre-Novo Nordisk studentship and an Ontario Graduate Scholarship.

The authors thank Loretta Lam for her excellent technical assistance.

Footnotes

  • Published ahead of print at http://diabetes.diabetesjournals.org on 6 August 2007. DOI: 10.2337/db07-0279.

    The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    • Received February 27, 2007.
    • Accepted July 28, 2007.

REFERENCES

| Table of Contents

This Article

  1. Diabetes vol. 56 no. 11 2722-2731
  1. All Versions of this Article:
    1. db07-0279v1
    2. 56/11/2722 most recent