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Department of Physiology, University of Saskatchewan College of Medicine, Saskatoon, Saskatchewan, Canada S7N 5E5
Address all correspondence and requests for reprints to: Dr. Joseph Fomusi Ndisang, Department of Physiology, University of Saskatchewan College of Medicine, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5. E-mail: joseph.ndisang{at}l.usask.ca.
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Abstract |
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B, activating-protein (AP)-1, AP-2, and c-Jun-N-terminal-kinase. Although hemeoxygenase (HO) suppresses oxidative insults, its effects on insulin-sensitizing agents like AMPK and GLUT4 remains unclear and were investigated using Goto-Kakizaki rats (GK), a nonobese insulin-resistant type-2 diabetic model. HO was induced with hemin or inhibited with chromium mesoporphyrin (CrMP). The application of hemin to GK rats evoked a 3-month antidiabetic effect, whereas the HO-inhibitor, CrMP, exacerbated hyperglycemia and nullified insulin-signaling/glucose metabolism. Interestingly, the antidiabetic was accompanied by a paradoxical increase of insulin alongside the potentiation of insulin-sensitizing agents such as adiponectin, AMPK, and GLUT4 in the gastrocnemius muscle. Furthermore, hemin enhanced mediators/regulators of insulin signaling like cGMP and cAMP and suppressed oxidative insults by up-regulating HO-1, HO activity, superoxide dismutase, catalase, and the total antioxidant capacity in the gastrocnemius muscle. Accordingly, oxidative markers/mediators including nuclear factor-B, AP-1, AP-2, c-Jun-N-terminal-kinase, and 8-isoprostane were abated, whereas CrMP annulled the cytoprotective and antidiabetic effects of hemin. Correspondingly, ip glucose tolerance, insulin tolerance, and homeostasis model assessment insulin resistance analyses revealed improved glucose tolerance, reduced insulin intolerance, enhanced insulin sensitivity, and reduced insulin resistance in hemin-treated GK rats. In contrast, CrMP, abolished the insulin-sensitizing effects and restored and/or exacerbated insulin resistance. Our study unveils a 3-month enduring antidiabetic effect of hemin and unmasks the synergistic interaction among the HO system, adiponectin, AMPK, and GLUT4 that could be explored to enhance insulin signaling and improve glucose metabolism in insulin-resistant diabetes. |
Introduction |
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B (NF-B), by a cGMP-dependent mechanism (12). Besides NF-B, c-Jun-N terminal kinase (JNK) mediates oxidative injury (13, 14). Recent evidence indicates that JNK regulates activating protein (AP)-1 (15) and mediates insulin resistance (14). Given that the reduction of JNK activity in diabetic mice was accompanied by reduced insulin resistance and attenuation of glucose tolerance (14), substances that block JNK could be useful against insulin resistance. On the other hand, elevated oxidative stress is known to deplete adiponectin mRNA and reduce circulating adiponectin levels (16, 17). Thus, the reduction of NF-B, AP-1, and JNK may attenuate the oxidative destruction of adiponectin (16). Recent studies have highlighted the emerging role of the hemeoxygenase (HO) system in insulin-resistant T2D (18, 19, 20). Up-regulating the HO system generates cytoprotective products such as bilirubin, biliverdin, and carbon monoxide (CO) (21). Interestingly, both CO and nitric oxide (NO) have been shown to regulate glucose metabolism (22). Furthermore, CO and NO stimulate cGMP, activate potassium channel, and inhibit cytochrome P450 (21). NO has recently been shown enhance GLUT4 expression in skeletal muscle, via a cGMP-dependent mechanism (23). Whether up-regulating the HO system will increase endogenous CO, which may in turn increase GLUT4, remains unclear. The stimulation of glucose uptake by insulin in muscle requires the translocation of GLUT4 from intracellular storage sites to the cell surface (24). Thus, investigating the effects of the HO system on GLUT4 may unveil a novel role of the HO system in the homeostatic control of blood glucose.
Although the HO system abates oxidative insults (21, 25), its effect on insulin-sensitizing agents such as AMPK and GLUT4 or inhibitor of insulin-signaling like JNK has not been fully characterized. Given the availability of motifs for inflammatory/oxidative transcription factors like NF-B, AP-1, AP-2, and a motif for glucocorticoid-responsive elements in the HO-1 gene promoter (26), HO-1 may have important regulatory role in many cellular activities including defense and glucose metabolism (19, 27, 28, 29). Thus, the pleiotropic effects of HO-1 may be explored pharmacologically to counteract hyperglycemia-induced oxidative insults, prevent JNK-mediated destruction of insulin, and improve insulin sensitivity/glucose metabolism in T2D. Therefore, one of the main objectives of the study was to study the effects of hemin therapy on insulin sensitivity in gastrocnemius skeletal muscles of Goto-Kakizaki rat (GK), a nonobese, insulin-resistant, type 2 diabetic model (18). Given that skeletal muscles accounts for 65–90% of the clearance of an oral or iv glucose challenge (23), and muscle contractility increases glucose clearance by improving insulin sensitivity in normal and insulin-resistant conditions by enhancing GLUT4 expression (30), investigating the effects of the HO system on GLUT4 and other pathways implicated in insulin-signaling like AMPK, cGMP, NF-B, AP-1, AP-2, and JNK in the gastrocnemius muscles of the GK rat may unmask a novel role of the HO system in glucose metabolism. In addition, the effect of hemin on cAMP was evaluated because cAMP has been shown to regulate insulin release (22) besides its implication in adiponectin-mediated suppression of NF-B (12). Therefore, this study was designed to unveil the enduring antidiabetic effects of hemin and the underlying mechanisms.
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Materials and Methods |
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Animal treatment and biochemical assays
The experimental protocol was approved by University of Saskatchewan Standing Committee on Animal Care and Research Ethics. Male GK rats (13 wk) were purchased from Taconi Farms (Germantown, NY), whereas age-matched euglycemic Wistar and Sprague Dawley rats (SD) from Charles River (Willington, MA). SD was used as an additional control for GK rat (31). Although the GK rat is a derived from nondiabetic Wistar colony, we included an additional SD control to avoid the numerous similarities (genotypic/phenotypic) due to same genetic background that may contrast less avidly with the distinct genetic features of SD and thus give our study a unique and important perspective in the pathophysiology of diabetes, which otherwise may remain unobserved. The animals were housed at 21 C with 12-h light, 12-h dark cycles, fed with standard laboratory chow, and had access to drinking water ad libitum.
Hemin (30 mg/kg ip; Sigma St. Louis, MO) was used to induce HO, whereas chromium mesoporphyrin (CrMP; 4 µmol/kg ip; Porphyrin Products, Logan, UT) was given to block HO. Although many HO inhibitors are nonspecific and may affect other hemoenzymes or even increase HO-1 expression (32), however, CrMP given at a dose of 4 µmol/kg is reportedly selective for HO (33). Hemin and CrMP were dissolved in 0.1 M NaOH, titrated to pH 7.4 with 0.1 M HCl and diluted 1:10 with phosphate buffer as we previously reported (34, 35). The study design included the following groups (n = 6–12/group): A, controls (Wistar, SD, and GK); B, GK+hemin; C, GK+hemin+CrMP; D, GK+CrMP; E, Wistar+hemin; F, SD+hemin; and G, vehicle-treated animals (vehicle dissolving hemin and CrMP). Each injection was 0.5 ml in volume, and was given daily for 4 wk, a period necessary for hyperglycemia to be normalized. We had previously shown that chronic hemin therapy was not toxic (34). During the entire 4 wk of treatment, fasting glucose was monitored weekly with a glucose-meter (BD Logic, Franklin Lakes, NJ). Hemin therapy began at 14 wk because the pathophysiological changes in tissue due to hyperglycemia, insulin resistance, and oxidative stress would be fully established by this age. At the end of the 4-wk regimen of hemin, the animals were 17 wk of age. After stoppage of therapy, fasting glucose was monitored on a weekly routine for the ensuing 3 months, after which the study was terminated, with the animals having 29 wk of age. A day before the animals were killed, the animals were fasted in metabolic cages for 24 h, urine samples collected, and after anesthesia (pentobarbital sodium, 50 mg/kg ip) the animals were killed, and plasma/tissues harvested. From the plasma ferritin was routinely measured by Saskatoon Royal University Hospital, Canada.
Gastrocnemius muscle HO activity was evaluated spectrophotometrically using our established method (34, 35, 36), whereas HO-1 concentration by ELISA (Stressgen-Assay Design, Ann Arbor, MI). Other biochemical parameters including urinary/gastrocnemius muscle 8-isoprostane, a noninvasive index of oxidative stress (37), gastrocnemius muscles superoxide dismutase (SOD) activity (total), catalase activity, cGMP, cAMP, and the total antioxidant capacity were measured by enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) (34).
Total RNA isolation and quantitative RT-PCR for p65-NF-B, AP-1, AP-2, GLU4, and JNK
The gastrocnemius muscle was homogenized in Trizol reagent as we previously described (34). Briefly, triplicate samples of 1 µl of cDNA each was ran using a template of 3.2 pmol of primers for NF-B (forward, 5'-CATGCGTTTCCGTTACAAGTGCGA-3' and reverse, 5'-TGGGTGCGTCTTAGTGGTATCTGT-3'); AP-1 (forward, 5'-AGCAGATGCTTGAGTTGAGAGCCA-3' and reverse, 5'-TTCCATGGGTCCCTGCTTTGAGAT-3'); AP-2 (forward, 5'-TAAAGTGGGATCGAGGAGGCCAGAAA-3' and reverse, 5'-AGTCACAAAGACTCCAAGAGGGCA-3'); JNK (forward, 5'-AAGCAGCAAGGCTACTCCTTCTCA-3' and reverse, 5'-ATCGAGACTGCTGTCTGTGTCTGA-3'); GLUT4 (forward, 5'-AATGAGCGGTTTGAATGGGACCTG-3' and reverse, 5'-AACCGCCCTTGTCTCTGTCATCTA-3'); and β-actin (forward, 5'-TCATCACTATCGGCAATGAGCGGT-3' and reverse, 5'-ACAGCACTGTGTTGGCATAGAGGT-3') in a final volume of 25 µl. The sequences of all primers used were confirmed by the National Research Institute of Canada, Saskatoon.
Western immunoblotting
The gastrocnemius muscle was homogenized and proteins extracted and resolved on a sodium dodecyl sulfate gel as we previously described (34, 36). The fractionated proteins were electrophoretically transferred to nitrocellulose blot and incubated with primary antibody against AMPK (Cell Signaling Technology Inc., Danvers, MA). After washing, the blot was incubated with antirabbit-IgG conjugated to horseradish peroxide and the immunoreactivity visualized with enhanced horseradish peroxide/luminol chemiluminescence reagent. β-Actin antibody was used as a control.
Determination of glucose tolerance, insulin tolerance, and insulin resistance [homeostasis model assessment insulin resistance (HOMA-IR) analyses]
Glucose tolerance was evaluated by ip glucose tolerance test (IPGTT) after 16 h fasting. A bolus of glucose (2 g/kg ip) was injected, and blood samples were collected from the tail vein after anesthesia at intervals of 0, 30, 60, 90, and 120 min and tested for glucose and insulin (38). To evaluate insulin tolerance, ip insulin tolerance test (IPITT) was done. A bolus of insulin (2 U/kg ip) was injected and blood samples taken sequentially at various time points from 0–120 min for glucose measurement. Insulin resistance (HOMA-IR) was used to further assess glucose tolerance. HOMA-IR was calculated from the product of fasting plasma glucose (milligrams per deciliter) and insulin (microunits per milliliter) divided by 22.5 (39).
Statistical analysis
All data were expressed as means ± SEM from at least four independent experiments unless otherwise stated. Statistical analyses were done using unpaired Student’s t test, analyses of variance in conjunction with Bonferroni test, and analyses of variance for repeated measures where appropriate. Group differences at the level of P < 0.05 were considered statistically significant.
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Results |
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, A and B). To ascertain the implication of the HO system in the antidiabetic effects, some GK rats were treated together with hemin and the HO blocker, CrMP, or with CrMP alone. The cotreatment of hemin and CrMP reversed the antidiabetic effect of hemin, whereas CrMP alone further exacerbated hyperglycemia in GK rats (Fig. 1A), suggesting the involvement of basal HO activity in glucose metabolism and blood sugar homeostasis.
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). Interestingly, the antidiabetic effect prevailed for up to 3 months after stoppage of therapy (7.2 ± 0.2 mmol/liter). The vehicle had no effect on blood glycemia. The application of hemin therapy to Wistar and SD also lowered glucose levels, although the antidiabetic effect was less intense than in GK rats. In Wistar, fasting and postprandial glucose levels were 6.53 ± 0.4 vs. 6.12 ± 0.3 (P < 0.05, n = 8) and 9.72 ± 0.6 vs. 7.21 ± 0.5 (P < 0.05, n = 8), respectively. Similarly, in SD, fasting/postprandial glucose was 9.3 ± 0.4 vs. 7.1 ± 0.3 (P < 0.01, n = 8) and 7.0 ± 0.6 vs. 6.05 ± 0.4 (P < 0.05, n = 8).
The 4-wk hemin regimen slightly affected body weight (Table 1). However, the animals quickly recovered and by 1 month after therapy, hemin-treated animals had comparable body weights as age-matched untreated controls. The loss of body weight may not account for the antidiabetic effect given that at 1, 2, and 3 months after therapy, the hemin-treated GK had similar body weights as age-matched untreated GK but significantly lower glucose levels (millimoles per liter) [6.7 ± 0.3 vs. 10.7 ± 0.3 (P < 0.01), 6.9 ± 0.4 vs. 11.2 (P < 0.01), and 7.2 ± 0.2 vs. 11.9 ± 0.6 (P < 0.01), respectively].
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, A and B). The 4-wk hemin regimen markedly enhanced HO activity and cGMP in GK rats by 10.7- and 8.2-fold, respectively. Hemin also enhanced HO-1, HO activity, and cGMP in Wistar and SD control rats, although the effects were significantly lower than in GK rats. To further evaluate the contribution of basal HO input in glucose metabolism, we gave the HO blocker, CrMP alone or with hemin and measured HO activity. Treatment with CrMP+hemin abolished the hemin-induced increase of HO-1, HO activity, and cGMP, with corresponding reversal of the antidiabetic effects, whereas treatment with CrMP alone further increased hyperglycemia. Importantly, the CrMP-induced exacerbation of hyperglycemia was accompanied by more pronounced reduction of HO-1, HO activity, and cGMP in the GK rat (Fig. 2), suggesting an important role of basal HO-cGMP signaling in glucose homeostasis. Interestingly, at 3 months after therapy, HO activity, and cGMP remained significantly elevated. Because cGMP enhances skeletal muscle GLUT4 (23) to improve insulin sensitivity and glucose metabolism, the 3-month enduring antidiabetic effect of hemin may be attributed, at least in part to the sustained elevation of cGMP.
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, A and B). In hyperglycemic GK rats, the basal urinary/gastrocnemius muscle 8-isoprostane were 2.4- and 3.9-fold higher and were reduced to control levels by hemin, whereas CrMP nullified the hemin effect. Moreover, the hemin-dependent reduction of 8-isoprostane was still evident 3 months after termination of therapy.
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, C–E) but were increased by hemin by 5.1-, 4.4-, and 3.4-fold, respectively, and abolished by CrMP. Correspondingly, hemin increased the total antioxidant capacity in GK rats to the levels observed in euglycemic Wistar control (Fig. 3D). The antioxidant effect of hemin remained evident throughout the 3-month posttreatment observation period. Hemin therapy also reduced 8-isoprostane and enhanced ferritin, SOD, catalase, and the total antioxidant status in Wistar control rats, although the effect was less intense compared with GK rats.
Hemin reduces proinflammatory/oxidative mediators including NF-B, AP-1, AP-2, and JNK but enhances insulin and adiponectin
Many transcriptions factors reduce insulin signaling in diabetes (14, 15). In hyperglycemic GK rats, quantitative real-time RT-PCR analyses indicated that the levels of NF-B, AP-1, and AP-2 in the gastrocnemius muscle were significantly elevated (Fig. 4, A–D) but were reduced by hemin by 3.2-, 2.2-, 1.9-, and 4.9-fold, respectively, whereas CrMP abolished the effects of hemin.
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B to control levels, whereas AP-1 AP-2, and JNK were partially reduced, suggesting greater sensitivity for NF-B. The reasons for this selective effect remain unclear and should be investigated further.
Because JNK inhibits insulin biosynthesis (14), we investigated how the reduction of JNK in hemin-treated animals would affect insulin levels. Interestingly, the abrogation of JNK was accompanied by a corresponding increase of plasma insulin (Fig. 4E), whereas CrMP reversed the effects of hemin. Given that oxidative stress depletes adiponectin (16, 17) and adiponectin deficiency leads to insulin resistance (4, 5), we investigated whether the antioxidant effect of an up-regulated HO system by hemin would alter plasma adiponectin. The levels of plasma adiponectin in GK rats were markedly reduced compared with the control (Fig. 4F). Interestingly, hemin significantly enhanced adiponectin by 4.9-fold, whereas CrMP abolished the hemin-induced increase of adiponectin.
Importantly, the hemin-induced enhancement of adiponectin/insulin and the concomitant reduction of NF-B, AP-1, AP-2, and JNK in GK rats were sustained for 3 months after therapy, suggesting a role of these pathways in the long-lasting antidiabetic effect. Hemin also reduced NF-B, AP-1, AP-2, and JNK and increased insulin/adiponectin in Wistar control rats, although to a lesser extent compared with GK rats.
Hemin therapy enhances cAMP, AMPK, and GLUT4 of the gastrocnemius muscles
AMPK, cAMP, and GLUT4 are important agents that enhance insulin-signaling and glucose metabolism (9, 11, 22). In insulin-resistant GK rat, cAMP, AMPK, and GLUT4 were significantly depressed (Fig. 5, A–C); however, assessment by enzyme immunoassay, Western blot, and quantitative RT-PCR indicated that hemin therapy robustly enhanced cAMP, AMPK, and GLUT4 by 3.2-, 3.1-, and 3.3-fold, respectively, whereas CrMP abrogated the antidiabetic effects of hemin.
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, A–C) would act in concert to sustain the enduring antidiabetic effect.
Hemin therapy reduces glucose and insulin intolerance in the GK rat
Because insulin resistance is accompanied by glucose and insulin tolerance, we investigated the effects of hemin on IPGTT, IPITT, and HOMA-IR. Interestingly, in untreated GK and GK+hemin+CrMP, IPGTT analyses revealed significantly elevated plasma glucose compared with GK+hemin and GK-posthemin groups at all time points tested (Fig. 6A), suggesting that hemin improved glucose tolerance. Furthermore, challenge with a bolus injection of glucose markedly stimulated insulin release in hemin-treated and posthemin-GK, suggesting improved glucose tolerance (Fig. 6B). However, only a modest level of glucose-stimulated insulin was observed in untreated GK and GK+hemin+CrMP and was significantly lower at all time points during the IPGTT analyses. In hemin-treated animals, two distinct phases of insulin release were observed: an acute-phase or first-phase response that peaked at 30 min and a second-phase response that gradually decreased to baseline by 90 min. On the other hand, in untreated GK and GK+hemin+CrMP, the two phases of insulin response were less evident, suggesting impaired glucose tolerance (Fig. 6B). Similarly, assessment by IPITT indicated a higher percentage change of glucose levels in GK+hemin and GK-posthemin after insulin challenge than untreated GK and GK+hemin+CrMP at all time points tested, suggesting the enhancement of insulin sensitivity by hemin (Fig. 6C).
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). However, hemin therapy markedly reduced insulin resistance to comparable levels as the Wistar rat. In contrast, the HO inhibitor, CrMP, abolished the effect of hemin. Importantly, CrMP alone further exacerbated insulin resistance in GK rats, suggesting the involvement of basal HO activity in the regulation of insulin-signaling and glucose metabolism. The reduction of HOMA-IR in GK+hemin and GK-posthemin groups may be indicative of greater insulin sensitivity. |
Discussion |
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B, AP-1, AP-2, and JNK, a substance that inhibits insulin biosynthesis. Accordingly, by up-regulating the HO system, hemin paradoxically increased plasma insulin and concomitantly improves glucose tolerance (IPGTT) and insulin tolerance (IPITT) but reduced insulin resistance (HOMA-IR index). Contrarily, the HO inhibitor, CrMP, suppressed HO activity and blocked the antidiabetic effects of hemin. Although many HO inhibitors are nonspecific and may affect other hemoenzymes or even increase HO-1 (32); however, CrMP given at a dose of 4 µmol/kg is reportedly selective for HO (33). Therefore, our study unveils a novel and unique characteristic in that up-regulating the HO system with hemin increases plasma insulin, potentiates skeletal muscle (gastrocnemius) insulin sensitivity, and improves glucose metabolism through the sustained increase of adiponectin, cAMP, cGMP, AMPK, and GLUT4 that lasted for 3 months after therapy.
The effect of hemin on AMPK and GLUT4 is a novel and intriguing observation. GLUT4 is important protein for glucose uptake. Reduced GLUT4 is implicated in insulin resistance and impaired glucose metabolism (24, 41). Therefore, the increased GLUT4 expression and reduced glucose/insulin tolerance are important antidiabetic mechanisms that could account for the sustained antidiabetic effect of hemin. Moreover, hemin also increased the levels of adiponectin, an insulin-sensitizing protein. Interestingly, the hemin-mediated increase of adiponectin was accompanied by enhanced AMPK signaling, a pathway that enhances glucose transport by stimulating the translocation of GLUT4 (9, 10, 11). However, it is not fully understood how hemin enhances GLUT4 expression. Some reports indicate that NO enhanced GLUT4 expression via a cGMP-dependent mechanism (23). Given that we also observed increased cGMP in our study, this cyclic nucleotide may be involved. Alternatively, the hemin-induced suppression of NF-B may improve GLUT4 signaling. Consistently, the reduction of NF-B has been associated with insulin-stimulated phosphorylation of Akt and GLUT4 translocation (42). Thus, our study unveils the multifaceted interaction between the HO system, insulin-sensitizing (adiponectin, AMPK, and GLUT4), and insulin-inhibitory pathways like NF-B and JNK (14). Our study also indicates loss of body weight in hemi-treated GK rats. Although the 4-wk hemin regimen slightly decreased body weight, the animals quickly recovered. Importantly, by 1 month after therapy, no difference in body weight was observed among the treated and untreated GK rats, although the antidiabetic effect was evident in the hemin-treated animals. Furthermore, at 2 and 3 months after therapy, comparable body weights were observed between hemin-treated/untreated GK rats, although the antidiabetic effect persisted only in hemin-treated animals, suggesting that the hemin-dependent antidiabetic effect is an intrinsic characteristic rather than an effect caused by body weight loss.
Hemin therapy also enhanced the HO signaling in euglycemic Wistar and SD, although to a lesser extent than in the GK rat. Because Wistar and SD rats are healthy animals with normal glycemic levels, the HO system may be acting with other functional pathways to regulate glucose homeostasis. Therefore, the less intense effect of the HO system might not have altered other healthy/functional pathways that act in concert with the HO system to regulate glucose metabolism in euglycemic Wistar and SD. Whether this is an intrinsic homeostatic and/or defensive mechanism to maintain healthy conditions within a certain physiological range remains unclear and needs to be clarified in future studies. On the other hand, the greater potentiation of HO signaling in GK rats was accompanied by more intense antidiabetic effect, suggesting that the HO system in the GK rat is more susceptible to pharmacological manipulations. Thus, hemin therapy may be selective in diabetic or diseased conditions. Given that GK rats are diabetic/unhealthy, the greater increment of HO signaling may be useful to surmount the threshold that initiates the restoration of glucose metabolism. This notion is consistent with previous studies in which up-regulating the HO system improved glucose metabolism (18).
Hyperglycemia-induced oxidative stress is a common phenomenon in insulin-resistant T2D (3). Moreover, the oxidative destruction of adiponectin and insulin (14, 16) aggravates glucose metabolism. Many signal transduction pathways including JNK is activated by oxidative stress (13, 14), and JNK is implicated in insulin resistance (14, 43). Accordingly, the suppression of JNK leads to reduced insulin resistance and improved glucose tolerance in diabetic mice (14). Moreover, the highly oxidative environment that characterizes diabetes activates JNK, which in turn, blocks insulin biosynthesis (14). Thus, the attenuation of JNK by hemin is another important antidiabetic mechanism. The role of the HO system in the enhancing insulin release (18) and the abating JNK activity (44) have been reported. On the other hand, oxidative stress depletes adiponectin mRNA with subsequent reduction of adiponectin (16, 17), suggesting the susceptibility of adipocytes to oxidative insults. Therefore, the potentiation of the overall antioxidant status from the relative inputs of different antioxidants like SOD, catalase, and ferritin may prevent the oxidative destruction of adiponectin and insulin in hemin-treated GK rats. The hemin-mediated increase in adiponectin is consistent with recent studies showing the enhancement of adiponectin by another HO inducer, cobalt protoporphyrin (20, 45, 46). The insulin-sensitizing role of adiponectin has been widely acknowledged (4, 5). The levels of adiponectin are low in patients with obesity, atherosclerosis, and insulin resistance (4). Furthermore, an adiponectin knockout mouse develops insulin-resistant T2D (5). Therefore, the hemin-mediated enhancement of adiponectin and insulin would greatly contribute in improving glucose metabolism in GK rats.
The regulation of insulin release by the HO system has been well acknowledged (18, 22, 27), and the central role of CO in glucose metabolism is becoming increasingly clear. In the human body, CO is formed at a rate of 16.4 µmol/h and daily production may reach 500 µmol (47). Interestingly, under normal physiological conditions, islets of Langerhans produce CO (27) and NO (22) to regulate insulin and glucagon secretion (22, 27). Whereas NO is a negative modulator of glucose-stimulated insulin release, CO stimulates insulin secretion (22, 27). Therefore, glucose stimulates islets to produce CO, which in turn triggers insulin release (22, 27). Moreover, previous studies have underscored the critical role of the HO system in insulin release and glucose metabolism in GK rats (18). Accordingly, impaired functional status of the HO system coupled to low CO production was detected in islets from GK rats (18). Interestingly, treatment with hemin or CO corrected the defective HO system, improved insulin release, and enhanced glucose metabolism (18), suggesting that impaired CO production by β-cells might lead to the development of T2D. The present study is a further testimony of the important role of the HO system in the homeostatic control of glucose.
Many other factors including pharmacokinetics and pharmacodynamics may underlie the sustained antidiabetic effects of hemin. For example, the slow absorption of hemin may be implicated. After killing the animals at 3 months after therapy, a small amount of unabsorbed hemin was present in the peritoneum. The slow absorption of hemin from the site of injection (peritoneum) may have contributed in maintaining a steady and consistent level of plasma hemin to sustain the potentiation of the HO signaling for several months with subsequent prolongation of the antidiabetic effect. Therefore, the suppression of hyperglycemia-induced oxidative stress along with the concomitant potentiation of insulin-sensitizing pathways may represent only the tip of an iceberg and does not profoundly address many unsettling questions. For a more-comprehensive understanding, further studies examining pharmacokinetics and pharmacodynamics of hemin are needed to shed more lights on the half-life, metabolites, distribution, and interaction with active proteins in biological milieu. Nevertheless, our study unveils important mechanisms that may account for the antidiabetic effect of the HO system. In the hemin-treated GK rat, enhanced cGMP was detected in the gastrocnemius muscles. Both cAMP and cGMP increase insulin levels (22). In addition, cGMP inhibits NF-B- and AP-1-induced inflammatory/oxidative insults (48). Similarly, adiponectin suppresses the activation of NF-B by a cAMP-dependent mechanism (12).
Seen in this light, the concomitant reduction of JNK, NF-B, AP-1, and AP-2 by hemin highlights the protective role of the HO system. Moreover, the HO-1 gene is pleiotropic with multiple physiological functions (26). Accordingly, the presence of binding sites for NF-B, AP-1, AP-2, and other motifs including glucocorticoid-responsive elements (26) in HO-1 gene promoter may be indicative of important regulatory role in many cellular events (28). Because glucocorticoids are involved in glucose metabolism and insulin resistance (49), it is possible that the HO system may suppress inflammatory/oxidative transcription factors to limit tissue insults (28) and regulate glucose metabolism (19, 27) through glucocorticoid-responsive elements (26) of the HO-1 gene. Moreover, HO-1 is induced by different stimuli including high glucose levels (50, 51). Accordingly, the diversity of HO inducers may be indicative of multiple regulatory elements for the HO-1 gene with binding sites for different transcription factors or genes. This array of genes may account for the diverse and important role of HO-1 in cellular homeostasis and defense. However, more in-depth studies are still needed to explain how HO-1 and/or the genes it modifies modulate glucose metabolism, insulin secretion, and insulin sensitization.
Although many studies have shown that the HO system increases insulin levels (18, 27, 7), some reports indicate otherwise because copper protoporphyrin, another HO inducer, did not increase the levels of insulin in obese diabetic mice (20). Whether this discrepancy is strain dependent and/or due to type of HO inducers needs further investigations. This is among the many challenges that have to be addressed for a greater understanding of the role of the HO system in glucose metabolism. Should these hurdles be cleared, then we could look to the future of HO inducers as antidiabetic agents with certain degree of optimism and hence increase their translational potential, given that hemin therapy is already used clinically against porphyria.
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Acknowledgments |
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Footnotes |
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Disclosure Summary: The authors have nothing to disclose.
First Published Online February 19, 2009
Abbreviations: AMPK, AMP-activated protein kinase; AP, activating protein; CrMP, chromium mesoporphyrin; GK, Goto-Kakizaki rat; GLUT4, glucose transporter-4; HO, hemeoxygenase; HOMA-IR, homeostasis model assessment insulin resistance; IPGTT, ip glucose tolerance test; IPITT, ip insulin tolerance test; JNK, c-Jun-N terminal kinase; NF-B, nuclear factor-B; NO, nitric oxide; SD, Sprague Dawley rats; SOD, superoxide dismutase; T2D, type 2 diabetes.
Received September 24, 2008.
Accepted for publication February 12, 2009.
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References |
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-glucoside hydrolase activities in parallel with glucose-stimulated insulin secretion. J Endocrinol 190:681–693B and AP-2 in the promoter region of the human heme oxygenase 1 gene. Proc Natl Acad Sci USA 91:5987–5991B and AP-1 activation induced by hemorrhagic shock via heme oxygenase-1 induction. Med Chem 2:271–274[CrossRef][Medline] generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo. Circulation 95:2492–2499B activation via NADPH oxidase. Am J Physiol Endocrinol Metab 294:E345–E351 and interleukin-6 in Zucker rats and reduce adipogenesis in human mesenchymal stem cells. J Pharmacol Exp Ther 325:833–840 mRNA during reperfusion of the rat liver upon decreased activation of NF-B and AP-1. J Hepatol 33:236–246[CrossRef][Medline]This article has been cited by other articles:
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  J. F. Ndisang, N. Lane, N. Syed, and A. Jadhav Up-Regulating the Heme Oxygenase System with Hemin Improves Insulin Sensitivity and Glucose Metabolism in Adult Spontaneously Hypertensive Rats Endocrinology, February 1, 2010; 151(2): 549 - 560. [Abstract] [Full Text] [PDF]
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 J. F. Ndisang and A. Jadhav The heme oxygenase system attenuates pancreatic lesions and improves insulin sensitivity and glucose metabolism in deoxycorticosterone acetate hypertension Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2010; 298(1): R211 - R223. [Abstract] [Full Text] [PDF]
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, P < 0.05 vs. all groups; *, P < 0.01 vs. all groups.
, P < 0.05 vs. all groups.
