AJP - Endo Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 296: E1029-E1041, 2009. First published February 10, 2009; doi:10.1152/ajpendo.90241.2008
0193-1849/09 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
296/5/E1029    most recent
90241.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ndisang, J. F.
Right arrow Articles by Jadhav, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ndisang, J. F.
Right arrow Articles by Jadhav, A.

Upregulation of the heme oxygenase system ameliorates postprandial and fasting hyperglycemia in type 2 diabetes

Joseph Fomusi Ndisang, Nina Lane, and Ashok Jadhav

Department of Physiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Submitted 18 February 2008 ; accepted in final form 9 February 2009


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In type 2 diabetes (T2D), postprandial and fasting hyperglycemia are important predictors of cardiovascular diseases; however, few drugs are currently available to simultaneously suppress these conditions. Here, we report an enduring antidiabetic effect of the heme oxygenase (HO) inducer hemin on Goto-Kakizaki rats (GK), a nonobese insulin-resistant T2D model. HO breaks down the heme-moiety-generating antioxidants (biliverdin/bilirubin and ferritin) and carbon monoxide, which stimulate insulin secretion. Hemin induces HO-1 to potentiate HO activity and the HO-derived products. Chronically applied hemin (30 mg/kg ip) for a month reduced and maintained fasting glucose at physiological levels for 3 mo. Before therapy, glucose levels were 9.3 ± 0.3 mmol/l (n = 14). At 1, 2, and 3 mo posttherapy, we recorded 6.7 ± 0.13, 5.9 ± 0.2, and 7.2 ± 0.2 mmol/l, respectively. Hemin was also effective against postprandial hyperglycemia (14.6 ± 1.1 vs. 7.5 ± 0.4 mmol/l; n = 14; P < 0.01), and the effect remained sustained for 3 mo after therapy. The reduction of hyperglycemia was accompanied by enhanced HO-1, HO activity, and cGMP of the soleus muscle, alongside increased plasma bilirubin, ferritin, SOD, total antioxidant capacity, and insulin levels, whereas markers/mediators of oxidative stress like urinary-8-isoprostane and soleus muscle nitrotyrosine, NF-{kappa}B, and activator protein-1 and -2 were abated. Furthermore, inhibitors of insulin signaling including soleus muscle glycogen synthase kinase-3 and JNK were reduced, while the insulin-sensitizing adipokine, adiponectin, alongside AMPK were increased. Correspondingly, hemin improved glucose tolerance, suppressed insulin intolerance, reduced insulin resistance, and overturned the inability of insulin to enhance glucose transporter 4, a protein required for glucose uptake. Hemin also upregulated HO-1/HO activity and cGMP and lowered glucose in euglycemic Sprague-Dawley control rats albeit less intensely, suggesting greater selectivity of the HO system in diabetic conditions. In conclusion, reduced oxidative stress alongside the concomitant and paradoxical enhancement of insulin secretion and insulin-sensitizing pathways may account for the 3-mo-enduring antidiabetic effect. The synergistic interaction among HO, adiponectin, and GLUT4 may be explored against insulin-resistant diabetes.

adiponectin; insulin resistance; oxidative stress; glucose transporter 4; glycogen synthase kinase-3; nuclear factor-{kappa}B; activating protein-1; c-Jun NH2-terminal kinase

EPIDEMIOLOGICAL STUDIES (19, 53) indicate that predictors of cardiovascular complications in diabetics include fasting/postprandial hyperglycemia and postglucose challenge hyperglycemia (2 h postload glucose). In diabetics and individuals with reduced glucose tolerance, transient glycemic rise after a meal increases the formation of advanced glycation end products (AGE) and oxidative/inflammatory events (10, 19, 53). Moreover, oxidative stress enhances AGE, which may potentiate the oxidative destruction of β-cells (15, 53).

Insulin insensitivity is a hallmark of type 2 diabetes (T2D; Ref. 57), and many factors, including oxidative stress, adiponectin deficiency, and over activity of glycogen synthase kinase-3 (GSK-3), are involved (17, 23, 36, 57, 59). GSK-3 is a serine/threonine kinase with two isoforms (GSK-3{alpha} and GSK-3β, and the 47-kDa isoform GSK-3β is known to inhibit insulin signaling; Ref. 23). In contrast, adiponectin is an insulin-sensitizing protein (36) that improves glucose metabolism and reduces insulin resistance by stimulating AMPK (16, 63), which in turn increases glucose transport by stimulating the translocation of glucose transporter-4 (GLUT4; Refs. 16, 26). Conversely, adiponectin suppresses the activation of NF-{kappa}B by a cAMP-dependent mechanism (51). In addition to NF-{kappa}B, JNK is activated by oxidative stress (11, 32), and it regulates activating protein-1 (AP-1; Ref. 7) and mediates insulin resistance (32). In diabetes, oxidative stress activates JNK, which in turn suppresses insulin biosynthesis (32). Thus the reduction of NF-{kappa}B, AP-1, and JNK may attenuate the oxidative destruction of insulin (32) and adiponectin (31).

Since hyperglycemia induces oxidative stress (53), the suppression of fasting/postprandial hyperglycemia may prevent cardiovascular complications in T2D. Therefore, substances that concomitantly suppress fasting/postprandial hyperglycemia, enhance adiponectin, and improve insulin sensitivity would be beneficial in T2D. The emerging role of the heme oxygenase (HO) system against T2D has been widely acknowledged (8, 45). HO is a microsomial enzyme with inducible (HO-1) and constitutive (HO-2) isoforms (2, 48). HO catalyzes the breakdown of the heme moiety to generate cytoprotective products including bilirubin, ferritin, and carbon monoxide (CO) with effects against, oxidative stress, inflammation hypertension, and apoptosis (1). In addition, the HO system has been shown to modulate other cellular activities such as insulin release and glucose metabolism (42, 45). In a related study (45), the HO inducer hemin and CO were shown to increase glucose-stimulated insulin release by pancreatic β-cells. Interestingly, glucose stimulates islets to produce CO, which in turn triggers insulin release (20, 22) as well as protection for β-cells (21, 64). On the other hand, emerging evidence indicates that the HO system enhances adiponectin levels (35, 37, 39). Whether the concomitant increase of adiponectin and insulin would be accompanied by improved insulin sensitivity will be investigated.

Although the HO system abates oxidative stress (25, 58), its effect on inflammatory/oxidative transcription factors like NF-{kappa}B, AP-1, and AP-2 in the soleus muscle are not fully characterized. Therefore, investigating the effects of the HO system in a highly oxidative tissue like the soleus muscle (6) may unveil important insights about HO in tissues with high susceptibility to oxidative destruction, especially in conditions characterized by elevated oxidative stress like T2D. Given that adiponectin stimulates AMPK, which in turn enhances and glucose transport, we studied whether the HO system improves glucose metabolism via stimulation of the soleus muscle AMPK and GLUT4. Whether the enhancement of insulin signaling will be accompanied by the suppression of inhibitors of insulin signaling, such as JNK and GSK-3, will also be investigated. Therefore, this study will unveil the effects of the HO inducer hemin on fasting/postprandial hyperglycemia and the multifaceted interaction among the HO system, adiponectin, AMPK, GLUT4, GSK-3, and oxidative/inflammatory mediators.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal treatment and plasma measurements. The experimental protocol was approved by University of Saskatchewan Standing Committee on Animal Care and Research Ethics. Thirteen-week-old (male) Goto-Kakizaki rats (GK) were purchased from Taconic Farms (Germantown, NY), while age-matched euglycemic Sprague-Dawley rats (SD) were purchased from Charles River (Willington, MA). The GK rat is a model of nonobese insulin-resistant T2D (45). Although the GK rat is a derived from a nondiabetic Wistar colony, many studies (52, 56) have used euglycemic SD as control for GK rats. The animals were housed at 21°C with 12-h light-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-1. To ascertain the involvement of HO-1 in the hemin-induced antidiabetic effect, the HO blocker chromium mesoporphyrin (CrMP; 4 µmol/kg ip; Porphyrin Scientific, Logan, UT) was given to some animals together with hemin. Although many HO inhibitors are nonspecific and affect other hemo-enzymes or even increase HO-1 expression (60), CrMP is reportedly selective against HO at a dose of 4 µmol/kg (61). 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 (27, 47, 50). As an additional control, some animals received the vehicle dissolving hemin and CrMP. Each injection was 0.5 ml and was given daily for 4 wk. The experimental design was made up of the following groups (n = 6-14 per group): 1) control GK; 2) GK + hemin; 3) GK + hemin + CrMP; 3) GK + CrMP; 4) control SD; 4) SD + hemin; and 5) GK + vehicle. Hemin therapy began at 14 wk, as the pathophysiological changes in tissues due hyperglycemia, insulin resistance, and oxidative stress are fully established by this age. At the end of the 4-wk hemin regimen, the animals were 17 wk of age.

During the entire 4-wk treatment, fasting/postprandial glucose was monitored weekly with a glucose meter (BD Biosciences, Franklin Lakes, NJ). Glucose was measured after the animals were fasted overnight in metabolic cages, while postprandial levels were taken at the same time (4 PM) each day throughout the study. Before postprandial glucose was measured, great attention was made to ensure that the animals had eaten. Since postprandial glycemic levels are highest 1 h after a meal (53), each animal was given 2 g of crushed chow by gavage 1 h before measurement. The mean daily food intake by GK rats was 24.9 ± 0.6 g (44). After therapy was stopped, fasting/postprandial glucose was monitored weekly for the ensuing 3 mo, at which time the study was terminated, with the animals at 29 wk of age. At this time, the study was terminated and the animals were killed after anesthesia (pentobarbital sodium; 50 mg/kg body wt). Plasma was collected by heart puncture, and tissues were harvested. From the plasma, bilirubin and ferritin were routinely measured by Saskatoon Royal University Hospital (Saskatoon, Canada).

Determination of HO activity and HO-1 concentration in the soleus muscle. HO activity in the soleus muscle was measured as bilirubin production using our established method (27, 47, 49). Briefly, the soleus muscle were homogenized on ice in 4 vol of 5:1 K/Na 100 mmol/l phosphate buffer (pH 7.4) with 2 mmol/l MgCl2 (HO-activity buffer) and centrifuged at 13,000 rpm for 15 min. Aliquots of 100 µl were collected from the supernatant and transferred into another beaker containing 500 µl of a mixture of 0.8 mmol/l nicotinamide dinucleotide phosphate, 20 µmol/l hemin, 2 mmol/l glucose-6-phosphate, 0.002 U/µl glucose-6-phosphate dehydrogenase, and 100 µl liver cytosol as a source of biliverdin reductase. The reaction was carried out in darkness for 1 h at 37°C and was stopped by addition of 500 µl of chloroform. Subsequently, bilirubin was extracted by vigorously agitating the tubes and centrifuging at 13,000 rpm for 5 min. The chloroform layer was collected and read on a spectrophotometer at 464 nm minus the background at 530 nm. The amount of bilirubin in each sample was expressed as nanomoles per milligram of protein per hour. The protein content was measured using Bradford assay. As a positive control, spleen tissue was used.

Soleus muscle HO-1 was determined by ELISA (Stressgen-Assay Design, Ann Arbor, MI) according to the manufacturer's instruction. This is a quantitative assay in which mouse monoclonal antibody specific for HO-1 was precoated on wells of a plate to capture HO-1 when the secondary rabbit polyclonal antibody conjugated to horseradish was added. The assay uses tetramethylbenidine substrate, which produces a blue color in proportion HO-1 levels. The addition of an acid converts the endpoint color to yellow, and the absorbance is read in a microplate at 540 nm (SpectraMax 340PC; Molecular Devices, CA). The concentration of HO-1 in the samples was calculated from a standard curve generated with calibrated HO-1 standards (0.195–12.5 ng/ml) that was provided by the manufacturer.

Total RNA isolation and quantitative RT-PCR for p65-NF-{kappa}B AP-1, AP-2, and JNK. This was done as we previously described (27, 47). Briefly, soleus muscles were homogenized in 0.5 ml TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). Reverse transcription was carried out using the First-Strand cDNA synthesis kit (Novagen, Madison, WI) with 0.5 µg Oligo (dT)6, 50 mM Tris·HCl (pH 8.3 at 25°C), 75 mM KCl, 75 mM KCl, 3 mM MgCl2, 50 mM DTT, 10 mM each free dNTP, and 100 U of Moloney murine leukemia virus reverse transcriptase, following the manufacturer's instruction. Quantitative PCR was done using Applied Biosystems 7300 Real Time PCR system (Foster City, CA) and iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) containing 50 mM KCl, 20 mM Tris·HCl (pH 8.4), 0.2 mM each free dNTP, 25 U/ml hot start enzyme iQTaq DNA polymerase, 3 mM MgCl2, SYBR Green 1, and 10 nM fluorescein as passive reference. Triplicate samples of 1 µl cDNA each were ran using a template of 3.2 pmol of primers for p65-NF-{kappa}B: forward, 5'-CATGCGTTTCCGTTACAAGTGCGA-3' and reverse, 5'-TGGGTGCGTCTTAGTGGTATCTGT-3'; AP-1: forward, 5'-AGCAGATGCTTGAGTTGAGAGCCA3' 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'; and β-actin: forward, 5'-TCATCACTATCGGCAATGAGCGGT-3' and reverse, 5'-ACAGCACTGTGTTGGCATAGAGGT in a final volume of 25 µl. The sequences of all primers used were confirmed by the National Research Institute of Canada (Saskatoon, SK, Canada).

Determination of plasma adiponectin. The concentration of adiponectin was determined by ELISA (Phoenix Pharmaceuticals, Burlingame, CA) following the manufacturer's instruction. Briefly, blood samples were collected and centrifuged, and the plasma was aliquoted into wells of a microplate containing adiponectin antibody. After treatment with horseradish peroxidase-conjugated secondary antibody and streptavidin, the samples were read at 450 nm using a microplate (SpectraMax 340PC).

Determination of plasma insulin. Plasma insulin was assessed by ELISA [Mercodia Ultrasensitive Rat Insulin kit (10-113-01), Mercodia, Uppsala, Sweden] according to the manufacturer's instructions. The assay is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule. During incubation, insulin in the samples reacts with peroxidase-conjugated anti-insulin antibodies and anti-insulin antibodies bound to microtitration well. After the unbound antibody is removed by washing, the bound conjugate is detected by reaction with 3,3',5,5'-tetramethylbenzidine. The reaction is stopped by the addition of acid to give a colorimetric endpoint that is read spectrophotometrically at 450 mn with a microplate (SpectraMax 340PC).

Determination of glucose tolerance and insulin tolerance. Glucose tolerance was evaluated by intraperitoneal glucose tolerance test (IPGTT), after overnight fasting. A bolus of glucose (2 g/kg ip) was injected, and blood samples were collected sequentially from the tail vein at intervals of 0, 30, 60, 90, and 120 min, and tested for glucose and insulin. Glucose was measured with a glucose meter (BD Biosciences), and insulin was measured by ELISA (Mercodia). To evaluate insulin tolerance, an intraperitoneal insulin tolerance test (IPITT) was done. A bolus of insulin (2 U/kg ip) was injected, and blood samples were taken sequentially at various time points from 0–120 min for glucose measurement.

Determination of insulin resistance by homeostasis model assessment of insulin resistance analyses. The homeostasis model assessment of insulin resistance (HOMA-IR) was used to further assess the effect of hemin therapy on glucose tolerance in GK rats. After overnight fasting, the animals were injected intraperitoneally with 2 mg/g glucose in 0.9% saline, and glucose and insulin were measured from blood obtained from tail vein at 0, 30, 60, 90, and 120 min. Values for HOMA-IR were calculated from the product of fasting plasma glucose (mg/dl) and insulin (µU/ml) divided by 22.5 (43).

Western immunoblotting. The soleus muscle was homogenized with a cocktail of protease inhibitors and centrifuged as we previously described (27, 47, 49). Briefly, proteins were extracted and quantified by Bradford assay, and aliquots of 50 µg were loaded on a 10% SDS-polyacrylamide gel for GSK-3, AMPK, and nitrotyrosine, a marker of oxidative stress (12, 30), and 12.5% for GLUT4. The fractionated proteins were electrophoretically transferred to nitrocellulose paper and nonspecific bindings blocked with 3% nonfat milk dissolved in PBS and were incubated overnight with primary antibodies against nitrotyrosine (Santa Cruz Biotechnology), GSK-3 (Stressgen Biotech), AMPK (Cell Signaling Technology, Danvers, MA), and GLUT4 (Abcam, Cambridge, MA). Anti-mouse β-actin (Sigma) was used as control. Densitometric scanning and analyses were done using UN-SCAN-IT software (Silk Scientific).

Measurement of cGMP content in the soleus muscle. The concentration of cGMP was assessed by enzyme immunoassay (EIA; Cayman Chemical, Ann Arbor, MI) as we previously described (27, 47, 49). Briefly, the soleus muscles were homogenized in 6% TCA at 4°C in the presence of IBMX to inhibit phosphodiesterase activity and were centrifuged at 2,000 g for 15 min. The supernatant was recovered and washed with water-saturated diethyl ether, and the upper ether layer was aspirated and discarded while the aqueous layer containing cGMP was recovered and lyophilized. The dry extract was dissolved in 1 ml of cGMP-assay buffer, and the cGMP content was measured using the manufacturer's protocol and expressed as picomoles of cGMP per milligrams of protein.

Determination of 8-isoprostane, an index of oxidative stress. Urinary 8-isoprostane is a noninvasive index of oxidative stress (13). This was determined by EIA (Cayman Chemical) as we previously reported (27). In brief, urine samples collected over a 24-h period were diluted 1:15 with ultrapure water, applied to a reverse-phase C-18 column at pH 3, and eluted with 1:1 (vol/vol) ethyl acetate/heptane. Thereafter, the eluent was further purified on a silica column and eluted with 1:1 (vol/vol) ethylacetate-methanol and aliquoted in a 96-well plate that was precoated with monoclonal antibody. Next, 8-isoprostane tracer and isoprostane antiserum were added to each well and the wells were incubated, and after they were washed, Ellaman's reagent containing the substrate of acetylcholinesterase was added. The absorbance were read at 412 nm in a plate reader (SpectraMax 340PC), and the values of 8-isoprostane were calculated from a standard curve.

Measurement of plasma SOD activity. Plasma SOD was determined by EIA (Cayman Chemical) as we have previously reported (27, 47), following the instructions of the manufacturer. The absorbance were read at 450 nm in a plate reader (SpectraMax 340PC), and the values of SOD were calculated from a standard curve. This assay detects both cytosolic and mitochondrial SOD activity and thus measures total SOD activity.

Total antioxidant capacity assay. The total antioxidant capacity assay was done by EIA (Cayman Chemical) as we previously reported (27, 47). This assay is based on antioxidants inhibiting the oxidation of 2,2'-azino-di-[3-ethylbenzthiazoline sulphonate] (ABTS) to ABTS plus metmyoglobin. In brief, plasma samples were treated with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), metmyoglobin, and chromogen, and the absorbance was read at 750 nm using Synergy Microplate Reader (BioTek Instruments, Winooski, VT). The results were expressed as Trolox equivalent antioxidant capacity per milligrams of protein.

Statistical analysis. All data are expressed as means ± SE from at least six independent experiments unless otherwise stated. Statistical analyses were done using unpaired Student's t-test, ANOVA in conjunction with Bonferroni test, and ANOVA for repeated measures where appropriate. Group differences at the level of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hemin therapy reduced fasting and postprandial hyperglycemia for 3 mo. The 4-wk regimen of hemin progressively reduced hyperglycemia and restored fasting glucose to physiological levels in GK. Before treatment, the fasting levels were 9.3 ± 0.3 mmol/l. After 1 wk of therapy, the levels were significantly reduced (7.7 ± 0.2 vs. 10.4 ± 0.2 mmol/l, P < 0.01) compared with age-matched untreated GK. After the second, third, and fourth week of therapy, fasting levels were as follows (in mmol/l): 6.6 ± 0.2 vs. 10.6 ± 0.2, P < 0.01; 6.4 ± 0.3 vs. 10.9 ± 0.4, P < 0.01; and 6.3 ± 0.4 vs. 10.9 ± 0.3, respectively. Interestingly, the effect of hemin on hyperglycemia prevailed for up to 3 mo after stoppage of therapy (7.2 ± 0.2 mmol/l), while age-matched untreated GK were 11.2 ± 0.2 mmol/l (Fig. 1, A and B). To ascertain the implication of the HO system in the antidiabetic effects, some GK rats were given hemin with or without the HO blocker, CrMP. The cotreatment of hemin and CrMP reversed the antidiabetic effects, whereas CrMP alone further exacerbated fasting hyperglycemia (Fig. 1A), suggesting the involvement of basal HO activity in blood sugar homeostasis.


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 1. Effect of the heme oxygenase (HO) inducer hemin and the HO blocker chromium mesoporphyrin (CrMP) on fasting and postprandial hyperglycemia in Goto-Kakizaki rats (GK). A: hemin therapy lowered and maintained fasting glucose at significantly low levels for 3 mo after discontinuation of therapy, while CrMP abolished the effects of hemin, exacerbating hyperglycemia (*P < 0.01 vs. control; {dagger}P < 0.01 vs. all groups). Bars represent means ± SE; n = 6–14 rats per group. B: fasting glucose levels of hemin-treated GK were significantly lower than in age-matched untreated GK (*P < 0.01 vs. control). Bars represent means ± SE; n = 10–14 rats per group. C: hemin regimen lowered postprandial hyperglycemia, and this effect was sustained for up to 3 mo after stoppage of therapy; however, CrMP annulled the effect of hemin (*P < 0.01 vs. control; {dagger}P < 0.01 vs. all groups). Bars represent means ± SE; n = 6–14 rats per group. D: postprandial glycemic levels of hemin-treated GK were significantly lower than in age-matched untreated GK (*P < 0.01 vs. control). Bars represent means ± SE; n = 10–14 rats per group.

 
The hemin regimen was also effective against postprandial hyperglycemia. During the period of hemin administration, there was a consistent and significant reduction of the postprandial glycemic levels. Before treatment the glucose levels were 14.6 ± 1.1 mmol/l, and at the end of therapy, there was a marked decrease at 7.5 ± 0.4 mmol/l. Subsequently, at 1, 2 and 3 mo posttherapy, postprandial hyperglycemia remained significantly lower than in the age-matched untreated GK as follows (in mmol/l): 9.2 ± 0.3 (n = 10) vs. 14.9 ± 0.9 (n = 14), P < 0.01; 9.4 ± 0.5 (n = 10) vs. 15.3 ± 1.4 (n = 14), P < 0.01; and 9.8 ± 0.6 (n = 10) vs. 15.8 ± 1.3 (n = 14), P < 0.01 (Fig. 1, C and D). On the contrary, hemin + CrMP reversed the effect of hemin against postprandial hyperglycemia, while CrMP alone further increased postprandial hyperglycemia in GK (Fig. 1C).

The 4-wk hemin therapy also lowered fasting (6.9 ± 0.4 vs. 6.03 ± 0.4, P < 0.05; n = 6) and postprandial glucose in SD (9.5 ± 0.7 vs. 7.2 ± 0.5, P < 0.01; n = 6), but the effect was less intense compared with GK rats. On the other hand, the vehicle had no effect on fasting (9.5 ± 0.4 vs. 9.6 ± 0.3 mmol/l; n = 10) and postprandial hyperglycemia (14.3 ± 0.8 vs. 14.2 ± 0.9 mmol/l; n = 10).

The hemin regimen affected body weight. During treatment, reduced body weight was observed in GK and SD (Table 1). The reduction of body weight was temporary, as the animals quickly regained weight after therapy. By the end of the first month posttherapy, the hemin-treated and control GK had comparable weights, but interestingly, fasting and postprandial hyperglycemia remained significantly lower in the hemin-treated animals. Therefore, it is unlikely that the loss of body weight is responsible for the sustained antidiabetic effect. Similarly, after 1 mo posttherapy body weight in hemin-treated and untreated SD were comparable (479 ± 10.5 vs. 481 ± 9.4 g; n = 6; Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Effect of hemin therapy on body weight

 
Hemin upregulates HO-1, HO activity, and cGMP in the soleus muscle and enhances plasma bilirubin and ferritin. In hyperglycemic GK, the basal HO-1 and HO activities were depressed compared with the euglycemic SD control. Interestingly, hemin increased the HO-1 concentration and HO activity in GK by 13.2- and 15.4-fold, respectively (Fig. 2, A and B), while the HO inhibitor CrMP abolished the increase. Hemin also enhanced HO-1 and HO activity in SD by 5.1- and 5.4-fold, respectively. The HO-induced increase of HO-1 and HO activity was significantly greater in GK than SD. Interestingly, after 3 mo posttherapy, HO-1 and HO activity in hemin-treated GK was still 10.9- and 11.8-fold higher than control GK (Fig. 2A). The interaction between the HO system and cGMP is an important mechanism by which hemin elicits its effects (48). cGMP is the downstream target of CO generated by the HO system (49), and cGMP regulates insulin release (46), GLUT4 (40), and thus glucose metabolism. Our results indicate that the basal cGMP in GK was depressed (Fig. 2C). Interestingly, the enhanced HO-1 and HO activity in GK was accompanied by a 10.1-fold parallel increase of cGMP, and after 3 mo posthemin therapy, cGMP content was still 7.5-fold higher than control GK. On the other hand, the HO inhibitor CrMP blunted the effects of hemin on cGMP. Although hemin therapy also enhanced cGMP in SD, the magnitude of the increment in GK was greater. The greater increase of HO activity and cGMP in GK might account for the more intense antidiabetic effect in GK. On the other hand, the less preponderant increase in HO activity in SD may suggest greater stability of the HO system in euglycemic SD than diabetic GK.


Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 2. Effect of hemin and the HO blocker CrMP on soleus muscle HO-1, HO activity, cGMP content, plasma ferritin, and bilirubin in GK rats. A: basal HO-1 in GK was depressed compared with euglycemic Sprague-Dawley (SD) control rats. Hemin therapy greatly increased HO-1, while CrMP abolished the effect of hemin ({dagger}P < 0.05 vs. all groups; *P < 0.01 vs. all groups). Bars represent means ± SE; n = 6 rats per group. B: basal levels of HO activity in GK were lower than in euglycemic SD control. Hemin therapy greatly increased HO activity, while CrMP reduced it ({dagger}P < 0.05 vs. all groups; *P < 0.01 vs. all groups). Bars represent means ± SE; n = 6 rats per group. C: treatment with hemin markedly increase of cGMP content, but CrMP blocked the effect of hemin (*P < 0.01 vs. all groups). Bars represent means ± SE; n = 6 rats per group. D: basal levels of bilirubin in GK were lower than in SD. Hemin increased biliirubin to levels even beyond those observed in euglycemic SD control, whereas CrMP abolished the increase ({dagger}{dagger}P < 0.01 vs. all groups). Bars represent means ± SE; n = 6 rats per group. E: basal ferritin levels in GK were lower than in SD control but were increased by hemin to similar levels as in SD. CrMP abolished the increase by hemin ({dagger}{dagger}P < 0.01 vs. all groups). Bars represent means ± SE; n = 6 rats per group.

 
Since bilirubin and ferritin are among the antioxidants produced by the HO system (25, 58), we assayed and detected significantly lower basal levels of bilirubin and ferritin in GK rats (Fig. 2, D and E). Interestingly, hemin therapy greatly enhanced plasma bilirubin and ferritin by 66.7 and 71%, respectively, whereas the HO blocker CrMP abrogated the hemin-induced increase. Importantly, the hemin-induced increase of bilirubin and ferritin remained sustained at significantly elevated levels for 3 mo after stoppage of therapy. The concomitant potentiation of HO activity, cGMP, bilirubin, and ferritin constitutes a potent protective axis against tissue insult (25, 49, 58).

Hemin therapy suppresses oxidative stress. Oxidative insult is a major contributor to insulin resistance (59), and hyperglycemia is a major trigger of oxidative stress. Interestingly, the enhancement of bilirubin and ferritin by hemin was associated with a significant depletion of important markers of oxidative stress like nitrotyrosine and urinary 8-isoprostane (12, 13, 30; Fig. 3, A and B). While the levels of urinary 8-isoprostane reflect systemic oxidative stress, the expression of nitrotyrosine in the soleus muscle indicates tissue-specific oxidative stress. In hyperglycemic GK rats, the basal levels of urinary 8-isoprostane were 2.4-fold higher than age-matched SD. Hemin therapy greatly reduced 8-isoprostane, reinstating levels similar to those in control SD. The reduction of 8-isoprostane was sustained for 3 mo after stoppage of therapy. Hemin also reduced 8-isoprostane in SD, although to a lesser extent compared with GK. Importantly, the normalization of glucose levels in GK coincided with the restoration of 8-isoprostane to similar levels as in the euglycemic SD rat (Fig. 3A). Interestingly, the expression of soleus muscle nitrotyrosine was reduced by hemin to levels even lower than those in age-matched SD (Fig. 3B).


Figure 3
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 3. Effect of hemin therapy on 8-isoprostane, nitrotyrosine, SOD, and total antioxidant status. A: basal urinary 8-isoprostane levels in GK were significantly elevated compared with age-matched euglycemic SD. Hemin therapy reduced 8-isoprostane in GK to comparable levels as observed in age-matched SD (*P < 0.05 vs. all groups; **P < 0.01 vs. all groups; {dagger}P < 0.01 vs. all groups). Bars represent means ± SE; n = 8 rats per group. B: representative Western immunoblot of soleus muscle nitrotyrosine and quantitative analyses of relative nitrotyrosine levels normalized by β-actin reveals that hemin therapy significantly abrogated the high levels of nitrotyrosine in GK to levels even below the control (*P < 0.05 vs. all groups; {dagger}P < 0.01 vs. all groups). Bars represent means ± SE; n = 4 rats per group. C: basal levels of plasma SOD in GK were lower than SD control but were increased by hemin to levels even beyond the control (**P < 0.01 vs. all groups; {dagger}P < 0.01 vs. all groups). Bars represent means ± SE; n = 8 rats per group. D: basal levels of plasma total antioxidant capacity of GK were reduced compared with SD control but were increased by hemin to levels even beyond the control (**P < 0.01 vs. all groups; {dagger}P < 0.01 vs. all groups). Bars represent means ± SE; n = 8 rats per group.

 
Since the HO system is known to enhance antioxidant enzymes (60), we measured SOD activity. The basal levels of plasma SOD was significantly depressed in GK (Fig. 3C). Hemin therapy greatly increased SOD activity by 2.6-fold, and the levels remained sustained for 3 mo after termination of therapy. Correspondingly, hemin increased the total antioxidant capacity of GK to levels even higher than in the nondiabetic SD (Fig. 3D). Interestingly, the suppression of markers of oxidative stress such as urinary 8-isoprostane and nitrotyrosine, alongside the concomitant enhancement of SOD activity, bilirubin, and ferritin coincided with the elevation of the total antioxidant status in hemin-treated GK.

Hemin therapy abates proinflammatory/oxidative signaling agents, including NF-{kappa}B, AP-1, AP-2, and JNK in the soleus muscle. Many transcriptions factors are implicated in tissue damage and insulin resistance in diabetes. These include NF-{kappa}B, AP-1, AP-2, and JNK (7, 32). Interestingly, quantitative real-time RT-PCR indicates that hemin therapy attenuated these transcription factors (Fig. 4). Accordingly, the elevated basal AP-1 mRNA levels in GK rats were significantly abated by 77.1% (Fig. 4A). Similarly, hemin reduced the basal AP-2 mRNA expression by 50.4% (Fig. 4B). Furthermore, the high basal NF-{kappa}B and JNK in GK rats were reduced by 73.4 and 84.9%, respectively, by hemin (Fig. 4, C and D). It is important to note that hemin therapy effectively restored NF-{kappa}B and JNK to the levels of SD, while AP-1 and AP-2 were partially reduced. The reasons for this selective effect remain unclear and should be further investigated.


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 4. Effect of hemin therapy on oxidative/proinflammatory mediators, activating protein (AP)-1, AP-2, NF-{kappa}B, and JNK in the soleus muscle of GK rats. A: quantitative real-time RT-PCR reveals that the basal AP-1 mRNA in GK was significantly elevated but was abated by hemin therapy (**P < 0.01 vs. all groups; {dagger}P < 0.01 vs. all groups). Bars represent means ± SE; n = 8 rats per group. B: similarly, elevated AP-2 levels detected in GK rats were abated by hemin therapy (*P < 0.01 vs. all groups; {dagger}P < 0.01 vs. all groups). Bars represent means ± SE; n = 8 rats per group. C: hemin therapy reduced the high levels of NF-{kappa}B in GK to similar levels as in SD control (*P < 0.01 vs. all groups). Bars represent means ± SE; n = 8 rats per group. D: application of hemin significantly reduced the enhanced mRNA expression of JNK in GK to comparable levels as in the SD control (*P < 0.01 vs. all groups). Bars represent means ± SE; n = 8 rats per group.

 
Hemin therapy enhances plasma adiponectin and insulin levels and potentiates soleus muscle GLUT4 in GK rats. The GK rat exhibits a markedly reduced insulin response to glucose, a defect that is coupled to an impaired glucose uptake (65). Both adiponectin and GLUT4 are important for insulin sensitivity and glucose uptake (17, 65). Our data indicate that the basal levels of plasma adiponectin in GK were lower than in age-matched SD (Fig. 5A). However, hemin increased adiponectin to comparable levels as in SD. Interestingly, the increased adiponectin in hemin-treated animals was associated with a parallel increase of plasma insulin (Fig. 5B). Importantly, the low basal levels of insulin in GK were increased by 2.7-fold. Our results also indicate that the depressed soleus muscle GLUT4 expression in GK was robustly upregulated by hemin (Fig. 5C).


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 5. Effect of hemin therapy on adiponectin, insulin, and GLUT4 protein expression. A: basal levels of adiponectin were lower in GK compared with age-matched SD. Hemin therapy greatly enhanced adiponectin to comparable levels as observed in SD (*P < 0.01 vs. all groups). Bars represent means ± SE; n = 6 rats per group. B: basal insulin levels in GK were significantly lower than in euglycemic SD control. Interestingly, hemin therapy greatly enhanced insulin levels in GK to similar levels as in SD (*P < 0.01 vs. all groups). Bars represent means ± SE; n = 6 rats per group. C: representative Western immunoblot of GLUT4 protein and relative desitometric analyses. Basal expression of GLUT4 protein was significantly depressed in GK compared with age-matched SD; however, hemin therapy enhanced the expression of GLUT4 proteins to levels even beyond those observed in age-matched SD ({dagger}P < 0.01 vs. all groups; *P < 0.01 vs. all groups). Bars represent means ± SE; n = 6 rats per group.

 
Hemin therapy improves glucose and insulin tolerance in GK. In GK, insulin resistance was accompanied by glucose and insulin tolerance, so we investigated whether the antidiabetic effect of hemin would improve glucose/insulin tolerance. Interestingly, IPGTT indicated that plasma levels of glucose in control GK were significantly elevated compared with hemin-treated animals at all time points tested (Fig. 6A), suggesting improved glucose tolerance in hemin-treated animals. In control GK, glucose concentration increased from 14.5 ± 1.8 to 25.8 ± 3.1 mmol/l by 30 min and remained beyond the baseline for 120 min (14.5 ± 1.8 vs. 21.9 ± 1.1 mmol/l) after the glucose challenge. In contrast, glucose levels in hemin-treated GK increased from 6.9 ± 0.7 to 8.9 ± 0.8 mmol/l, quickly returned to the basal level, and remained constant at baseline throughout the experiment. In GK 3 mo posthemin, the effects were similar to the hemin-treated-GK killed at the end of the 4-wk therapy, albeit less intense. Accordingly, in GK-3-mo-posthemin glucose levels increased from 9.3 ± 0.6 to 12.9 ± 1.5 mmol/l and quickly returned to baseline. By the end of 120 min, the levels were 7.8 ± 0.6 mmol/l.


Figure 6
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 6. Effect of hemin therapy on glucose and insulin tolerance in GK. A: hemin therapy improved glucose intolerance (*P < 0.01 vs. all groups; n = 6 per group). B: hemin therapy increased glucose-stimulated insulin release in GK (*P < 0.01 vs. all groups; n = 6 per group). C: hemin therapy improved insulin tolerance (IPITT) in GK after insulin challenge (*P < 0.01 vs. all groups; n = 6 per group). Bars represent means ± SE; n = 6 rats per group. D: hemin therapy reduced insulin resistance [homeostasis model assessment of insulin resistance (HOMA)-IR index] and thus enhanced insulin sensitivity in GK (*P < 0.01 vs. all groups; n = 6 per group). Bars represent means ± SE; n = 6 rats per group.

 
Interestingly, the lower glucose levels in hemin-treated GK and GK 3 mo posthemin were associated with a parallel increase of insulin levels (Fig. 6B), whereas the levels of insulin in control GK remained significantly low at all time points during the IPGTT. Importantly, two distinct phases were observed: an acute-phase or first-phase response (0–30 min) and a S-phase response characterized by a gradual decrease that subsequently returned to fasting levels by 90 min and remained constant at that level for the entire duration of the experiment. In hemin-treated GK and GK 3 mo posthemin, the insulin released during the first 30 min (acute phase) response after glucose challenge might arise from preformed insulin that had been packaged in the secretory vesicles of β-cells, while the insulin at the later stage may come from both preformed and newly synthesized insulin. In GK, reduced insulin production has been attributed to a dysfunctional HO system (20, 42, 45). Therefore, the lack of adequate first-phase insulin secretion in control GK underscores the pivotal role of the HO system in insulin release and glucose metabolism. Importantly, hemin has been shown to increase glucose-stimulated insulin release by pancreatic β-cells (45).

We investigated the effects of hemin on insulin sensitivity by performing IPITT. Interestingly, the administration of a bolus injection of insulin evoked a greater change of glucose levels in GK + hemin and GK-3-mo-posthemin groups (Fig. 6C), suggesting increased insulin sensitivity in hemin-treated animals. In contrast, insulin challenge evoked only a marginal change of glucose in control GK, suggesting reduced insulin sensitivity. To further evaluate the effect of hemin on insulin sensitivity, the HOMA-IR assay was done. Interestingly, HOMA-IR analyses indicated that insulin resistance in hemin-treated GK and GK 3 mo posthemin was significantly reduced compared with control (untreated) GK (Fig. 6D), suggesting improved insulin sensitivity in hemin-treated animals.

Hemin therapy enhances AMPK but suppresses abated GSK-3 in the soleus muscle of GK. AMPK and GSK-3 are important proteins implicated in glucose metabolism and insulin resistance, and thus we investigated the effect of hemin therapy on these proteins. These proteins have opposite effects on insulin signaling. AMPK mediates the insulin-sensitizing effects of adiponectin, whereas GSK-3 inhibits insulin signaling (23, 36, 57, 59).

Western immunoblotting and relative densitometric analyses indicated that the basal levels of GSK-3 in the soleus muscle of GK were significantly elevated (Fig. 7A). However, the application of hemin suppressed the diabetic status in GK with a corresponding reduction of GSK-3 expression. Indeed, hemin reduced and restored the expression of GSK-3 to the same level of age-matched euglycemic SD. In contrast, the levels of AMPK in the soleus muscle of GK were markedly reduced (Fig. 7B). However, hemin therapy robustly increased AMPK expression in GK by 3.4-fold, reinstating comparable levels as observed in the SD control. The concomitant upregulation of AMPK, adiponectin, and GLUT4 by hemin is an important finding by which the HO system combats insulin-resistant T2D. Interestingly, adiponectin improves insulin sensitivity by activating AMPK signaling, which in turn increases glucose transport by stimulating the translocation of GLUT4 (Refs. 16, 26, 63).


Figure 7
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 7. Effect of hemin therapy on the expression of GSK-3 and AMPK. A: representative Western immunoblot of GSK-3 protein and relative desitometric analyses. Basal expression of GSK-3 protein was significantly elevated in GK compared with SD. Hemin therapy significantly abated the expression of GSK-3 to similar levels as observed in age-matched SD (*P < 0.01 vs. all groups). Bars represent means ± SE; n = 6 rats per group. B: representative Western immunoblot of AMPK and relative densitometric analyses of expressed proteins normalized by β-actin revealed that hemin therapy greatly enhanced the depressed AMPK expression in GK rats (*P < 0.01 vs. all groups). Bars represent means ± SE; n = 6 rats per group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our results demonstrate the enduring effect of hemin against fasting and postprandial hyperglycemia. The mechanisms involved include the potentiation of the HO system alongside the concomitant and paradoxical increase of both insulin production and insulin sensitivity. The enhanced insulin sensitivity was accompanied by a parallel increase of GLUT4 expression. The effect of hemin on GLUT4 is a novel and intriguing observation. GLUT4 is an important protein for glucose uptake. Reduced GLUT4 signaling is implicated in insulin resistance and impaired glucose metabolism (14, 65). Hemin therapy also increased adiponectin levels with corresponding enhancement of AMPK, a protein known to increases glucose transport by stimulating the translocation of GLUT4 (16, 26, 63). The hemin-induced increase of GLUT4 protein expression may be due to increased cGMP, given that a cGMP-dependent mechanism has been previously reported to enhance GLUT4 (40). Alternatively, the hemin-induced suppression of NF-{kappa}B may improve GLUT4 signaling, since the reduction of NF-{kappa}B is associated with insulin-stimulated phosphorylation of Akt and the translocation of GLUT4 (62). Whether these two mechanisms act in concert or independently to modulate GLUT4 should be clarified in future studies. The enhancement of promoters of insulin signaling was accompanied by the concomitant attenuation of insulin-inhibitory pathways like GSK-3 and JNK (23, 32). The inhibition of GSK-3 has been shown to improve insulin signaling and glucose transport (23), while JNK is known to inhibit insulin biosynthesis (32) Thus our study unveils the multifaceted interaction between the HO system and insulin-sensitizing and insulin-inhibitory pathways.

The involvement of the HO system in the antidiabetic effect of hemin was confirmed by blocking HO activity. The HO inhibitor CrMP suppressed HO-1 and HO activity GMP, and it exacerbated hyperglycemia in GK, suggesting an important role of the HO system in glucose metabolism. Although many HO inhibitors are nonspecific and may affect other heme-derived enzymes or even increase HO-1 (60); however, CrMP given at a dose of 4 µmol/kg is reportedly selective against HO activity (61). Our study also indicates that hemin therapy slightly decreased body weight in GK. However, the weight loss may not account for the sustained antidiabetic effects because the hemin-treated animals quickly regained weight, and by 1 mo posttherapy there was no significant difference in body weight between the hemin-treated and control GK, although the antidiabetic effect was observed only in the hemin-treated animals. A similar observation was made at 2 and 3 mo posttherapy.

Hyperglycemia-induced oxidative stress is a common phenomenon in insulin-resistant T2D (57), and the oxidative destruction of important agents of glucose metabolism like adiponectin and insulin (31, 32) has been reported. Interestingly, hemin therapy abated oxidative stress by suppressing 8-isoprostane and nitrotyrosine, two important markers of oxidative stress (12, 13, 30). The attenuation of oxidative stress was accompanied by the enhancement of antioxidants, including SOD, bilirubin, and ferritin, with subsequent potentiation of the total antioxidant status in GK rats. In tissues, the antioxidant system comprises a variety of different compounds such as SOD, catalase, glutathione peroxidase, ferritin, ascorbic acid, {alpha}-tocopherol, β-carotene, reduced glutathione, uric acid, biliverdin, and bilirubin (3, 4, 25, 28, 58), and the sum of endogenous and food-derived antioxidants represents the total antioxidant activity of the system (3). The additive effect of all the different antioxidants provides greater protection against oxidative stress than any single compound alone. Given that oxidative stress depletes adiponectin mRNA with a subsequent reduction of adiponectin levels (31, 55), adipocytes may be very susceptible to oxidative destruction. Therefore, by suppressing oxidative stress, hemin attenuated the oxidative destruction of adiponectin and insulin. Consistently, increased plasma adiponectin and insulin were observed in hemin-treated GK, and the levels remained elevated for up to 3 mo after stoppage of therapy. The hemin-mediated increase in adiponectin is consistent with recent studies (35, 37, 39) in which another HO inducer, cobalt protoporphyrin (CoPP), enhanced adiponectin levels in Zucker fatty rats and obese diabetic mice. The insulin-sensitizing role of adiponectin has been widely acknowledged (17, 36). The levels of adiponectin are low in patients with obesity, atherosclerosis, and insulin resistance (17). Furthermore, adiponectin knockout mouse develops insulin-resistant T2D (36). Therefore, the concomitant increases of adiponectin and insulin in GK are important factors that contributed to the sustained effect of hemin against hyperglycemia.

An interesting observation from our study is that the enduring antidiabetic effect of hemin was accompanied by the paradoxical enhancement of both insulin sensitivity and insulin secretion. The regulation of insulin release by the HO system has been well acknowledged (20, 45, 46), and hemin as well as CO has been shown to play a central role. Interestingly, under physiological conditions, islets of Langerhans produce CO (20) and NO (46) to regulate insulin and glucagon secretion (20, 46). While NO is a negative modulator of glucose-stimulated insulin release, CO stimulates insulin secretion (20, 46). Moreover, dysfunctional HO-2 and low production of CO by islets resulted in insulin-resistant T2D in GK rats (45), and treatment with hemin or CO corrected the defective HO system by increasing insulin release (45). Moreover, the hemin-induced increase of cGMP may also increase insulin levels, since previous studies (46) have shown that insulin levels are increased by activating cGMP. Therefore, the present study is a further testimony to the important role of the HO system in glucose homeostasis, since hemin therapy not only increased insulin levels but also improved IPGTT and IPITT and enhanced insulin sensitivity (HOMA-IR index). Seen in this light, our study unveils a unique characteristic of hemin that is evidenced by the paradoxical and concomitant increase of insulin release and insulin sensitivity. These findings could be explored in the design of a novel remedy against diabetes, especially in cases where the destruction and progressive reduction of β-cell mass by apoptosis/necrosis and oxidative stress (5, 8, 9) in insulin-resistant type-2 diabetic patients lead to insulin insufficiency. Moreover, the HO system also possess antioxidant and anti-apoptotic/necrotic properties (8, 18) besides its insulin-sensitizing effects. The hemin-mediated improvement of insulin sensitivity reported in the present study is consistent with previous observations in which another HO inducer, CoPP, was shown to improve insulin sensitivity in obese mice (39). Although the present study alongside other reports (20, 42, 45) in the literature supports the notion that the HO system increases insulin levels, a recent study (39) showed that CoPP did not increase insulin in obese diabetic mice. Whether the discrepancy is strain and/or HO-inducer dependent has to be further investigated.

Another important observation from our study is the reduction of soleus muscle NF-{kappa}B, AP-1, and AP-2 that was accompanied by increased cGMP. Importantly, cGMP is known to blunt NF-{kappa}B- and AP-1-induced inflammatory/oxidative insults (34). Moreover, the presence of binding sites for NF-{kappa}B, AP-1, AP-2, and other motifs, including serum-, metal-, heat-shock-, and glucocorticoid-responsive elements (38), in the HO-1 gene promoter may be indicative of an important regulatory role in many cellular events (54). Since glucocorticoids are involved in glucose metabolism and insulin resistance (41), the HO system may suppress inflammatory/oxidative transcription factors to limit tissue insults (54) and regulate other glucose metabolism (8, 20) through glucocorticoid-responsive elements (38) of the HO-1 gene. Given that HO-1 is induced by different stimuli including high glucose levels (29, 33), 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 processes.

Our study also revealed increased HO-1, HO activity, and cGMP in euglycemic SD, although the increment was less intense than in the GK rat. The greater magnitude of HO signaling in the GK rat was associated with a more-pronounced antidiabetic effect, suggesting that hemin therapy may be less effective under euglycemic conditions but more selective in a diabetic environment. Therefore, the HO system in SD may be more stable and less susceptible to pharmacological manipulations. Whether this is an intrinsic homeostatic and/or defensive mechanism to maintain healthy status within a certain physiological range remains unclear and should be further investigated. Given that the GK rat is unhealthy with dysfunction of critical pathways involved in glucose metabolism like the HO system, the more preponderant increase of HO signaling might be necessary to surmount the threshold that triggers the restoration of glucose metabolism. This notion is consistent with previous studies (45) in which an upregulated HO system improved glucose metabolism. On the other hand, since SD rats are healthy animals with normal glycemia, the HO system may be acting in conjunction with other functional pathways in maintaining the homeostatic control of blood glucose. Therefore, the less intense effect of the HO system would not alter the healthy/functional pathways in SD. However, these notions have to be investigated profoundly in future studies.

Although the present study unveils the sustained antidiabetic effects of hemin, the findings should be cautiously interpreted because of the limitations in the study design. Examples include the use of euglycemic SD instead of Wistar as control GK rats as well as the choice of tissue (soleus muscle). Although the soleus muscle is widely used to investigate insulin resistance (24), it is a very oxidative tissue (6) and may not ideally represent the physiology of the major muscle fibers in the body. However, studying the soleus muscle in GK will advance our understanding of the antioxidant effects of the HO system on the pathophysiology of this muscle in a diabetic model characterized by impaired HO system and elevated hyperglycemia-induced oxidative stress.

Collectively, our findings unveil the antidiabetic effect of hemin and suggest that reduced oxidative stress and the concomitant enhancement of both insulin and insulin sensitivity are among the mechanisms involved in the sustained reduction of fasting/postprandial hyperglycemia. These were coupled to improved insulin tolerance, reduced glucose intolerance, increased insulin sensitivity, and the potentiation of promoters of insulin signaling such as adiponectin, AMPK, and GLUT4. Therefore, our study unmasks the multifaceted interaction among the HO system, adiponectin, AMPK, GLUT, GSK-3, and JNK in glucose metabolism and insulin-resistant T2D. Given the high incidence of insulin-resistant T2D and related cardiovascular complications, upregulating the HO system may constitute a novel strategy to combat these plights.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Heart and Stroke Foundation of Saskatchewan, Canada, and the Canadian Institute of Health Research/University of Saskatchewan College of Medicine Bridge funding.


   ACKNOWLEDGMENTS
 
We thank James Talbot for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. F. Ndisang, Dept. of Physiology, Univ. of Saskatchewan College of Medicine, 107 Wiggins Road, Saskatoon, SK, Canada S7N 5E5 (e-mail: joseph.ndisang{at}usask.ca)


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abraham NG, Kappas A. Pharmacological and clinical aspects of heme oxygenase. Pharmacol Rev 60: 79–127, 2008.[Abstract/Free Full Text]
  2. Abraham NG, Tsenovoy PL, McClung J, Drummond GS. Heme oxygenase: a target gene for anti-diabetic and obesity. Curr Pharm Des 14: 412–421, 2008.[CrossRef][Web of Science][Medline]
  3. Apak R, Guclu K, Ozyurek M, Karademir SE, Altun M. Total antioxidant capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: the CUPRAC method. Free Radic Res 39: 949–961, 2005.[CrossRef][Web of Science][Medline]
  4. Balla G, Jacob HS, Balla J, Rosenberg M, Nath K, Apple F, Eaton JW, Vercellotti GM. Ferritin: a cytoprotective antioxidant strategem of endothelium. J Biol Chem 267: 18148–18153, 1992.[Abstract/Free Full Text]
  5. Baumgartner HK, Gerasimenko JV, Thorne C, Ashurst LH, Barrow SL, Chvanov MA, Gillies S, Criddle DN, Tepikin AV, Petersen OH, Sutton R, Watson AJ, Gerasimenko OV. Caspase-8-mediated apoptosis induced by oxidative stress is independent of the intrinsic pathway and dependent on cathepsins. Am J Physiol Gastrointest Liver Physiol 293: G296–G307, 2007.[Abstract/Free Full Text]
  6. Beha A, Juretschke HP, Kuhlmann J, Neumann-Haefelin C, Belz U, Gerl M, Kramer W, Roden M, Herling AW. Muscle type-specific fatty acid metabolism in insulin resistance: an integrated in vivo study in Zucker diabetic fatty rats. Am J Physiol Endocrinol Metab 290: E989–E997, 2006.[Abstract/Free Full Text]
  7. Bennett BL, Satoh Y, Lewis AJ. JNK: a new therapeutic target for diabetes. Curr Opin Pharmacol 3: 420–425, 2003.[CrossRef][Web of Science][Medline]
  8. Bruce CR, Carey AL, Hawley JA, Febbraio MA. Intramuscular heat shock protein 72 and heme oxygenase-1 mRNA are reduced in patients with type 2 diabetes: evidence that insulin resistance is associated with a disturbed antioxidant defense mechanism. Diabetes 52: 2338–2345, 2003.[Abstract/Free Full Text]
  9. Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 52: 102–110, 2003.[Abstract/Free Full Text]
  10. Ceriello A, Davidson J, Hanefeld M, Leiter L, Monnier L, Owens D, Tajima N, Tuomilehto J. Postprandial hyperglycaemia and cardiovascular complications of diabetes: an update. Nutr Metab Cardiovasc Dis 16: 453–456, 2006.[CrossRef][Web of Science][Medline]
  11. Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature 410: 37–40, 2001.[CrossRef][Medline]
  12. Darwish RS, Amiridze N, Aarabi B. Nitrotyrosine as an oxidative stress marker: evidence for involvement in neurologic outcome in human traumatic brain injury. J Trauma 63: 439–442, 2007.[CrossRef][Web of Science][Medline]
  13. Delanty N, Reilly MP, Pratico D, Lawson JA, McCarthy JF, Wood AE, Ohnishi ST, Fitzgerald DJ, FitzGerald GA. 8-Epi PGF2 alpha generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo. Circulation 95: 2492–2499, 1997.[Abstract/Free Full Text]
  14. Desrois M, Sidell RJ, Gauguier D, King LM, Radda GK, Clarke K. Initial steps of insulin signaling and glucose transport are defective in the type 2 diabetic rat heart. Cardiovasc Res 61: 288–296, 2004.[Abstract/Free Full Text]
  15. Devangelio E, Santilli F, Formoso G, Ferroni P, Bucciarelli L, Michetti N, Clissa C, Ciabattoni G, Consoli A, Davi G. Soluble RAGE in type 2 diabetes: association with oxidative stress. Free Radic Biol Med 43: 511–518, 2007.[CrossRef][Web of Science][Medline]
  16. Guo Z, Xia Z, Yuen VG, McNeill JH. Cardiac expression of adiponectin and its receptors in streptozotocin-induced diabetic rats. Metabolism 56: 1363–1371, 2007.[CrossRef][Web of Science][Medline]
  17. Haluzik M. Adiponectin and its potential in the treatment of obesity, diabetes and insulin resistance. Curr Opin Investig Drugs 6: 988–993, 2005.[Medline]
  18. Harder Y, Amon M, Schramm R, Rucker M, Scheuer C, Pittet B, Erni D, Menger MD. Ischemia-induced up-regulation of heme oxygenase-1 protects from apoptotic cell death and tissue necrosis. J Surg Res 150: 293–303, 2008.[CrossRef][Web of Science][Medline]
  19. Held C, Gerstein HC, Yusuf S, Zhao F, Hilbrich L, Anderson C, Sleight P, Teo K. Glucose levels predict hospitalization for congestive heart failure in patients at high cardiovascular risk. Circulation 115: 1371–1375, 2007.[Abstract/Free Full Text]
  20. Henningsson R, Alm P, Ekstrom P, Lundquist I. Heme oxygenase and carbon monoxide: regulatory roles in islet hormone release: a biochemical, immunohistochemical, and confocal microscopic study. Diabetes 48: 66–76, 1999.[Abstract]
  21. Henningsson R, Alm P, Lundquist I. Evaluation of islet heme oxygenase-CO and nitric oxide synthase-NO pathways during acute endotoxemia. Am J Physiol Cell Physiol 280: C1242–C1254, 2001.[Abstract/Free Full Text]
  22. Henningsson R, Alm P, Lundquist I. Occurrence and putative hormone regulatory function of a constitutive heme oxygenase in rat pancreatic islets. Am J Physiol Cell Physiol 273: C703–C709, 1997.[Abstract/Free Full Text]
  23. Henriksen EJ, Dokken BB. Role of glycogen synthase kinase-3 in insulin resistance and type 2 diabetes. Curr Drug Targets 7: 1435–1441, 2006.[Web of Science][Medline]
  24. Henriksen EJ, Kinnick TR, Teachey MK, O'Keefe MP, Ring D, Johnson KW, Harrison SD. Modulation of muscle insulin resistance by selective inhibition of GSK-3 in Zucker diabetic fatty rats. Am J Physiol Endocrinol Metab 284: E892–E900, 2003.[Abstract/Free Full Text]
  25. Hintze KJ, Theil EC. DNA and mRNA elements with complementary responses to hemin, antioxidant inducers, and iron control ferritin-L expression. Proc Natl Acad Sci USA 102: 15048–15052, 2005.[Abstract/Free Full Text]
  26. Holmes BF, Sparling DP, Olson AL, Winder WW, Dohm GL. Regulation of muscle GLUT4 enhancer factor and myocyte enhancer factor 2 by AMP-activated protein kinase. Am J Physiol Endocrinol Metab 289: E1071–E1076, 2005.[Abstract/Free Full Text]
  27. Jadhav A, Torlakovic E, Ndisang JF. Interaction among heme oxygenase, nuclear factor-{kappa}B, and transcription activating factors in cardiac hypertrophy in hypertension. Hypertension 52: 910–917, 2008.[Abstract/Free Full Text]
  28. Jeney V, Balla J, Yachie A, Varga Z, Vercellotti GM, Eaton JW, Balla G. Pro-oxidant and cytotoxic effects of circulating heme. Blood 100: 879–887, 2002.[Abstract/Free Full Text]
  29. Jonas JC, Guiot Y, Rahier J, Henquin JC. Haeme-oxygenase 1 expression in rat pancreatic beta cells is stimulated by supraphysiological glucose concentrations and by cyclic AMP. Diabetologia 46: 1234–1244, 2003.[CrossRef][Web of Science][Medline]
  30. Julius U, Drel VR, Grässler J, Obrosova IG. Nitrosylated proteins in monocytes as a new marker of oxidative-nitrosative stress in diabetic subjects with macroangiopathy. Exp Clin Endocrinol Diabetes 117: 72–77, 2008.[CrossRef][Web of Science][Medline]
  31. Kamigaki M, Sakaue S, Tsujino I, Ohira H, Ikeda D, Itoh N, Ishimaru S, Ohtsuka Y, Nishimura M. Oxidative stress provokes atherogenic changes in adipokine gene expression in 3T3–L1 adipocytes. Biochem Biophys Res Commun 339: 624–632, 2006.[CrossRef][Web of Science][Medline]
  32. Kaneto H, Nakatani Y, Kawamori D, Miyatsuka T, Matsuoka TA, Matsuhisa M, Yamasaki Y. Role of oxidative stress, endoplasmic reticulum stress, and c-Jun N-terminal kinase in pancreatic beta-cell dysfunction and insulin resistance. Int J Biochem Cell Biol 38: 782–793, 2006.[CrossRef][Web of Science][Medline]
  33. Keyse SM, Tyrrell RM. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc Natl Acad Sci USA 86: 99–103, 1989.[Abstract/Free Full Text]
  34. Kiemer AK, Vollmar AM, Bilzer M, Gerwig T, Gerbes AL. Atrial natriuretic peptide reduces expression of TNF-alpha mRNA during reperfusion of the rat liver upon decreased activation of NF-kappaB and AP-1. J Hepatol 33: 236–246, 2000.[CrossRef][Web of Science][Medline]
  35. Kim DH, Burgess AP, Li M, Tsenovoy PL, Addabbo F, McClung JA, Puri N, Abraham NG. Heme oxygenase-mediated increases in adiponectin decrease fat content and inflammatory cytokines tumor necrosis factor-alpha and interleukin-6 in Zucker rats and reduce adipogenesis in human mesenchymal stem cells. J Pharmacol Exp Ther 325: 833–840, 2008.[Abstract/Free Full Text]
  36. Kubota N, Terauchi Y, Yamauchi T, Kubota T, Moroi M, Matsui J, Eto K, Yamashita T, Kamon J, Satoh H, Yano W, Froguel P, Nagai R, Kimura S, Kadowaki T, Noda T. Disruption of adiponectin causes insulin resistance and neointimal formation. J Biol Chem 277: 25863–25866, 2002.[Abstract/Free Full Text]
  37. L'Abbate A, Neglia D, Vecoli C, Novelli M, Ottaviano V, Baldi S, Barsacchi R, Paolicchi A, Masiello P, Drummond GS, McClung JA, Abraham NG. Beneficial effect of heme oxygenase-1 expression on myocardial ischemia-reperfusion involves an increase in adiponectin in mildly diabetic rats. Am J Physiol Heart Circ Physiol 293: H3532–H3541, 2007.[Abstract/Free Full Text]
  38. Lavrovsky Y, Schwartzman ML, Levere RD, Kappas A, Abraham NG. Identification of binding sites for transcription factors NF-kappa B and AP-2 in the promoter region of the human heme oxygenase 1 gene. Proc Natl Acad Sci USA 91: 5987–5991, 1994.[Abstract/Free Full Text]
  39. Li M, Kim DH, Tsenovoy PL, Peterson SJ, Rezzani R, Rodella LF, Aronow WS, Ikehara S, Abraham NG. Treatment of obese diabetic mice with a heme oxygenase inducer reduces visceral and subcutaneous adiposity, increases adiponectin levels, and improves insulin sensitivity and glucose tolerance. Diabetes 57: 1526–1535, 2008.[Abstract/Free Full Text]
  40. Lira VA, Soltow QA, Long JH, Betters JL, Sellman JE, Criswell DS. Nitric oxide increases GLUT4 expression and regulates AMPK signaling in skeletal muscle. Am J Physiol Endocrinol Metab 293: E1062–E1068, 2007.[Abstract/Free Full Text]
  41. Liu Y, Nakagawa Y, Wang Y, Liu L, Du H, Wang W, Ren X, Lutfy K, Friedman T. Reduction of hepatic glucocorticoid receptor and hexose-6-phosphate dehydrogenase expression ameliorates diet-induced obesity and insulin resistance in mice. J Mol Endocrinol 41: 53–64, 2008.[Abstract/Free Full Text]
  42. Lundquist I, Alm P, Salehi A, Henningsson R, Grapengiesser E, Hellman B. Carbon monoxide stimulates insulin release and propagates Ca2+ signals between pancreatic β-cells. Am J Physiol Endocrinol Metab 285: E1055–E1063, 2003.[Abstract/Free Full Text]
  43. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28: 412–419, 1985.[CrossRef][Web of Science][Medline]
  44. Moreira T, Malec E, Ostenson CG, Efendic S, Liljequist S. Diabetic type II Goto-Kakizaki rats show progressively decreasing exploratory activity and learning impairments in fixed and progressive ratios of a lever-press task. Behav Brain Res 180: 28–41, 2007.[CrossRef][Web of Science][Medline]
  45. Mosen H, Salehi A, Alm P, Henningsson R, Jimenez-Feltstrom J, Ostenson CG, Efendic S, Lundquist I. Defective glucose-stimulated insulin release in the diabetic Goto-Kakizaki (GK) rat coincides with reduced activity of the islet carbon monoxide signaling pathway. Endocrinology 146: 1553–1558, 2005.[Abstract/Free Full Text]
  46. Mosen H, Salehi A, Henningsson R, Lundquist I. Nitric oxide inhibits, and carbon monoxide activates, islet acid alpha-glucoside hydrolase activities in parallel with glucose-stimulated insulin secretion. J Endocrinol 190: 681–693, 2006.[Abstract/Free Full Text]
  47. Ndisang JF, Lane N, Jadhav A. Crosstalk between the heme oxygenase system, aldosterone, and phospholipase C in hypertension. J Hypertens 26: 1188–1199, 2008.[CrossRef][Web of Science][Medline]
  48. Ndisang JF, Tabien HE, Wang R. Carbon monoxide and hypertension. J Hypertens 22: 1057–1074, 2004.[CrossRef][Web of Science][Medline]
  49. Ndisang JF, Wu L, Zhao W, Wang R. Induction of heme oxygenase-1 and stimulation of cGMP production by hemin in aortic tissues from hypertensive rats. Blood 101: 3893–3900, 2003.[Abstract/Free Full Text]
  50. Ndisang JF, Zhao W, Wang R. Selective regulation of blood pressure by heme oxygenase-1 in hypertension. Hypertension 40: 315–321, 2002.[Abstract/Free Full Text]
  51. Ouchi N, Kihara S, Arita Y, Okamoto Y, Maeda K, Kuriyama H, Hotta K, Nishida M, Takahashi M, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Funahashi T, Matsuzawa Y. Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-kappaB signaling through a cAMP-dependent pathway. Circulation 102: 1296–1301, 2000.[Abstract/Free Full Text]
  52. Patriti A, Aisa MC, Annetti C, Sidoni A, Galli F, Ferri I, Gulla N, Donini A. How the hindgut can cure type 2 diabetes. Ileal transposition improves glucose metabolism and beta-cell function in Goto-kakizaki rats through an enhanced proglucagon gene expression and L-cell number. Surgery 142: 74–85, 2007.[CrossRef][Web of Science][Medline]
  53. Rendell MS, Jovanovic L. Targeting postprandial hyperglycemia. Metabolism 55: 1263–1281, 2006.[CrossRef][Web of Science][Medline]
  54. Sasaki T, Takahashi T, Maeshima K, Shimizu H, Toda Y, Morimatsu H, Takeuchi M, Yokoyama M, Akagi R, Morita K. Heme arginate pretreatment attenuates pulmonary NF-kappaB and AP-1 activation induced by hemorrhagic shock via heme oxygenase-1 induction. Med Chem 2: 271–274, 2006.[CrossRef][Medline]
  55. Soares AF, Guichardant M, Cozzone D, Bernoud-Hubac N, Bouzaidi-Tiali N, Lagarde M, Geloen A. Effects of oxidative stress on adiponectin secretion and lactate production in 3T3–L1 adipocytes. Free Radic Biol Med 38: 882–889, 2005.[CrossRef][Web of Science][Medline]
  56. Song MK, Hwang IK, Rosenthal MJ, Harris DM, Yamaguchi DT, Yip I, Go VL. Anti-hyperglycemic activity of zinc plus cyclo (his-pro) in genetically diabetic Goto-Kakizaki and aged rats. Exp Biol Med (Maywood) 228: 1338–1345, 2003.[Abstract/Free Full Text]
  57. Song P, Wu Y, Xu J, Xie Z, Dong Y, Zhang M, Zou MH. Reactive nitrogen species induced by hyperglycemia suppresses Akt signaling and triggers apoptosis by upregulating phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in an LKB1-dependent manner. Circulation 116: 1585–1595, 2007.[Abstract/Free Full Text]
  58. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science 235: 1043–1046, 1987.[Abstract/Free Full Text]
  59. Tang C, Han P, Oprescu AI, Lee SC, Gyulkhandanyan AV, Chan GN, Wheeler MB, Giacca A. Evidence for a role of superoxide generation in glucose induced β-cell dysfunction in vivo. Diabetes 56: 2722–2731, 2007.[Abstract/Free Full Text]
  60. Turkseven S, Kruger A, Mingone CJ, Kaminski P, Inaba M, Rodella LF, Ikehara S, Wolin MS, Abraham NG. Antioxidant mechanism of heme oxygenase-1 involves an increase in superoxide dismutase and catalase in experimental diabetes. Am J Physiol Heart Circ Physiol 289: H701–H707, 2005.[Abstract/Free Full Text]
  61. Vreman HJ, Ekstrand BC, Stevenson DK. Selection of metalloporphyrin heme oxygenase inhibitors based on potency and photoreactivity. Pediatr Res 33: 195–200, 1993.[Web of Science][Medline]
  62. Wei Y, Sowers JR, Clark SE, Li W, Ferrario CM, Stump CS. Angiotensin II-induced skeletal muscle insulin resistance mediated by NF-{kappa}B activation via NADPH oxidase. Am J Physiol Endocrinol Metab 294: E345–E351, 2008.[Abstract/Free Full Text]
  63. Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, Yamashita S, Noda M, Kita S, Ueki K, Eto K, Akanuma Y, Froguel P, Foufelle F, Ferre P, Carling D, Kimura S, Nagai R, Kahn BB, Kadowaki T. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med 8: 1288–1295, 2002.[CrossRef][Web of Science][Medline]
  64. Ye J, Laychock SG. A protective role for heme oxygenase expression in pancreatic islets exposed to interleukin-1beta. Endocrinology 139: 4155–4163, 1998.[Abstract/Free Full Text]
  65. Zisman A, Peroni OD, Abel ED, Michael MD, Mauvais-Jarvis F, Lowell BB, Wojtaszewski JF, Hirshman MF, Virkamaki A, Goodyear LJ, Kahn CR, Kahn BB. Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance. Nat Med 6: 924–928, 2000.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
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]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
296/5/E1029    most recent
90241.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ndisang, J. F.
Right arrow Articles by Jadhav, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ndisang, J. F.
Right arrow Articles by Jadhav, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2009 by the American Physiological Society.