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Endocrinology Vol. 148, No. 12 5925-5932
Copyright © 2007 by The Endocrine Society

Des-Aspartate-Angiotensin I Exerts Hypoglycemic Action via Glucose Transporter-4 Translocation in Type 2 Diabetic KKAy Mice and GK Rats

Meng-Kwoon Sim, Xiao-Guang Xu, Yong-Chiat Wong, Sai-Zhen Sim and Kok-Onn Lee

Departments of Pharmacology (M.-K.S., X.-G.X., Y.-C.W., S.-Z.S.) and Department of Medicine (K.-O.L.), Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Address all correspondence and requests for reprints to: Meng-Kwoon Sim, Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. E-mail: phcsimmk{at}nus.edu.sg.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study investigated the hypoglycemic action of des-aspartate-angiotensin I (DAA-I), a metabolite of angiotensin I, in two animal models of type 2 diabetes. The rationale was based on our earlier studies demonstrating that DAA-I acts on the angiotensin AT1 receptor and exerts responses opposing those of angiotensin II and on recent reports that curtailment of angiotensin II formation by angiotensin converting enzyme inhibitors and blockade of the AT1 receptor attenuate hyperglycemia in type 2 diabetics and diabetic animals. Diabetic KKAy mice and GK rats were administered orally (by gavage) one of the following doses of DAA-I: 400, 600, or 800 nmol/kg·d for 4 and 6 wk, respectively. Control diabetic animals were similarly administered water. Blood glucose of each animal was determined fortnightly by oral glucose tolerance test and blood insulin on the last day of treatment. Animals were killed, and the levels of plasma membrane glucose transporter-4 and cytosolic tyrosine-phosphorylated insulin receptor substrate-1 in hind limb skeletal muscles were determined by Western blot in insulin-challenged and nonchallenged animals. Orally administered DAA-I had no effect on blood insulin level but exerted dose-dependent hypoglycemic action in KKAy mice and GK rats after 4 and 6 wk of treatment, respectively. At the maximal effective dose of 600 nmol/kg, insulin induced a significant increase in plasma membrane glucose transporter-4 and cytosolic tyrosine-phosphorylated insulin receptor substrate-1. These findings show that DAA-I is not an insulin secretagogue and exerts hypoglycemic action by attenuating insulin resistance, the first such demonstration indicating that the nonapeptide is involved in glycemic regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OF LATE, CLINICAL TRIALS have shown that angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers reduce the development of type 2 diabetes in persons with essential hypertension (1, 2, 3). ACE inhibitors have also been shown to improve insulin resistance in mouse and rat models of type 2 diabetes (4, 5) and valsartan (an angiotensin receptor blocker) in type 2 diabetic mice (6). At the cellular level, angiotensin II impairs two insulin-stimulated processes, namely phosphorylation of insulin receptor substrate-1 (IRS-1) tyrosine and coupling of the insulin receptor pathway to phosphatidylinositol (PI) 3-kinase, suggesting that activation of the renin-angiotensin system may lead to insulin resistance in the vasculature (7, 8). Imidapril, an ACE inhibitor, has been shown to significantly enhance the activity of insulin-stimulated PI 3-kinase (9). These findings support the clinical observations that interruption of tissue renin-angiotensin system reduces the development of type 2 diabetes in persons with essential hypertension.

Recent studies on angiotensin II and insulin resistance show that angiotensin II attenuates insulin-induced translocation of glucose transporter-4 (GLUT4) from cytoplasmic vesicles to the plasma membrane of skeletal muscle (10, 11). The angiotensin AT1 receptor is coupled to a variety of intracellular second messengers, which cross-talk with the pathway of GLUT4 translocation (12). Through these second messengers, angiotensin II impairs tyrosine phosphorylation of IRS-1 and activation of protein kinase B by PI 3-kinase (12). Specific drugs that are able to act on these cross-talks are not yet available, and it appears that upstream curtailment of angiotensin II formation by ACE inhibitors (9, 13) and blockade of the AT1 receptor by blockers (6, 10) remain the avenues for drugs that act via the angiotensin system to halt hyperglycemia in type 2 diabetics and animal models of type 2 diabetes. In this respect, des-aspartate-angiotensin I (DAA-I, an endogenous angiotensin peptide), which acts on the AT1 receptor and induces responses opposing those of angiotensin II (15, 16, 17), was studied for its hypoglycemic action and effects on insulin resistance in two animal models of type 2 diabetes. The results demonstrated that DAA-I exerts significant hypoglycemic action by enhancing insulin-induced GLUT4 translocation in the diabetic animals. The findings also support the pleiotropic nature of the AT1 receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Six- to 7-wk-old male C57BL/6J mice and Wistar rats were purchased from the National University of Singapore Animal Centre. Seven- to 9-wk-old type 2 diabetic KKAy mice and 6- to 7-wk-old GK rats were purchased from CLEA Inc., Tokyo, Japan. Diabetic animals were individually housed to avoid aggression. Animals were fed on standard rat chow and water ad libitum. Temperature of the animal house was set at 25 ± 1 C with lighting from 0700–1900 h daily. Animals were allowed 1 wk acclimatization before experimentation. All experimental protocols were approved by our Institutional Animal Care and Use Committee and carried out with its guidelines.

Administration of DAA-I
Diabetic animals (KKAy mice and GK rats) were randomly assigned to groups of eight animals and administered orally (by gavage) DAA-I solution or water. The doses used were 400, 600, and 800 nmol/kg and were based on the dose range that effectively attenuated experimentally induced cardiac hypertrophy in rats (18). The volumes and duration of DAA-I and vehicle administration were 0.1 ml and 4 wk for diabetic mice and 0.2 ml and 6 wk for diabetic rats. Water was used as a vehicle because autoclaved DAA-I solution was found to be remarkably stable. Shelf life, at ambient temperature, was greater than 6 months. Nondiabetic control animals (C57BL/6J mice, BALB/c mice, and Wistar rats) were similarly grouped and administered either 600 nmol/kg DAA-I or water. The dose of 600 nmol/kg was chosen because this dose exerts maximal hypoglycemic effect in the diabetic animals in this study.

Oral glucose tolerance test (OGTT) and serum insulin determination
After every 2 wk of vehicle or DAA-I treatment, animals were fasted overnight (16 h) and the weight of each animal was recorded. Each animal was then administered orally 2 g/kg glucose solution. Blood was obtained from the orbital sinus of each fasted animal using a glass capillary at 0 min (just before glucose administration), and at 30, 60, and 120 min after glucose administration. Glucose concentration in serum of the clotted blood samples was determined using a commercial kit from Thermo Electron Corp., Noble Park, Victoria, Australia. In preliminary experiments that were performed to determine the onset of hypoglycemic action of DAA-I, only one blood sampling at 30 min was taken. Insulin in the serum was determined using a commercial insulin kit from Crystal Chem Inc., IL.

Correlation of serum glucose and insulin with insulin resistance
The whole-body insulin sensitivity was determined by using an equation defined as an index of whole-body insulin sensitivity [10,000/square root of (fasting glucose x fasting insulin) x (mean glucose x mean insulin during OGTT)]. This index has been shown to correlate with the rate of whole-body glucose disposed during the euglycemic insulin clamp (19). Similarly, the hepatic insulin resistance and muscle insulin sensitivity were determined by the respective indexes, which have also been shown to strongly correlate with measurement of hepatic and muscle insulin resistance obtained directly with hyperinsulinemic-euglycemic clamp (20) (see Table 2Go for formulas of indexes).


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  		TABLE 2. Effects of DAA-I on insulin resistance in KKAy mice and GK rats

 
Skeletal muscle preparation
After an overnight fast, vehicle- and DAA-I-treated animals were ip administered 0.5 U/kg insulin or equivalent volume of PBS. Animals were killed by cervical dislocation at 30 min after insulin or PBS administration, and hind limb skeletal muscles were excised and frozen in liquid nitrogen. Muscles were stored at –80 C until used for GLUT4 translocation study by Western blotting.

Skeletal muscle for the IRS-1 tyrosine phosphorylation study were similarly prepared except that fasted animals were administered 40 U/kg insulin and animals were killed at 5 min after insulin administration.

Membrane preparation for GLUT4 study
Skeletal muscle plasma membrane was prepared according to the method described by Dombrowski et al. (21). Briefly, 3 g frozen skeletal muscle was allowed to thaw over an ice pack, minced with a pair of fine scissors, and homogenized at a low-speed setting with a Polytron in ice-cold lysis buffer containing 10 mM sodium bicarbonate, 0.25 M sucrose, 5 mM sodium azide, and 100 µmol phenylmethylsulfonyl fluoride at a ratio of 1 g muscle/15 ml buffer. The homogenate was spun at 1200 x g for 10 min, and the supernatant was respun at 9000 x g for 10 min. The second-time supernatant was spun at 190,000 x g for 60 min to obtain the pellet, which contained the crude membranes. A sample of the crude membrane was reserved for Western blot analysis. The remaining pellet was resuspended in 2 ml buffer and subjected to discontinuous sucrose gradient (25, 32, and 35% wt/wt) centrifugation for 16 h at 150,000 x g. Fractions were collected and resuspended in sucrose-free buffer and subjected to 190,000 x g for 1 h. The pelleted membrane fractions were resuspended in 200 µl buffer and used for protein assay, alkaline phosphodiesterase-1 activity assay, and Western blot assay for GLUT4 protein.

Protein, alkaline phosphodiesterase-1, and Western blot assays
Sample protein concentrations were determined using the Bio-Rad (Hercules, CA) DC Protein Assay with BSA as a standard. Alkaline phosphodiesterase-1 is a plasma membrane-bound enzyme, and its activity was used for determining the plasma membrane concentration in sucrose gradient pelleted membrane fractions. Its activity was measured by the release of p-nitrophenol from a buffered thymidine 5'-monophosphate p-nitrophenol ester solution (22). For Western blot assay, plasma membrane fractions were normalized to either alkaline phosphodiesterase-1 activity or soluble protein. Antibody for GLUT4 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and a dilution of 800 times in 5% nonfat milk solution was used. Alkaline phosphodiesterase-1-enriched plasma membranes were found in the 25% sucrose gradient fraction (see Fig. 4Go). This fraction was used for GLUT4 determinations. The plasma membrane recovery in terms of total alkaline phosphodiesterase-1 activity was 9–13% for all groups, which is similar in range to that reported by Dombrowski et al. (21).


Figure 4
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  		FIG. 4. Distribution of GLUT4 protein and alkaline phosphodiesterase-1 activity in membranes of different sucrose gradient fractions. Membranes were prepared from skeletal muscles of fasted C57BL6J mice 30 min after administration with 0.5 U/kg insulin. Top, Protein (30 µg) from each fraction and the total membrane was subjected to Western blotting; bottom, specific activity of alkaline phosphodiesterase-1 in each membrane fraction. Each value is the mean ± SEM of eight animals. Similar distribution patterns of GLUT4 protein and enzyme activity were obtained with muscles from diabetic animals.

 
Immunoprecipitation of IRS-1
Frozen muscles were minced and homogenized in 12 vol (wt/vol) lysis buffer containing 50 mM HEPES/NaOH (pH 7.5), 150 mM NaCl, 2 mM EDTA, 10% glycerol, 10 mM NaF, 2 mM Na4VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 mM Na4P2O7 using a Polytron (at high speed) as described by Hirose et al. (23). The homogenate was centrifuged at 10,000 x g for 10 min, and the supernatant was stored at –20 C until used for IRS-1 and protein determination.

Protein A-agarose beads in suspension (Invitrogen, Carlsbad, CA) were washed with PBS and incubated with monoclonal IRS-1 antibody (Santa Cruz) at a ratio of 6:1 (vol/vol) at room temperature for 3 h with end-over-end rotational mixing. The antibody-conjugated protein A-agarose was washed free of untagged antibody and incubated with muscle supernatant with similar rotational mixing. Beads were then washed in RIPA buffer, 0.5 M LiCl, and Tris-HCl to remove nonspecific binding proteins. The beads were denatured at 95 C in Laemmli sample buffer for 5 min, and the immunoprecipitated products were assayed for IRS-1 and its phosphotyrosine residue by Western blotting. Mouse monoclonal antibodies against phosphorylated tyrosine and IRS-1 (Santa Cruz) were used at dilution of 400 and 800, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hypoglycemic action of DAA-I
Figure 1Go shows that DAA-I, when administered orally, exerts dose-dependent hypoglycemic action in KKAy mice after 4 wk of treatment. Based on this experiment to find dose range and treatment duration, a repeat experiment was carried out with a dose of 600 nmol/kg and a treatment duration of 4 wk (Fig. 2Go). Both experiments show that DAA-I is an effective oral hypoglycemic agent at a dose of 600 nmol/kg. The range of blood glucose in our present batches of KKAy mice was similar to reported ranges (6, 24, 25). The hypoglycemic action was not accompanied by an increase in serum insulin level of DAA-I-treated diabetic animals (1.9 ± 0.2, 2.1 ± 0.5, and 2.0 ± 0.4 ng/ml for animals treated with water and 400 and 600 nmol/kg DAA-I, respectively). The blood insulin level was comparable to the reported level (6). Similar dose-dependent hypoglycemic action was also seen in GK rats that were orally treated with DAA-I for 6 wk (there was no significant effect at 4 wk of treatment, data not shown). The OGTT profile for GK rats treated with 400 and 600 nmol/kg DAA-I for 6 wk is shown in Fig. 3Go. There was also no significant increase in level of serum insulin of DAA-I-treated GK rats (1.4 ± 0.1, 1.3 ± 0.2, 1.2 ± 0.01 ng/ml for animals treated with water and 400 and 600 nmol/kg DAA-I, respectively). The blood glucose and insulin levels observed in our batch of GK rats were comparable to those reported by Koyama et al. (26), and the slightly higher blood glucose level observed in our animals was probably due to the older age (14 wk) of our rats. Wistar rats and BALB/c mice that were administered 600 nmol/kg DAA-I for 6 wk did not show any significant difference in blood glucose level from those that were similarly treated with water (data not shown). However, C57BL/6J mice administered 600 nmol/kg showed significantly lower serum glucose level compared with water-treated animals at 4 and 6 wk. Values of serum glucose at 4 wk determined at 30 min of the OGTT were 13.75 ± 0.45 and 17.50 ± 1.0 mM for DAA-I- and water-treated animals, respectively. Although the hypoglycemic action of DAA-I in the C57BL/6J mice was unexpected, these mice have been found to be sensitive to metabolic manipulation (27, 28, 29).


Figure 1
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  		FIG. 1. Effect of orally administered DAA-I on serum glucose level in type 2 diabetic KKAy mice (dose- and temporal range-finding experiment). Animals were divided into four groups of eight animals per group. Animals in the control group were administered daily, by gavage, 0.1 ml water. Animals in the second, third, and fourth groups were similarly administered 0.1 ml DAA-I solution containing 400, 600, and 800 nmol/kg, respectively. OGTT (at 30 min) was performed after 2 and 4 wk of water or DAA-I treatment. Each pointis the mean ± SEM obtained from eight animals. *, Significantly different from the value of the corresponding control (P < 0.05, ANOVA post hoc Tukey test).

 

Figure 2
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  		FIG. 2. Effect of orally administered DAA-I on serum glucose profile in type 2 diabetic KKAy mice. This was a repeat of the previous experiment (Fig. 1Go). In this experiment, a multitemporal OGTT (i.e. at 0, 30, 60, and 120 min, respectively) was carried out on the control and DAA-I treated (600 nmol/kg) animals after 4 wk of treatment. For each group, n = 8. *, Significantly different from the corresponding values of the control untreated mice (P < 0.05, ANOVA followed by post hoc Tukey test).

 

Figure 3
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  		FIG. 3. Effect of orally administered DAA-I on serum glucose profile in type 2 diabetic GK rats In this experiment, a multitemporal OGTT was performed after 6 wk of treatment with either 400 or 600 nmol/kg DAA-I (in 0.2 ml solution). Control animals were similarly treated with 0.2 ml water. Each value is the mean ± SEM of eight animals. *, Significantly different from the value of the corresponding control (P < 0.05, ANOVA followed by post hoc Tukey test).

 
Body weight of diabetic animals
Table 1Go shows that the average body weight in the different groups of diabetic animals was not significantly different among the groups. The data show that DAA-I has no effect on the body weight of the diabetic animals.


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  		TABLE 1. Average body weight of KKAy mice and GK rats during the period of oral DAA-I treatment

 
Insulin resistance indexes of diabetic animals
Table 2Go lists the parameters and formulas of the indexes. The data show that DAA-I significantly increases whole-body and muscle insulin sensitivity and attenuates hepatic insulin resistance of diabetic KKAy mice and GK rats.

Cellular mechanism of DAA-I actions
Western blot data (Fig. 5Go) show that 1) insulin increases the amount of GLUT4 at the plasma membrane in skeletal muscles of the Wistar rat, 2) the increase was impaired in the GK rats, and 3) DAA-I attenuates the impairment. GLUT4 content in total membrane was not affected by DAA-I. Similar quantitative data were also obtained from the skeletal muscles of KKAy mice (Fig. 6Go). In the upstream pathways of GLUT4 translocation, DAA-I enhances the tyrosine phosphorylation of IRS-1 in the skeletal muscles of GK rats and KKAy mice (Figs. 7Go and 8Go). There was no significant difference in the total GLUT4 protein between the diabetic and nondiabetic animals. Similar data have also been reported between the GK and Wistar rats (30) and between the KKAy and C57BL/6J mice (31).


Figure 5
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  		FIG. 5. Effects of DAA-1 on insulin-induced GLUT4 translocation to plasma membrane and total GLUT4 content in skeletal muscle of GK rats. Animals were fasted for 16 h (overnight) and ip administered either insulin solution (0.5 U/kg) or an equivalent volume of PBS. Animals were killed by cervical dislocation 30 min later. The hind limb muscles were excised and its plasma membranes isolated by sucrose gradient. Thirty micrograms of protein from the 25% fraction of a discontinued sucrose gradient (which contained plasma membranes, A) and the total membrane (crude membrane, B) were subjected to SDS-PAGE, transferred to polyvinylidene difluoride sheets, incubated with anti-GLUT4 polyclonal primary antibody (1:800), and washed, followed by incubation with antigoat secondary antibody (1:10,000). Top, Representative Western blots of GLUT4 protein from plasma membrane and total membrane of insulin and non-insulin-treated animals; bottom, plasma membrane GLUT4 content (relative to plasma membrane GLUT4 in the corresponding non-insulin-treated animals) and total GLUT4 (relative to the total membrane GLUT4 of the non-insulin-treated Wistar rat). The vertical bars represent the SEM of samples obtained from three individual animals. *, Significantly different from the corresponding content in non insulin-treated animals (P < 0.05, two-tailed t test).

 

Figure 6
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  		FIG. 6. Effects of DAA-1 on insulin-induced GLUT4 translocation to plasma membrane and total GLUT4 content in skeletal muscle of KKAy mice. Animals were fasted for 16 h (overnight) and ip administered either insulin solution (0.5 U/kg) or equivalent volume of PBS. Animals were killed by cervical dislocation 30 min later. The hind limb muscles were excised and its plasma membranes isolated by sucrose gradient. Thirty micrograms of protein from the 25% fraction of a discontinued sucrose gradient (which contained plasma membranes, A) and the total membrane (crude membrane, B) were subjected to SDS-PAGE, transferred to polyvinylidene difluoride sheets, incubated with anti-GLUT4 polyclonal primary antibody (1:800), and washed, followed by incubation with antigoat secondary antibody (1:10,000). Top, Representative Western blots of GLUT4 protein from plasma membrane and total membrane of insulin and non-insulin-treated animals; bottom, plasma membrane GLUT4 content (relative to plasma membrane GLUT4 in the corresponding non-insulin-treated animals) and total GLUT4 (relative to total membrane GLUT4 in non-insulin-treated C57BL/6J mice). The vertical bars represent the SEM of samples obtained from three individual animals. *, Significantly different from the corresponding content in non-insulin-treated animals (P < 0.05, two-tailed t test).

 

Figure 7
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  		FIG. 7. Effects of DAA-1 on insulin-induced tyrosine phosphorylation of IRS-1 and total IRS content in skeletal muscle of GK rats. Animals were fasted overnight (16 h) and ip administered either insulin solution (40 U/kg) or equal volume of PBS. Animals were killed by cervical dislocation 5 min later. The hind limb muscles were excised, and IRS protein was immunoprecipitated from muscle supernatant with IRS-1 antibody-conjugated protein A-agarose beads. Isolated IRS-1 protein (1000 µg) was then resolved through SDS-PAGE, transferred to polyvinylidene difluoride sheets, and incubated using anti-phosphotyrosine antibody (1:400). The sheets were then washed followed by incubation with antimouse secondary antibody (1:10,000). The immunoreactive bands were detected by enhanced chemiluminescence. Top, Representative Western blot of p-Y IRS-1 protein (A) and total IRS-1 protein (B) from muscle of insulin-treated and non-insulin-treated animals; bottom, phosphotyrosine IRS-1 protein content (relative to content in the corresponding non-insulin-treated animals) and total IRS-1 (relative to total IRS-1 in non-insulin-treated Wistar rats). The vertical bars represent the SEM of samples obtained from three individual animals. *, Significantly different from the corresponding content in the non-insulin-treated animals (P < 0.05, two-tailed t test).

 

Figure 8
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  		FIG. 8. Effects of DAA-1 on insulin-induced tyrosine phosphorylation of IRS-1 and total IRS content in skeletal muscle of KKAy mice. Animals were fasted overnight (16 h) and ip administered either insulin solution (40 U/kg) or equal volume of PBS. Animals were killed by cervical dislocation 5 min later. The hind limb muscles were excised, and IRS-1 protein was immunoprecipitated from muscle supernatant with IRS-1 antibody-conjugated protein A-agarose beads. Isolated IRS-1 protein (1 mg) was then resolved through SDS-PAGE, transferred to polyvinylidene difluoride sheets, and incubated using anti-phosphotyrosine antibody (1:400). The sheets were then washed, followed by incubation with antimouse secondary antibody (1:10,000). The immunoreactive bands were detected by enhanced chemiluminescence. Top, Representative Western blot of phosphotyrosine IRS-I protein (A) and total IRS-1 protein (B) from muscle of insulin-treated and non-insulin-treated animals; bottom, phosphotyrosine IRS-1 protein content (relative to content in the corresponding non-insulin-treated mice) and total IRS-1 (relative to the total IRS-1 in non-insulin-treated C57BL/6J mice). The vertical bars represent the SEM of samples obtained from three individual animals. *, Significantly different from the corresponding content in non-insulin-treated animals (P < 0.05, two-tailed t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Concurrent to the writing of the present manuscript, Giani and co-workers (32) reported the ability of angiotensin-(1–7) to overcome the inhibition of insulin-induced phosphorylation of Akt phosphorylation by angiotensin II in rat hearts. The authors postulated that angiotensin-(1–7) could be a positive physiological contributor to the actions of insulin in the heart. However, the involvement of other angiotensin peptides in glycemic control and insulin resistance in type 2 diabetes is not known. This has somehow restricted the role of the renin-angiotensin system in blood glucose handling to the deleterious actions of angiotensin II. The present findings, showing that DAA-I is an effective oral hypoglycemic agent that attenuates insulin resistance in two animal models of type 2 diabetes, have several implications. DAA-I is endogenously formed from angiotensin I (33, 34) and may act as a physiological antagonist to angiotensin II with regard to glycemic control. At the doses used in this study, DAA-I has no effect on the blood pressure (unpublished data). In hypertensive animals and human, the level of circulating DAA-I is attenuated, and treatment with ACE inhibitors elevated the level to a normal value in the hypertensive animals and more than twice the normal value in hypertensive human with a longer period of treatment (35). This increase in circulating DAA-I could have hitherto contributed to the reduced development of type 2 diabetes in hypertensive patients treated with ACE inhibitors (13).

The absence of a significant DAA-I effect on serum insulin level in diabetic animals shows that the nonapeptide is not an insulin secretagogue. It exerts hypoglycemic action in diabetic animals by attenuating insulin resistance. Although the molecular basis for insulin resistance is not fully understood, defects in the early and intermediate steps of the insulin signaling cascade are likely causes (36). Substantial decreases in insulin-stimulated receptor kinase activity and a defect in receptor-mediated IRS-1 phosphorylation or PI 3-kinase activation have been found in muscle or fat tissue from type 2 diabetic patients and rodent models of the disease (36, 37, 38). Based on this premise, DAA-I attenuates insulin resistance in skeletal muscle of diabetic animals by enhancing insulin-induced tyrosine phosphorylation of IRS-1 via the angiotensin AT1 receptor, which leads to a corresponding increase in the translocation of cytoplasmic GLUT4 to plasma membrane. This is supported by the fact that angiotensin AT1 receptors are present in skeletal muscles of animals and human (39, 40) and the de novo renin-angiotensin system plays functional roles in muscle physiology (41) and pathology (42). The exact pathways coupling DAA-I to GLUT4 translocation is not known. In vitro and in vivo studies show that DAA-I acts as an agonist on the AT1 receptor and exerts actions opposing those of angiotensin II (15, 16, 17, 18, 43, 44). A notable characteristic in some of these studies is the susceptibility of DAA-I actions to indomethacin inhibition, which indicates that prostaglandins transduce its actions. Although the effect of indomethacin on the hypoglycemic action of DAA-I could not be studied (in preliminary studies, animals became sick and lost weight when treated with indomethacin for periods longer than 2 wk), the findings of Leighton et al. (46, 47) showing that prostaglandins of the E series increase and indomethacin decreases the sensitivity of glucose uptake to insulin in isolated skeletal muscle of the rat could support the possibility that DAA-I exerts its hypoglycemic action via prostaglandins.

We have shown that DAA-I specifically released prostaglandin E2 and prostaglandin I2 from human umbilical vein endothelial cells at sub-nanomolar range, and the release was blocked by indomethacin and losartan (an AT1 receptor antagonist) but not PD123319 (an AT2 receptor antagonist) (unpublished data). This finding suggests that DAA-I acts via the AT1 receptors and releases prostaglandins as second messengers. With regard to induction of hypoglycemia, the prostaglandins could act as ligands for peroxisome proliferator-activated receptors (PPARs) (48), potentiate insulin action (46, 47, 49, 50), and attenuate inflammatory cytokines and reactive oxygen species formation. The latter is an implied pathway because insulin resistance has been shown to be associated with inflammation (51) and reactive oxygen species formation (10, 52), and prostaglandins have been shown to attenuate proinflammatory cytokines (53, 54) and protect cardiac myocytes from oxidative stress in the presence of H2O2 (55).

The onset of the hypoglycemic action of DAA-I is 4 and 6 wk for the KKAy mice and GK rats, respectively, which is comparable to the onset of action of the thiazolidinediones. The latter compounds act via the steroid nuclear receptor PPAR-{gamma} to affect gene regulation involved in glucose and lipid metabolism, processes that require a long onset of weeks to months. Cyclooxygenase products are ligands for PPARs (48), and the scenario that prostanoids mediate the hypoglycemic action of DAA-I by acting on PPAR-{gamma} could account for the long onset of action seen with the nonapeptide. On this note, it is of interest to note that other experimental hypoglycemic compounds too have a similar long onset of action (45, 56, 57).

A second characteristic of DAA-I action is its biphasic nature (see Fig. 1Go), and doses higher than the maximal efficacious dose were progressively less effective (44). The exact mechanism for this biphasic phenomenon is not known, but receptor down-regulation by different mechanisms could occur at high doses of DAA-I and cause the biphasic response (43). DAA-I, being an endogenous angiotensin, could at higher doses also activate the classical angiotensin II inositol triphosphate-generating AT1 receptor and annul its more specific action mediated by the indomethacin-sensitive pathway. Although DAA-I is a peptide consisting of nine amino acids, it has been shown to be effective when administered orally in the present and earlier studies (18, 44). Part of the oral dose of DAA-I would probably be digested by intestinal peptidases including ACE. However, because the effective oral doses were in the nanomolar range, an effective amount of DAA-I would have escaped the enzymatic degradation considering that the Michaelis-Menten constant (Km) of most enzymes is within the micromolar range. In addition, DAA-I is stable to nonenzymatic hydrolysis and is broken down to a limited extent (30%) by enzymes produced by the Caco-2 monolayer. It has been shown to be transported across the Caco-2 monolayer by diffusion (14). DAA-I also exerts significant hypoglycemic effect at doses of 100–400 nmol/kg when administered by the ip route (unpublished finding), demonstrating that digestion products are unlikely to be the active hypoglycemic compounds.

In conclusion, the present study demonstrates for the first time the hypoglycemic action of DAA-I and its possible use as an oral hyperglycemic agent with a new mechanism of action. That DAA-I, like angiotensin II, is also a direct enzymatic product of angiotensin I degradation opens up new vistas in the intense research linking angiotensin II to diabetes and related cardiovascular diseases, two highly prevalent and overlapping maladies of the present time.


   Footnotes
 
The study was supported by a Proof of Concept Grant from the Economic Development Board (Singapore) and Agency for Science, Technology and Research-Biomedical Research Council Grant (04/1/21/19/306).

Disclosure Statement: All of the authors have nothing to disclose.

First Published Online September 6, 2007

Abbreviations: ACE, Angiotensin converting enzyme; DAA-I, des-aspartate-angiotensin I; GLUT4, glucose transporter-4; IRS-1, insulin receptor substrate-1; OGTT, oral glucose tolerance test; PI, phosphatidylinositol; PPAR, peroxisome proliferator-activated receptor.

Received May 8, 2007.

Accepted for publication August 28, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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