Current findings of renin-angiotensin-aldosterone system (RAAS) bring about the connection with diabetes mellitus, and how important angiotensin IV (Ang IV) in diabetic research and treatment. Ang IV physiologically antagonizes angiotenisn II (Ang II), and gives benefits to human system and prolonging action of peptides such as vasopressin, lys-bradykinin, and oxytocin. Therefore, Ang IV is believed to reduce aggravations in macrovascular and microvascular associated diabetes. However, Ang IV causes insulin-resistance in glucose transporter (GLUT4) due to binding on insulin-regulated aminopeptidase (IRAP) or angiotensin subtype IV receptor (AT4). Thus, insulin signalling in GLUT4 translocation reduced and glucose could not be uptake into cell. Research has been done to increase sensitivity, such as AL-11 development, which has more potent and specific compared with the current drug, divalinal-Ang IV. In conclusion, Ang IV has good and bad properties, and further research is needed to understand its actions in patients suffer from diabetes mellitus.
Angiotensin IV (Ang IV), Renin-Angiotensin-Aldosterone-System (RAAS), Diabetes Mellitus, Insulin-regulated Aminopeptidase (IRAP), Angiotensin Subtype IV Receptor (AT4)
The renin-anigotensin-aldosterone system (RAAS) is the major regulatory pathway mediated by secretion of renin due to the high levels of aldostrone and to certain extent, cortisol. The system is responsible in water and sodium retention, as well as maintaining blood pressure of the body . However, new discoveries led to the detection of angiotensin IV (Ang IV) and its receptor known as angiotensin subtype IV (AT4) receptor, which later is also known as insulin-regulated aminopeptidase (IRAP) enzyme that is found in glucose transporter known as GLUT4 [1, 2]. Several findings indicate that IRAP may have relationship with glucose levels in the body via GLUT4, and thus, may have connection with diabetes mellitus . Apart from that, there are also other findings which are related to Ang IV and linked with other systems . Therefore, the report will cover the properties and relationship between RAAS, AT4, IRAP, and diabetes mellitus in general, as well as the importance of Ang IV in diabetic research and treatment [1, 3, 4].
1. Renin-Angiotensin-Aldosterone System (RAAS)
Figure 1: The RAAS System and Mechanism of Action of Angiotensins I, II, and IV, and Ang (1-7) on the Human System. Source: 
The classical renin-angiotensin-aldosterone system (RAAS) is involved in synthesis of angiotensin I (Ang I) and angiotensin II (Ang II), when angiotensinogen, an inactive peptide which produced from liver is converted to Ang I by renin, and subsequently, Ang II produced becomes the main peptide responsible for the regulation of RAAS in renal system. However, for the past ten years, there are discoveries which led to the changes in the classical concept of RAAS.
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The newer discoveries include the presence of local or tissue RAAS such as in heart, blood vessels, pancreas, central nervous system (CNS), reproductive system, lymphatic system, and adipose tissue which act independently or interact together with adrenal glands and kidney . Not only that, based on other research, uterus, placenta, and, brain also have a local RAAS . These localized systems could affect intracrine, autocrine, paracrine, and endocrine functions [1, 2].
Other important findings for the newer RAAS concept are such as characterization of angiotensin II (1-7) or Ang II-(1-7) or Ang (1-7), angiotensin-(3-7) or Ang (3-7), and angiotensin A (or Ang A) as a biological active metabolite of RAAS and degraded by aminopeptidase or carboxypeptidase, the connection and evidence that angiotensin subtype IV receptor (AT4) is the insulin-regulated aminopeptidase (IRAP) enzyme [1, 2], the discovery of renin/prorenin receptor, findings of angiotensin-converting enzyme II (ACE2) as an angiotensin peptide processing enzyme and homologue of angiotensin-converting enzyme (ACE), and presence of Ang (1-7) and Ang (3-7) as substrates for both G-coupled receptor Mas and AT4 receptor respectively. These discoveries lead to the understanding of the RAAS as cascade that has multiple mediators and receptors, as well as multi-functional enzymes .
Usually, RAAS is associated with local regulatory mechanisms which involved in homeostatic pathways. Cellular growth, extracellular matrix formation, vascular proliferation, endothelium function, and also apoptosis can be induced via the action of Ang II (and Ang III which act mainly in brain) . Other involvements of RAAS include the repression of the system without medication through antibody suppression or gene therapy for long time as well as involvement of ribozyme or by interfering messenger ribonucleic acid (mRNA). The examples can been seen in rat models whereby vectors expressing antisense mRNA of angiotensin subtype I (AT1) receptor or angiotensinogen antisense mRNA stabilizes blood pressure in hypertensive rat models. Not only that, rats actively immune with Ang I analogues or anti-angiotensinogen antibody reduced the repressor’s response to exogenous Ang I and thus, caused a fall in blood pressure. However, in humans, hypotensive effect can only be seen at higher antibody titres. This shows that the RAAS is a localised system that able to produce multiple effects dependent on their substrates and pathways .
2. Angiotensin IV
2.1 Biochemical Properties of Angiotensin IV and its Related Ligands
Ang IV or Ang (3-8) is a hexapeptide which consist of the third until eighth peptide of the original angiotensinogen peptide [1, 6, 7]. It is formed in two ways. One of it is from the Ang II via aminopeptidase A and aminopeptidases B, M, and N and thus, aspartic acid and arginine is removed from the first and second position of the peptide . It can also be converted directly from Ang II via D-aminopeptidase [1, 2].
Figure 2: The angiotensins’ production pathway. Source: 
The pharmacological properties of Ang IV is mainly induced onto its receptor known as AT4 receptor or IRAP and sometimes, AT1 and angiotensin subtype II (AT2) receptors at higher concentrations [1, 2, 4, 7, 9]. The findings are based on affinity and specificity of its radioligands to the receptor found in various organs . Other ligands which can bind on AT4 receptor is LVV-hemorphin 7 (LVV-H7) which is biochemically unrelated with Ang IV, divalinal-Ang IV (which can also produce antagonistic effects in certain occasions), and norleucine1-Ang IV (Nle1-Ang IV). Meanwhile, statin drugs such as amastatin are potent inhibitor of aminopeptidases A and N which is responsible in production of Ang IV but has no direct effect on Ang IV towards endothelial cells [1, 4, 6, 8, 9, 10]. Ang IV could not be cleaved by both ACE and ACE II which cleaved most angiotensin peptides and other mediators such as bradykinins [7, 8, 11].
Figure 3: The ACE and ACE2 cleaveage sites of angiotensin peptides, apelin, bradykinins, dynorphin A, and Ï-casomorphin. Source: 
2.2 Cellular Mechanism of Angiotensins IV
Due to the similarity of action with insulin, Ang IV can activate certain protein kinases pathway especially phosphoinositol III kinase (PI3 K) and PI-dependent kinase-1 (PDK-1) activities in the endothelial cells where it can enhance the action of insulin, promote action of bradykinin and nitric oxide, and causes vasodilatation [1, 4, 10]. It also caused intracellular calcium release and activates phospholipase C via the increase of inositol phosphate, causing muscle contraction [4, 10].
However, it also triggers mitogenic activated protein kinase (MAPK) pathway which leads to little vasoconstriction action, apart from activating mitogenesis and tropic activity of cell [1, 7]. It also increases expression of plasminogen activator inhibitor (PAI-1) to prevent myocardial infarction but may increase risk of thrombosis, in which is due to the incresase of Ang IV via increase in Ang II and plasma renin activity [1, 8].
Other pathways that are activated via Ang IV action are such as nuclear factor kappa-B (NF-κB) and the plasminogen activator inhibitor I (PAI-1) pathways [8, 10].
2.3 Pharmacological Actions and Effects of Angiotensins IV and its Related Ligands on Human System
Due to its ability to cause many biological actions, scientists had focused on Ang IV although initially considered being biologically inactive. The general actions of Ang IV are basically the opposite with Ang II [4, 6].
Ang IV has a distinct intracerebroventricular effect and increased in memory and learning by acting on CNS [2, 4, 7]. This triggers neurotransmitter release in hippocampus and pons via induction of potassium (K+) . There are three hypotheses proposed concerning memory-potentiating effects of Ang IV and LVV-hemorphin 7 in which they are potent inhibitors of IRAP, prolong action of endogenous peptides, and also modulate glucose uptake by modulating GLUT4 trafficking .
As for the vascular system, it has predominant vasodilatation effect. For example, some studies shows vasodilatation of pulmonary artery provided the endothelium is intact although it is pre-constricted. Besides that, it also causes the increase of endothelial nitric oxide (eNO) activity and cyclic guanyl monophosphate (cGMP) content via bradykinin-nitric oxide-cGMP vasodilator pathway which had manifested in porcine pulmonary arterial endothelial cells [1, 5, 7]. It also showed vasoprotective effect, reduces superoxide formation, and improve aortic endothelial function in Apolipoprotein E (Apo-E) deficient mice . However, hypertension can occur due to action on AT1 receptor at high concentrations but it can be reversed via AT1 receptor blockers [2, 4, 7].
For renal, Ang IV could increase renal cortical blood flow and decreases sodium (Na+) transport in renal proximal tubule without change in systemic blood pressure. Apart from vasodilator effect, studies also show decrease total and regional renal blood flow in rats. 
In cardiovascular system, there is reduction in pressure-development and ejection capabilities, and increase sensitivity of left ventricular heart wall during systole and speeded relaxation . However, due to expression of PAI-1, it may increase the risk of atherosclerosis and affect fibrinolytic system based on studies involving ACE inhibitors and angiotensin receptor blockers (ARB), and comparing PAI-1 expression with other angiotensin peptides [8, 13].
Meanwhile, Ang IV is also involved in metabolism and may extent half-life of biologically active neuropeptides such as neuromedin B and neurokinin A through competitive inhibition and prevent cleavage by AT4 receptor [4, 8].
As for other Ang IV ligands such as divalinal-Ang IV, it can attenuate the effect of Ang IV in biological systems. Thus, there is involvement of other mechanism which possibly due to antagonism to the activation of second messenger system [4, 8]. As for LVV-H7, the action is much similar to Ang IV except in peripheral actions. However, it do not cause increase in intracellular activity, and Ang IV could act as partial antagonist against LVV-H7 [1, 6].
Overall, Ang IV is involved in interfering of the cleavage of other bio-active peptides from AT4 or IRAP, inhibiting its allosteric mechanism [4, 10]. Ang IV can be useful to explain the role of IRAP in CNS and insulin responsive tissues due to its high-affinity to AT4 receptors as well as importance in cognition, cardiovascular, and renal metabolism as well as pathophysiological conditions such as diabetes, atherosclerosis, and hypertension . It could also contribute to inflammatory events in cardiovascular diseases via NF-kÏ and Jak/Tak kinase pathways mediated by tyrosine phosphorylation and involvement in regulation of pro-inflammatory genes [1, 10].
2.4 Binding Affinity and Potency of Angiotensin IV on AT4 Receptor
The AT4 receptor is known as insulin-regulated membrane aminopeptidase (IRAP). IRAP is previously known as placental leucine aminopeptidase (PLAP) and oxytocinase (OTase) based on their mechanism of action to cleave oxytocin and are homologues with 87% similarity in the amino acid sequence. It can be further proof by similar regional distribution of mRNA, immunoreactivity, and binding of [125I]-Nle1-Ang IV in thin sections of mouse brain [1, 3, 6, 10, 14]. It belongs to Type II integral membrane and M1 family of aminopeptidases, particularly zinc metallopeptidases [1, 14, 15]. It has a zinc binding motif and an exopeptidase motif where the catalytic site is extracellular .
It was first cloned in 1995 and originally identified in adipocytes and skeletal muscle cells which is associated with insulin-responsive cells as a major protein in vesicles containing the insulin regulated glucose transporter (GLUT4) [1, 3, 4, 6]. There is similarity between sub-cellular distributions of IRAP with GLUT4 under basal and insulin-stimulated conditions .
Ang IV binds at high affinity to AT4 receptor in pharmacologically-distinct binding site such as brain compared to Ang III which has ten times lower affinity as well as Ang (3-7) [1, 4]. Meanwhile, Ang II with its analogue [Sar1-Ile8] Ang II has distinctly lower affinity, and other drugs such as losartan, PD 123177, and CGP 42112A show no affinity to it . The potency between Ang IV with its analogue is in order of Ang IV > Nle1-Ang IV > LVV-H7 > divalinal-Ang IV (D1318) .
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The substrates of AT4 receptor or IRAP are such as vasopressin (an anti-diuretic hormone), oxytocin, met-enkephalin (met-encephalon), lys-bradykinin, Ang III and IV, dynorphin A, neurokinin A, and neuromedin B [3, 14, 15]. Some studies shows the importance of soluble IRAP in oxytocin regulation levels during pregnancy whereas in other studies, Ang IV, and its ligands such as LVV-H7 could inhibit catalytic activity of IRAP and the ligands resist proteolytic cleavage, and thus, identified as enzyme-inhibitor interaction [4, 14].
Vasopressin, oxytocin, and met-enkephalin which rapidly cleaved by IRAP can be interrupted by the Ang IV and its ligands, which prolonging the effects of these peptides . Meanwhile, insulin, calcitonin, and endothelin are not IRAP substrates while vasopressin is a unique substrate which can be cleave N-terminal cysteine in vitro apart from oxytocin [14, 15]. It is believed that Ang IV may have actions connected with diabetes mellitus or other metabolic syndromes [1, 16].
3. Diabetes Mellitus
Diabetes mellitus is a multisystem disorder characterised by excess glucose level in blood circulation. It is mainly due to the lack of insulin production or resistance to insulin sensitivity while, other factors such as polymorphism, inflammatory mediators, cytokines, and oxidative stress could also contribute. These can be made worse with patient’s unawareness and non-compliance . Diabetes mellitus often results in hyperglycaemia, hypertension, polydypsia, polyuria, and polyphagia, increased hypersensitivity and inflammation, and also hypercholestrolaemia . There are microvascular and macrovascular associated diabetes such as diabetic nephropathy, neuropathy, and retinopathy, and also cardiovascular associated diseases .
3.1 Relationship between RAAS, Angiotensin IV, AT4 Receptor, Insulin, GLUT4, and Diabetes Mellitus
The involvement of renal RAAS system could contribute in the association both vascular and renal associated diabetes mellitus [1, 3, 5]. As far as we know, Ang II is directly involved in the aggravations of symptoms in microvascular and macrovascular diseases via vasoconstriction, reduction in eNO content, increased thrombin and fibrin formation, increased sodium and water retention, decreased in blood flow, bradykinin degradation, and in diabetic patients. These actions are physiologically antagonized by Ang IV and thus, could reduces aggravation of symptoms in diabetic patients [1, 16]. Thus, there may be possible that Ang IV is connected with Ang II, AT4 receptor or IRAP, and also its substrates, insulin action, as well as diabetes mellitus . Another minor association is the ACE2-Ang-(1-7)-Mas axis which show improvement in diabetic patients through RAAS blockade during clinical trials and, could also play a role in diabetes by countering the vasoconstriction effect of ACE2-Ang II-AT1 axis [1, 5]. Moreover, there is indirect connection whereby there are actions of Ang II on both AT1 and AT2 receptors to activate the bradykinin-nitric oxide-cGMP vasodilator pathway .
The result of hyperglycaemic conditions in diabetic patients further triggers RAAS and cause further vasoconstriction and increase blood pressure in diabetic patients . At the same time, the RAAS activation in diabetic patients associated with myocardial infarction causes viscous cycle, where the cardiac output continues at a lower rate, causing continuous action of the angiotensin peptides . Moreover, arthrosclerosis which result in free radical formation in hyperglycaemic conditions could trigger macrovascular complications . Therefore, diabetic mellitus are usually associated with hypertension and myocardial infarction due to the involvement of RAAS and the inhibition of IRAP is involved in the regulation of GLUT4 trafficking by reducing glucose uptake and causing hyperglycaemia [15, 17].
Figure 4: Connection between Ang II, Ang IV, and diabetes mellitus. Source: [1, 16]
Since there is connection between RAAS, Ang II, and Ang IV, there is also relationship between diabetes mellitus and Ang IV with insulin, IRAP, and GLUT4 whereby the presence of IRAP is needed to maintain normal GLUT4 levels regardless of tissue type, sex, and age . Ang IV antagonises insulin response on IRAP and may develop insulin resistance, reducing response of insulin to IRAP, and at the same time prolonging actions of IRAP substrates. Therefore, decreasing Ang IV may decrease insulin-resistance but may also down-regulate vasodilatation effect, cognitive efficiency, eNO activity, mitogenic and tropic, as well as the cellular mechanism pathways [1, 2, 3, 4, 17].
A study by Keller (2004) explained the cause of GLUT4 decrease which is due to the changes of the action of IRAP substrates. Prior to the lack of GLUT4 or increase in GLUT4 degradation, heart size may increase and it may cause hyperglycaemia or diminished glucose disposal in fat or muscle tissue due to decreased translocation of GLUT4 onto the surface. This study also showed that GLUT4 impairment is similar with those who are having diabetes mellitus type 2 or insulin resistance and since GLUT4 is diminished, it is expected that IRAP action at the cell membrane are also declining in these patients. Thus, the impaired IRAP function may play a role in development of complications in insulin-resistant individuals and may be linked to the decrease of peptide substrate cleavage and change in hormone action [2, 3, 17].
Figure 5: Connection between insulin signalling and Ang IV. Source: 
As we know, the current mechanism of glucose uptake is believed to be triggered by insulin whereby it activates to cause translocation of GLUT4-IRAP complex to the cell surface which enables IRAP to degrade its substrates such as vasopressin. Thus, insulin could promote inactivation of IRAP substrates and controls its actions and at the same time, promote insulin-dependent glucose uptake into the cell . Meanwhile, as have been discussed before, Ang IV has antagonistic effect on IRAP and allows prolonging the action of its substrates such somatostatin and substance P [1, 4, 15]. With the current understanding of its mechanism, there are studies related to diabetic treatment via the usage of AT4 receptor blocker such as AL-11 .
4. Current Research Related to the Importance of Angiotensin IV in Diabetic Research and Treatment
Based on the effect of both Ang IV and insulin on IRAP and GLUT4 translocation, Beckie et. al. (2009) have came out with one solution to treat diabetic patients by increasing the insulin sensitivity . AL-11 is a synthetic compound which have similar action with divalinal-Ang IV which antagonizes the binding of Ang IV, allowing insuilin to act on IRAP and promote translocation of IRAP [1, 18]. The difference between these two compounds is that, AL-11 has higher potency and more specific in its action compared with divalinal-Ang IV. The prototype drug is patented in the United States, and the team has also suggested a principal whereby Ang IV up-regulates angiotensinogen expression and AT1 receptors at high doses but down-regulates the uptake of glucose and IRAP expression in endothelial blood vessels .
It has been tested in human smooth muscle cell cultures and found out that MAPK activation could cause inhibition of p85 AKT pathway which is responsible of GLUT4 translocation and glucose uptake . Therefore, it is suggested that Ang IV promote insulin resistance at certain concentrations but could be prevented if by reducing its maximum effect [1, 4, 15, 18]. Moreover, findings also suggested that Ang IV has the opposite effect of Ang II. Therefore, AL-11 can be used to treat diabetes mellitus Type II, insulin resistance, or cardiovascular risk related with metabolic syndrome .
Summary and Conclusion
Vasodilatation (via increased cGMP and eNO content, bradykinin action, and decreased in sodium and water retention)
May increase atherosclerosis (via PAI-1)
Improve memory cognition
May increase insulin resistance
Reduce fibrin and thrombin formation
Increased mitogenic and tropic action (via MAPK pathway)
Reduces oxidative stress (due to increased eNO content and reduced free radical formation)
Table 1: Summary of Ang IV Action in Human System. Source: [1, 2, 4]
The pharmacological actions of Ang IV in vascular system, as well as renal and cardiovascular systems are the opposite of Ang II and the binding of Ang IV towards IRAP are highly specific compared with other angiotensin peptides [1, 4, 14]. This causes various effects which can be either good or bad to the human system and these effects are crucial as it involves the aggravations in diabetic patients. [1, 2, 4] Therefore, further research is needed in order to fully understand the mechanism of Ang IV action in patients suffering from diabetes mellitus [1, 5].
Special thanks to Dr. Dharmani Devi and all the staffs and lecturers from Pharmacology Department, Faculty of Medicine, Universiti of Malaya for support and guidance. Not to forget, coursemates, staffs, and lecturers of the Biomedical Science Programme, Faculty of Medicine, Univerisity Malaya.
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