Type 1 Diabetes Mellitus: Redefining the Future of Cardiovascular Complications with Novel Treatments

Diabetes Mellitus (DM) is a disease that was identified centuries ago, in around 1500 BC (King and Rubin, 2003). The word ‘Diabetes’ means “running through” (Holt, 2004) which is used to describe the excessive output of urine in this disease. “Mellitus” meaning “sweet” identifies the nature of the urine in patients suffering from DM (Widmaier et al., 2004). Thomas Willis was the first to differentiate DM from other polyurias in 1674 (Eknoyan and Nagy, 2005). In 1776, Matthew Dobson demonstrated that the sugar present in the urine was also present in the blood and was associated with its rise (Holt, 2004). In 1848, Claude Bernard’s experiments on the liver showed that glycogen stored in the liver produced sugar and he hypothesized that glycogenolysis caused the disease. Although his discovery that “sugar production is a normal function of the animal” was revolutionary, it did not quite explain the etiology of the disease. In 1889, Oskar Minkowski confirmed that the ablation of the pancreas in dogs resulted in DM (Farmer, 1952). Frederick Banting and Charles Best were the medical scientists that discovered insulin, later in 1921, the lack of which, it was made clear, caused DM (Voet and Voet, 2004). They used the extract of a dog’s fresh pancreas and demonstrated that upon administration of 10mL of extract to blood, blood glucose level is decreased from 0.3% to 0.17% (Rosenfeld, 2002). Some 30 years later, in 1953, Frederick Sanger was able to determine the complete amino acid sequence of the protein for the first time in history (Boron and Boulpaep, 2009). Today, DM is defined as a carbohydrate disorder characterized by impaired insulin secretion and/or peripheral insulin resistance leading to hyperglycemia (Beers and Berkow. 1999). It is considered to be the third leading cause of death after heart disease and cancer in the United States (Voet and Voet, 2004) and its incidence is expected to rise to 366 million people by the year 2030 (Wild et al., 2004). Diabetic patients have symptoms such as, thirst, polyuria, blurring of vision and weight loss. In extreme cases, ketoacidosis may develop leading to coma and ultimately death (Alberti and Zimmet, 1998). Diabetes Mellitus is classified according to etiology to two major types:


Introduction
Diabetes Mellitus (DM) is a disease that was identified centuries ago, in around 1500 BC (King and Rubin, 2003). The word 'Diabetes' means "running through" (Holt, 2004) which is used to describe the excessive output of urine in this disease. "Mellitus" meaning "sweet" identifies the nature of the urine in patients suffering from DM (Widmaier et al., 2004). Thomas Willis was the first to differentiate DM from other polyurias in 1674 (Eknoyan and Nagy, 2005). In 1776, Matthew Dobson demonstrated that the sugar present in the urine was also present in the blood and was associated with its rise (Holt, 2004). In 1848, Claude Bernard's experiments on the liver showed that glycogen stored in the liver produced sugar and he hypothesized that glycogenolysis caused the disease. Although his discovery that "sugar production is a normal function of the animal" was revolutionary, it did not quite explain the etiology of the disease. In 1889, Oskar Minkowski confirmed that the ablation of the pancreas in dogs resulted in DM (Farmer, 1952). Frederick Banting and Charles Best were the medical scientists that discovered insulin, later in 1921, the lack of which, it was made clear, caused DM (Voet and Voet, 2004). They used the extract of a dog's fresh pancreas and demonstrated that upon administration of 10mL of extract to blood, blood glucose level is decreased from 0.3% to 0.17% (Rosenfeld, 2002). Some 30 years later, in 1953, Frederick Sanger was able to determine the complete amino acid sequence of the protein for the first time in history . Today, DM is defined as a carbohydrate disorder characterized by impaired insulin secretion and/or peripheral insulin resistance leading to hyperglycemia (Beers and Berkow. 1999). It is considered to be the third leading cause of death after heart disease and cancer in the United States (Voet and Voet, 2004) and its incidence is expected to rise to 366 million people by the year 2030 (Wild et al., 2004). Diabetic patients have symptoms such as, thirst, polyuria, blurring of vision and weight loss. In extreme cases, ketoacidosis may develop leading to coma and ultimately death (Alberti and Zimmet, 1998). Diabetes Mellitus is classified according to etiology to two major types:

Type 1 diabetes mellitus
Although both type 1 Diabetes Mellitus (type 1 DM) and type 2 Diabetes Mellitus (type 2 DM) are characterized clinically by hyperglycemia, they have their differences. Type 1 DM  ). These injuries cause on the long run acceleration in atherosclerotic formation . In type 1 diabetic patients, hyperglycemia has been correlated with a variety of events in cardiovascular pathology, initiated with endothelial dysfunction and progressed to develop arterial stiffness (Tabit et al., 2010). Thus, a relation has been clearly established to link glycemia with cardiovascular events in type 1 DM, based on the fact that glycemia itself is the only factor mediating a risk factor for cardiovascular risk in the absence of other risk factors (Retenakaran and Zinman, 2008). One way of reducing cardiovascular risk and renal outcomes in type 1 DM is to initiate an early intensive therapy for its management.

Microvascular complications
Microvascular complications include nephropathy, retinopathy and neuropathy (Coccheri, 2007;Fowler, 2008;. There is a direct clinical practice to link these complications with DM type 1 rather than type 2 (Retenakaran and Zinman, 2008). Of note, type 2 DM may manifest development of microvascular complication 7 years preceding diagnosis (King et al., 2005).

Macrovascular complications
These define the pathophysiology of cardiac disease in type 1 DM which includes atherosclerosis, coronary artery diseases, stroke and peripheral arterial disease (Retenakaran and Zinman, 2008;. These cardiovascular complications are mostly focused in clinical practice to type 2 DM (Fowler, 2008), and increased in rate as well in type 1 DM (Mcphee et al., 2008); however, the mortality impact of cardiovascular diseases in both DM types 1 and 2 is similar (Juutilainen et al., 2008) contributing to 80 % of the mortality and morbidity (Coccheri, 2007). More alarmingly, development of macrovascular complication might not manifest in type 1 DM until 10 years proceeding diagnosis (King et al., 2005). Endothelial and smooth muscle dysfunction has been associated with type 1 diabetic patients, which in turn might cause hypertension, a major risk factor for developing cardiovascular diseases (Retenakaran and Zinman, 2008). Also lipid abnormalities in type 1 DM, decrease in HDL and increase in LDL compositions, are being incidents of causing cardiovascular diseases (Retenakaran and Zinman, 2008). High levels of ketone bodies are produced from the body as an alternate fuel in the absence of glucose as a main source of energy in severe starving conditions, mainly hyperglycemia , or an acute multiplication of type 1 but not type 2 DM . Three types of ketones can be generated in the body these are acetone whose odor can be detected overtly via breath, acetacetic acid andhydroxybutyric acid whose levels may elevate in the blood and be excreted in urine, which is followed by cations and fluid loss ultimately leading to coma ).

Renin angiotensin system
With the discovery of renin in 1898, Robert Tigerstedt and Per Bergman were able to fill a gap present in the understanding of fluid balance, hypertension and cardiovascular disease (Basso and Terragno, 2001;Phillips and Schmidt-Ott, 1999). The Renin Angiotensin System (RAS) plays an important role in maintaining normal blood volume and blood pressure. When salt and water intake is reduced, the role of RAS becomes critical (Rhoades and Bell, 2008). Upon low plasma volume, intrarenal baroreceptors found in the afferent arteriole walls sense a decrease in stretch and neighboring granular cells increase renin synthesis and release . The granular cells of the juxtaglomerular apparatus of the kidney produce renin (Widmaier et al., 2004). At first, preprorenin is synthesized in the granular cells; its 23 amino acid signal peptide is then cleaved to form prorenin (Sepehrdad et al., 2007). This proenzyme undergoes further modification-clipping the 43 amino acid segment from the N-terminal -to produce renin (Pool, 2007). Despite the conversion of prorenin to renin, prorenin remains the predominant form in the systemic circulation and represents 90% of the total plasma renin in humans (Pimenta and Oparil, 2009). Renin belongs to the family of aspartyl proteases, including pepsin, cathepsin D and chymosin (Verdeccia et al., 2008). Unlike other aspartyl proteases however, renin is able to work at neutral pH. Moreover, renin is specific for one substrate only-angiotensinogen (Sepehrdad et al., 2007). Made up of two lobes with a cleft in between renin accommodates the liverderived angiotensinogen in its active site, where a peptide bond of angiotensinogen is hydrolyzed and the decapeptide Angiotensin I (Ang I) is released (Verdeccia et al., 2008). Furthermore, this conversion is catalyzed 4-fold upon the binding of renin to its receptor (Pool, 2007). Angiotensin converting enzyme (ACE) is the principal enzyme that converts Ang I to the octapeptide Ang II (60% conversion); some other enzymes such as chymases, cathepsin G and other serine proteases account for the rest of the conversion (Weir, 2007).

Cross talk between RAS and litus
Angiotensin II was always considered in close proximity with diseases and vascular complications. This is implied from data linking high levels of Ang II to DM and vasoconstriction (causing hypertension) , in addition to glomerular damaging ending up with nephropathy (Coccheri, 2007). Other than its direct effect as a potent vasoconstrictor, it was denoted to alter Endothelin-1 (ET-1) production , remodel cardiovascular structure (Parmar and Jadav, 2007), modulate heart and vessels (Nuwayri-salti et al., 2007), augment transforming growth factor-beta (TGF-) and boost proliferative and inflammatory events . In conjunction; modulation of insulin significance is obtained , either by directly halting signaling mechanism of insulin per se or damaging structure and function of -cells via local pancreatic RAS (Coccheri, 2007). Angiotensin II has 2 receptors: Ang II Type 1 receptor (AT1R) and Ang II Type 2 receptor (AT2R). But until now AT1R rather than AT2R is assessed in the pathophysiology Rao, 2010) directing therapeutic agents either to target Ang II formation or block its binding to AT1R via Angiotensin converting enzyme inhibitors (ACEIs) and Ang II Receptor Blocker(ARB) respectively . AT1R blockers improved vascular smooth muscle cell vasoconstriction and declined the hypertrophy of cardiomyocytes (CM) counteracting Ang II deleterious effect  once bound to the G-coupled protein receptor (AT1 R) (Parmar and Jadav, 2007;. Quite interestingly, ACEIs ameliorated insulin sensitivity and mitigated new onset of DM type 2 (Hadi and Suwaidi, 2007;Scheen, 2004). Pooled together, these G-coupled transmembrane proteins mimic insulin receptor action (Nuwayri-Salti et al., 2007). Among several evidences, AT2R is more related to apoptotic actions thereby antagonizing insulin's growth effect, resulting in one way or another to boost cardiac hypertrophy mainly in type 1 DM . To complete this picture, a balance should be assured between both Ang II receptors to modulate a normal cardiac status in type 1 DM along the endothelial cells and cardiomyocytes . The idea of blocking RAS at its point of origin was initiated around 50 years ago (Pool, 2007). The origin of these direct renin inhibitors (DRIs) was either analogues of the prosegment peptide of renin or of the AGT's N-terminal amino acid sequence (Gradman and Kad, 2008). Many peptidomimetic synthetic drugs used for renin inhibition have been launched including pepsatitn (the first renin inhibtor) and other oral drugs, including remikiren, enalkiren and zankiren (Pimenta and Oparil, 2009), but their low efficiency related to large molecular size, first-pass metabolism, incomplete intestinal absorption, hydrophobicity and short half-lives attributed their poor oral activity and bioavailability, besides the high production cost (Pool, 2007;Waldmeier et al., 2007). The drug Tekturna ® (United States) or Rasilez ® (Europe) , known with the generic name of Aliskiren, ascribed to Dr. Alice Huxley (Gradman and Kad, 2008), manufactured by Novartis Pharmaceuticals, was the first of the nonpeptide DRIs to be approved by the Food and Drug Administration (FDA) (Azizi, 2008). Thereby becoming the commercially available renin blocker in markets, prescribed through United States and Canada for an effective essential hypertension treatment in March 2007 (Azizi, 2008), and approved by European Medicine Agency (EMEA) in August 2007 (Pool, 2007;Azizi, 2008).

Endothelin system
In 1985, Hickey et al. discovered a potent vasoconstricting substance from cultured endothelial cells and named it endotensin or endothelial contracting factor. Later on, Yanagisawa et al. isolated the same substance from cultured porcine aortic and endothelial cells and renamed it endothelin. Endothelin (ET) is a naturally occurring polypeptide (Prasad et al., 2009) produced by the endothelium (Shah et al.,2009;Wikes et al., 2003;Ying wu, 2003) and consists of 21 amino acids. It is present in three isoforms ET-1, ET-2 and ET-3 (Agapitov et al., 2002;Prasad et al., 2009). These three isoforms are encoded by different genes but have similar structure and function (Wilkes et al., 2003). They have conserved sequences of amino acids mainly at the 6 C terminus and 4 cysteine residues that form 2 disulfide bridges between residues 1 to 15 and 3 to 11 and the main difference is at the Nterminus (Prasad et al., 2009). They are vasoconstrictors synthesized by vascular, right atrial and left ventricular endothelial cells, vascular smooth muscle cells and fibromyocytes. In addition, they are synthesized in extra vascular tissues such as the lungs, spleen, pancreas and nervous system (Penna et al., 2006). All studies done on endothelial systems focused on ET-1, because represents the majority of the circulating endothelins (Prasad et al., 2009;Schneider et al., 2002) and has important role in the regulation of the cardiovascular system (Penna et al., 2006;Prasad et al., 2009).

Endothelin-1
Endothelin-1 is the most common isoform correlated with the cardiovascular system (Kalani, 2008). It was identified in the late 1980s by Dr. Yanagisawa and his colleagues and found to be a very potent vasoconstrictor (Prasad et al., 2009;Steiner and Preston, 2008;Thorin and Webb, 2009). It has ionotropic, chemotactic and mitogenic activities. It influences salt and water retention through its effect on RAS, vasopressin release and stimulation of sympathetic nervous system. So the ultimate role of ET-1 is to increase blood pressure (Agapitov et al., 2002) and to maintain vascular tone (Thorin and Webb, 2009). It is produced mostly from endothelial cells in addition to fibroblasts, cardiacmyocytes (Agapitov et al., 2002;Schorlemmer et al., 2008), leukocytes, macrophages (Thorin and Webb, 2009), kidney, central nervous system and posterior pituitary (Agapitov et al., 2002). Endothelin-1 exerts a paracrine/autocrine effect. In the circulation, the levels of ET-1 are in picomolars lower than that needed to cause vasoconstriction (Prasad et al., 2009). Also endothelial cells secrete more ET-1 toward the vicinity of vascular smooth muscle cells than into the lumen of the blood vessels (Agapitov et al., 2002).

Endothelin-1 synthesis and clearance
Endothelin-1 synthesis begins with the cleavage of a 200 amino acid peptide called preproendothelin-1 (preproET-1) to form preendothelin-1 (preET-1) (Agapitov et al., 2002;Penna et al., 2006). PreET-1 is then cleaved by furin endopeptidase to form big endothelin-1 (big ET-1) of 38-39 amino acids that is further metabolized by Endothelin Converting Enzyme-1 (ECE-1) to generate ET-1 of 21 amino acids (Agapitov et al., 2002;Penna et al., 2006). Moreover, chymase enzyme was found to produce ET(1-31) by breaking down big ET-1 at Tyr31-Gly32 bond (Agapitov et al., 2002). Furthermore, the regulation of ET-1 synthesis is at the gene level which means at the level of ET-1 messenger RNA (mRNA). The ET-1 mRNA is up-regulated by several factors including: interleukins, insulin, Ang II, tumor necrosis factor alpha and growth factor while it is down regulated by hypoxia, shear stress and nitric oxide (Thorin and Webb, 2009). The clearance of ET-1 from the plasma involves different mechanisms including: (1) endocytosis in the lungs, (2) enzymatic degradation in the liver and the kidney and (3) ET-1 binding to ET B receptor thus forming ET B Receptor-Legand complex that is broken down by endocytosis (Kalani, 2008).

Cross talk between endothelin system and renin angiotensin system
The Renin Angiotensin and the Endothelin Systems are the most potent identified vasoconstrictory systems and has been suggested to be correlated with each other (Rossi et al., 1999;McEwan et al., 2000). This was demonstrated by several cell culture studies in cardiac fibroblasts. These studies showed that AngII increases preproET-1 mRNA expression, ET-1 levels and thus causes cardiomyocyte hypertrophy through regulation of ET A R. Also increased levels of ET B R via Ang II have also been identified in cultured CM (McEwan et al., 2000). The potential sites at which RAS increases ET-1 levels may be at the preproET-1 gene that possesses a jun sequence which is defined as the regulatory region of preproET-1. It is the site at which transcriptional regulation takes place by factors acting through G-proteinphospholipase and C-protein kinase C pathway. Another site for interaction of RAS and ET system is at the chymase enzyme which is abundant in the myocardium where it was found to produce ET (1-31) from big ETs, bearing in mind that the main function of chymase enzyme in the heart is to produce Ang II from Ang I (Rossi et al., 1999). Therefore, elevated levels of ET-1 can be reduced by blocking the RAS system. Studies on patients suffering from hypertension or diabetes showed decrease in the level of ET-1 when ACEI was administered (Schneider et al., 2002). Therapies targeting these complications include the use of ARBs which proved their value as antihypertensive drugs. Losartan, an ARB, was found to decrease the destructive changes caused by Ang II on cardiac muscle beside its (Berk, 1999;Fiordaliso et al., 2000) capacity to reduce blood pressure. Recently, Al Jaroudi et al. proved that losartan in diabetic normotensive rats supplemented with insulin was able to prevent nearly totally myocardial degeneration caused by diabetes   (Fig. 1). Later, Karam et al. reported that the supplementation with insulin and administration of pharmacologic doses of losartan could improve cardiac contraction as well as coronary blood flow in the same normotensive rat model for type 1 DM as the one used by Jaroudi et al. through the modification of the affinity of ET type 1 receptor subtypes ET A R and ET B R to ET-1, a potent vasoconstrictor largely stimulated by Ang II . In fact, Kakoki et al. have demonstrated that the ET B Rs are down-regulated by Ang II in the endothelial cells of the renal artery of diabetic rats through stimulation of increased ET-1 production (Kakoki et al., 1999). On the other hand, these effects of Ang II are prevented by the ACE inhibitor Imidapril (Kakoki et al., 1999). ACE inhibitors reduced also the plasma ET-1 levels in type 1 DM (Schneider et al., 2002). Administration of Losartan to Ang IItreated rats restored the vasoconstrictive effect of ET-1 and decreased its tissue levels (D' Uscio et al., 1997). Mc Ewan et al. showed that the blockade of the AT1Rs by Losartan, in the presence of a high plasma Ang II levels, is accompanied by an increase in ET-1 production by the CM of Ang II-treated rats. In addition to this, an increase in the ET B Rs mRNA but a decrease in the ET A Rs mRNA was also noted (Mc Ewan et al., 2000). On the other hand, treatment with the selective ET A R blocker LU135252 normalized the increased ET-1 level in the aorta, femoral artery and kidney, and the ECE activity in isolated aorta and femoral artery of rats treated with Ang II (Barton et al., 1997). In cultured heart endothelial cells, Ang II stimulates ppET-1 mRNA and ET-1 production via Protein Kinase C (PKC) -dependent pathway. Calphostin, a PKC inhibitor, blocks Ang II effects (Chua et al., 1993). Taken together, these results show that the ET and the RA systems are related to each other. They also demonstrate the beneficial role of ARBs and ACE inhibitors in the amelioration of the blood flow which can be altered in some disease states due to the ET-1 and Ang II constrictor effects.

Insulin
The discovery of insulin in 1921 was one of the most dramatic events in the history of diabetes therapy. Insulin is a small globular protein comprising of two polypeptide chains A (21 amino acid residues) and B (30 amino acid residues) held together by two disulfide bonds (Docherty and Steiner. 2003;Joshi et al. 2007). It is produced in the -cells of the islets of Langerhans in the form of preproinsulin which is rapidly cleaved by the action of proteolytic enzymes into proinsulin. Further catalysis of proinsulin results in insulin and a 31 amino acid connecting peptide, C-peptide, both of which are stored for secretion in secretory vesicles in -cells (Docherty and Steiner. 2003). The biosynthesis and secretion of insulin by -cells primarily occurs in response to increased circulating glucose levels. During feeding, elevated glucose concentrations in the blood increase the plasma insulin level, which facilitates glucose uptake through GLUT-4 into muscle tissues for utilization as a source of energy and into adipose tissues for synthesis of glycerol. Insulin also exerts its action on liver cells as well thus promoting glycogen formation. Consequently, glucose utilization by these different tissues contribute to the decrease in the concentration of glucose in blood. On top of its profound effect in carbohydrate metabolism, insulin has a fatsparing effect. Not only does it promote the synthesis of fatty acids in the liver, it also inhibits the breakdown of fats in adipose tissue thereby inducing fat accumulation. Hence, insulin is the major determinant of carbohydrate and lipid metabolism and has significant effects on protein metabolism. Insulin acts by binding to a receptor molecule embedded in the plasma membrane of its target tissues (Docherty and Steiner. 2003

Insulin receptor
Insulin receptor is a member of the receptor tyrosine kinase family (De Meyts and Whittaker, 2002;Kanzaki, 2006;Klarlund et al., 2003;Lawrence et al., 2007;Stern, 1995;Ward et al., 2008). It is a large cell-surface multi-domain glycoprotein that consists of two extracellular -subunits (MW~125 kDa) and two transmembrane -subunits (MW~95 kDa) (Kanzaki, 2006;Klarlund et al., 2003;Lawrence et al., 2007;Stern, 1995;Ward et al., 2008) linked by disulfide bonds (Kanzaki, 2006;Lawrence et al., 2007) into an 2 2 heterotetrameric complex (Kanzaki, 2006). Extracellular -subunits contain the insulin-binding site (Klarlund et al., 2003) while intracellularly transmembrane -subunits contain the insulin-regulated tyrosine kinase catalytic domain (De Meyts and Whittaker, 2002;Klarlund et al., 2003;Ward et al., 2008). Two nearly identical isoforms (A and B) of the insulin receptor exist due to tissue specific alternative splicing of the receptor m-RNA at exon 11. Isoforms A and B differ by the presence or absence of a 12 amino acid sequence at the carboxyl-terminus of thesubunit (Anderson et al. 1993;De Meyts and Whittaker. 2002;Klarlund et al., 2003;Lawrence et al., 2007). Despite the known biochemical and physiologic differences of these isoforms, changes in their expression levels have not been consistently found in patients with DM (Klarlund et al., 2003). Once insulin binds to the -subunit of the insulin receptor, it induces a conformational change in the receptor that subsequently leads to the stimulation of the intrinsic tyrosine kinase activity in the -subunits (Kanzaki, 2006;Klarlund et al., 2003;Stern, 1995). This results in the transfer of phosphate groups from ATP to several tyrosine residues on the insulin receptor itself as well as phosphorylation of cellular proteins such as insulin receptor substrate-1 (IRS-1) and Shc (Klarlund et al., 2003;Stern, 1995). The stimulation of the receptor tyrosine kinase allows transmission of the insulin signal to metabolic pathways such as glucose uptake upon translocation of GLUT-4 glucose transporters to the plasma membrane, glycogen synthesis, protein synthesis, and lipid metabolism within the cell. The various biological responses generated upon insulin receptor activation through insulin binding granted insulin a role in DM treatment due to its glucose lowering effects. Nevertheless, daily insulin injections continue to be a treatment for diabetic patients since 1922 despite the increasing worldwide prevalence of this disease (Sparre et al., 2005). Furthermore, insulin treatment dramatically prolongs survival, but does not cure diabetes (Myers and Zonszein, 2002). Insulin treatment does not seem to be beneficial for many patients and is associated with weight gain, hypoglycemia, and failure of their glycemic control (Halimi, 2008). Therefore, new treatment modalities are intensively studied mainly the incretins, exemplified by GLP-1.

Definition of incretins
The intravenous infusion of glucose at a constant rate results in a biphasic insulin secretory response, in which the first peak rises rapidly followed by a slower second peak. In contrast, an oral administration of glucose followed by its gastrointestinal absorption, triggers a hormonal pathway that eventually leads to a far greater response of insulin secretion which can last as long as glucose is administered. This phenomenon is termed as the 'Incretin Effect' (Rhoades and Bell, 2008). Incretins are hormones that are secreted by the gut upon feeding; their release alerts the pancreatic islets that nutrients will come through the gastrointestinal tract and islets start priming by vagal stimulation (Boron and Boulpaep, 2009). Glucagon-Like Peptide-1 (GLP-1) and Glucose Dependent Insulinotropic Peptide (GIP) are the major incretin hormones www.intechopen.com Type 1 Diabetes Complications 228 produced by the L cells of the ileum and colon, and K cells of the duodenum and jejunum, respectively (Inzucchi and McGuire, 2008).

Effects of GLP-1
GLP-1 has numerous effects on glucose homeostasis. Upon interaction with its receptor on -cells, GLP-1 increases the intracellular levels of cAMP and calcium, thereby releasing insulin (Drucker, 2007). Furthermore, sustained GLP-1 receptor (GLP-1R) signaling leads to enhanced gene transcription, insulin biosynthesis and -cell proliferation (Elahi et al., 2008;Drucker, 2007). GLP-1 was shown to increase the expression of glucose transporter 2 (GLUT2) (Elahi et al., 2008), a hepatic glucose transporter that facilitates glucose transport in or out of the liver regulated by insulin (Eisenberg et al., 2005) and glucokinase (Elahi et al., 2008) the enzyme that phosphorylates glucose to glucose-6-phosphate (G6P) (Voet and Voet, 2004). GLP-1 has also inhibitory effects on Glucagon secretion from the -cells, gastric emptying and food ingestion (Drucker, 2007). The role of GLP-1 in relaxing the proximal stomach and increasing gastric capacity has been demonstrated (D' Alessio and Vahl, 2004

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GLP-1 also exerts effects on the central nervous system: it promotes satiety and weight loss, inhibits food and water intake and improves memory and neuronal survival (Mannucci and Rotella, 2008;Drucker, 2007).

GLP-1 receptor
The GLP-1R belongs to the Guanine Nucleotide-Binding Protein (G-protein) coupled receptor family (GPCR) (Drucker, 2007). This seven transmembrane receptor protein is 90% identical to the rat GLP-1R and shows a 95% homology in amino acid. Its gene is found on the long arm of chromosome 6p21 in humans (Doyle and Egan, 2007). GLP-1 receptor is made up of 463 amino acid residues and has a molecular weight of approximately 65 kDa (Hui et al., 2005). GLP-1 receptors are expressed in many tissues including, the central and peripheral nervous systems, heart, kidney, lungs and the gastrointestinal tract (Drucker and Nauk, 2006). However, GLP-1 receptor expression in the pancreas is controversial. Studies have confirmed the detection of GLP-1 receptors in , and δ cells of the islets of Langerhans, whereas other studies indicate their expression exclusively in cells (Doyle and Egan, 2007). In the heart, GLP-1 receptor expression was shown to exist in CM, microvascular endothelium, coronary smooth muscle cells and the highest in endocardium. In cardiac fibroblasts, however, there was no evidence in GLP-1R expression (Ban et al., 2009). Low detection of GLP-1 receptor gene expression proves the existence of GLP-1 receptor in liver and muscles (MacDonald et al., 2002). Abundant GLP-1R specific transcripts were found in lungs and detectable amounts in the heart (Ban et al., 2009). When fragments of the Nterminus are denatured, GLP-1R loses its affinity for GLP-1. This asserts that GLP-1 receptor's N-terminus plays an important role in recognizing and binding of the GLP-1 (Doyle and Egan. 2007). Upon binding of the GLP-1 to its receptor numerous signaling messengers are activated; first, GLP-1 receptor can couple to G proteins, including G s , G q , G i or G o (Baggio and Drucker, 2007). Adenylate cyclase uses ATP to form cAMP via the stimulatory G protein. Following the increase of cAMP is a chain of events is triggered: ATP-sensitive K + channels shut and L-type voltage gated Ca 2+ channels open; together with the efflux of Ca 2+ molecules from intracellular Ca 2+ stores, these ion channel alterations result in a subsequent intracellular rise in Ca 2+ ions (Gomez et al., 2002) and the ultimate exocytosis of granules that contain insulin. A sustained receptor signaling results in the activation of Protein Kinase A (PKA), gene transcription, insulin biosynthesis and -cell proliferation (Drucker and Nauk, 2006). Additionally, the binding of GLP-1 to its receptor activates protein kinase B, which is linked to glucose transporting in muscles, glycogen synthesis and lipolysis in various tissues (Peyot et al., 2009).

GLP-1 in diabetes mellitus and the heart
A recent study showed that GLP-1 receptor expression is downregulated in -cells exposed to high glucose concentrations in vitro and hyperglycemia in vivo (Xu et al., 2007). Diabetic individuals' -cells exhibit attenuated sensitivity to GLP-1. In both type 1 and type 2 DM, there is a marked reduction in the incretin effect (Knop et al., 2007). On the other hand, a study reported that GLP-1 levels are not decreased in type 2 diabetic patients (Lee et al., 2010). The European GLP-1 Club held a meeting recently and debated this controversial issue and came to a conclusion that more data is required to determine the exact effect of DM on GLP-1 secretion from L-cells (Burcelin, 2008). GLP-1 has been suggested to ameliorate left ventricular function, because of its antiapoptotic and insulin-like properties (Inzucchi and McGuire, 2008). In fact, one study confirmed that GLP-1 enhances the regulation of phosphatydil inositol 3 kinase (PI3K), that plays a key role in activating the antiapoptotic pathway, thus promoting cardioprotection in the ischemic rat hearts. Therefore, a direct effect of GLP-1 against apoptosis in cardiac cells is possible (Bose et al., 2005).

Dipeptidyl-peptidase IV
DPP-IV grasped a great deal of the interest of the scientific, pharmaceutical, and medical community (Lambeir et al., 2003). Every year, an increasing number of publications tend to elucidate the diverse compelling questions concerning the various properties of DPP-IV and its multiple functions in the different fields of Biology (Lambeir et al., 2003). DPP-IV is a 766 amino acid serine protease that belongs to the prolyloligopeptidase family. It is a widely distributed cell surface peptidase expressed to different degrees in a variety of tissues such as the kidney, intestine, liver, placenta, uterus, prostate, skin, and capillary endothelium. Another soluble form of DPP-IV (sDPP-IV) also exists in the plasma and other body fluids (Drucker, 2007;Idris and Donnelly, 2007;Lambeir et al., 2003). This proteolytic enzyme acts by specifically cleaving the N-terminal dipeptide of peptide hormones containing proline or alanine in the second position (Drucker, 2007;Idris and Donnelly, 2007;Lambeir et al., 2003). Hence, DPP-IV truncates several biologically active peptides of medical importance. Furthermore, it has been implicated in glucose homeostasis through proteolytic degradation of the incretins (Drucker, 2007). In the case of GLP-1(7-36), proline and alanine are key determinants in incretin receptor activation therefore DPP-IV-mediated proteolysis results in the biologically inactive truncated molecules GLP-1(9-36) (Drucker, 2007;Idris and Donnelly, 2007). DPP-IV is a critical determinant of incretin inactivation (Drucker, 2007). Thus chemical inhibition of DPP-IV activity results in increased level of biologically active GLP-1 (Drucker, 2007). Therefore, extensive research studies were carried to create highly selective DPP-IV inhibitors.

DPP-IV inhibitors
DPP-IV inhibitors can be used as a potential treatment for diabetes due to their capability to potentiate the levels of active incretins (Lambeir et al., 2003;Halimi, 2008;Inzucchi and McGuire, 2008) by reducing the enzymatic activity of DPP-IV by more than 80% for duration up to 24 hours (Inzucchi and McGuire, 2008). Oral inhibitors of DPP-IV reduce glycosylated hemoglobin (HbA 1C ) (Drucker, 2007;Halimi, 2008;Moritoh et al., 2008). Various studies reported the multiple metabolic effects of DPP-IV inhibitors including enhancement of glucose-dependent stimulation of pancreatic insulin release as well as attenuation of glucagon secretion (Drucker, 2007;Halimi, 2008;Lambeir et al., 2003;Moritoh et al., 2008). Furthermore, DPP-IV inhibitors demonstrated modestly effective glucose-lowering actions without being associated with hypoglycemia (Halimi, 2008;Lambeir et al., 2003;Moritoh et al., 2008) due to the fact that they are remarkably able to specifically end the insulinsecreting effect and glucagon inhibition once glycemic levels are normalized (Halimi, 2008). Moreover, loss of DPP-IV activity is associated with improved glucose tolerance, reduced glycemic excursion following oral glucose challenge, and increased pancreatic insulin content (Drucker, 2007). A cardinal role of DPP-IV inhibitors is their potential to significantly augment -cell mass in streptozotocin (STZ)-injected diabetic rats ( al., 2008) and enhance pancreatic islet cell function in animal models of type 2 diabetes and in diabetic patients (Lambeir et al., 2003). In contrast to GLP-1 mimetics, there are no data indicating inhibition of gastric emptying or appetite or weight reduction due to a treatment with DPP-IV inhibitors (Inzucchi and McGuire, 2008). Thus, chronic treatment with orally administered DPP-IV inhibitors has neutral effects on body weight and food consumption (Halimi, 2008).

GLP-1 receptor agonists and GLP-1 analogues
In April 2005, the FDA approved the subcutaneous injectable exenatide with the brand name Byetta ® to be launched as a GLP-1 analogue (Inzucchi and McGuire, 2008; with longer half life to maintain its glycemic control as diabetic medication (Behme et al., 2003). Exenatide exhibits a half-life of 60-90 minutes and a single injection of exenatide makes its concentration in the blood last for 4-6 hours (Drucker and Nauk, 2006). Exenatide exhibits antidiabetic effects similar to those of GLP-1 and is shown to improve cell functioning (Baggio and Drucker, 2007). It also reduces the glycated Hemoglobin-HbA1c-levels that reflect mean blood glucose levels of the last 6-8 weeks. In addition, exenatide affects gastric emptying, appetite and consequently causes weight loss (Inzucchi and McGuire, 2008). GLP-1 agonists have exhibited vasodilating and diuretic effects. Recently, Laugero et al. showed that Exenatide may display antihypertensive effects mediated by pathways independent from glucose control, but possibly by altering steroid hormones (Laugero et al., 2009).

GLP-1 analogue, exendin-4
The short half-life of GLP-1 limits itself from performing as a good therapeutic agent, as it is rapidly degraded by the serine protease DPP-IV (Hirata et al., 2009). The search for biologically active peptides in lizard venom, led to the discovery of Exendin-4, a naturally occurring GLP-1 analogue (Drucker and Nauk, 2006). Exendin-4 is a peptide naturally found in the saliva of heloderma suspectum -the Gila monster (Laugero et al., 2009;Zhou et al., 2008). Exendin-4 is a peptide made up of 39 amino acid residues that shares a 53% structural homology to GLP-1 (Zhou et al., 2008).This long acting GLP-1 receptor agonist has an Nterminus almost identical to that of the GLP-1, except that the second amino acid residue is glycine in exendin-4 while alanine in GLP-1 (Hui et al., 2005). This one amino acid difference makes it resistant to degradation by the enzyme DPP-IV, hence explaining its long half-life in vivo (Lovshin and Drucker, 2009). Studies have shown that GLP-1 receptor agonists like exendin-4, exhibit cardioprotective effects such as modifications in contractility, cardiac output and blood pressure (Ban et al., 2009).

Mode of action of exendin-4
Endogenous GLP-1 action is inhibited upon degradation by DPP-IV (De Koning et al., 2008;Mann et al., 2007), an enzyme expressed in many organs mainly kidney, small intestine, liver, and lung (Inzucchi and McGuire, 2008). More apt, exendin-4 is synthetic, thus not recognized by DPP-IV enzyme, thereby not degraded ). This resistance allows it to stay in the circulation longer mimicking GLP-1 role in bolstering insulin secretion but with 5 to 10 times more powerful insulinotrpic effect (Hantouche et al., 2010;Xu et al., 1999). Moreover, it binds to GLP-1R with high affinity even after truncation of the first 8 amino acids at the N-terminal of the peptide unlike the endogenous GLP-1 (Mann et al., 2007). GLP-1R is a G-coupled transmembrane receptor (GPCR) that is found in many organs, most importantly the pancreatic -cells and ducts, heart, lung, kidneys (Ban et al., 2008), brain and stomach (Hantouche et al., 2010). Conceptual understanding of this receptor delineates its advantages in cardiac management (Ban et al., 2008). On the initiation of treatment, slight adverse effects might arise as nausea and vomiting (Inzucchi and McGuire, 2008), which are dose-dependent and will vanish with time (Mcphee et al., 2008). It can be used efficiently in type 1 DM (Behme et al., 2003) in conjunction with insulin irrespective of hypoglycemic effects attributed to its glucose-dependent action; in spite of insulin low efficiency with type 2 DM (Inzucchi and McGuire, 2008).

Exendin-4 in the treatment of diabetes mellitus correlated with cardiovascular risk
In conjunction to insulinotropic action of GLP-1 role, exendin-4 also mimics the action of insulin itself, through reinforcing the heart to uptake glucose irrespective of insulin level in both diabetic and non-diabetic cases (Hantouche et al., 2010) STZ-induced diabetic rats acquire cardiomyopathic damage on day 1 starting with apoptosis and ending with hypertrophied hearts , particularly myocardial atrophy (Poornima et al., 2006). Thereby, associated with its glucose lowering effect, certain data emphasized a potential benefit of exendin-4 in recovery of heart failure and function of left ventricle (Inzucchi and McGuire, 2008). Just similar to that of GLP-1 in improving ventricular contractions (Hantouche et al., 2010) upon activation of pancreatic GLP-1R to boost insulin synthesis and secretion (De Koning et al., 2008). Treatment with exendin-4 was concomitant with no cardiovascular complications, inexorable renal progression, or escalating plasma lipid (De Fronzo et al., 2005). In type 2 DM exendin-4 has completed a novel fruition in the arena of organ rejuvenation especially at the level of both  and cells mass, implementing a new born anti-hyperglycemic drug for solving the diabetes conundrum (Xu et al., 1999). This increase in pancreatic mass happens as a post hoc to -cells neogenesis from either existing duct cells and/or replication of the already present -cells (hyperplasia) and not from hypertrophy (escalating cell size) (De Koning et al., 2008). Pooled together, this regimen was found to be concomitant with cardioprotection, judicious vascular and cardiac actions based on the fact that endogenous GLP-1 has the same sequence in humans, rats and mice (Ban et al., 2008). Recently, it was found that exendin-4 ameliorates the sensitivity of insulin receptors towards insulin at both the CM and coronary endothelium (CE) level (Hantouche et al., 2010). This effect is achieved by either enhancing the sensitivity of insulin towards insulin or augmenting -cells to release more of the hypoglycemic hormone (De Koning et al., 2008). Valuable efficacy of treatment with both exendin-4 and insulin alleviates cardiomypathic regressions associated in type 1 DM at the level of receptor affinity enhancement and insulin secretion bolstering (Hantouche et al., 2010) (Fig. 1 and 2). Insulin affinity (τ) to its receptor was shown to be decreased in the diabetic state at the level of the CE as compared to normal. Exendin-4 treatment increases insulin receptor affinity at the CE in both diabetic and normal rat groups; whereas insulin tends to normalize the affinity irrespective of exendin-4 absence or presence (Table 1). Exendin-4 treatment probably augments insulin receptor affinity both in normal and diabetic state through exerting its insulinotropic actions (Nikolaidis et al., 2005) and/or by improving insulin sensitivity (Ebinger et al., 2000). The dramatic increase in the affinity of insulin to its receptor in the normal group treated with exendin-4 might be attributed to both of the previously mentioned causes. Yet the rise in insulin receptor affinity in exendin-4-treated diabetic group is not as remarkable as in NE which may be due to the lack of -cells in diabetic type 1 state thus abolishing the insulinotropic effect of exendin-4.  Therefore, in diabetes type 1, exendin-4 increases the insulin receptor affinity only by improving insulin sensitivity. As a conclusion, exendin-4 seems to have supplementary effects which might explain the increase in τ value at the level of the CE. First, it augments insulin release from pancreatic -cells when present in the normal state. This is its insulinotropic effect. Second, it improves insulin sensitivity possibly by inducing a conformational change in the insulin receptor (Ebinger et al., 2000). On the other hand, DPP-IV inhibitor (KR-62436, Sigma Chemical Company) treatment solely does not seem to be implicated in modulating insulin receptor affinity at the endothelial site in diabetic rats treated with DPP-IV inhibitor as compared to diabetics. Yet the effect of DPP-IV inhibitor at the CE becomes obvious in the presence of insulin, as seen in the diabetic group co-treated with insulin and DPP-IV inhibitor (DI-Dp) compared to diabetics (D) and in the normal group treated with DPP-IV inhibitor (N-Dp) with respect to normal. This could be due to some kind of a cross-talk between insulin and DPP-IV inhibitor which results in an increase of τ value in both N-Dp and DI-Dp. Moreover, insulin receptor affinity is not altered at the level of the CM in diabetics treated with DPP-IV inhibitor (D-Dp) and/or insulin (DI-Dp) when compared to diabetic rats (D) ( Table 2). The major difference between the CE and CM upon DPP-IV inhibitor treatment could be attributed to the fact that the already limited increase of GLP-1 by DPP-IV inhibitor is primarily imposed on endothelial cells which are the first site of encounter with systemic GLP-1. The difference in affinities observed between DPP-IV inhibitor and exendin-4 treatment might be due to the fact that DPP-IV inhibitor increases the systemically available GLP-1 levels; whereas exendin-4, besides its quantitative systemic effect, has a higher physiologic quality in term of being 5-to 10-fold more potent than GLP-1 (Saraceni and Broderick, 2007). Western blotting was performed on protein extracts from rat heart homogenates in order to assess the variation in the insulin receptor subunits density among the different treated and untreated normal and diabetic rat groups. Both insulin receptor -subunit (IR-) (Fig. 2) and insulin receptor -subunit (IR-) (Fig. 3) levels are augmented in diabetic state indicating that there is an increase in the level of cardiac insulin receptor protein in diabetics (D) compared with normal controls (N). These results indicate that cardiac insulin receptors are up-regulated in the heart of diabetic rats as a feedback mechanism probably due to the lack of insulin. This is consistent with the results demonstrated by Al  in the study done on insulin receptor regulation in the diabetic heart. Our results indicate that insulin administration to diabetic rats reduces the number of IR-and IR-to near normal values. Interestingly, exendin-4 treatment normalizes insulin receptor subunits density in diabetic rat hearts ( Fig. 2 and 3). The regression in insulin receptor density with exendin-4 treatment is suggested to be attributable to the insulinomimetic effects of exendin-4 (Sokos et al., 2006) which result in a cross-talk between GLP-1 and insulin signaling pathways (Ebinger et al., 2000). There is no further attenuation in the level of insulin receptor upon insulin administration in combination with exendin-4 in diabetic rat groups. Thus, it is conceivable that GLP-1 is a key player in insulin receptor regulation in the diabetic state. GLP-1 also induces its insulinomimetic effects in the normal state thereby resulting in a slight decrease of insulin receptor density in NE. DPP-IV inhibitor induces similar effects as exendin-4 on IR-and IR-levels and this could be attributed as well to the insulinomimetic effects of GLP-1 ( Fig. 2 and 3). There is a similar attenuation in receptor density in non-diabetic DPP-IV inhibitor treated rats (N-Dp). This reflects the actions of DPP-IV inhibitor treatment on the modulation of insulin receptor density not only in the diabetic but also in the normal state.

Conclusion
In conclusion, insulin, Losartan, exendin-4 and DPP-IV inhibitor have demonstrated their ability as promising candidates for treating type 1 diabetic patients. Moreover, exendin-4 and DPP-IV inhibitor appear to be promising in their efficacy and prolonged antidiabetic properties. Their actions on cardiovascular function, in both the preclinical and clinical realms, warrant future investigation.

Acknowledgement
The authors would like to acknowledge funding from the University Research Board and Medical Practice Plan of the American University of Beirut.