5 Diabetes Mellitus

4

Introduction

Introduction

5 Diabetes Mellitus

Diabetes mellitus is a pandemic metabolic disorder characterized by high levels of blood glucose, resulting from defects in insulin production, insulin action, or both, and is accompanied by altered metabolism of lipids, carbohydrates and proteins. Two different types of diabetes can be recognized, classified as insulin- dependent diabetes mellitus (Type 1 Diabetes or IDDM) or non- insulin-dependent diabetes (Type 2 Diabetes or NIDDM).

The incidence of each type of diabetes varies widely throughout the world. Actually, this pathology is recognized as a public health problem, as it affects a significant portion of the population worldwide and is rising to pandemic proportions. According to epidemiological studies, approximately 135 million of adults worldwide were diagnosed with diabetes in 1995, and this number is expected to rise to at least 300 million by 2025, with a 122% overall increase in the worldwide prevalence ¹ (Figure 1).

Figure 1. Distribution of Diabetic Subjects in the World.

Introduction

6 Type 1 diabetes (IDDM) is characterized by a deficiency of insulin production due to the destruction of pancreatic β-cells caused by an autoimmune T-cell attack. There is no known preventive measure which can be taken against type 1 diabetes; it represents about 10% of diabetes mellitus cases in North America and Europe.

Sensitivity and responsiveness to insulin are usually normal, especially in the early stages. Type 1 diabetes can affect children or adults but is traditionally termed "juvenile diabetes" because it mainly affects children.

The vast majority of diabetic patients are affected by non-insulin- dependent diabetes (NIDDM).

Genetic and environmental components are the leading causes of

both types of diabetes, but a positive family history is predictive for

the disease and also people more than 20% over ideal body weight

show a greater risk of developing diabetes. In addition, previously

identified impaired glucose tolerance, gestational diabetes,

hypertension, or significant hyperlipidemia are associated with an

increased risk of type 2 diabetes. These data suggest that there is a

strong genetic basis for non-insulin-dependent diabetes, but the

genetic mechanisms are still not well known. Recent works have

demonstrated that a pancreatic β-cell defect and a reduction in

tissue sensitivity to insulin are both required before phenotypic

NIDDM is apparent. However, NIDDM is an extremely

heterogeneous disease, and it is likely that a variety of different

genes are involved and also environmental factors could play a

crucial role. From a physiological point of view most studies

demonstrate that there is a drastic reduction of β-cell mass: the

earliest manifestation is a loss of the regular periodicity of insulin

secretion. At diagnosis, virtually all persons have a profound defect

in first-phase insulin secretion in response to an intravenous glucose

Introduction

7 challenge. Abnormalities of the β-cells probably are secondary to desensitization by chronic hyperglycemia.

Long-term Diabetic Complications

Patients with diabetes are at a higher risk for cardiovascular events, including strokes, and show accelerated formation of severe atherosclerotic lesions in peripheral, coronary, and cerebral arteries.

They quickly develop visual impairment and blindness due to cataract and severe retinopathy, as well as terminal renal diseases and different forms of nervous system damage, including impaired sensation of pain and physical disability. ² Actually, hyperglycemia is strictly connected with microvascular diseases, and therefore with long term diabetic complications, through different mechanisms:

polyol pathway activation, advanced glycated end product (AGE) formation, protein kinase C (PKC) activation, and hexosamine pathway action (Figure 2). These mechanisms are especially activated in those tissues that don’t need insulin for the uptake of glucose and that, therefore, are exposed to high levels of blood glucose. ^3,4

Figure 2. Pathways Implicated in Diabetic Complications.

Glucose Hexokinase Glucose-6-P

Glucose-6-phosphate

isomerase Fructose-6-P Glyceraldehyde-3-P

ATP ADP

Mg

GAPDH

NAD

NADH

1,3-Diphosphoglycerate

Glycolisis

ALR2 Sorbitol Fructose

NADPH

NADP

NAD

NADH

Polyol Pathway

GFAT Gln

Glu Glucosamine-6-P

UDP-GlcNAc Hexosamine

Pathway

DHAP NADH

NAD

-Glycerol-P

DAG

PKC Activation

PKC Pathway

Fructose, trioses, dicarbonyl compounds

AGEs

AGE Pathway

SD

Introduction

8 Polyol Pathway

Aldose reductase (ALR2) is the first enzyme of the polyol pathway and reduces glucose to sorbitol in the presence of NADPH as cofactor. Sorbitol dehydrogenase (SD), the second enzyme of the polyol pathway, subsequently oxidizes sorbitol to fructose (Figure 3).

Polyol pathway was first identified in the seminal vesicles by Hers, who demonstrated the conversion of glucose into fructose, an energy source of sperm cells. Later, the existence of this pathway was also reported in diabetic rat lenses.

Figure 3. Polyol Pathway.

CHO OH H

OH H

H HO

OH H

CH ₂ OH

CH ₂ OH OH H

OH H

H HO

OH H

CH ₂ OH

CH ₂ OH O OH H

H HO

OH H

CH ₂ OH NADPH

NADP ⁺

ALR2

NAD ⁺

NADH

SD

Glucose Sorbitol Fructose

Under normoglycemia conditions , since the affinity of aldose reductase for glucose is low, most of the cellular glucose is phosphorylated into glucose-6-phosphate by hexokinase. In the presence of hyperglycemic states such as diabetes, however, there is an increased flux of glucose through the polyol pathway because of the saturation of the hexokinase.

Since the reduction of glucose by ALR2 is particular significant

during hyperglycemia, the polyol pathway hyperactivity has been

linked to the development of diabetic complications. In these

conditions sorbitol is formed more rapidly than it is converted to

Introduction

9 fructose, so it accumulates increasing cellular osmolarity, which causes tissue damage. ^5,6

In the ocular lenses, for example, osmotic stress imposed by sorbitol accumulation has long been suggested to be the major cause of diabetic cataract, since sorbitol was demonstrated to be significantly elevated in cataractous lenses from diabetic animals, such as rats, rabbits, dogs, and also from diabetic patients.

Increased formation of sorbitol causes an intracellular osmotic imbalance which promotes the occurrence of a cascade of events leading to lens opacification.

Advanced Glycated End Products (AGEs)

Reducing sugars, such as glucose, react non-enzymatically and reversibly with free amino groups of proteins to form small amounts of stable Amadori products through Schiff base adducts. During aging, spontaneous further irreversible modification of proteins by glucose results in the formation of a series of compounds termed AGEs, a heterogeneous family of biologically and chemically reactive compounds with cross-linking properties. In diabetes, this continuous process of protein modification is magnified by high ambient glucose concentrations (Figure 4).

AGEs bind to specific receptors on endothelial cells and after

cellular attachment these products have been shown to increase

vascular permeability, procoagulant activity, adhesion molecule

expression and monocyte influx, actions that may contribute to

vascular injury. AGEs probably contribute to the rapidly progressive

atherosclerosis that develops in patients with diabetes and renal

insufficiency. In fact, within the atherosclerotic lesion, collagen-

linked AGEs bind plasma proteins, interact with macrophage

receptors to induce cytokine and growth factor release; there is

Introduction

10 also evidence that they modify LDL, making it less susceptible to clearance by LDL receptors.

Study of coronary arteries obtained from patients with type 2 diabetes, using immonohistochemical analysis, detected high levels of AGE reactivity within atherosclerotic plaques stained with anti- AGE antibodies. This observation is consistent with a link between hyperglycemia, hyperlipidemia, and atherosclerosis in diabetes.

AGE formation also contributes to the development of diabetic complications by changing the structure and function of the extracellular matrix in the glomerular mesangium and elsewhere causing changes in matrix integrity and functions. These alterations affect biological functions important to normal vascular tissue integrity supporting the development of diabetic complications.

Figure 4. Advanced Glycated End Products.

Introduction

11 Protein Kinase C (PKC) Activation

The activation of the PKC pathway is associated with many vascular abnormalities in the retinal, renal, and cardiovascular tissues in diabetic and insulin resistant states. The activation of the PKC pathway contributes to vascular function in many ways, such as regulation of endothelial permeability, vasoconstriction, extracellular matrix synthesis and turnover, cell growth, angiogenesis, cytokine activation and leucocyte adhesion.

PKC activation could affect the functions of several vasoactive factors within the kidney like prostaglandins, whose concentration is increased causing the release of arachidonic acid and leading to an inflammatory response.

At the same time most studies have demonstrated that PKC activation causes an alteration also in retinal blood flow, increasing vascular permeability, impairing vascular tone and increasing microaneurysm formation. Ischemia of retinal tissues will cause an increase in the expression of angiogenic growth factors such as vascular endothelial growth factor (VEGF), leading to macular edema and proliferative retinopathy. PKC activation can directly increase permeability of macromolecules across the endothelial or epithelial barriers by phosphorylating cytoskeletal proteins or by regulating expression or activity of various growth factors, as we have already seen. Elevated expression of VEGF has been reported in the retina of diabetic patients and animals, thus implicated in the neovascularization process of proliferative diabetic retinopathy.

Another effect of PKC activation is connected with glomerular

mesangial matrix expansion and capillary basement membrane

thickening, characterizing diabetic nephropathy, due to expression

of extracellular matrix collagen, fibronectin and laminin. Different

Introduction

12 studies demonstrated that in diabetes there is stimulation in the transcription of collagen IV in mesangial cells.

In summary, PKC activation plays an important role in multiple diseases involving the inflammatory process and complications of diabetes and actually, specifical isoforms of PKC inhibitors, such as PKCβ inhibitors, exhibited very few side effects and demonstrated to be therapeutically useful in the treatment of diabetic complications.

Hexosamine Pathway Action

The last mechanism activated during hyperglycemia is the increased flux through the hexosamine pathway. When glucose concentration rises, it is mainly metabolized through glycolysis, going first to glucose-6 phosphate, then to fructose-6-phosphate, and then on through the rest of the glycolytic pathway. However, some of that fructose-6-phosphate gets diverted into a signaling pathway in which an enzyme called GFAT (glutamine: fructose-6- phosphate amidotransferase) converts the fructose-6-phosphate to glucosamine-6-phosphate and finally to UDP (uridine diphosphate) N -acetyl glucosamine. What happens after that the N -acetyl glucosamine gets put onto serine and threonine residues of transcription factors, just like the more familiar process of phosphorylation, and overmodification by this glucosamine often results in pathologic changes in gene expression.

Oxidative Stress

In diabetic subjects, the hyperglycemia causes oxidative stress due

to an increase of reactive oxygen species (ROS) production,

particularly superoxide anion and hydrogen peroxide. These are

above all subproducts of glucose auto-oxidation, but they are

Introduction

13 present also in non-enzymatic glycosylation process performed by glucose. Structural proteins undergo change when exposed to this process that consists of two steps. In the first one, glucose binds structural protein by the condensation with the amino group of a basic amino acid (usually a Lys); in the second one, the so-formed Schiff base is converted into a very reactive carbonylic species by a rearrangement reaction. This intermediate binds another amino group generating cross-linked bonds with the consequent alteration of the structure and the function of the proteins. For instance, Cu,Zn- superoxide dismutase is inactivated by the glycosylation of two Lys residues present on the enzyme. As this enzyme provides for the removal of superoxide anions transforming it in H 2 O 2 and O 2 , its denaturation contributes to oxidative stress increase and to cellular deterioration.

As fructose is more potent than glucose as glycosylating agent, its intracellular concentration increase due to ALR2 catalytic activity promotes glycosylation processes.

In the meantime, polyol pathway activation alters oxido-reductive potentials, modifying the pyridine cofactor availability for different enzymes. Particularly, Glutathione Reductase exploits NADPH to retrieve GSH from its oxidative form GSSG. Therefore, the increased activity of ALR2 involves an increase of GSSG, thus leading to a lower capacity of cells to face oxidative stress. The reduced form of GSH is very important for metabolism cell as it acts as scavenger against many toxic substances. ⁷

On the whole, the mechanisms leading to long-term diabetes

complications are tightly correlated and directly dependent by the

polyol pathway.

Introduction

14

Therefore, the inhibition of ALR2 represents a good therapeutic

strategy to prevent the beginning or to delay the progression and

the gravity of diabetes complications.

Introduction

15 Aldose Reductase (ALR2)

ALR2 is a small, cytosolic, monomeric protein of 315 amino acid residues, 36 kDa, identified first in the seminal vesicles by Hers but present in most of mammalian cells. It belongs to the aldo-keto reductase (AKR) superfamily and catalyzes the NADPH-dependent reduction of a wide variety of carbonyl compounds to their corresponding alcohols, exhibiting broad substrate specificity.

ALR2 is the key enzyme of the polyol pathway and catalyzes the

reduction of glucose to sorbitol, which is then oxidized to fructose by

sorbitol dehydrogenase (L-iditol:NAD ⁺ , 5-oxidoreductase, EC

1.1.1.14, SD). As ALR2 has a low substrate affinity for glucose, the

conversion of glucose to sorbitol through the polyol pathway is

generally non-significant in normoglycemic conditions, accounting

for no more than 3% of total glucose utilization. In fact, ALR2 must

compete directly with the hexokinase of the glycolytic pathway,

and as the substrate affinity of hexokinase is greater than that of

ALR2, glucose is preferentially phosphorylated into glucose-6-

phosphate. Conversely, under hyperglycemic conditions,

hexokinase is rapidly saturated and the polyol pathway becomes

activated. Sorbitol is formed more rapidly than it is converted to

fructose, and its polarity hinders an easy penetration through

membranes and subsequent removal from tissues by diffusion. The

resulting elevated intracellular concentration of sorbitol increases

cellular osmolarity, which in turn initiates a cascade of events that

lead to the development of disabling complications, peculiar of

diabetic disease and affecting the nervous, cardiovascular, renal

and visual systems. In addition to the osmotic imbalance, an

increase in the activity of ALR2 during hyperglycemia causes a

substantial imbalance in the free cytosolic coenzyme ratios

NADPH/NADP ⁺ and NAD ⁺ /NADH. This alteration in the redox state of

Introduction

16 pyridine nucleotides induces a state of pseudohypoxia, which contributes to the onset of hyperglycemic oxidative stress through the accumulation of reactive oxygen species (ROS). ROS, in turn, trigger activation of downstream mechanisms, namely, protein kinase C (PKC) isoforms, mitogen-activated protein kinases (MAPKs) and poly(ADP-ribose)polymerase (PARP), as well as the inflammatory cascade, which sustain the pathogenesis of diabetic complications.

The X-ray crystallographic analysis of the holo-enzyme reveals that ALR2 adopts a triose phosphate isomerase TIM-barrel conformation, folding up in a typical eight-stranded α/β-barrel to which a small β- sheet capping the N-terminal end, residues 2–14, and a C-terminal extension, residues 275–315, are added. ⁸ The active site is located at the C-terminal side of the barrel, deeply buried into it. The cofactor binds to ALR2 in an extended conformation, straddling the barrel and projecting its nicotinamide moiety to the centre of the protein where it becomes part of the catalytic site contributing to the reduction mechanism. ⁹ Actually, NADPH transfers the 4-pro-R hydride from the C4 atom of the pyridine ring to the re face of the carbonyl group of the substrate, allowing its reduction. The reaction has been exhaustively described by different authors as a sequential and ordered ensemble of steps, in which NADPH binds first, followed by the binding of the aldehyde substrate. After conversion of the aldehyde into alcohol, the reaction product is released and the oxidized cofactor disengages from the enzyme.

Kinetic and spectroscopic experiments showed that both bond and

release of NADPH are accompanied by conformational changes of

the enzyme–cofactor complex, the second modification being the

overall rate-limiting step of the whole enzymatic cycle.

Introduction

17 ALR2 Active Site

The catalytic active site of ALR2 is highly hydrophobic. With the only

exception of three polar residues, namely Gln49, Cys298 and His110,

the cavity is bordered by aromatic residues, such as Trp20, Tyr48,

Trp79, Trp111, Phe121, Phe122 and Trp219, and apolar residues, such

as Val47, Pro218, Leu300, Leu301. Therefore, ALR2 shows a marked

orientation toward lipophilic substrates. Considering its compelling

role in aldo-sugar metabolism, such a preference would seem

rather unusual. Actually, D-glucose is a well recognized

physiological substrate for ALR2 but it is not the preferred one. The

catalytic efficiency showed by the enzyme for its reduction is low, as

an apparent K m value ranging from 50 to 100 mM testifies, thus

suggesting that ALR2 metabolizes this sugar only when its

concentration increases to pathological levels, such as under

diabetic conditions. Extensive studies by different research groups

clearly demonstrated that ALR2 is highly efficient in reducing short-

to long-chain aldehydes, both saturated and unsaturated, aliphatic

or aromatic, either of exogenous origins or arising in large quantities

from lipoproteins and membrane phospholipids as a consequence

of pathological conditions connected with oxidative stress. ALR2

also catalyzes the reduction of glutathione conjugates of

unsaturated aldehydes, showing in most cases efficiency higher

than that of the parent compounds, free aldehydes. Moreover,

steroid metabolites such as 3,4-dihydroxyphenylglycoaldehyde,

isocorticosteroids, isocaproaldehyde, progesterone and 17α-

hydroxyprogesterone, as well as noradrenalin catabolites, have

been identified as further endogenous ALR2 substrates. These

findings imply that, besides its role in glucose metabolism, ALR2 plays

a significant aldehydes removal function, ensuring a complete and

efficient elimination of the major electrophilic end products of

Introduction

18 metabolism. As aldehydes exert cytotoxic, mutagenic and carcinogenic activities through reaction with biomolecules such as DNA base adducts and proteins, ALR2 performs a physiological detoxification role. Therefore, under normoglycemic conditions, this enzyme represent a key component of the complex antioxidant cell defense system including aldehyde dehydrogenases (ALDHs), which catalyzes the oxidation of aldehydes to their corresponding acids, glutathione-S-transferases (GSTs), which catalyze the conjugation of aldehydes with glutathione, and others aldo-keto reductases (AKRs), above all aldehyde reductase, ALR1, (EC 1.1.1.2, ALR1), which shows a high degree of structural homology with ALR2, possessing a 65% identity in the amino acid sequences.

The least conserved residues are located at the C-terminal end of the proteins, in a region lining the hydrophobic specificity pocket.

This portion of the active site is responsible for the substrate and inhibitor specificity of the two enzymes, and can therefore be usefully exploited for the design of selective compounds, which must be able to inhibit ALR2 without affecting the detoxification activity of ALR1.

The broad substrate specificity shown by ALR2 is achieved through a high degree of plasticity of its catalytic binding site.

A lot of crystal structures of ALR2, obtained in the presence of different compounds, mainly inhibitors, concurred to prove the ability of ALR2 to modify the conformation of this site as a result of an induced-fit adaptability to the ligand. A careful comparison of these conformations led to consider the ALR2 binding site as composed by two distinct portions, commonly named ‘anion binding pocket’ and ‘specificity pocket’, endowed with different flexibility.

The ‘anion binding pocket’, which is bordered by the pyridine ring

Introduction

19 of the cofactor and the surrounding amino acids (Figure 5), takes part actively to the catalytic machinery appearing rather stiff.

Actually, it represents the fixed portion of the site which anchors the

ligand, either substrate or inhibitor, through its functional group. With

the exception of Trp111, which exposes its π-face to the remaining

part of the site and shows slight changes in the position of its side

chain, residues lining the ‘anion binding pocket’, represented by

Trp20, Tyr48, Val47, His110, and Trp79, preserve their positions quite

unaltered across all the known crystal structures of ALR2. On the

contrary, the so-called ‘specificity pocket’ is highly elastic and

shows frequent changes in its conformation. Residues going from

Val297 to Leu300, the flanking Trp219, Cys303 and Tyr309, and, to a

minor extent, the far Thr113 and Phe122 (Figure 5), host the lipophilic

portion of the ligand exhibiting a marked adaptation to it. In

particular, Leu300 plays a determining role of gatekeeper. Indeed,

depending on the rearrangement of its side chain, as well as on the

resulting position of the neighboring Cys303 and Tyr309, Leu300

makes accessible the ‘specificity pocket’ expanding its width.

Introduction

20 Figure 5. Simplified Representation of ALR2 Catalytic Binding Site.

To date, a number of different conformations of the active binding site have been identified, both different and recurrent. The most frequent one is the so called ‘holo-conformation’, shown not only by the holo-enzyme, but also by the enzyme complexed with different ligands such as glucose-6-phosphate, cacodylate, citrate, Sorbinil, Alrestatin and IDD384.

In this conformation the binding site is quite limited as Leu300,

turning its side chain toward Trp111 (Figure 5), shorten the extent of

the ‘specificity pocket’. The ligand, anchored to the ‘anion binding

pocket’, mainly occupies this part of the site and, if its size exceeds

the pocket, possibly protrudes above the protein as in the case of

Alrestatin and IDD384.