NON ENZYMATIC GLYCATION OF MACROMOLECULES

IN DIABETES PART-1

DEPARTMENT OF BIOCHEMISTRY

BY; Dr JYOTI SHARMA

WORLD-WIDE ESTIMATED NUMBER OF ADULTS WITH DIABETES BY AGE

GROUP AND YEAR

DIABETES MELLITUS is a group of metabolic diseases characterized by

hyperglycemia

resulting from• defects in insulin secretion, • defects in insulin action

(“insulin resistance”), • or both.

long-term damage, dysfunction, and failure of various organs, especially the eyes

kidney

nerves

heart

blood vessels.

“Complications”

Pancreas beta cells

Insulin actions

Glucose entry and utilization (oxidation,

storage)

Glucose entry and oxidation

TG synthesis

INSULIN ACTIONS: A REVIEW

TYPES AND CAUSES OF DIABETES

Type 1 ( was called “insulin- dependent DM”/ juvenile type of DM) ( 5-10%)

Type 2 (was called “non insulin- dependent DM” /adult onset DM ) (90-95%)

The “pre-diabetic stage” (impaired glucose tolerance)

Gestational DM -any degree of glucose intolerance with onset or first recognition during pregnancy [in 2-5% of all pregnancies]

Other specific types (1% - 2%)– genetic syndromes (affecting insulin secretion or action)– endocrinopathies (Acromegaly, Cushing’s syndrome, glucagonoma,

pheochromocytoma, thyrotoxicosis)– diseases of pancreas (chronic pancreatitis, cancer)– drug- or chemical-induced (corticosteroids, beta-blockers, thiazide

diuretics)– infections (viral)

The main focus of this plenary

DIABETES MELLITUS PATHOGENESIS

N O N -IN S U L IN -D E P E N D E N TC E L L S

E X C E S SG L U C O S E D E P O S IT S

IN S U L IN -D E P E N D E N TC E L L

D E F IC IE N T IN G L U C O S E

G L U C O S E L O S TIN U R IN E

H Y P E R G L Y C E M IA

A B S O L U T E /R E L A T IV EL A C K O F IN S U L IN

• Metabolic complications of low blood glucose levels

(hypoglycaemia) and of high blood glucose levels (hyperglycaemia). – e.g. diabetic symptoms(3P’s ), infections, Diabetic coma

• Chronic complications:Microvascular

retinopathynephropathyneuropathy

Macrovascularcerbrovascular, cardiovascular, peripheral vascular disease

COMPLICATIONS OF DIABETES MELLITUS

CLINICAL FEATURES OF DM DUE TO LACK OF INSULIN : MC

Polyphagia(decr. leptin?)

Starvation in the midst of plenty

Hyperosmolar hyperglycemic syndrome (HHS)

Lactic acidosis

Lactic acidosis

Muscle protein breakdown

Acetoacetate,0H-butyrate, acetone

CHRONIC COMPLICATIONS OF DIABETES MELLITUS

Microvascular And consequently, macrovascular complications are specific to diabetes and do not occur without longstanding hyperglycaemia.

Other metabolic, environmental and genetic factors are undoubtedly involved in their pathogenesis. Both T1DM and T2DM are susceptible to microvascular complications, although patients with T2DM are older at presentation and may die of macrovascular disease before microvascular disease is advanced.

Prolonged exposure to elevated glucose concentrations damages tissues by causing either acute, reversible metabolic changes (mostly related to increased polyol polyol pathway activity and glycosylation of proteins) or cumulative irreversible changes in longlived molecules (formation of advanced glycosylation end products /AGE/ on matrix proteins such as collagen and on nucleic acids and nucleoproteins).

In this presentation we will study that how non enzymatic glycation of macromolecules causes damage to the body.

Starting with non enzymatic glycation of protien today we proceed further;

Nonenzymatic glycation is a process by which glucose is chemically bound to amino groups of proteins but without the help of enzymes.

It is a classical covalent reaction in which, by means of N-glycoside bonding, the sugar-protein complex is formed through a series of chemical reactions described by a chemist Maillard.

Maillard reactions are complex and multilayer, and can be analyzed in three steps.

1) The sugar-protein complex is formed first (Amadori rearrangement). It is an early product of nonenzymatic glycation, an intermediary which is a precursor of all later compounds.

2) The formation of numerous intermediary products, some of which are very reactive and continue with glycation reaction.

3) Polymerization reaction of the complex products formed in the second step, whereby heterogeneous structures named advanced glycation endproducts (AGE) are formed.

Other processes that contribute to the formation of AGES are summarised in Figure .

Schematic presentation of potential pathway leading to AGE formation

1.AGE arise from decomposition of Amadori products2.fragmentation products of polyol pathway3.as glycooxidative products,which all react with amino groups of protein

GLO=glyoxal; MGO=methylglyoxal; 3-DG=3deoxyglucosone; CML=carboxymethyl-lysine

Glycation of proteins take place at ε-amino groups of lysine or hydroxylysine residues as well as at α-amino groups of amino terminal residues .

Specific lysine residues in hemoglobin, human serum albumin and α-crystallins have been identified as preferential sites of glycation.

Other lysine-rich proteins, IgG and IgM, were found to be glycated in diabetes patients.

Glycation also takes place on arginine residues and that of histidine, tryptophan and cysteine residues

Endogenous glycations occur mainly in the bloodstream to a small proportion of the absorbed simple sugars:Glucose , fructose, and galactose .

It appears that fructose and galactose have approximately ten times the glycation activity of glucose, the primary body fuel.

Cross-linking potency is variable among the sugars, with a rank order of glucose < fructose < ribose, and phosphorylated sugars being more potent than their unphosphorylated counterparts.

In spite of the fact that sugars are the main precursors of AGE compounds, numerous intermediary metabolites, i.e. a -oxoaldehydes, , glyoxal, methylglyoxal and 3-deoxyglucosone also creatively participate in nonenzymatic glycation reactions. Such intermediary products are generated during glycolysis (methylglyoxal) or along the polyolic pathway, and can also be formed by autooxidation of carbohydrates (glyoxal).

Alpha-oxoaldehydes modify AGEs surprisingly fast, in contrast to classical Maillard reactions, which are very slow.

GLYCATION HAS BOTH PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL SIGNIFICANCE.

In physiological conditions, glycation can be detected in the process of aging, and the reactions are significantly faster and more intensive, with frequently increased glucose concentrations , such as in diabetes.

In diabetology, the importance of these processes manifests in two essential issues:

1) effect of protein glycation on the change of their structure and function, and

2) use of glycated protein level as a parameter of integrated glycemia.

A classical example of nonenzymatic glycation is the

a) Formation of glycated hemoglobin, HbA1c. As the life of Hgb is 120 days, HbA1c,reflects the status of

glucose in diabetes over a long time. b) Fructosamine a glycated product,depicts short term

status as short half life

The excessive cleavage of glucose, especially with important protein amino groups, can affect cell function and structure and create an imbalance which leads to cell destabilization.  This condition seems to target organs and tissues that are not dependent on insulin for their absorption of glucose.

Kidneys, blood vessels, peripheral nerves and lenses of the eye are more susceptible to damage from periods of hyperglycemia than other organs due to their lack of insulin dependence.

EFFECT OF GLYCATION ON PROTEIN

Due to glycation there is change in the structure and function of a protein that can be studied by;

1) U.V.2) Florescence3) PAGE4) CD5) MALDI6) HPLC7) ELISA

ADVANCED GLYCATION ENDPRODUCTS (AGES)

During the process of glycation, early glycation products are formed first, which subsequently rearrange into final AGE structures through a series of very complex chemical reactions.

Protein modification with AGE is irreversible, as there are no enzymes in the body that would be able to hydrolyze AGE compounds. These structures then accumulate during the lifespan of the protein on which they have been formed. Examples include all types of collagen , albumin, basic myelin protein, eye lens proteins,lipoproteins, and nucleic acid.

So, AGE is the result of years of accumulated glycated damage to molecules that are not replaced regularly, but have a low turnover rate.

The major biological effects of excessive glycation include:

1) Inhibition of regulatory molecule binding2) Crosslinking of glycated proteins3) Trapping of soluble proteins by glycated extracellular

matrix4) Decreased susceptibility to proteolysis5) Inactivation of enzymes6) Abnormalities of nucleic acid function7) Increased immunogenicity in relation to immune complex formation.

It has been well documented that AGEs progressively accumulate on tissues and organs developing chronic complications of diabetes mellitus, i.e. retinopathy, nephropathy, neuropathy, and progressive atherosclerosis.

Age-corrected levels of glycoxidation products in collagen correlate with the severity of diabetic complications

Also, cross-linking of collagen proteins, for example, contributes both to the rigidity and the loss of elasticity of tissues, and to the thickening of capillary walls observed in diabetes and during the aging process.

This protein modification is also responsible for crystalline lenses becoming opaque in cataracts, a degenerative disease that is also frequent in diabetic or aged persons.

Also glycation substances may be involved in the pathogenesis of Alzheimer's disease, since an accumulation of these substances is observed at the sites of neuronal degeneration during the course of this disease

In addition to the cross-linking of long-lived molecules, AGE are able to stick the rapidly renewable plasma Molecules together, whether albumin, antibodies, or LDL cholesterol.

AGE RECEPTORS

The body does have a defense against cross-linked proteins. The immune system has macrophages with special receptors for AGEs. The macrophages engulf AGEs and Eventually the products are excreted in the urine

The level of AGE proteins reflects kinetic balance of two opposite processes: the rate of AGE compound formation, and the rate of their degradation by means of receptors.

AGE receptors participate in the elimination and change of aged, reticular and denatured molecules of extracellular matrix as well as of other AGE molecules.

.

However, in diabetes mellitus AGE protein accumulation may exceed the ability of their elimination due to chronic hyperglycemia and excessive glycation process.

AGE protein binding to macrophage receptors causes a cascade of events in the homeostasis of blood vessel walls and their milieu by mediation of cytokines and tissue growth factors.

These are sites on cell membranes that bind AGE ligands. The abbreviation used to denote them in the literature is RAGE, they belong to immunoglobulin receptor family, and predominate in tissues. Can variations in AGE level explain differences in the susceptibility to development of complications?

It is not known, however, theoretical reflections indicate that gene diversity in AGE receptors could offer an explanation.

Thus ,,,,,

Glycotoxins (AGE peptides)

Tissue macrophages with AGE receptors represent the major pathway of tissue AGE alteration and cell degradation. In this process, AGE peptides are released as degradation products, which partly occurs through proteolysis of the matrix Component, commonly named glycotoxins.

Glycotoxins (AGE peptides) are very reactive on entering blood circulation. In case they have not been eliminated through the kidneys, recirculating AGE peptides can generate new AGE products that react with other plasma or tissue components.

At this stage, glycation becomes an autonomic process which significantly accelerates the progress of the complication

CLINICAL SIGNIFICANCE OF ADVANCED GLYCATION

A variety of human tissues interact with the products of advanced glycation during normal homeostasis.

Clinical implications of the phenomenon of advanced glycation are discussed below.;

Role of AGEs and AGE receptors in the pathogenesis of diabetic complications

AGEs in diabetic vasculopathy and atherosclerosis

Atherosclerotic cardiovascular disease is the major cause of morbidity and mortality in diabetes.

The mechanisms by which diabetes so dramatically increases atherosclerosis are yet poorly understood.

AGEs also play a significant role in atherosclerosis.

For instance, reticulated and irreversible LDL from the circulation binds to AGE-modified collagen of the blood vessel walls. (Vascular tissue AGE accumulation cause protein crosslinking & oxidative damage)

Increased endothelial cell permeability and procoagulant activity thrombosis

Mononuclear cell chemotaxis/activation cytokine and growth factor release

In the majority of blood vessels, reticular binding delays normal outflow of LDL particles that have penetrated the vessel wall, thus enhancing cholesterol deposition in the intima.(Increased vascular matrix thickening and narrowing of lumen) Such AGE reticulation increases lipoprotein deposition regardless of the plasma LDL level.( Increased macrophage uptake of AGE-LDL atheroma)

This is followed by an accelerated development of atherosclerosis.

This lead to High Blood Pressure

The presence of many AGE compounds in the atheroma has been demonstrated by immunohistochemistry techniques.

It has been well documented that lipids and lipoproteins are deeply involved in the atherogenic process.

AGEs and renal failure

Persistent hyperglycemia has a central role in the development of diabetic nephropathy that is clinically manifested by proteinuria progressing to renal insufficiency, and histopathologically by mesangial expansion and glomerular basement membrane thickening.

A possible link between elevated glucose level and diabetic nephropathy resides in the glycation process producing AGEs.

This modification may impair the original function of either protein and may affect normal processes of turnover and clearance.

AGEs can induce an excess crosslinking of collagen molecules in the glomerular plasma membrane affecting the .

assembly and architecture of the glomerular basement membrane and mesangial matrix, and can potentially act on mesangial cells via growth factors, causing cells to synthesize more extracellular matrix. All these processes may lead to enhanced deposition of extracellular matrix proteins in the mesangium, interfere with the mesangial clearance of macromolecules, and alter macrophage function, thus contributing to mesangial expansion and glomerular occlusion.

Circulating serum AGE level is markedly increased in patients with diabetes and renal insufficiency.

Serum AGEs include both serum proteins that have been modified by advanced glycation and low molecular weight AGE peptides.

Using specific immunoassay, serum AGE peptide levels have been found to correlate with renal function.

In fact, close correlation has been demonstrated between serum AGE levels and creatinine clearance.

In normal controls, AGE peptide clearance has been estimated to 0.72 ml/min.

Diabetic persons with normal glomerular filtration rate can clear AGE peptides at the same rate.

However, progressive loss of renal function is associated with increasing circulating AGE peptide levels.

Current renal replacement therapies, hemodialysis or peritoneal dialysis, are relatively inefficient in removing AGEs from the serum of diabetic patients.

In these patients, AGE peptides persist at up to 8-fold normal level.

Diabetic patients with renal failure are known to be particularly susceptible to cardiovascular complications due to accelerated atherosclerosis.

AGES AND DIABETIC RETINA

Histologic features of early diabetic retinopathy are characterized by acellular capillaries and resultant areas of capillary nonperfusion.

These microvascular alterations are associated with the accumulation of AGEs of long-lived extracellular matrix components.

Such structural abnormalities are detectable by specific autofluorescence and by anti-AGE antibodies.

In an experimental animal model, treatment with aminoguanidine, an ihibitor of AGE formation, was shown to reduce the formation of acellular capillaries and to prevent proliferation of endothelial cells, suggesting that AGE may play a major role in the pathogenesis of diabetic retinopathy.

It was also demonstrated that AGE-modified albumin co-localizes with the component of AGE receptors in the retinal vasculature of both diabetic rats and AGE-infused rats, suggesting that progressive accumulation of AGE may well be the underlying mechanism for the loss of pericytes and endothelial cells in early diabetic retinopathy.

This is supported by the finding that AGE receptor is localized in human and rat retinal microvessels.

.

AGES IN DIABETIC NEUROPATHY

The major causative link between clinical diabetic neuropathy and peripheral nerve changes is hyperglycemia.

One of the important biochemical pathways involved, with a potential role in diabetic neuropathy, is glycation leading to AGE modification of nerve proteins.

AGEs have been stained in the endoneurium, particularly on the axons, endoneurial capillaries, and perineurium of diabetic patients with neuropathy.

Axonal cytoskeletal proteins have essential roles in axonal structure and function.

Nonenzymatic glycation of axonal proteins causes alteration in structure and transport, leading to axonal atrophy and degeneration..

Additionally, studies have shown that glycation of myelin occurs in both peripheral nerve and brain.

To more precisely define the role of nonenzymatic glycation in diabetic neuropathy, it is important to identify exactly which proteins are being glycated.

Their localization is also of immense value in determining the relative contribution of these glycated proteins to neuropathy

The main aim of the studies is to contribute to open new lines for basic research on the compelling issue of establishing a common molecular basis for the mechanisms of diabetic complications.

This, in turn, should pave the way for clinical investigation & looking for appropriate targets for a therapeutic counteraction aiming to retard the formation chronic complication.

THANK YOU