END2.4 - Diabetes mellitus 4Biochemistry of diabetic complications ©Dr S Nussey
Hyperglycaemic hypothesis • DM is associated with 2 types of complication - • Macrovascular - i.e. accelerated atherosclerosis • Microvascular - affecting predominantly the eye, nerves and kidneys • Microvascular complications are specific to diabetes • Although subject to genetic influences, microvascular complications are related to the duration and quality of glucose control
Protein structure • Protein structure determines function • Changes in structure alter function • In these examples, lens crystal is rendered opaque and collagen inflexible Cataract Cheiro- arthropathy
Cell metabolism • Alterations in cellular metabolism affect cell function • In this example, disturbances in axoplasmic transport affect nerve function Neuropathic ulcer on heel
Extracellular matrix structural changes • Deposition of interstitial materials may affect function • In this case, glomerular function is severely affected leading to renal failure Normal Diabetic
Cellular proliferation • Changes in cell signalling may lead to cell division • In this example, proliferation of new blood vessels in the retina lead to blindness Proliferative retinopathy
Vascular function • Changes may lead to an increase in vascular permeability • In this example, production of exudates or haemorrhages in the retina lead to blindness
Four major hypotheses* • Increased activity of aldose reductase (sorbitol pathway) • Formation of reactive oxygen species (‘free-radicals’) • Increased production of advanced glycation end-products (AGE) • Activation of protein kinase C (PKC) *Note - these are potentially linked in complex ways
Principle of hyperglycaemic ‘memory’ • Experimentally, the damage done to tissue during periods of hyperglycaemia is ‘remembered’. • Thus, after a period of poor diabetic control complications occur at an accelerated rate even though subsequent glycaemic control is excellent.
Q - Why is an understanding of the biochemistry of diabetic complications important? A - Because it leads to therapeutic opportunities.
Sorbitol pathway • Increased fructose leads to: osmotic changes; non-enzymic fructosylation and AGE (via 3-deoxyglucasone) • Decreased NADPH/NADP+ leads to: alteration in redox state(decreased ability to deal with oxidative stress); increased activity of pentose phosphate shunt (PPP) • Increased NADH/NAD+ leads to: increased activity of PPP • Increased triose phosphate intermediates leads to increased second messenger diacylglycerol (DAG) and, thus, Protein Kinase C activity. • Increase PKC activity leads to a wide variety of changes (see later) • Arachidonate in DAG may be a substrate for the synthesis of eicosanoids including prostaglandins, prostacyclins, thromboxanes and leukotrienes all of which have potent vascular (and other) actions
Pentose phosphate pathway • Important in the generation of: • NADPH as source of reducing power in biosynthesis • ribose phosphate to synthesize RNA, DNA, FAD, CoA • The red loop indicates the reversibility via transketolase and transaldolase and the intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate thus linking the PPP with glycolysis
Aldose reductase inhibitors • Good results in animal models • Less good in human trials • Why? • AR not expressed in all tissues affected by DM e.g. endothelial cell • most trials short-term but complications accrue over many years • other pathways more important?
Free radicals in DM - ‘Oxidative stress’ • Increased production e.g. by auto-oxidation of glucose, superoxide production from mitochondrial oxidation of NADH to NAD+ • Decreased clearance via action of catalase or glutathione peroxidase. Regeneration of reduced glutathione requires NADPH, levels of which are decreased (in tissues containing aldose reductase)
‘Carbonyl stress’ • The idea of carbonyl stress arose from the recognition that not all damaging processes required oxidation. • Carbonyl products (e.g. methylglyoxal and 3-deoxyglucasone) are obtained non-oxidatively via the PPP and inhibit glutathione reductase. • These may form AGE or, in the presence of membrane lipids, form lipid dialdehydes that form glycoxidated adducts. • Note that AR has apparently deleterious effects at 1 but potentially beneficially ones at 2 and 3
AGE • Maillard reaction ‘browning’ of food described in 1912 • Non-enzymatic glycation of amines
AGE CML Pentosidine MOLD
AGE • Receptors for AGE include: macrophage scavenger receptor for AGE (types I and II); oligosaccharyl transferase-48; 80-K H phosphoprotein and galectin • The scavenger receptors belong to the immunoglobulin superfamily • Expressed on wide variety of cells and expression is increased in animal models of DM • AGE-RAGE interactions are important in a number of inflammatory conditions • They also occur in atherosclerosis and this draws both micro- and macro- vascular complications together pathophysiologically
AGE directed therapies • Aminoguanidine an inhibitor of AGE formation has good results in animal nephropathy models • Undergoing phase III clinical trials • Others include phenacylthiazolium
PKC X = processes inhibited by LY333531
PKC directed therapy • LY333531- an orally active inhibitor of PKC-bII isoform- good results in animal models of both retinal and renal disease