Diabetes - Plaque, Periodontal Disease 3.03

Diabetes

1/50 to 1/100 patients you will see may be diabetic

May present with

1. Increased caries

2. Candidal infection

3. Xerostomia (salivary hypofunction)

4. Increased frequency of severe, rapidly progressing forms of periodontitis

“diabetes is an acquired risk factor” for chronic periodontal disease

• Activity of microbes in the plaque

• Conditions prevailing in the plaque environment

Microflora in Health and Disease

• Change in habitat

  • Alter stability of bacterial population

e.g. “unusual” nutrients – sugar, GCF components, defences decreased (e.g. AIDs)

• “Opportunistic pathogens”

  • Potential to cause disease

• This is an example of the “Ecological Plaque Hypothesis”

Potential Contributory Factors in Plaque Accumulation in Diabetes

• Elevated [glucose] in saliva, blood, GCF

  • Resting levels 4-5mmol, in diabetics 9+mmol

• Impaired neutrophil function

• Dry mouth – reduced salivary flow

• Low tissue oxygen levels

• Increased tissue carbon dioxide levels

• AGE – Advanced Glycation End-products e.g. haemoglobin, albumin, etc.

• AGE-Receptors of AGE (AGE-RAGE) interactions

Reduced Saliva Flow

Host defences in saliva

• Saliva flow (can’t wash things away)

• Mucins/agglutinins (can’t bind bacterial for effective removal)

• Lysozyme (can’t bust cell walls)

• Lactoferrin (can’t bind iron)

• Histatins – antimicrobial cationic peptides

• Secretory IgA

• Complement

Glucose Levels in Saliva

• Only slightly elevated in diabetes vs normal

• Mirrors plasma [glucose]

  • Normal 3-6mM

  • Diabetic 10mM

• Predisposes supra-gingival plaque build-up

• Increases buccal caries

• GCF [glucose]

Environmental Change and Sub-gingival Ecology

• Diabetes leads to vascular thickening which leads to reduced blood flow

  • Lead to poor oxygen perfusion and increased [carbon dioxide]

• Increased carbon dioxide is directly proportional to the number of “capnophiles” (carbon dioxide-loving)

  • Capnocytophaga spp

  • Known to be proteolytic

  • Peptidases – render peptides down to amino acids

  • Can damage the tissues

  • Helps growth of other pathogens

Phagocyte Function in Diabetes

Hyperglycaemia impacts:

• Impaired chemotaxis

• Impaired phagocytosis

• Impaired bactericidal activity

• Are proportionally decreased and correlate with [AGEs]

  • [AGEs] goes up if hyperglycaemic

Chemotaxis: movement toward or away from a chemical stimulus

Chemo-attractants:

• Cytokines (stimulate this)

• Bacterially-derived

• Peptides (kick it off)

• Complement factors

These functions are depressed when you are chronically hyperglycaemic

Accepted Paradigm

• Plaque causes periodontal disease

Problem?

• Plaque load does not appear to correlate with disease severity

AGEs

AGEs are major pathophysiological features of diabetes

• Proteins (& lipids) react with reducing sugars

e.g. albumin, haemoglobin, etc

• Non-enzymic glycation

With 1. Glucose

2. Fructose (more so than glucose)

• Occurs when there is long-standing hyperglycaemia

• Long-lived proteins

• Extra-cellular proteins

• Glycation can alter molecular shape/function

• [AGE-proteins] – a measure of diabetic glucose control

  • E.g. HbA1c

Formulation of Advanced Glycation End-products

Examples of AGEs: chips, crisps, bread, roast chicken, roasted chestnuts, sausages, etc

• Essentially the same reactions involved in browning of chips, crisps, cooked meat, baked bread

• Maillard Reaction

General Properties/Effects of AGEs

• AGEs form via a chemical/non-enzymic reaction

Sugar + protein = protein-sugar

• Glucose and fructose bind to certain amino acids

e.g. valine and lysine (more likely due to long side-chain with an extra amino group) in haemoglobin (and other proteins)

• Molecular rearrangements

• Factors dictating the formation of AGEs

(Law of Mass Action)

Sugar + protein = AGE-protein

1. Concentrations of reactants

2. Temperature

3. Time

4. Type of protein/sugar

Properties of AGEs and their Effects

• Form slowly

• Cross-link with other proteins

• Turned over slowly i.e. because they are broken down slowly

• Lead to thickening/occlusion in vessels

• Can result in chemical radical generation and fragmentation damage to proteins/DNA

Collagen (Tropocollagen)

• 3 polypeptide chains in helix

• Each chain is approx. 1000 amino acids

• General sequence repeated

  • 1/3 glycine

• Abundant in lysine – approx. 106 per tropocollagen

  • Has an ε amino side chain

  • Important target for glycation

Collagen Glycation

Glucose attaches to lysine and then over a number of hours and days, you get modifications to an end result of an AGE.

• Some of these modifications of glucose can cross-react with others in other peptides. So adjacent tropocollagens can get linked by that.

• Structural/functional alteration

• Increases thermal unfolding of the triple helix

• Glycation decreases turnover and repair

Why is collagen important?

• Is within dentine

Some Medical Consequences of Protein and Lipid Glycation

• Most common cause of blindness

• Increased proliferation of blood vessels

• Vascular occlusion, angiogenesis

• Micro-aneurysms, haemorrhages and retinal infarction

• AGEs contribute towards vascular occlusion

Diabetic Nephropathy

• [AGE] in kidney tissues

  • Correlates with severity of nephropathy

• AGE promote

  • Increase release of transforming growth factor-b

  • Occurs in the glomerular basement membrane collagen (type 4 collagen)

    • This can be thickened and affect perfusion which can have a knock-on effect on kidney function

  • Stimulates collagen synthesis

    • Thickening of glomerular basement membrane

    • Traps plasma proteins

Consequences?

• Reduce filtration

• Loss of glomerular function

• Kidney failure – death!

Diabetic Neuropathy

• Pain or numbness of limbs

(impotence in men)

• Axonal degeneration of peripheral neurones

• Reduced nerve conduction and blood flow

• Increased glycation of myelin

Stimulates macrophages to secrete proteases and phagocytose myelin

• AGEs on myeline trap plasma proteins

e.g. IgG, IgM and complement C3 to elicit immunological reactions contributing to demyelination

AGEs and Periodontal Disease

• Thickening of vasculature in periodontal tissues

• Poor perfusion of tissues

• Poor protein turnover and repair of tissues

• Elevated glycated haemoglobin

Glycated Haemoglobin (HbA1c)

• Glucose can glycate haemoglobin as it has several lysin residues but not all of them are targets due to a number of physiochemical reasons

• If you modify haemoglobin, you have this drastic effect on the functioning of haemoglobin

• 10% Hb glycosylation in diabetic red cells

(3-6% in normal)

• Periodontal disease severity

Directly proportional to [HbA1c] in GCF

• [HbA1c] in the blood is a measure of the degree of hyperglycaemia

• Higher affinity for oxygen

• Erythrocytes do not unload oxygen

  • Reduced tissue oxygenation

• Low oxygen levels

  • Decrease oxygen-mediated neutrophil killing

  • Encourages anaerobic bacteria

[HbA1c] in GCF is directly proportional to increased P. gingivalis in plaque

A KNOWN PERIODONTOPATHOGEN

Neutrophil Reactive Oxygen Intermediates

NADPH + 2O₂ → H² + NADP + 2O₂^- Superoxide

2O₂^- + 2H⁺ → O₂ + H₂O₂ Hydrogen peroxide

H₂O₂ + Cl^- → OH^- + HOCl Hypochlorous acid

ClO^- + H₂O₂ → Cl^- + H₂O + ¹O₂ Singlet oxygen

O₂^- + H₂O₂ → OH^- + O₂ + OH^* Hydroxyl radical

Respiratory Burst in Phagocytes

• Likely to be depressed

• Is a set of processes that are meant to kill organisms

RAGE – Receptors for AGE

AGE proteins are recognised by various cells in the body

• Immunoglobulin-related proteins

• Cell surface

• Mononuclear phagocytes

• React with AGEs

AGE-RAGE Interactions

Hyperglycaemia

• Proteins get glycated to form AGEs

• These bind to the surface of monocytes via RAGE

• This activates monocytes to become a bit more aggressive

Activated macrophage:

• Reactive oxygen species

• Tissue destruction

• Release varies pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α

• These cytokines increase the inflammatory response

• This results in the chemotactic attraction of more neutrophils and white cells.

• Host inflammation causes tissue breakdown and remodelling

• This can lead to bone resorption and inflammation

• If you over-stimulate inflammation, without removing the source, then you get chronic inflammation.

• When this happens, you get increased levels of GCF

• Increased bacterial growth stimulation and therefore, increased protein-degrading organisms.

• More bacteria means more inflammation and the cycle continues

• Plaque can also lead to tissue destruction and indirectly through the actions of bacteria-making proteases, destroy tissue.

Bacterial Virulence Factors

• Lipopolysaccharide (LPS; endotoxin)

• Usually gram-negative, anaerobic and proteolytic

• Has long chains of carbohydrates on the surface which are pro-inflammatory

Activated Macrophages

• Have toll receptors on them

  • Innate defence

  • Transmembrane

  • Signalling molecules

• LPS can interact with toll receptors

• This brings a more amplified inflammatory cascade

• This then leads to tissue breakdown

Anti-AGE Proteins

There is glucose attached to proteins where rearrangements then occur. These keto groups can react with aminoguanides.

Normally, you get the glucose that modifies the protein and these proteins will crosslink and become difficult to break down.

You can use aminoguanide to stop the AGE-protein cross-linking effect which are in high quantities in garlic, blueberry and pomegranate.

Is there a reverse relationship – i.e. does periodontitis predispose towards diabetes?

1. Moderate (3.5-5.5mm) and severe (pockets >5.5mm) are significantly associated with an increased risk of diabetes incidence.

2. Patients with advanced periodontitis at baseline show approx. 5x greater increase in HbA1c 0.106±0.03% vs 0.023±0.02%

What are the Mechanisms?

Periodontitis:

• Dysregulated secretion of host inflammatory mediators and tissue breakdown

• IL-1β, IL-6, prostaglandin E2, TNF-α,

Receptor Activator of Nuclear Factor _KB Ligand (RANKL)

Matrix Metalloproteinases (MMPs)

T-cell regulatory cytokines (e.g. IL-12, IL-18)

(Periodontal lesion area = 8-20cm²)

Diabetes is characterised by:

• Elevated serum levels of IL-6, TNF-α → CRP

• Predictive of future occurrence of type 2 diabetes

Mechanisms: IL-6, TNF-α, and CRP impair intracellular insulin signalling and contribute to insulin resistance

Reasoning: periodontal inflammation state influences diabetes

Collective evidence: effective periodontitis treatment

→ Improvements in metabolic control

Summary

• Increased saliva/GCF [Glucose]

  • Augments growth supra/sub-gingival bacteria

• Hyperglycaemia → Impairs neutrophil function → plaque build-up

AGE proteins → vascular thickening → poor perfusion → CO₂

• AGE-RAGE interactions

Monocytes → macrophages → tissue damage

↘Cytokines → stimulate bacterial growth

↘ Inflammation → increased GCF → plaque → tissue destruction

• Low tissue O₂ levels – promotes anaerobes

• Glycated Hb → poor O₂ unloading → anaerobiosis

• Low saliva flow rate – reduced bacteria clearance/killing

• A two-way relationship thought to exist

  • Periodontitis predisposes to diabetes

  • Inflammation affects the response of cells to insulin