obesity, diabetes and cardiovascular disease

Obesity, diabetes andcardiovascular disease

http://www.bmb.leeds.ac.uk/illingworth/bioc3600/index.htm

Dr John IllingworthUniversity of Leeds

BIOC3600 / BMSC3145 / BIOL5215M / BIOL5235M (2010)

all slides

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• Most adults in the UK are already overweight. Modern living ensures every generation is heavier than the last – `Passive Obesity’.

• By 2050 60% of men and 40% of women could be clinically obese. Without action, obesity-related diseases will cost the United Kingdom an extra £50 billion per year (almost double the UK Trade Gap).

• The obesity epidemic cannot be prevented by individual action alone and demands a societal approach.

• Tackling obesity requires far greater change than anything tried so far, and at multiple levels: personal, family, community and national.

• Preventing obesity is a societal challenge, similar to climate change. It requires partnership between government, science, business and civil society.

Tackling Obesities: Future Choices

Government Office for Science http://www.foresight.gov.uk

October 2007 and subsequent updates

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definitions of obesity

Body mass index = weight (kg) / height (m)2

• 20-25 normal• 25-30 overweight• >30 obese

• BMI does not adequately distinguish fat from lean muscle mass.

• Separate norms should be used for men, women, children and for different races, but this is rarely done.

• Dual-wavelength X-ray absorptiometry is the best method to measure body composition, but this is rarely available.

• For many purposes a simple waist measurement is optimal, because this measures abdominal fat, which caries the highest risk.

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obesity increases the risk of disease

Willet et al. Guidelines for Healthy Weight (1999) NEJM 341, 427 - 433

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other conditions associated with obesity

• asthma • cancers (reproductive organs and gastro-intestinal tract) • Cushing's syndrome (excessive production of ACTH)• dyslipidaemias (abnormal blood lipids), hyperlipidaemia • dyspnoea (breathlessness) snoring and sleep apnoea • hiatus hernia and gastro-oesophageal reflux disease • kidney diseases • osteoarthritis / degenerative joint diseases • psychological problems • reproductive problems • varicose veins

Associations need not imply causality, and several of them are disputed, especially the asthma and cancer risks.

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the most serious links:

• obesity

• hypertension

• type 2 diabetes

• cardiovascular diseases

Heart attacks and strokes are major causes of death and disability in the general population.

Low grade inflammation is the common factor.

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Age Most common cause of death Second most common cause90+

Heart attack and chronic heart disease

Pneumonia85—89

Cerebrovascular disease (strokes)

80—8475—7970—7465—69

Lung cancer60—6455—59

50—5445—49

Breast cancer40—4435—3930—34

Motor vehicle accidentsDeaths due to drugs25—29

20—24

15—19

Other nervous disorders10—14Pedestrian hit by vehicle

5—91—4 Congenital heart defects

0 Perinatal conditions Sudden death, cause unknown

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age at death 1981 – 200414.8 million deaths analysed by age and sex.

Data from Shaw et al “The Grim Reaper’s Road Map” page 2 ISBN 978 1 86134 824 1 Health Sciences Library WA 900 SHA Very few Britons die <50. Transport accidents and suicides cause few deaths overall, but these are preventable deaths.

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loss of life expectancy

• Here is some information about the next slide, which shows the loss of life expectancy caused by different diseases.

• Diseases that kill early in life will obviously receive a greater weighting in this analysis.

• The following table underestimates female premature mortality because it uses the same “normal” life expectancy of 75 years for both women and men. [It would have been better to use 72 years for men and 78 for women.]

• Numerous individually rare diseases account for the missing 30% in the following table.

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million years of life lost through diseaseCause of death before age 75 Males Females Totals % lost

Heart attack and chronic heart disease 12.40 4.08 16.48 19.16

Lung cancer 3.83 1.89 5.72 6.66

Cerebrovascular disease 2.37 2.03 4.41 5.13

Breast cancer 0.01 3.25 3.27 3.80

Motor vehicle accidents 1.97 0.50 2.48 2.88

Chronic lower respiratory diseases 1.40 0.88 2.29 2.66

Perinatal conditions 1.29 0.91 2.20 2.56

Pneumonia 1.19 0.80 1.99 2.31

Chronic liver disease 1.06 0.63 1.69 1.96

Sudden death, cause unknown 0.99 0.60 1.59 1.85

all other cancers 9.07 7.35 16.42 19.08

TOTALS (Britain, 1981-2004) 53.13 32.91 86.06 100.0011

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the metabolic syndrome

This is a statistical association between six important clinical findings:

• Hypertension (high blood pressure)

• Dislipidaemia (raised LDL cholesterol)

• Type 2 diabetes (insulin resistance)

• Abdominal obesity (“beer belly”)

• Low-grade, systemic vascular inflammation

• Cardiovascular disease (atherosclerosis, neuropathies, retina, kidney, amputations, heart attacks, strokes)

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inflammation – a common thread

Most terminal, disabling and debilitating diseases in older patients have substantial inflammatory components, for example:

• Most varieties of cardiovascular disease

• Neurodegenerative diseases – Alzheimers

• Respiratory diseases – asthma, emphysema

• Arthritis, osteoporosis and sarcopenia

• Kidney diseases, gut diseases (IBD, coeliac)

• Many types of cancer

Obesity may lead to an inflammatory state 13

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cachexia• Greek word pronounced “ca –hexia” which means “poor

condition”.

• Cachexia is a hypermetabolic state where patients lose lean muscle mass.

• It is a common feature of terminal or life threatening diseases: chronic heart failure, AIDS, serious burns, tuberculosis, cancer…

• Characterised by insulin resistance and increased pro-inflammatory cytokines such as TNF-, IL-1 and IL-6

• Cachexia is an immediate threat to life, in contrast to the long-term threat from the metabolic syndrome, but these two conditions share several biochemical, physiological and immunological characteristics. 14

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inflammation basics

• The classic signs are redness, heat, swelling, pain and loss of function.

• Initiated by a few immune cells resident in the tissue which detect foreign materials or tissue damage and release inflammatory cytokines.

• Cytokines cause vasodilation and stimulate blood flow (redness, heat), increase capillary permeability leading to the loss of plasma proteins and fluid to the interstitial space (swelling) and attract other immune cells into the tissue from the blood (recruitment, positive feedback).

• Lymphatic drainage flushes antigens into lymph nodes, initiating an adaptive immune response.

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types of inflammation

Acute• Needs stimulation• Lasts only a few days• Pre-programmed “task

and finish” system

Chronic• Self-perpetuating • May persist for years• Simultaneous tissue

destruction and repair

There are dozens of inflammatory mediators, which alter in importance as the process develops, may have stimulatory or inhibitory actions. Release of histamine from mast cells is an early event, also eicosanoid production (prostaglandins, prostacyclins, thromboxanes and leucotrienes) from long chain poly-unsaturated fats. At least 20 protein cytokines are involved, also reactive oxygen species (ROS). CRP (C-reactive protein) is a marker measured in blood.

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reactive oxygen species (ROS)• ROS are free radicals produced during inflammation,

and also at other times

• Includes superoxide (O2.-) hydroxyl (OH.) and related

molecules such as hydrogen peroxide, peroxynitrite, hypochlorite …

• ROS are used by macrophages for killing bacteria, they may also damage tissues and have a signalling role.

• ROS destroy nitric oxide, which otherwise increases blood flow in arterioles.

• ROS are destroyed by superoxide dismutases (SOD) a diverse group of unrelated enzymes of whose genetic absence causes serious disease, assisted by catalase and glutathione peroxidase. 18

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matrix metalloproteinases (MMPs)

• MMPs are a family of zinc-dependent endopeptidases

• Degrade extracellular matrix proteins, such as collagen, they also release various messengers that are tethered to the plasmalemma

• Secreted initially in an inactive form, they may be activated by proteolytic cascades

• Heavily involved in tissue remodelling

• Major roles in inflammation, metastasis

• Cause tissue damage in chronic disease

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other cardiovascular risk factors• Blood clotting disorders [fibrinogen / plasminogen activator].

• Familial hypercholesterolaemia and dyslipidaemias(caused by genetics rather than lifestyle choices)

• Old age [over 45 years for men, 55 years for women].

• South Asian ancestry, family history [affected male relatives under 55 years, females under 65 years].

• Male gender, post menopausal women (oestrogens protect).

• Homocysteinaemia [strong but non-causal association].

• Oral contraceptives [minor risk with low dose pills].

• Poverty and low socio-economic status.

• Tobacco smoking [very high risk].

• Lack of physical exercise. }modifiable factors

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racial differencesThere are significant racial differences in the incidence of diabetes and cardiovascular disease:

• South Asians (from Bangladesh, India & Pakistan) are particularly susceptible to type 2 diabetes and coronary artery disease, but have a lower incidence of peripheral arterial disease.

• Blacks have a higher incidence of strokes, but a lower incidence of coronary artery disease.

• Whites appear to have a higher incidence of abdominal aortic aneurysm, though the data for other ethnic groups may be incomplete.

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‘all-cause’ mortality in Leeds

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men women

These maps show age-standardised ‘all-cause’ mortality statistics from NHS Leeds, broken down by electoral wards. The data are displayed on a spectral scale, where dark green is best, red is worst. Death rates near Leeds city centre are three times higher than those in the leafy suburbs, corresponding to a ten-year difference in life expectancy.

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cardiovascular diseases• Microvascular disease

– typically affects small blood vessels supplying the kidneys, retina and nervous system

– very sensitive to blood glucose control, but unaffected by blood lipid profiles.

– causes glomerulonephritis, blindness, neuropathies

• Macrovascular disease– typically affects large arteries such as the aorta, coronary

arteries, carotids, major limb vessels

– very sensitive to blood lipid profiles, but relatively insensitive to blood glucose control

– atherosclerosis, leading to heart attacks, strokes, angina, intermittent claudication, ulcers, gangrene, amputations

– abdominal aortic aneurisms (distinct pathology)23

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pathogenic mechanisms

• endothelial dysfunction, including major inflammatory or auto-immune components, is a common mechanism in cardiovascular diseases.

• diabetes: non-enzymic glycation of connective tissue proteins leads to microangiopathy and kidney, retinal and neurological problems.

• diabetics also have adverse blood lipid profiles causing atherosclerosis and large vessel disease.

• hypertension: mechanical damage to vascular endothelium increases the risk of clot formation.

• hypertension also increases myocardial oxygen demand because the heart has more work to do.

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Endothelial Dysfunction: The Common Consequence in Diabetes and Hypertension.Wong, Wing; Wong, Siu; Tian, Xiao; Huang, Yu

Journal of Cardiovascular Pharmacology. 55(4):300-307, April 2010. DOI: 10.1097/FJC.0b013e3181d7671c

Vasodilative and vasoconstrictive pathways in the vascular wall. * decrease # increase

ACh, acetylcholine; M, muscarinic receptor; ONOO-, peroxynitrite; sGC, guanylate cyclase; AC, adenylate cyclase; AA, arachidonic acid; IPR, IP receptor; TPR, TP receptor; FPR, FP receptor; EPR, EP receptor; DPR, DP receptor.

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drugs for obesity, diabetes & cardiovascular diseaseexample type principal targets typical indication

ramipril ACE inhibitor arteriolar smooth muscle hypertension

valsartan AR blocker arteriolar smooth muscle hypertension

metoprolol -blocker cardiac muscle hypertension

nifedipine Ca++ antagonist cardiac & smooth muscle hypertension

frumil diuretic kidney tubules (loop of Henle) heart failure

nitroglycerine organic nitrate venous smooth muscle angina

aspirin NSAID non-specific COX1 & COX2 old age?

clopidogrel anti-platelet platelet ADP receptor atherosclerosis

heparin anti-coagulant vascular endothelium atherosclerosis

warfarin anti-coagulant liver (clotting factor synthesis) atherosclerosis

streptokinase clot dissolution blood clots acute MI

lovastatin statin liver HMG-CoA reductase dislipidaemia

gemfibrozil fibrate PPAR- in many tissues dislipidaemia

metformin biguanide liver AMPK type 2 diabetes

exenatide incretin pancreatic -cells type 2 diabetes

glipizide sulphonylurea pancreatic -cells type 2 diabetes

pioglitazone thiazolidinedione PPAR- in many tissues type 2 diabetes

acarbose amylase inhibitor small intestinal lumen type 2 diabetes

orlistat lipase inhibitor small intestinal lumen diabetes, obesity

sibutramine SSRI central nervous system obesity

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drug side effectsMany of these drugs are useful, but may bring unwanted side effects. Several have been withdrawn for safety reasons, for example:

• Dexfenfluramine – improves weight loss, but causes pulmonary hypertension and heart valve defects

• Rimonabant – improves weight loss, but causes depression and increases the risk of suicide

• Sibutramine – improves weight loss, but increases the overall risk of heart attacks and strokes

• Rosiglitazone – improves diabetes, but increases the risk of heart failure

• Increased physical activity costs little, has very few side effects, and is more effective than most drugs.

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exercise – a common therapy• The health benefits from physical activity were known to

the Ancient Greeks, but for centuries this knowledge was ignored.

• Interest has re-awakened over the last fifty years, which have seen an enormous increase in research papers on exercise and health.

• Lean, physically active individuals suffer less depression and live significantly longer than their less active, obese peers.

• Moderate exercise has anti-inflammatory actions, reduces blood viscosity and improves vasodilation, which may partly explain the effect.

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exercise and “all cause” mortality• Katzmarzyk et al (2003)

Obesity Reviews 4, 257–290

• Meta-analysis of 57 previous studies, world wide.

• Measured ‘all cause’ mortality with and without adiposity measurements

• Physically active people had a 20% lower death rate than inactive peers, regardless of adiposity

• Raised BMI > 25 kg/m2 resulted in a 24% increase in mortality, regardless of physical activity.

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dose-response curve for exercise

Kokkinos et al (2009) Am J Hypertens 22, 735-741

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effect of leisure-time physical activity

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Byberg, L. et al. BMJ 2009; 338:b688

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macrovascular disease• Atherosclerosis is an inflammatory process, strongly

associated with adverse blood lipid profiles.• Desirable fasting lipid levels:

– Total cholesterol <5.2mM (<200mg/dL)

– LDL cholesterol <3.4mM (<130mg/dL)

– HDL cholesterol >1.6mM (>60mg/dL)

• Inflammation is Th1 based, exacerbated by TNF- and angiotensin II, and suppressed by Tregs

• Inadequate nitric oxide production may be important• Aneurysms are also inflammatory lesions that weaken &

remodel the outer regions of the blood vessel wall. They are exacerbated by smoking and angiotensin II, and are more closely associated with ROS and oxidative stress.

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atherosclerotic plaques

Galkina & Ley (2009) Annu. Rev. Immunol. 27, 165-197.

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atherosclerosis (1)• Progressive, inflammatory process mainly affecting the walls of the

larger arteries. Healthy vessels with an intact endothelium are highly resistant to the formation of clots.

• Atherosclerosis starts with intimal thickening and “fatty streaks” in the vessel wall, which gradually transform into atheromatous plaques.

• Macrophages invade the structure, phagocytose and partly degrade the lipid, forming foam cells. Partially oxidised lipid accumulates within the plaque and the normal intimal lining of the blood vessel is transformed into a collagen-rich “cap”.

• The plaque enlarges and may partially calcify. There is damage to the underlying smooth muscle in the vessel wall. Necrotic interior, unless new vessels develop in the plaque and supply the interior with blood.

• Eventually the cap ruptures, or the internal vessels burst, exposing the highly thrombogenic interior directly to the bloodstream.

• A thrombus (clot) forms, which may either obstruct the vessel locally, or detach and embolise in a narrower vessel further down.

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atherosclerosis (2)

Intimal thickening is very common in older subjects, and may be a natural adaptation to ageing.

The thickening “P” may be asymmetric.

IEL = inner elastic lamina.

From Wheater’s Basic Histopathology, page 88, figure 8.3

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atherosclerosis (3)

C = collagen cap; F = fibrous tissue; L = free lipidfrom Wheater’s Basic Histopathology, p 90

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atherosclerosis (4)F = foam cells; C = free lipid

Wheater’s Basic Histopathology p. 89

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atherosclerosis (5)

A = plaque

T = thrombus

C = p.m. clot

from

Wheater

p. 94

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acute myocardial infarction (AMI)• Caused by obstruction of a coronary artery by thrombus

leading to death of the downstream muscle tissue.

• AMI may be painless in elderly patients, but is usually suspected after intense crushing chest pain, nausea, vomiting, sweating and extreme distress.

• Diagnosis is confirmed from electrocardiogram changes and the release of cardiac-specific troponin variants into the blood stream from dying cells.

• The key treatment objective is rapid thrombolysis with “clot buster” enzymes, or surgical removal of the obstruction.

• Cardiac patients may subsequently develop chronic heart failure and / or cachexia [means “poor condition”].

• Obesity is a risk factor for both conditions, BUT cachectic patients survive better if they are fat.

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basic cardiovascular physiology (1)• Each beat, the heart normally expels most of the blood

that is presented to it for pumping. Atrial filling pressures rise as the venous flow to the heart increases, and the chambers become distended during diastole by the returning blood. Greater initial distension stimulates more forceful contractions. This is Starling’s Law.

• When cardiac contractility is poor, central venous blood pressures increase to abnormally high levels before they can adequately stretch the ventricles to maintain cardiac output. This situation is known as heart failure. It does not mean that the heart has stopped beating.

• High venous pressures lead to fluid accumulation in the tissues. Swollen ankles indicate right side failure, while left side failure is manifest by fluid in the lungs.

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basic cardiovascular physiology (2)

• We regulate blood volume, blood osmolarity and blood pressure, but these control systems overlap.

• The atria sense total blood volume from the venous filling pressure and resulting atrial stretch. They secrete atrial natriuretic peptide in response to stretching, which increases the loss of salt and water via the kidneys.

• The hypothalamus senses blood osmolarity and responds by secreting vasopressin via the posterior pituitary. This restricts water losses via the kidney.

• Arterial baroceptors monitor blood pressure, and trigger vasodilation and a slower heart rate via the autonomic nervous system when the blood pressure is too high.

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defence of salt and water balance

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basic cardiovascular physiology (3)• In addition, the autonomic nervous system directly

modulates the renin – angiotensin system, which normally maintains an adequate renal blood flow.

• Sensory cells in the kidney respond to low perfusion by secreting a protease, renin, which cleaves an 2 globulin produced by the liver to generate angiotensin I.

• Angiotensin converting enzyme (ACE) in the lungs cleaves more amino acids to generate angiotensin II.

• Angiotensin II causes vasoconstriction and increases blood pressure. It also stimulates adrenal production of aldosterone, a mineralocorticoid which favours sodium retention by the kidneys and expands the blood volume. Angiotensin II acts on the hypothalamus to stimulate vasopressin release and increases thirst.

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preload, afterload & contractility• The venous pressure which returns blood to the heart,

fills and distends the ventricles during diastole is often referred to as preload.

• The aortic pressure seen by the heart during systole (contraction) is often referred to as afterload.

• The relationship between preload and afterload depends on the cardiac contractility.

• Drugs such as diuretics and nitroglycerine reduce the preload, and thereby relieve many symptoms of angina and heart failure.

• Drugs such as ACE inhibitors, -blockers and calcium blockers reduce the afterload and / or contractility.

• Different classes of cardiac drug may achieve similar effects, but rely on different mechanisms.

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peripheral vascular disease

In addition to a much greater incidence of strokes and coronary artery disease, diabetic patients are prone to atherosclerotic obstruction and poor circulation through the small arteries supplying the lower limbs and feet. This under-perfusion results first in diabetic foot ulcers, which may progress to gangrene, requiring amputations. This is another manifestation of macrovascular disease.

Intermittent claudication is pain from poorly perfused skeletal muscles. Like angina (which it closely resembles) this pain is exacerbated by exercise and relieved by rest.

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microvascular disease• Small vessel disease most often presents as sensory and

autonomic neuropathies, kidney and retinal disease.

• All these conditions are major causes of ill health in the general population.

• Thickening of the capillary basement membrane is a consistent early feature of microvascular disease, and contributes to tissue hypoxia and poor wound healing.

• Small vessel disease can be minimised by good glycaemic (blood glucose) control.

• Large vessel disease is little affected by glycaemic control.

• Inflammation plays a major role in both microvascular and macrovascular disease.

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diabetic retinopathy

Fragile new blood vessels develop in the proliferative form, which is more severe.

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NFB – nuclear factor kappa B (1)

• NFKB are a group of proteins regulating gene expression that are expressed in many types of cells that signal via cytokines

• NFKB are activated by numerous inflammatory stimuli, and inhibited by corticosteroids

• NFKB activation typically (ideally?) produces a transient, time limited nuclear response

• NFKB activation generates further inflammatory messengers

• NFKB regulation can therefore get “stuck in a rut” leading to persistent inflammation

• Over-activity of NFKB is associated with chronic inflammatory diseases such as atherosclerosis, lung fibrosis, arthritis, asthma, septic shock, and glomerulonephritis.

• Prolonged inhibition of NFKB is associated with apoptosis, inappropriate immune cell development and delayed cell growth.

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NFKB activators examples

cytokines tumor necrosis factor (TNF),interleukin-1 (IL-1),interleukin-17 (IL-17)

protein kinase C activators

phorbol esters, platelet-activating factor

oxidants hydrogen peroxide, ozone

viruses rhinovirus, influenzavirus, Epstein–Barr virus, cytomegalovirus, adenovirus

immune stimuli antigens, phytohemagglutinin,anti-CD3 antibodies (by means of T-lymphocyte activation)

other lipopolysaccharide, ultraviolet radiation

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NFKB targets examples

pro-inflammatory cytokines

various granulocyte / macrophage colony stimulating factors, TNF , IL-1, IL-2, IL-6

chemokines IL-8, gro-, gro-, gro-, eotaxin, macrophage inflammatory protein 1, macrophage chemotactic protein 1,

inflammatory enzymes

inducible nitric oxide synthase (iNOS), inducible cyclo-oxygenase-2, 5-lipoxygenase, cytosolic phospholipase A2

cell adhesion molecules

intracellular adhesion molecule 1, vascular cell adhesion molecule (VCAM), E-selectin

receptors IL-2 receptor ( chain)T-cell receptor ( chain)

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• NFKB1 / NFKB2 bind either REL / RELA / RELB to form the NFKB complex

• I- inactivates NFKB by trapping it in the cytoplasm

• Kinases IKBKA / IKBKB phosphorylate I-, causing ubiquitination and destruction, and thereby activate NFKB

• NFKB complex translocates to the nucleus and binds motifs such as 5’ GGGRNNYYCC 3’ (where R is an A or G purine; and Y is a C or T pyrimidine).

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• Barnes & Karin (1997) NEJM 336, 1066-1071

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NFB – nuclear factor kappa B (6)• In mice, obesity activates hepatic NFKB causing chronic low level

inflammation. Activation of NFKB by other routes produces a very similar outcome, resembling type 2 diabetes. See: Cai et al (2005) Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappa-B. Nature Med. 11, 183-190.

• Both NFKB genes produce alternate products with antagonistic functions. The short (amino terminal) versions p50 & p52 are pro-inflammatory, but the full length p105 & p100 are anti-inflammatory IKBs that sequester the short forms in the cytosol. See: Pereira & Oakley (2008) Nuclear factor-B1: Regulation and function. Int. J. Biochem. & Cell Biology 40, 1425–1430.

• Outputs from the NFKB system can be oscillatory, and oscillatory stimuli generate various outcomes, depending on the stimulation frequency. See: Ashall et al (2009) Pulsatile stimulation determines timing and specificity of NF-kappa-B-dependent transcription. Science 324, 242-246.

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peroxisome metabolism

• Peroxisomes handle big, awkward shaped lipids and degrade them to more manageable chunks that are processed by the mitochondria.

• Peroxisomal metabolism does not directly yield any ATP

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PPARs: peroxisome proliferator activated receptors • Peroxisome proliferators are a diverse group of compounds including

various natural lipids, anti-diabetic drugs & plasticisers (including some used in food containers) that increase peroxisomes in cells.

• PPARs are transcription factors that bind peroxisome proliferators and form heterodimers with retinoic acid binding proteins.

• All PPARs regulate genes involved in fat metabolism & inflammation.

• PPAR- active in liver, skeletal muscle, heart & kidney; regulates fatty acid uptake and oxidation, inflammation & vascular function.

• PPAR-/ is ubiquitously expressed, regulates fatty acid metabolism, muscle fibre types & suppresses macrophage-triggered inflammation.

• PPAR- active in white & brown adipose tissue, colon, endothelial & vascular smooth muscle cells and to a lesser extent in immune cells. Regulates adipocyte proliferation and differentiation. Improves insulin sensitivity by promoting fat storage and inhibiting adipokine synthesis. Requires PGC-1 to activate brown fat thermogenesis.

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Duan et al (2009) PPARs: the vasculature, inflammation and hypertension. Current Opinion in Nephrology & Hypertension 18(2), 128–133.

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PPARs & cardiovascular drugs• PPARs are the intracellular targets for two important groups of

cardiovascular drugs:

• Fibrates (e.g. gemfibrozil, bezafibrate, fenofibrate) activate PPAR- They reduce blood triglycerides & raise HDL cholesterol in men & post-menopausal women, may help to prevent both macrovascular (atherosclerosis & strokes) and microvascular (kidney, nerves & retina) diabetic complications.

• Thiazolidinediones (e.g. rosiglitazone, pioglitazone, troglitazone) activate PPAR-They improve insulin sensitivity of target tissues, with microvascular benefits in diabetes, but also cause unwanted weight gain, and problems with bone loss.

• In addition, compounds such as oleoylethanolamide (OEA) bind to intestinal PPAR- and reduce food intake by prolonging eating latency. (OEA is closely related to the endocannabinoids, but is not itself a cannabinoid.)

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Effects of TZDs pioglitazone / rosiglitazone on cardiovascular risk factors and surrogate markers

lipid profile endothelial function vascular parameters inflammation

↓ free fatty acids ↑ flow-mediated dilation

↑ coronary reserve ↓ c-reactive protein

↑ LDL particle size ↑ insulin-dependent endothelial NO release

↓ pulse wave velocity ↓ white cell count

↑ lipoprotein a ↓ oxidative stress ↓ wall thickness ↓/0 interleukin-6

↑/(↑) HDL ↓ oxidized LDL ↑ endothelial function ↓ ICAM and VCAM

↓/0 LDL / HDL ratio blood clotting ↓ albumin excretion ↓ TNF-α and MCP-1

↓/↑ triglycerides ↓ PAI-1 fat distribution ↓ MMP-9

hemodynamics ↓ fibrinogen ↓ visceral fat ↓ soluble CD40 ligand

↑ blood volume ↓ platelet aggregation ↑ subcutaneous fat ↓ adiponectin

↓ blood pressure ↓ endothelin-1 ↓ hepatic fat content ↓ serum amyloid A

but TZDs increase the risk of heart failure, MAY increase risk of MI.

ICAM — intercellular adhesion molecules; MCP-1 — monocyte-chemoattractant protein-1; MI — myocardial infarction; MMP-9 — Matrix metalloproteinase-9; PAI-1 — plasminogen activator inhibitor 1;VCAM — vascular cell adhesion molecules.

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normal blood glucose regulation

bloodglucose

physiologicassessment

short termresponse

long termresponse

>6mM too high:

secrete insulin

make liver glycogen, let glucose into cells

make triglyceride (lipogenesis)

~5mM OK ongoing turnover and metabolism

<4mM too low:

secrete glucagon & adrenalin

split liver glycogen, exclude glucose from most cells, except in emergencies

make glucose (gluconeogenesis)

mobilise fat and protein stores

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What if blood [glucose] is wrong?

• Too low: people feel weak, bad tempered, cold and hungry – unpleasant sensation.

• Too high: (diabetes) causes no sensations, unless it is enormous, when patients start to urinate uncontrollably because their kidneys can’t cope.

• BUT – high blood glucose reacts non-enzymically with amino groups on blood and tissue proteins.

• This protein glycation causes serious damage.

• The essence of successful diabetes therapy is good glycaemic control.

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glycated haemoglobin reflects risk

Glycated Hgb Diabetes CAD Stroke Death

< 5 % 0.53 0.95 1.09 1.48

5.0 - 5.5% 1.00 1.00 1.00 1.00

5.5 - < 6.0% 1.80 1.25 1.16 1.19

6.0 - < 6.5% 4.03 1.88 2.19 1.61

< 6.5% 10.40 2.46 2.96 1.71

• Selvin et al (2010) N Engl J Med. 362, 800-811 followed long term outcomes after measuring glycated Hb

• Low glycated Hb meant less risk of developing diabetes• BUT overall survival was better when glycated Hb was in

the normal range.64

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advanced glycation end-products (1)

• Elevated blood glucose causes protein glycation.

• Rearrangement and oxidation produces a variety of cross-linked Advanced Glycation End-products [AGEs] which are highly immunogenic

• Glycated haemoglobin is used for diagnosis, BUT

• Glycated endothelial proteins do the real damage.

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advanced glycation end-products (2)

Nessar Ahmed (2005) Advanced glycation endproducts — role in pathology of diabetic complications. Diabetes Research and Clinical Practice 67(1), 3-21

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Soro-Paavonen et al (2008) Receptor for Advanced Glycation End Products (RAGE) Deficiency Attenuates the Development of

Atherosclerosis in Diabetes Diabetes 57: 2461 – 2469.

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AGE inhibitors

• Figarola et al (2003) LR-90 a new advanced glycation endproduct inhibitor prevents progression of diabetic nephropathy in streptozotocin-diabetic rats. Diabetologia 46(8), 1140-1152.

• Figarola et al (2007) Anti-inflammatory effects of the advanced glycation end product inhibitor LR-90 in human monocytes. Diabetes 56(3), 647-655.

• Figarola et al. (2008) LR-90 prevents dyslipidaemia and diabetic nephropathy in the Zucker diabetic fatty rat. Diabetologia 51(5), 882-891.

• Bhatwadekar et al. (2008) A new advanced glycation inhibitor, LR-90, prevents experimental diabetic retinopathy in rats. Brit. J. Ophthalm. 92(4), 545-547.

7272Copyright ©2009 American Society of Nephrology

Coughlan, M. T. et al. J Am Soc Nephrol 2009;20:742-752

7373

what is diabetes mellitus?

● Heterogeneous group of disorders of carbohydrate metabolism that result in hyperglycaemia

● WHO diagnostic criteria for diabetes (2002)

Symptoms of diabetes1 plus casual1 plasma [glucose] >11.1 mmol/L

– OR fasting2 plasma [glucose] > 7.0 mmol/L

– OR plasma [glucose] >11.1 mmol/L 2h post 75g oral glucose load

1 Classic symptoms include polydipsia, polyuria and unexplained weight loss. Casual means any time of day without regard for last meal

2 Fasting means no caloric intake for at least 8 h

Diabetes from Greek for “siphon”

Mellitus from Latin for “sweet like honey”

7474

why is diabetes important?

● ~ 2.5 million cases in UK (October 2008 GP figures, yearly increase up more than 2-fold over 2006/07 increase)*

● ~ 750,000 cases in UK still undiagnosed

● 246 million cases worldwide (International Diabetes Foundation figures for 2006)

● Predicted worldwide incidence 380 million by 2025

*Comments from Chief Executive of Diabetes UK on 20th October 2008:

“ .... there is no getting away from the fact that this large increase is linked to the obesity crisis. Diabetes is one of the biggest health challenges facing the UK today. It causes heart disease, stroke, amputations, kidney failure and blindness, and more deaths than breast and prostate cancer. The NHS already spends one million pounds an hour on diabetes.”

7575

history of diabetes and insulin

LangerhansLangerhans

Ebers papyrus, 1550 BCFirst description

Charaka Samhita, ~300 BC2 types of diabetes

Paul Langerhans, 1869Islets of Langerhans

Banting, Best & Collip, 1922Isolation of insulin for patient treatment

Margery

Fred Sanger, 1953Insulin sequence

Dorothy Hodgkin, 1969Insulin structure

Ullrich, Ebina et al., 1985Insulin receptor cloning

Many, 1985-2008Signal transduction

7676

two types of diabetes mellitus

● Type 1 [mainly juvenile onset]

– 5-15% of cases

– inability to produce insulin

● Type 2 [mainly maturity onset]

– 85-95% of cases

– resistance to the normal action of insulin, and / or inability to produce sufficient insulin

– MODY = “maturity onset diabetes in the young”

Rosalyn Yallow & Solomon BersonRadioimmunoassay (RIA) 1959

7777

type 1 diabetes mellitus (T1DM) ● < 20% of diabetics (approx 1 in 300)

● Age at onset usually < 30 years (peaks at 12-15 years, hence the formerly-used term “Juvenile Onset Diabetes Mellitus”)

● Appearance of symptoms rapid, may be life-threatening:

– Hyperglycaemia leading to osmotic diuresis (glycosuria) and unquenchable thirst

– Ravening hunger & weight loss (up to 15 lbs in 2 weeks), tiredness

– Tendency to life-threatening ketosis (hyperventilation, vomiting, drowsiness and coma)

● Blood [insulin] low, always needs treatment with insulin

● Increased prevalence in relatives, association with MHC genes (involved in antigen presentation), but <50% concordance in identical twins

7878

Non-Obese Diabetic (NOD) mouse model

Type 1 Diabetes Mellitus (70% of females, 10% of males) BUT the incidence depends on the microbial environment in which the mice raised

6 months

MHC class II

antigenic peptide mutations

T-cell mediated autoimmune destruction of pancreatic -cells

(For immunology afficionados only, see Wen et al. (2008) Nature 455, 1109-1113)

7979

pathogenesis & treatment of T1DM● Apparent rapid onset of symptoms comes after several years gradual

destruction of -cells

● 95% of IDDM patients carry at least one HLA-DR3 or HLA-DR4 allele, in comparison to 50% of normal individuals

● Possible association with virus infection may reflect similarity between -cell proteins and viral proteins:

● Current treatment – insulin injections, insulin pumps

● Future treatments – inhaled insulin (but Exubera withdrawn in late 2007), insulin pumps, islet transplantation, production of -cells from stem cells, etc.

Coxsackievirus protein PC-2

-cell glutamic acid decarboxylase (GAD)

AMLIARYKMFPEVKEKGMAAVPRL

FIEWLKVKILPEVKEKHEF-LSRL

8080

type 2 diabetes mellitus (T2DM)

● > 80% of diabetics

● Age at onset usually > 30, appearance of symptoms slow. Ketosis rare.

● Often associated with obesity. No HLA association but family history of diabetes common (usually >70% concordance in identical twins): i.e. stems from combination of environmental & genetic factors.

● Often associated with peripheral insulin resistance - insulin is less able to stimulate glucose uptake or to inhibit hepatic glucose production.

● Insulin secretion by pancreas persists (initially insulin levels may be elevated and receptors downregulated), but insulin release in response to glucose is impaired and cannot compensate for insulin resistance.

● Commonly managed by diet, exercise and oral hypoglycaemic agents:

– sulphonylureas and meglitinides - promote insulin release;

– biguanides and thiazolidinediones - increase sensitivity to insulin.

8181

pathogenesis of T2DM

Insulin sensitivity

Insulin secretion

Years before onset of diabetes

25 15 5 01020

Decreased sensitivity to insulin precedes fall in insulin secretion

normal

normal

patient

patient sen

sitiv

ity

% Ideal body weight

Offspring of Type 2 DM

Controls (no relatives with Type 2DM)

100 160

Impact of “diabetogenes” on obesity-induced insulin resistance

Obesity affects sensitivity to insulin

– genes plus environment, but we can’t find the genes...

82

diabetes treatment strategies

therapy type 1 type 2

Insulin injections (usually a mixture of short-acting and long-acting varieties)

always needed

avoid if possible

Reduce the need for insulin by cutting out easily digested carbohydrates

helpful helpful

Lifestyle changes: increased exercise, calorie or carbohydrate restricted diet

helpful first choice

Increase insulin sensitivity using oral hypoglycaemic drugs (e.g. metformin)

helpful second best

Stimulate insulin production using oral hypoglycaemic drugs (e.g. glimepiride)

useless third best

8383

reducing the need for insulin• Foods high in sugar or simple polysaccharides break down quickly in

the gut, and generate large peaks of free glucose in the portal vein.

• Large quantities of insulin are then required to stabilise the blood glucose concentration.

• If digestion is delayed the glucose peak becomes longer and flatter (although no smaller in total volume) but less insulin is needed to maintain blood glucose near 5mM.

• Digestion can be delayed or prevented with enzyme inhibitors like acarbose. Bacterial fermentation may become a problem.

• Alternatively, patients may be advised to eat foods with a low glycaemic index, which break down more slowly in the gut.

• Such foods often include “wholegrain” products, containing complex carbohydrates, which are less easily hydrolysed, and less physically accessible to digestive enzymes. Reduced cooking and less refined sugar may also be helpful.

84

lifestyle changes are effective

Parameter Lifestyle

intervention Support & education

p

Weight loss (%) −6.15 −0.88 <0.001

Treadmill fitness (% METS) 12.74 1.96 <0.001

Hemoglobin A1c (%) −0.36 −0.09 <0.001

Systolic pressure (mm Hg) −5.33 −2.97 <0.001

Diastolic pressure (mm Hg) −2.92 −2.48 <0.01

HDL-C (mg/dL) 3.67 1.97 <0.001

Triglycerides (mg/dL) −25.56 −19.75 <0.001

• Wing et al (2010) Long-term Effects of a Lifestyle Intervention on Weight and Cardiovascular Risk Factors in Individuals With Type 2 Diabetes Mellitus. Archives of Internal Medicine 170, 1566-1575.

• Exercise + low fat diet, but would low carbs have been better? 84

85

gestational diabetes• All types of diabetic mothers often produce excessively

large babies – this is known as “macrosomia”.• High maternal glucose stimulates fetal insulin production

which acts as a growth factor.• Insulin resistance sometimes develops transiently during

pregnancy, especially during the third trimester, and may progress to “gestational diabetes mellitus” (GDM).

• Both conditions normally resolve after the baby has been born, but these mothers face increased long term risk of developing type 2 diabetes.

• Maternal obesity increases the risk of GDM.• Diet and resistance exercise improve glycaemic control

and reduce the need for insulin in GDM, but if necessary metformin and insulin are safe to use in pregnancy.

86

periodontal disease• Gingivitis – inflammation of the gums with bleeding and

sponginess caused by pathogens in dental plaque.• Periodontitis – inflammation of the tissues supporting the

teeth, leading to destruction of the periodontal ligament, damage to alveolar bone and tooth loss.

• Periodontal bacteria readily enter the circulation, causing transient bacteraemia. They sometimes colonise artificial heart valves or cause bacterial endocarditis.

• Treatment of periodontal inflammation reduces circulating levels of CRP and IL-6.

• Gum disease and heart disease correlate with social class.• There is a clear two-way relationship between diabetes and

gum disease, but the correlation between gum disease and cardiovascular disease is still disputed.

8787

pancreatic islets• the pancreas has two functions

• exocrine acinar cells manufacture and secrete a variety of digestive enzymes into the gut

• endocrine islet cells manufacture and release a variety of peptide hormones into the portal vein

• islet stem cells have a local gut origin, but the autonomic nerves that control them develop from neural crest

• all varieties of islet cell are ultimately derived from the same type of stem cell

types of islet cells:

• 20% α cells – glucagon• 70% β cells – insulin + amylin• 5% δ cells – somatostatin• 5% other minor cell types

8888

insulin basics• Insulin and amylin are both manufactured by the β cells, and

released primarily in response to raised blood glucose, although there are many other stimuli.

• Insulin suppresses glucose output from the liver, and promotes glucose metabolism by most tissues other than brain, thereby returning blood glucose to the target value near 5mM.

• Insulin has numerous actions on carbohydrate, fat and protein metabolism, which differ from one tissue to another:

– More glycogen synthesis in liver, stops glycogenolysis.

– More glycolysis + lipogenesis in liver, stops lipolysis + gluconeogenesis.

– More glucose transport into most peripheral tissues, except for red blood cells and brain.

– Net synthesis of glycogen, protein and fat in most tissues.

– Long term changes in gene expression in all target tissues.

• Amylin acts on the brain to regulate appetite and body weight.

8989

insulin secretagogues• β cells contain a glucose sensing system, which requires glucose to

be metabolised. Islet cells have a membrane potential. They are excitable and communicate with one another.

• Other stimuli for insulin release include amino acids (alanine, leucine, arginine), 2-keto acids, peptide hormones (glucagon, GIP, GLP, CCK) and acetyl choline (autonomic nervous system).

• Free fatty acids have a biphasic effect: a short-term stimulation of insulin secretion is superseded by a long-term inhibition.

• Catecholamines (adrenaline, noradrenaline) inhibit insulin release via -receptors, weaker stimulation via -receptors.

• Glucagon is secreted by α cells in response to low blood glucose. It has widespread anti-insulin effects, but it is not a complete opposite and glucagon actually stimulates insulin release.

• Somatostatin from the δ cells suppresses both insulin and glucagon release. This hormone is also produced by the hypothalamus and suppresses growth hormone release from the pituitary.

9090

insulin microcrystals in secretory granules

insulin green, glucagon red, nuclei blue

Mouse pancreatic islet1

1Source: Solimena Lab, Med. Fac., University of Technology, Dresden, Germany

Mouse pancreatic beta cell1

insulin hexamers(with zinc)

9191

biphasic response to glucose

100

50

0

Insu

lin s

ecre

tion

(pg/

min

/isle

t)

11 mM glucose

0 15 60

Secondphase

Firstphase

Time (min)

100

50

0

Insu

lin s

ecre

tion

(pg/

min

/isle

t)

11 mM glucose

0 15 60

Secondphase

Firstphase

Time (min)

transient phase:

Release and depletion of “readily releasable pool” (RRP) – about 50 granules per cell (from 13,000 total). Caused by increased [Ca2+]i.

sustained phase:

Slow replenishment of RRP by priming granules from a reserve pool. 5-10 granules primed per cell per min. Requires glucose metabolism, possibly increasing [glutamate].

9292

glucose transporters

porter mechanism glucose Km

cellular location

tissues features

GLUT-1 passive 20 mM - brain, red cells, endothelium, β cells

constitutive porter

GLUT-2 passive 42 mM mobile kidney, ileum, liver, pancreatic β cells

low-affinity porter

GLUT-3 passive 10 mM apical neurones, placenta (trophoectoderm)

high-affinity porter

GLUT-4 passive 2 - 10 mM - skeletal muscle, heart, adipocytes

insulin-responsive

GLUT-5 passive - both widely distributed fructose transport

SGLT-1 sodium dependent

high affinity

apical small intestine, kidney tubules

high affinity uptake

SGLT-2 sodium dependent

low affinity

apical kidney proximal tubule

high capacity uptake

9393

glucose sensor (1)• The ATP concentration in most cells is high and constant, and it is

very difficult to detect any variations. Glucose metabolism in β cells is inefficient, so that ATP levels in β cells depend on glucose availability. This the essence of the sensing mechanism.

• Glucose entry into β cells uses low-affinity GLUT2 transporters, so that the uptake rate varies with the blood glucose concentration. Intracellular glucose concentration tracks the external supply.

• Glucose phosphorylation in β cells uses low-affinity glucokinase. The enzyme is not saturated with its substrate. The phosphorylation rate varies with intracellular glucose concentration, so in β cells the glycolytic rate ultimately depends on the glucose concentration in arterial blood.

• Glucose starvation affects the mitochondrial fuel supply, so in β cells the ATP concentration increases when blood glucose is high.

• Potassium efflux channels in β cells are inhibited by ATP. The channels close as ATP rises, depolarising and activating the cells.

9494

-cell glucose sensor (2)Glucose

Glucose

Glucose 6-phosphate

ATP:ADP

K+-

Ca2+

+

+

Glucokinase(Km 8 mM)

Metabolism

GLUT2(Km42 mM)

Insulin

K+ATP channel Voltage-gated

Ca2+ channel

ATP

ADP

H+

Glutamate

-+

priming

fusion

Glucose

Glucose

Glucose 6-phosphate

ATP:ADP

K+-

Ca2+

++

+

Glucokinase(Km 8 mM)

Metabolism

GLUT2(Km1 mM)

Insulin

K+ATP channel Voltage-gated

Ca2+ channel

ATP

ADP

H+

Glutamate

-+

priming

fusion

The -cell glucose uptake and phosphorylation systems are not saturated at physiological glucose concentrations.

9595

glucose sensor (3)• Depolarisation activates voltage-dependent calcium channels

(VDCCs), triggering calcium spikes and action potentials. This leads to exocytosis and insulin release from stored secretory granules.

• Islet tissue is also controlled by the autonomic nervous system. Many other compounds affect insulin release, using various signaling pathways:

– Leucine raises ATP through metabolism, but alanine and arginine directly depolarise the cells.

– Acetylcholine and cholecystokinin stimulate via phospholipase C, diacyl glycerol and IP3.

– Glucagon, GLP and GIP stimulate through G-proteins, adenyl cyclase and cyclic AMP.

– Catecholamines signal via β-receptors, G-proteins and adenyl cyclase, but inhibit the granule docking system via -receptors.

• Local gut hormones that stimulate insulin release are known as incretins.

9696

Oral hypoglycaemic drugs promoting insulin release

SO2 R2C

O

NHNHR1Sulfonylureas

R1 R2

Glimepiride(3rd generation drug)

H3CNCH3

CH3

O

CONHCH2CH2

N

NH

O

O

OH

O CH3

CH3

CH3

Repaglinide

Meglitinides

(short acting “prandial” blood glucose regulator)

(long duration of action)

9797

the sulphonylurea receptor

K+

sulphonylureas

ATP

Mutations in eitherFamilial persistanthyperinsulinemichypoglycemia of infancy

meglitinides

MgADP K+

sulphonylureas

ATP

Mutations in eitherFamilial persistanthyperinsulinemichypoglycemia of infancy

meglitinides

MgADP

NBF-1 NBF-2

SUR1 KIR6.2

(Photolabelled by the sulfonylurea glibenclamide)

(inward-rectifier K+ channel)

NBF-1 NBF-2NBF-1 NBF-2

SUR1 KIR6.2

This group of drugs all require functional islet tissue, and are therefore completely useless in patients with type 1 diabetes.

9898

model of the K+ATP channel

side view top view

ATP

From Mikhailov et al. (2005) EMBO J. 24, 4166-4175.

9999

the incretin effect

● Oral glucose causes 2-3-fold greater insulin secretion than equivalent intravenous load

● Two gut hormones are primarily responsible

– Glucose-dependent insulinotropic polypeptide (GIP), formerly known as gastric inhibitory polypeptide

– Glucagon-like peptide 1 (GLP-1), a 30-residue peptide

● GLP-1 stimulates insulin secretion and inhibits glucagon secretion

● In animals, GLP-1 stimulates -cell proliferation

100100

glucagon like peptide 1 is an “incretin”

From: Holst (2007) Physiol. Rev. 87, 1409-1439.

pancreasProglucagon

33 61

glucagon GLP-1

78 107

pancreas L-cell of intestine

Dipeptidylpeptidase IV in capillaries

t1/2 few minutes in circulation

GLP-1

secretion in response to meal

degradation

Sitagliptin and Vildagliptin, inhibitors of DPP4, are in clinical use

101101

mechanism of GLP-1 action on -cells

ATP cAMPPKA

Adenylate cyclaseGLP-1R

G-protein

N

cAMP-GEFII

Ion channels, exocytotic machinery

Enhanced insulin secretion

Gila monster

Exendin 4/ Exenatide /Byetta50% sequence identity to

GLP-1, full receptor agonist, resistant to DPP-IV

102102

insulin has many effects on cells

signals the fed state, promotes fuel storage

protein synthesis (translation)

mitogenesis (cell division)

Metabolism

• glycogen synthesis• glycolysis and fat biosynthesis• glucose uptake by muscle and fat

(Vmax increased 30-fold in minutes)

slow slowfast

103103

summary of insulin signalling

104104

summary of insulin signalling (1)

insulin binds to receptor, which auto-phosphorylates

105105


phosphorylation of insulin receptor substrate

106106


activation of phosphatidylinositol 3-kinase

107107


Recruitment of PDK1 and PKB (=Akt) to the membrane

108108


activation of PKB

109109


phosphorylation of synip promotes fusion of storage vesicles with plasmalemma

110110


without insulin, GSK3 keeps glycogen synthase inactive

111111


steps 1 – 5 are the same as for GLUT4 activation

112112


PKB phosphorylates and inactivates GSK3

113113


protein phosphatase activates glycogen synthase

114114


IRS binds to the small GTPase RAS through adaptor proteins

115115


activated RAS activates Raf-1

116116


Raf-1 activates MEK

117117


MEK activates mitogen activated protein kinase (MAPK) which enters the nucleus

recycling GLUT4 porters – 1

118

Shepherd & Khan (1999) Glucose transporters and insulin action. NEJM 341, 248-257.

recycling GLUT4 porters – 2

119

Exercise increases insulin sensitivity, and independently stimulates vesicle recycling.

120120

Structure of the membrane-bound insulin receptor

S

S

S SS

S

135 kDa

90 kDa

extracellular

Activation loops

oligosaccharide

tyrosine kinase active sites

insulin

P P

121121

Tyrosine kinase activity of the receptor subunit is essential for transduction

Activation loop Autophosphorylation of Y 1158, 1162 & 1163 greatly enhances kinase activity of receptor towards other substrates

ATP-binding motif:G-X-G-X-X-G……..K(1030)

V X

(patient) (site-directed mutagenesis)

OR

normal insulin bindingbut

no kinase activity and no biological effect of insulin

122122

Phosphorylation of tyrosine residues allows access for protein substrates

Before auto-phosphorylation After auto-phosphorylation

Activation loop

Catalytic loop

substrate

123123

PTBPH

IRS-1, 2 etc. are “docking” proteins that couple multiple signalling pathways

PATP

Insulin receptor substrate 1(IRS-1) 131 kDa

(and IRS2-4, Shc,Gab-1, Cbl, etc.)

PATHWAY 1 PATHWAY 2

Y Y Y YY

Y

P P P

PNH3+

CO2-

SH2 SH2

124124

SH2 (SRC homology 2) domains recognise phosphotyrosine motifs

C-terminal SH2 domain from p85 subunit of phosphatidylinositol 3-kinase

Peptide containingphosphotyrosine

tyrosine

phosphate

Complex of SH2 domainand phosphotyrosyl peptide

+

125125

PTBPH

P

ATP

Y Y Y YY Y

P P P

PNH3+

CO2-

110 kDa85 kDa

P

OHOHOH

41P

P5

PIP2

P

OHOH

41 P

3

P

P5

PIP3

ADP

Phosphatidylinositol 3-kinase(PI 3-K) wortmannin

A key pathway involves phosphorylation of phosphoinositides on the 3-position

126126

Recruitment of Akt2 to the plasma membrane leads to its activation

P

OHOH

41 P

3

P

P5

PIP3

P

OHOH

41 P

3

P

P5

PIP3

PDK1

PH domain PH domain

PT309

Akt2

S 474P

Insulin

mTORC2

Akti-1/2Akt is also called PKB – protein kinase BPDK1 is 3-phosphoinositide-dependent protein kinase 1mTORC2 is mammalian target of rapamycin complex 2

127127Cheng et al (2009) Targeting the phosphoinositide 3-kinase pathway in cancer. Nature Reviews Drug Discovery 8, 627-644.

128128

mTORC

mammalian target of

rapamycin complex 1

andcomplex 2

129129

Targets for activated Akt2?

AS160/TBC1D4 and TBC1D1

PTB PTB S T RAB GAP

14-3-3

PTB PTB S T RAB GAP

PP

Rab GDPRab GTP

more active less active

Rab GDP

Rab GTP

RAB GEF RAB GEF

Rab10 in adipocytes, Rab8A in muscle?

Akt2

GSK3

glycogen metabolism

glucose transport

Synip S

Synip S

P

S

GSK3 S

P

glucose transport

130130

Where do Rabs, Synip and other factors act?

Synip S

Synip S

P

Rab GTP

insulin

Insulin ?

Synip = syntaxin 4-interacting protein

insulin

Rab GDP

Synaptobrevin 2

SNAP-23

Syntaxin 4

GLUT4

131131

Insulin-induced translocation of GLUT4-GFP

From: Oatey et al. (1997) Biochem. J. 327, 637-642.

Basal Insulin

midsection midsection

top top

extracellular

CO2-

1 2 3 4 5 6 7 8 9 10 11 12 GLUT4

GFP

MPSGFQQIGSE ......... 1aromatic motif (needed for endocytosis)

Targeting/sorting motifs

12 ..........RGELEYLGPDEND

acidic motif (needed for targeting to intracellular compartment)

See: Song et al. (2008) J. Biol. Chem. 283, 12571-12585 for more details.

132132

Insulin increases the rate of exocytosis of recycling GLUT4

From: Fletcher et al. (2000) Biochem. J. 352, 267-276.

early endosome

GLUT4 storage compartment/vesicles

glucose glucose

= sites where insulin/contraction may exert an effect

+

+

+-?Docking/fusion

Translocation

Budding

Endocyticretrieval

Sorting Retention

+/-

+

+

+-?

-

+

+

+-?

133133

What becomes of the glucose that is taken up by peripheral tissues?

glucose

glycogen

+ -

glucose

+

triacylglycerol

fatty acid

VLDL

glycerol-P

glucose

+

-

glucose

Unlike liver glycogen, muscle glycogen cannot be converted into blood glucose.

Peripheral tissues cannot use the glycerol released during triglyceride hydrolysis.

In adipocytes, the glucose is converted into triglycerides.

134134

Oral antidiabetic drugs that increase insulin sensitivity

• very widely used drug that improves insulin sensitivity

• decreases hepatic glucose production

• decreases intestinal absorption of glucose

• increases peripheral glucose uptake and utilization

• unrelated to most other oral hypoglycaemic drugs

• activates AMP-dependent protein kinase (AMPK)long-acting biguanide

Metformin

wrong kind of AMP?• There are two different kinds of AMP in cells, which work

independently, and transmit different signals.

• 3’5’cyclic AMP is produced by adenyl cyclase in the plasmalemma, usually in response to some circulating hormone that signals via G-protein coupled receptors (GPCRs).

• 3’5’cyclic AMP often activates protein kinase A (PKA)

• linear 5’ AMP is produced by myokinase located in the mitochondrial intermembrane space:

ATP + AMP = ADP + ADP

• mitochondria actively export of ATP and take up ADP, driven by the mitochondrial membrane potential.

• [AMP] is vanishingly small in healthy cells, but rises rapidly on any threat to the cellular energy supply.

136136

AMPK• Most important cellular energy gauge.

• Activated by linear 5’ AMP & upstream kinases LKB1 and calmodulin-dependent protein kinase (CaMKK).

• Steers metabolism away from non-essential synthetic activities towards energy-yielding processes that are important for cell survival.

• Activity is normally low because [AMP] is kept low by the myokinase reaction: AMP + ATP = ADP + ADP

• Phosphorylates and inactivates HMG-CoA reductase (sterol biosynthesis), acetyl-CoA carboxylase (fatty acid biosynthesis) and TORC2 (PEPCK transcription).

• Activated by the fat cell hormone adiponectin, and also by metformin and the thiazolidinediones.

the hunting of the SNARK...• nonsense poem by Lewis Carroll 1874• paper by Koh et al (2010) Sucrose nonfermenting

AMPK-related kinase (SNARK) mediates contraction-stimulated glucose transport in mouse skeletal muscle. PNAS 107, 15541–15546.

• Insulin is required for glucose uptake by resting skeletal muscle expressing GLUT4 porters.

• During exercise, muscle takes up glucose without insulin, and the sensitivity to insulin is also enhanced.

• This effect is mediated by one member from a family of AMP dependent protein kinases that play a central role in the regulation of energy metabolism.

• Remember that AMPK responds to linear 5’-AMP, and differs from PKA which responds to cyclic 3’5’-AMP 137

138138

Oral antidiabetic drugs that increase insulin sensitivity

Thiazolidinediones (glitazones)

Rosiglitazone (Avandia, GSK)SNH

N N

CH3

O

O

O

• Increase insulin sensitivity of liver, skeletal muscle and adipose tissue.

• Nearly normalise rates of hepatic glucose production.

• Cause 40 - 60 % increase in insulin-mediated glucose disposal

• Rosiglitazone recommended for treatment of a subset of Type 2 diabetic patients by NICE in 2000, but concerns about the risk of cardiovascular disease raised in 2007.

139139

Thiazolidinediones stimulate transcription via PPAR

transcription

peroxisome proliferatoractivated receptor-

retinoid X receptor

DR-1 sites

PPAR RXR

Rosiglitazone in ligand-binding domain (LBD)

● Endogenous ligands: Unsaturated and oxidized fatty acids, eicosanoids and prostaglandins

● Ligand binding to PPAR or RXR promotes dimerization, interaction with DNA and conformational changes in the LBD that control recruitment of transcriptional regulators

Genes involved in adipogenesis and lipogenesis e.g. GLUT4, adiponectin, etc.

140140

How do thiazolidinediones work?

↑TG storage

FFA

PPAR activationTZDs

↓ lipid accumulation↑ FA oxidation liver

↓ lipid accumulation↑ glucose uptake

muscle

↓ TNF, resistin↑ adiponectin

141141

Targets for new antidiabetic drugs?

PI3K

PTEN

PI(4,5)P2 PI(3,4,5)P3

insulinreceptor

insulin

P

IRS-1 IRS-1

P

PTP1B

PTP1B

(protein tyrosine phosphatase 1B)

(phosphatase and tensin homologue on chromosome 10)

Y

P

IRS-1 S

P

JNK

142142

obesity and insulin-resistance

Obesity and type 2 diabetes mellitus is associated with:

● expression of GLUT4 in adipose tissue

● ~ normal expression of GLUT4 in muscle liver muscle

insulin

glucose

glucose

pancreas

adipocytes

glucose

Selective knockout of GLUT4 expression in adipocytes leads to insulin-resistance in liver and muscle.

Therefore intact adipocytes must secrete something in response to glucose which improves insulin sensitivity in other tisses.

143143

adipocytes secrete “adipokines”

COO-

Free fatty acids (FFA)

Tumour necrosis factor (TNF)

ResistinAdiponectin (Acrp30)

ObesityObesity

TNF inhibits insulin signalling

Transgenic mice lacking TNF gene, or genes for TNF receptors are protected from obesity-induced insulin resistance

Adiponectin potentiates the effect of insulin

Transgenic mice lacking adiponectin are moderately insulin resistant.

Resistin (10 kDa protein) impairs insulin action in rodents, but role in humans is moot.

144144

Adiponectin shares a signalling pathway with biguanides in liver

N C

NH

NH C

NH

NH2CH3

CH3

↑[AMP]:[ATP]

Metformin (glucophage)

↓hepatic gluconeogenesis

?

Mitochondrial respiratory complex 1

AMP-activated protein kinase

(AMPK)

P

T172

LKB1

AdipoR2

Decreased expression of gluconeogenic enzyme PEPCK

Hypoxia

Metformin:

● Suppresses hepatic gluconeogenesis ● Increases insulin-stimulated skeletal

muscle glucose uptake● Reduces gastrointestinal glucose

absorption (probably by stimulating conversion to lactate)

● Lowers plasma [triglyceride] and [FFA]● Useful for obese patients because, unlike

sulfonylureas, doesn’t cause weight gain

145145

Mechanism of TNF and FFA effects – serine phosphorylation of IRS-1

TRAF-2

Fil

am

in

PKC

TNF

TNF receptor

insulinreceptor

insulin

COO-

FFA

JNK

MAP2K4

MAPKKK

OBESITY

PTBPH

Y S Y Y Y

P P P

NH3+

CO2-

S S

P

S

P

S307

IRS-1

metabolism

146146

hunger, satiation and satiety

• We all understand hunger, but satiation and satiety cause confusion.

• Satiation refers to physiological processes that promote meal termination.

• Satiation results from a coordinated series of neural and hormonal signals produced by the gut in response to a meal.

• Satiation is normally pleasant, but includes feel bloated and sick.

• Satiety refers to physiological processes that delay the start of the next meal.

147147

weight regulation is very precise

• Annual food intake is ~ 4.4GJ per person year

• 20kg gained over 10 years is about 590MJ in total, but only 1.3% excess over requirements.

There are two regulatory components:

• Hunger varies inversely with body weight

• Metabolism varies directly with body weight

If people lose weight their desire to eat more food becomes very strong, while at the same time their physical activity and basal metabolic rate decline, which conserves their remaining fat stores.

148148

achieving energy balance

Schwartz et al (2000) Central nervous system control of food intake Nature 404, 661 - 671

149149

it is very difficult to lose weight…Successful weight loss offers clear health benefits for those most seriously overweight, but...

• The multiple interlocking feedback loops conspire against the patient.

• Gastric surgery is the most effective method, but it is expensive and impractical for whole populations.

• The available drugs have limited effectiveness and significant side-effects.

• It is very difficult to maintain more than 10% cut in body weight by diet and lifestyle changes.

• There is a marked tendency to regress.

• Lifestyle changes must be permanent. One cannot diet to a target, and then return to previous habits without regaining all the weight.

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regulatory systems are redundant

• It has proved difficult to dissect the mechanisms regulating body weight using gene knockout or inhibitors because the systems are highly redundant, with cross-talk between the channels, so that individual alterations have limited effect.

• Multiple sensors around the body report total energy stores and the current state of play.

• Multiple sensors in the gut accurately report the size and composition of the current meal.

• Multiple pathways within the central nervous system process the input data and decide what must be done.

• Multiple effector systems (conscious, autonomic, hormonal) control feeding patterns, reproductive behaviour and the storage and disposal of the food previously consumed.

151151

multiple layers of control

• The enteric nervous system within the gut can organise the efficient digestion and storage of an entire meal without needing external assistance.

• The autonomic nervous system (ANS) adds a subconscious supervisory layer. There are two key regions with the brain: (1) the hypothalamus and (2) the nucleus of the solitary tract, whose main communication channel is the vagus nerve.

• The forebrain adds a layer of conscious decision making and perception, but it has limited ability to over-ride the decisions of the hypothalamus and the solitary tract.

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central control systems

153153

hindbrain details

• The solitary tract matches the GI tract, front to rear, and communicates with GI tract via cranial nerves.

• Taste information is transmitted via the facial (VII) and glossopharyngeal (IX) nerves.

• Stomach & bowel signal via the vagus nerve (X).

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vagus nerves

• The paired vagi are the longest of the cranial nerves (the latin name means “wandering”).

• Left and right branches leave the base of the skull, and run close to the oesophagus to innervate the heart, viscera and reproductive tract.

• The two vagi carry afferent sensory information about meal size and chemical composition from the gut to the brain.

• The vagi also carry efferent “motor” commands from the brain that modulate secretion of hormones and digestive juices, peristalsis and metabolism in the target organs.

• Enteric neurones manage the detailed program.

155

enteric nervous system (1)

156156

enteric nervous system (2)

• The enteric nervous system is a complex, multi-layered network, which covers the entire length of the GI tract from oesophagus to anus.

• It contains about 100 million neurones – roughly as many neurones as the spinal cord.

• It receives inputs from numerous mechanical and chemical sensors within the gut, and sends outputs to smooth muscles and glands.

• It independently regulates peristalsis, secretion of mucus and enzymes, local blood flow and responses to noxious stimuli.

157157

monitoring total fat reserves

• The key hormone is leptin, a cytokine secreted by well-filled adipocytes.

• There are leptin receptors in the arcuate nucleus in the hypothalamus, which is outside the blood – brain barrier.

• Leptin signals via the JAK/STAT pathway controlling nuclear gene expression, and also via ATP-sensitive K+ channels.

• Leptin is also required for sexual maturation and fertility.

158158

monitoring the current position

• Insulin reflects more recent food ingestion and reinforces the effects of leptin on the arcuate nucleus.

• The hypothalamus and solitary tract contain their own glucose sensors, and receive data from other glucose sensors in the pancreas, carotid bodies, and (in rodents) portal vein.

• Although hypoglycaemia causes hunger and activates the sympathetic nervous system, hyperglycaemia has little effect.

159159

the solitary tract monitors the gut

160160

there are many signals from the gut

• The signals vary with time as food passes through the gut.

• Messages reflect the quantity and quality of the ingested food.

• These signals inform the rest of the body, but also control local gut reflexes that match secretion, peristalsis and absorption to the quantity and quality of the food that has been eaten.

161161

time course for hormone secretion

Adan et al (2008) Trends in Pharmacological Sciences 29, 208-217

162162

entero-endocrine cells

• A family of entero-endocrine cells line the gut wall from the mouth to the colon, and continually “taste” the gut contents.

• These cells also receive a wide range of “gating” input signals and send a variety of neural and hormonal output signals.

• There is considerable homology between the sensing and signalling mechanisms in different regions of the gut.

• Many different output molecules encode the information being transmitted.

163163

Berner-Hansen & Witte (2008) The role of serotonin in intestinalluminal sensing and secretion. Acta Physiologica 193, 311-323.

• Serotonin (5-hydroxy tryptamine, 5-HT) is one of many transmitters that are used within the gut.

• Cells that signal using 5-HT are known as entero-chromaffin (EC) cells.• These cells are particularly common in the duodenum.• One important signalling route involves capsaicin-sensitive TRPV1

channels. (TRP=transient receptor potential)• Capsaicin is an active ingredient in curries and hot chilli sauce.

164164

PeptideMain site of synthesis

Receptors mediating feeding effects

Sites of action of peripheral peptides germane to feeding

Effect on food intake

Hypothalamus Hindbrain Vagus

CCKProximal intestinal I cells

CCK1R X X X down

GLP1Distal-intestinal L cells

GLP1R X? X? X down

OxyntomodulinDistal-intestinal L cells

GLP1R etc X down

PYY3–36Distal-intestinal L cells

Y2R X X down

Enterostatin Exocrine pancreasF1-ATPase subunit

X down

APO AIVIntestinal epithelial cells

Unknown X X down

PP Pancreatic F cells Y4R, Y5R X X down

Amylin Pancreatic cells CTRs, RAMPs X X down

GRP and NMBGastric myenteric neurons

GRPR X X down

Gastric leptinGastric chief and P cells

Leptin receptor ? ? X down

GhrelinGastric X/A–like cells

Ghrelin receptor X X X up

165165

entero-endocrine cells

Cummings & Overduin (2007) J. Clin. Invest. 117, 13-23.

166166

TRP proteins

• TRP stands for “transient receptor potential” which is a mutation first observed in Drosophila visual pathways.

• Hundreds of additional TRP proteins have since been identified, and are now grouped into five major classes, with a wide range of functions.

• Many (but not all) of them function as sensory transducers or transmembrane ion channels.

• There is a recent review by Venkatachalam & Montell (2007) TRP Channels. Ann. Rev. Biochemistry 76, 387-417

• TRPM5 is involved in taste transduction.

167167

168168

AGRP

• Agouti-related protein (AGRP) is named after a similar gene that controls hair colour in rodents.

• Powerful antagonist of the MC3R and MC4R melanocortin receptors in the hypothalamus.

• Obese patients have elevated plasma levels of AGRP, and over-expression of AGRP in animal models leads to obesity.

• Brain injections of AGRP stimulate feeding

• No known drugs in development.

169169

amylin

• Amylin is a peptide hormone co-released with insulin by pancreatic cells.

• Produces a feeling of satiation, and assists in the regulation of food intake.

• Pramlintide acetate is an amylin analogue that was licensed by the FDA in 2005

• Inconvenient to use (several injections per day) but it is an effective drug with few side effects.

170170

CART

• Cocaine and amphetamine regulated transcript (CART) is a neuropeptide precursor protein that is abundant in the hypothalamus. It is up-regulated after cocaine or amphetamine administration.

• CART-derived peptides reduce food intake when injected into the third cerebral ventricle.

• CART probably has many other functions within the central nervous system.

• It has proved difficult to identify CART receptors.

• No known drugs in development.

171171

cholecystokinin

Cholecystokinin (CCK) is secreted by entero-endocrine cells in the duodenum in response to the partially digested output from the stomach, which is called chyme. Fatty high-protein meals are particularly effective.

• CCK delays gastric emptying• stimulates pancreatic enzyme output• causes the gall bladder to contract • has a weaker incretin effect than GLP-1• produces a sensation of fullness or satiation• signals to the brain via the vagus nerve• causes satiation rather than satiety and leads to small,

frequent meals but no weight loss

172172

endocannabinoids (1)

• Anandamide and 2-arichidonoyl glycerol are “genuine” cannabinoids, which bind to CB1 and CB2 receptors.

• CB1 agonists increase food intake: “getting the munchies”.

• Oleoyl ethanolamide suppresses food intake but it is not a “real” cannabinoid. It works through PPAR- .

• Palmitoyl ethanolamide has anti-inflammatory effects.

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endocannabinoids (2)

• Cannabinoids are not stored in secretory vesicles like most neurotransmitters, but are synthesised on demand from arachidonyl phospholipids.

• Their action is terminated by cellular uptake and hydrolysis to free arachidonic acid.

• Rimonabant is a CB1 receptor antagonist, which is effective against nicotine addiction.

• Rimonabant reduces appetite and achieves significant weight loss.

• Rimonabant was never approved for use in America. The European licence was withdrawn because patients were at increased suicide risk.

174174

ghrelin

• Ghrelin is a 28-residue peptide secreted by endocrine cells within the gastric sub-mucosa.

• The hormone is released by the empty stomach and produces sensations of hunger.

• It acts on the hypothalamus to stimulate growth hormone release by the pituitary.

• It is also produced locally by neurons within the hypothalamus, and in other parts of the intestine.

• It antagonises leptin, increases metabolic efficiency and stimulates eating behaviour, resulting in weight gain.

• It is thought that the effective weight loss achieved by gastric surgery may reflect a fall in ghrelin output.

175175

glucagon like peptide 1

• Wren & Bloom (2007) Gastroenterology 132, 2116-2130.• Differential processing of preproglucagon • GLP-1 = glucagon-like peptide 1• MPGF = major proglucagon fragment• GRRP = glicentin-related pancreatic peptide

176176

insulin• Insulin reduces food intake and plays a major part in

appetite regulation.

• Gene knockout shows that lack of either brain insulin receptors or insulin receptor substrate 2 (IRS2) results in hyperphagia, obesity and female infertility.

• Insulin promotes phosphorylation of leptin receptors and JAK2, which enhances the phosphorylation of STAT3 in the presence of leptin.

• Insulin levels are often raised in type 2 diabetes which is associated with insulin resistance and obesity.

177177

leptin

• Leptin is a cytokine released by bloated fat cells and also by the stomach.

• It acts on the arcuate nucleus within the hypothalamus to suppress eating behaviour and also increases energy expenditure.

• Leptin production rises with increasing fat cell mass.

• ob/ob mice fail to make leptin and are grotesquely obese, as are db/db mice that lack the leptin receptor.

178178

Leptin

Leptin can modulate the responses from enteroendocrine cells, autonomic nervous system, the hypothalamus and the solitary tract.

179179

POMC

• Pro-opiomelanocortin (POMC) Initial transcript is cleaved to yield MSH and ACTH, transmits many other messages within the brain.

• Four distinct 7-transmembrane G-protein linked receptors.

• MC1R controls skin pigmentation.

• MC2R is the ACTH receptor.

• MC3R controls the conversion of dietary fuel into fat.

• MC4R regulates food intake and energy expenditure.

180180

MSH and MCH

• MSH is melanocyte stimulating hormone,which darkens pigmentation in lower vertebrates.

• MSH is the downstream signal from POMC / CART neurones, and suppresses food intake.

• MCH is melanocyte concentrating hormone, which lightens pigmentation in lower vertebrates.

• MCH delivers an orexigenic signal

• MCHR1 receptor antagonists reduce food intake and increase energy expenditure in animal tests, but have not yet been approved for humans.

181181

NPY

• Neuropeptide Y (NPY) is a highly conserved 36-residue peptide, widely distributed in vertebrate nervous systems.

• Many neurosecretory and cardiovascular functions.

• At least five classes of NPY receptor distributed over a wide range of tissues.

• Powerful orexigenic (appetite-promoting) signal within the hypothalamus.

• Activates a neural pathway eventually leading to the nucleus of the solitary tract.

• NPY is over-produced in obese, leptin-deficient mice.

• Leptin + NPY double knockouts are less obese, which suggests that NPY mediates the over-feeding observed in leptin-deficient animals.

182182

orexins

Orexins A & B are neurotransmitters (otherwise known as hypocretins 1 & 2) derived from a common precursor.

• Discovered in 1998 with their G-protein linked receptors OX1-Rs and OX2-Rs.

• Major role in arousal and food seeking behaviour.

• Damage to the orexin signalling system leads to narcolepsy.

183183

peptide YY• Peptide YY (PYY) is a 36-residue peptide secreted by

endocrine cells in the lower small intestine, pancreas and colon.

• It slows digestion and reduces the consumption of food.

• PYY inhibits gastric acid secretion and gastric emptying, pancreatic enzyme secretion and gut motility.

• Acts on the arcuate nucleus in the hypothalamus to suppress appetite and reduce food intake.

• Reduces food intake by 33% in obese subjects, who are normally leptin resistant.

• PYY shows sequence homology to orexigenic NPY and to pancreatic polypeptide (PPY) from F cells in islets.

184184

signalling from gut to brain

185185

hypothalamic nuclei

186186

arcuate nucleus

187187

melanocortin signalling in the arcuate nucleus.

•Leptin activates POMC neurones releasing -MSH which binds to MC4R receptors.•Ghrelin activates NPY neurones, releasing

AGRP & NPY

diagnosing the metabolic syndrome

measurementhealthy range

metabolicsyndrome

comments pathology

body mass index 18 - 25 kg/m2 > 30 kg/m2 includes muscle

abdominalobesity

waist : hip ratio (male) 0.85 > 0.9alternativemeasures for abdominalobesity

waist : hip ratio (female) 0.7 > 0.85

waist (male) < 94 cm (IDF) > 102 cm (NCEP)

waist (females) < 80 cm (IDF) > 88 cm (NCEP)

fasting blood glucose 4.5 - 5.5 mM > 7 mM ideally 5 mM type 2diabetesfasting blood insulin 5 - 10 µU/mL 20 - 40 µU/mL 1 µU/mL = 6.94 pM

fasting triglycerides 0.5 - 1.5 mM > 1.7 mM raised after meals

dislipid-aemia

total cholesterol 3.5 - 5.5 mM > 6 mM ideally 5 mM

LDL-cholesterol 2.0 - 3.0 mM > 3.3 mM "bad" cholesterol

HDL-cholesterol 1.1 - 2 mM < 0.9 mM "good" cholesterol

blood pressure (mm Hg) 120 / 80 > 140 / 90 systolic / diastolic hypertension

C-reactive protein < 3 mg/L > 3 mg/L quantifies inflammation

IDF International Diabetes Federation; NCEP National Cholesterol Education Program

waist measurement

mostly white Europeans > 50 years

Waist circumference is increasingly seen as the best indicator for the metabolic syndrome. Ideally, it should be measured early morning, after an overnight fast.

Cameron et al

(2010)

Cut-points for Waist

Circumferencein Europids and South Asians

Obesity 18, 2039–2046.

doi: 10.1038/oby.2009.45

5

Note the log – linear relationship between waist measurement and diabetes risk

191191

energy stores for a 70kg male

5mM blood glucose is only sufficient for a few minutes normal activity

body composition

[all in kg] lean man obese man comments

weight 70 100

water 42 47

protein 12 13 about 50% can be metabolised

fat 12 35 almost all can be metabolised

rest 4 5 bones, glycogen etc

The bulk of our energy is stored as fat, but this occupies relatively little space because it has a very high energy density and the water content is low. This obese man has 23kg of superfluous fat, corresponding to a three-fold expansion of the “normal” energy store, but this brings with it only 1kg of additional protein and 5kg of body water.

energy content of foods and tissuesdietarycomponent

energy densitytypicalwatercontent

effectiveenergycontentkJ/g kcal/g

dietary fibre 8 2 variable maybe 1 kJ/g

ethanol 29 7 60 - 96% wine 3.6 kJ/g

oils and fats 37 9 zero 37 kJ/g

proteins 17 4 75% ~ 4 kJ/g

refined sugar 17 4 zero 16 kJ/g

starches 17 4 25 - 95% 2 - 8 kJ/gIn practice, the energy density (and flavour!) of real foods is hugely dependent on the fat and refined sugar contents, because these have very high energy densities. Composition varies with preparation and cooking methods. The overall water content is also important.

194194

strategies for weight lossThere are three basic approaches:

1. Decrease food absorption

– Lower energy meals

– Less efficient digestion

– Smaller meals (quicker satiation)

– Less frequent meals (longer satiety)

– Appetite-suppressing drugs

2. Block lipid biosynthesis

– Carbonic anhydrase inhibitors

3. Increase food oxidation

– Less efficient metabolism

– Increased physical activity

Examples exist of all three strategies.

maintaining muscle mass• Most people on a weight loss diet want to lose adipose

tissue mass, while retaining their muscles intact.• Unfortunately, sudden withdrawal of food in humans

produces a “profligate” phase of metabolism, where the individual searches avidly for food and blood glucose is initially maintained at the expense of muscle proteins.

• It takes several days to establish the long-term fasting pattern, where triglycerides provide the major source of energy, the glycerol moiety being converted into blood glucose, while some of the fatty acids are converted into acetoacetate and hydroxybutyrate which reduce cerebral glucose requirements.

• This is why “crash” diets are generally ineffective, unless the subject sticks to them for several weeks. 195

196196

hormonal control of metabolism

hormonal effects

on

insulin glucagon / adrenalin

growth hormone

cortisol TNF – α and

IL – 1

sugars glycolysis gluconeo-genesis

gluconeo-genesis

gluconeo-genesis

glycolysis

glycogen synthesis breakdown synthesis redistribute to muscle

breakdown

proteins synthesis breakdown synthesis breakdown breakdown

fats synthesis lipolysis lipolysis redistribute to abdomen

breakdown

In addition, cortisol has immunosuppressive effects and thyroidhormones increase general activity and basal metabolic rate.

conventional wisdom• In order to avoid the adverse effects of “yo-yo dieting”

the usual recommendation is that subjects reduce their energy intake by about 500 kcals/day and aim to shed about 10% of their body weight over several months.

• It is very difficult to stick to this, and most diet studies have large drop-out rates.

• Subjects are conventionally advised to reduce their fat intake below 30% of energy intake, with no more than 10% saturated fat, and to increase their intake of fruit and vegetables (providing bulk and vitamins) while obtaining most of their energy from slowly-digested carbohydrates with a low glycaemic index.

• This approach leaves many people feeling ravenously hungry and bad-tempered almost all the time.

197

the heretics• The problem with the conventional wisdom is that fats

provide much of the flavour and texture in foods, and many satiation mechanisms are largely based on fat.

• People also point out the application of the conventional wisdom has coincided with the largest obesity epidemic that the world has ever seen.

• An alternative school of thought recommends a high-fat, high-protein, low-carbohydrate diet. Although commonly associated with Dr Atkins, this approach is actually over a century old.

• High-fat, low-carb diets trigger the physiological satiation systems, so the subjects do not feel hungry and the diets can be eaten ad lib.

• Reduced need for insulin may help diabetics. 199

Mediterranean diets• Epidemiological studies show that many Mediterranean

countries have relatively low rates of diabetes and cardiovascular disease.

• This has stimulated interest in Mediterranean diets, which allegedly have a high content of raw vegetables, nuts, olive oil and red wine.

• Olive oil is mostly monounsaturated, but contains some polyunsaturates as well.

• The nuts contain a good mixture of nutrients and are also slow to digest.

• The red wine contains anti-oxidants, and is also known to raise HDL cholesterol.

200

so which diet is best?• The scientific dispute has taken on an almost religious

ferocity, with prophets of doom on both sides.• Volunteers are often reluctant to stick to their diets and

very few studies have continued for long enough to draw reliable conclusions.

• It is almost impossible in this area to achieve the gold standard of a “randomised, prospective, double-blind cross-over design”.

• Many studies examine “surrogate end points” which are easily and cheaply measured, but the only real test is to wait for several years and record whether the subjects are alive or dead.

• There has been considerable interest in an important study by Shai et al (2008) NEJM 359, 229- 241. 201

202202

low fat diet details

• The low-fat, restricted-calorie diet was based on American Heart Association guidelines.

• They aimed at an energy intake of 1500 kcal per day for women and 1800 kcal per day for men, with 30% of calories from fat, 10% of calories from saturated fat, and an intake of 300 mg of cholesterol per day.

• The participants were counselled to consume low-fat grains, vegetables, fruits, and legumes and to limit their consumption of additional fats, sweets, and high-fat snacks.

203203

Mediterranean diet

• The moderate-fat, restricted-calorie, Mediterranean diet was rich in vegetables and low in red meat, with poultry and fish replacing beef and lamb.

• They restricted energy intake to 1500 kcal per day for women and 1800 kcal per day for men, with a goal of no more than 35% of calories from fat; the main sources of added fat were 30 to 45 g of olive oil and a handful of nuts (five to seven nuts, <20 g) per day.

• The diet is based on the recommendations of Willett and Skerrett.

204204

low carbohydrate diet

• The low-carbohydrate, non–restricted-calorie diet aimed to provide 20 g of carbohydrates per day for the 2-month induction phase and immediately after religious holidays, with a gradual increase to a maximum of 120 g per day to maintain the weight loss.

• The intakes of total calories, protein, and fat were not limited.

• Participants were counselled to choose vegetarian sources of fat and protein and to avoid trans fat.

• The diet was based on the Atkins diet.

205205

Which is the best weight-loss diet?

206206

low carbs beats low fat

more on lifestyle...

• Two papers were recently published online before print which seem highly relevant to the prevention and treatment of type 2 diabetes:

• Uusitupa et al (2010) Impact of positive family history and genetic risk variants on the incidence of diabetes – the Finnish Diabetes Prevention Study. Diabetes Care doi:10.2337/dc10-1013

• Salas-Salvadó et al (2010) Reduction in the Incidence of Type 2-Diabetes with the Mediterranean Diet: Results of the PREDIMED-Reus Nutrition Intervention Randomized Trial. Diabetes Care doi:10.2337/dc10-1288

• Paste the doi into http://dx.doi.org/ to see the abstracts.

207

Uusitupa et al• Studied the effects of family history of diabetes and 19 known

genetic risk loci on the effectiveness of lifestyle changes.

• Subjects with impaired glucose tolerance were randomized into control (n=257) and intervention (n=265) groups. The mean follow-up was 6.2 years.

• Lifestyle intervention, aimed at weight reduction, healthy diet and increased physical activity, lasted for 4 years (range 1-6 years).

• Family history and genetics had no measurable effect on the outcome.

• The only factor with a significant effect was the lifestyle intervention (hazard ratio 0.55, 95% confidence limit 0.41 – 0.75, p<0.001)

• These results emphasize the effectiveness of lifestyle intervention in reducing the risk of diabetes in high risk individuals independent of genetic or familial risk of type 2 diabetes.

208

Salas-Salvadó et al• 418 nondiabetic subjects aged 55-80 years at high cardiovascular risk

were randomized to education on a low-fat diet (control group) or one of two Mediterranean diets, supplemented with either free virgin olive oil (1 litre/week) or nuts (30 g/day).

• Diets were ad libitum and no advice on physical activity was given.

• After a median follow-up of 4.0 years, diabetes incidence was 10.1% in the Mediterranean-diet with olive oil group, 11.0% in the nuts group, and 17.9% in the control group.

• Diabetes incidence was 52% lower in the Mediterranean groups.

• In all study arms, increased adherence to the Mediterranean-diet was inversely associated with diabetes incidence.

• Diabetes risk reduction occurred in the absence of significant changes in body weight or physical activity.

• Mediterranean diets without calorie restriction appear to be effective in the prevention of diabetes in subjects at high cardiovascular risk. 209

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childhood obesity

• There is great concern about increasing obesity among children, and the likely continuation into adult life.

• The Avon Longitudinal Study has followed a large cohort of parents and children living near Bristol from birth.

• Ness et al (2007) PLoS Medicine 4(3), e97 conclusively demonstrated that obesity was directly linked to physical activity quintiles in boys and girls aged 12.

211211

thermogenesis in brown fat

212212

Year Drug Mechanism Side effects

late 19th cent. Thyroid hormone Increases metabolic rate Hyperthyroidism

1920s Dinitrophenol Mitochondrial uncouplingCataracts, neuropathy, cardiac failure

1930sAmphetamines (phentermine, diethylpropion, phendimetrazine)

Dopamine–noradrenaline-reuptake inhibitor, releaser, sympathomimetic drugs

Addiction, myocardial infarction, stroke

1950s Phenylpropanolamine Sympathomimetic Stroke

1960sRainbow pills (mixture of digitalis, amphetamine and diuretics)

Mixed Fatalities: digitalis has a narrow therapeutic index

1990sFen-phen (mixture of fenfluramine and phentermine)

5-HT-reuptake inhibitor and releasing agent with sympathomimetic

Cardiac valvulopathy

Recently withdrawn

Rimonabant Cannabinoid CB1 receptor antagonist Depression, anxiety

Sibutramine 5-HT–noradrenaline-reuptake inhibitor Tachycardia, hypertension

Rosiglitazone Thiazolidinedione – PPAR- Heart failure

Currently usedOrlistat Gastric lipase inhibitor

Flatulence and diarrhoeaAcarbose Amylase inhibitor

[epilepsy] TopiramateAntiepileptic drug targeting multiple proteins, carbonic anhydrase inhibitor

Memory impairment, depressive symptoms

[depression] Fluoxetine 5-HT-reuptake inhibitor Nausea, diarrhoea

[ADHD] Atomoxetine Noradrenaline-reuptake inhibitor Dry mouth, palpitations

[epilepsy] ZonisamideAntiepileptic drug targeting multiple proteins, carbonic anydrase inhibitor

Memory impairment

[depression and smoking]

Bupropion Dopamine–noradrenaline-reuptake inhibitor Dry mouth, insomnia

213213

drugs that work• In contrast to the generally disappointing results from

appetite suppressants -

• ACE inhibitors and angiotensin receptor antagonists routinely deliver substantial reductions in blood pressure and improved survival

• Diuretics such as Frumil (furosemide + amiloride) reduce central venous blood pressure after heart failure

• HMG-CoA reductase inhibitors “statins” deliver roughly 10% fewer adverse vascular events for each 0.65mM reduction in LDL-C. [Delahoy et al (2009) Clinical Therapeutics 31, 236 – 244 provide a meta-analysis of 155,613 subjects, 6321 vascular deaths, 23791 major vascular events, 11357 coronaries and 4717 strokes]

214214

Ludman et al (2009) Pharmacology & Therapeutics 122, 30–43.

215215

cholesterol turnover in the body

216216

regulating HMG-CoA reductase

Espenshade & Hughes (2007) Regulation of Sterol Synthesis in Eukaryotes. Annu. Rev. Genet. 41, 401–27

• SREBP – sterol regulatory element binding protein (helix – loop – helix / leucine zipper)

• Insig – insulin induced gene (resident ER protein, binds oxysterols involved in bile acid synthesis)

• Scap – SREBP cleavage activating protein (binds cholesterol)

• S1P – site one protease (Golgi)

• S2P – site two protease (Golgi)

217217

In the absence of sterols, HMGR does not bind Insig and is present at high levels. In the presence of lanosterol or oxysterols, Insig mediates the ubiquitinylation (Ub) and subsequent degradation of HMGR by the proteasome through interactions with the E2 conjugating enzyme Ubc7, the E3 ubiquitin ligase gp78, and the ATPase VCP/p97. Geranylgeraniol (GG-OH) enhances HMGR degradation through an unknown mechanism that acts downstream of ubiquitinylation.

218218

219219

drugs for obesity, diabetes & cardiovascular diseaseexample type principal targets typical indication

ramipril ACE inhibitor arteriolar smooth muscle hypertension

valsartan AR blocker arteriolar smooth muscle hypertension

metoprolol -blocker cardiac muscle hypertension

nifedipine Ca++ antagonist cardiac & smooth muscle hypertension

frumil diuretic kidney tubules (loop of Henle) heart failure

nitroglycerine organic nitrate venous smooth muscle angina

aspirin NSAID non-specific COX1 & COX2 old age?

clopidogrel anti-platelet platelet ADP receptor atherosclerosis

heparin anti-coagulant vascular endothelium atherosclerosis

warfarin anti-coagulant liver (clotting factor synthesis) atherosclerosis

streptokinase clot dissolution blood clots acute MI

lovastatin statin liver HMG-CoA reductase dislipidaemia

gemfibrozil fibrate PPAR- in many tissues dislipidaemia

metformin biguanide liver AMPK type 2 diabetes

exenatide incretin pancreatic -cells type 2 diabetes

glipizide sulphonylurea pancreatic -cells type 2 diabetes

pioglitazone thiazolidinedione PPAR- in many tissues type 2 diabetes

acarbose amylase inhibitor small intestinal lumen type 2 diabetes

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obesity, diabetes and cardiovascular disease

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obesityrelated diseases

tackling obesity

different diseases

obesity epidemic

passive obesity

common cause90 heart

common factor

loss of life expectancyhere