DIABETES : THE GLUCOSE METABOLIC DISEASE

1. DIABETES : THE GLUCOSE METABOLIC DISEASE Class 7 Dr. Pittler

2. Diabetes (or diabetes mellitus) is a disease that has been known for millennia. The complete name has both Greek and Latin origins and was coined by Aretaeus, a physician of the 2nd century : diabetes - diabenein - “to pass through”. It refers to the constant urination of its victims. mellitus – a Latin adjective meaning “sweet” and refers to the high sugar content of the urine. It will be seen that urination in diabetes is a compensatory mechanism to rid the body of high levels of blood glucose.

3. Symptoms associated with diabetes (particularly type 1): • Polyuria – frequent urination. This occurs as an effort of • the body to decrease levels of blood sugar. • Polydipsia – excessive thirst. This is the compensatory • mechanism for polyuria and another way to • lower blood sugar. • Weight loss – involuntary weight loss can occur from a • failure to retain fat storage and a loss of • muscle tissues. • Chronic hunger – continued hunger due to a need of • the starved body to replenish lost • cells and tissues.

4. Some conditions that may develop as a result of diabetes: • Damage to or loss of limbs or organs – This • includes hands and feet; nerves, cardiac • tissues, bones, and kidneys. All of this is • related to damage/loss of blood vessel • walls. • Decrease or loss of sight - This is a result of blood • vessel damage and other factors to be • discussed. • Metabolic acidosis – This occurs with the production • of excessive amounts of ketone bodies.

5. In general, it can be said that diabetes is a disease that deals with disorders of glucose metabolism as well as toxic effects of excessive levels and insufficient levels of glucose both inside and outside of cells. At present the disease is divided into two major types: Type 1 and type 2. Type 1 was also known as: insulin dependent diabetes mellitus (IDDM) and juvenile onset diabetes. Type 2 was also known as mature onset diabetes. The lines between those distinctions have become less distinct in recent years – as you will see. That means that some of the properties of type 1 can be seen in type 2 and vice versa.

6. NORMAL GLUCOSE UPTAKE INTO CELLS Glucose must be taken up into cells before metabolism can begin. The mechanism by which this occurs is called facilitated diffusion and requires a transport protein known as GLUT. Presently, there are nearly a dozen different GLUT proteins. However, the main concern here focuses on GLUT-4 which is dependent on the hormone insulin.

7. The pancreas is shown on the right with its association with the duodenum. The islets of the pancreas contain three kinds of cells that produce hormones that affect glucose blood levels and the uptake of glucose into cells. Alpha cells produce glucagon. Beta cells synthesize insulin. Delta cells make soma- tostatin.

8. The three peptide hormones are shown below: Insulin is a dipeptide held together by disulfide bridges. Its principal function is to increase the overall rate of metabolism and one way that it does this is to increase the availability of GLUT-4 proteins on a cell surface. Glucagon is a peptide that increases blood glucose by causing the liver to break down glycogen & release glucose into the circulation. It also brings about increased synthesis of insulin. Somatostatin inhibits the release of insulin and glucagon – maintaining a steady state of glucose uptake.

9. Our principal concern here is how insulin acts to influence the uptake of glucose in those cells that depend upon it. The grey areas in the right hand figure are all areas that bind to the receptor protein on the cell. Cells that require insulin for glucose uptake include: muscle, adipose, and blood vessel endothelial cells. Those cells that are not insulin dependent include: brain, liver and notably-lens fiber cells.

10. When insulin binds to its receptor protein, it induces a conformational shift in the beta subunits of the receptor [as seen on the right]. The shift converts the beta subunit into an active kinase enzyme that adds phosphate to a nearby protein (IRS-1). This initiates a cascade mechanism within the cell that results in the delivery of GLUT-4 proteins to the plasma membrane of the cell. Understanding how this cascade works is crucial to understanding how one type of diabetes operates.

11. The cascade begins with the phosphorylation of the insulin receptor substrate (IRS-1). It continues with the repeated phosphorylations and activations of several enzymes that ultimately cause the release of GLUT-4 from a subcellular vesicle as seen below (blue arrow). Not all of the intermediate enzymes have been described.

12. TYPE 1 DIABETES is the form of diabetes in which the beta cells in the islets of the pancreas are destroyed. This effectively removes any source of insulin from the body. The destruction of the islet cells occurs as the result of an autoimmune attack on these cells. One theory explains this destruction to be due to the resemblance of the cell surface membranes to the structure of some viral coat proteins. The term “juvenile diabetes” is not longer used since the destruction can occur later in life. The term “insulin dependent diabetes” (although true) is not used either since some type 2 diabetics may become insulin dependent. The loss of insulin in the body also affects other functions such as lipid and protein synthesis. A genetic component (on chromosome 6) may be involved in the autoimmune problem.

13. It is important to emphasize again that two phenomena occur with the loss of insulin: starvation of insulin dependent cells and glucose toxicity in non-insulin dependent cells. Glucose toxicity can also occur to proteins that are extra-cellular. The emergency that occurs to insulin dependent cells: Cells that can no longer obtain glucose replace that metabolite with breakdown products from fatty acids and ultimately convert them to acetyl CoA. In the process, byproducts of the conversion appear as ketone bodies. These are produced when the supply of acetyl CoA cannot be used quickly enough. Acetoacetate and beta- hyroxybutyrate decrease the body pH (acidosis) and the condition may lead to coma and death. Acetone may be detected on the breath of severe diabetics.

14. The toxicity of high levels of glucose, as already shown, may activate the polyol pathway in some cells – particularly lens fiber cells – and lead to their osmotic destruction. High levels of glucose can also bring about binding of glucose to proteins, a reaction known as glycation. The initial reaction, shown below, causes an irreversible ketimineformation that goes on to more complex formations. LATER GLYCATION STAGES: Note that crosslinking occurs. AGE= advanced glycation end product. Schiff base Amadori product

15. TYPE 2 DIABETES may have more than one cause and is often associated with older individuals. One cause is due to an insufficient number of functional insulin receptors. Another cause is a failure of the cascade mechanism. The latter may be due to “defective” proteins in the cascade that may be insufficient in amount, mutated or denatured. The partial failure of glucose to be taken up under these conditions is called insulin resistance. The amount of insulin present (at least initially) is normal. Later it is possible that the pancreas begins to fail in its output of insulin, but usually this does not require insulin injections. Obesity is often associated with this form of diabetes. The cause is not known, but may be associated with a fat cell product: TNFalpha (tumor necrosis factor alpha). This cytokine has been shown to inhibit insulin receptor autophosphorylation.

16. TNFalpha is a large protein with a molecular weight of 51 kD. It has three polypeptide chains and a majority of beta pleated sheet structure. This protein cytokine has multiple known roles such as cell death (apoptosis) and extensive participation in enzyme pathways involving kinase enzymes. Consequently, its direct participation in type 2 diabetes must be viewed with some caution. tumor necrosis factor alpha The hyperglycemia (elevated blood levels) of type 2 diabetes takes longer to develop than type 1. The type 1 features such as decreased insulin production and elevated ketone bodies are generally less severe vs. type 1. It is usually, but not always, possible to control type 2 with diet (= weight control) alone. Another condition, called pre-diabetes, also exists in which blood glucose levels are borderline for diabetes and pre-diabetes may develop into type 2 diabetes.

17. LABORATORY TESTS FOR DIABETES: Some common laboratory tests for diabetes include the glucose tolerance test and assays for hemoglobin HbA1c. In the glucose tolerance test (which may be run for diabetes, insulin resistance, or reactive hypoglycemia), a pateint fasts for 8-14 hours. Then he/she blood is drawn for the fasting level (zero time). In the next step, the patient drinks a glucose solution and has their blood drawn for various times up to 5 to 6 hours. The most crucial time is considered to be 2 hours (see blue arrow) in which the diabetic has levels approaching 200 mg/ dL.

18. The test for levels of hemoglobin HbA1c depends on the glycation of the beta chain of hemoglobin at valine near the N-terminal end of the polypeptide chain. This test monitors this glycation during the average 120 day life span of red blood cells. It reflects blood glucose levels without the fluctuations that occur before and after meals. It also gives an indication of how well a patient is responding to insulin therapy. There are several methods to make this determination. One of them, an automated method, is shown below: oxidase peroxide Hb hydrolysis Val-glucose H2O2 color assay The valine-glucose (released from HbA1c) is oxidized to release hydrogen peroxide and a color reaction for the peroxide is run. Typical results: % HbA1c blood glc.(mg/cL) 5 97 6 126 7 154 8 183 9 212 10 240 11 269 12 298 In the results shown on the left, values of 5 through 7 would indicate good control of blood glucose. Values of 8-12 shows that there is deteriorating insulin control. This would indicate to the doctor a need to adjust the insulin dosage. The % of HbA1c are shown with the equivalent blood sugar levels.

19. SUMMARY: Your understanding of glucose uptake mechanisms and diabetes is essential to the disease processes that occur with ocular diabetes so this is an important lecture. • What is the significance of urination and excessive thirst in diabetes? • Can you name some of the conditions that may develop as a result of having • diabetes? • *What are the differences between type 1 and type 2 diabetes? • *What is a GLUT protein and why is it important for insulin dependent cells? • *What are the 3 pancreatic hormones made in the pancreas and why are they • important for glucose uptake and the maintenance of blood glucose? • *Besides glucose uptake, what other roles does insulin have for metabolism? • *Can you describe the process that occurs when insulin binds to its receptor all the • way up to the translocation of GLUT-4? • *What might cause type 1 diabetes? ….type 2 diabetes? • *What are ketone bodies and what effects might they have in type 1 diabetes? • *What is glycation and what effect does glycation have on proteins in the diabetic • state? • *What is TNFalpha and how might it be related to type 2 diabetes? • *What are 2 analytical procedures that may be used for diabetic patients?

20. OCULAR DIABETES& GALACTOSEMIA

21. GENERAL CONSIDERATIONS: The diabetic has three areas in his/her eye that may suffer from the consequences of the disease (shown below). Blood vessels of the retina may fail and produce partial or total blindness. The lens fiber cells may be destroyed and initiate cataract formation. The cornea is the least vulner- able of the three tissues, but even this tissue is subject to changes in corneal volume and nerve desensitization. Galactosemia (the inability to convert galactose to glucose) is another metabolic disease in which cataract formation may take place. CORNEA LENS RETINA

22. The Lens: To some extent, consideration of diabetic (or metabolic) cataract formation has already been made. That story deals with the formation of osmotic intermediates by way of the polyol pathway (shown below). In the pathway, the inter- mediate (sorbitol) is synthesized following the activation of aldose reductase. Sorbitol, an osmotic intermediate, remains in the lens for unsatisfactory periods of time due to the slow rate at which polyol dehydrogenase catalyzes the formation of fructose. You recall that lens fiber cells are subject to this problem since lens fiber cells are not insulin dependent.

23. Still another source of osmotic polyol intermediates related to cataract formation comes from the sugar: galactose. The disease known as galactosemia occurs in which one of three enzyme defects can lead to an accumulation of galactose and galactitol within lens fiber cells. Galactosemia is a comparatively rare disease (1:60,000 in one of the three forms). Infants that are affected typically have lethargy, vomiting, diarrhea and jaundice at birth. In the classic form of the disease, death can occur within days and mental retardation is common unless treated. The diagnosis is made by assay for one of the deficient enzymes. The disease is treated by withholding all sources of galactose and lactose (a galactose-glucose dimer). It should be noted that this disease is not a form of lactose intolerance in which there is a deficiency of the enzyme lactase. The latter is a disease that causes significant GI upsets, but does not have the serious consequences that galactosemia does.

24. The pathway on the right shows how galactose is converted to glucose 1-P (then to glucose 6-P) to enter the Embden- Meyerhoff pathway. Three enzymes: galacto- kinase (1); uridyl trans- ferase (2);and UDP-Gal epimerase (3) have been shown to be deficient in the known three forms of the disease. Enzymes 1 and 3 represent the lesser deficiencies while enzyme 2 (also called GALT) deficiency forms the so-called “classic form” of the disease. In general, deficiencies of enzyme 1 and 2 result in an accumulation of galactose and galactose 1-phosphate. The compound uridyl (uridine) is a co-substrate “handle” with which to transfer galactose and glucose. galactokinase uridyl transferase UDP-gal epimerase

25. THE POLYOL PATHWAY IN GALACTOSEMIA In the lens, the polyol formed from galactose is galactitol. This polyol is, essentially, a substrate that is not catalyzed by polyol dehydrogenase. As a result of this, there is much more galactitol accumulated in the lens causing earlier and more intense osmotic pressure. It has been shown that galactose cataracts are more quickly formed by deficiencies of enzymes 1 and 2 in galactosemia. HO HO GALACTOSE GALACTITOL NO PRODUCT

26. THE CORNEA Diabetes can cause three documented effects in the cornea: 1) decreased epithelial adhesion to the corneal stroma 2) a loss of neural sensitivity 3) increased cornel thickness in the stroma. The adhesion of the basal epithelial cells to the stroma is not usually apparent in diabetes until corneal surgery is performed. This implies that the adhesion is weakened in diabetes and separation occurs with trauma. The damaging effect then can be defined as failure to replace the collagen fibers (type VII collagen) of those that were damaged in surgery. There is also a suggestion that glycation of the fibers present prevent satisfactory repair. The condition is known clinically as recurrent corneal erosion (RCE).

27. The neuropathy that occurs in the corneal in diabetics (type 1 and insulin-deficient type 2) involves a loss of corneal sensitivity. The • exact cause of this is uncertain, but evidence has suggested either • the formation of AGEs at the nerves; or • a degeneration of the nourishing Schwann cells (surrounding the nerves) due to their insulin dependence. • Swelling of the cornea in diabetes is generally not marked, but may • affect the ability to wear contact lenses. The exact cause may be • related to a decrease in Na,K-ATPase activity. For example, corneal • endothelial cells exhibit the polyol pathway and may produce • sorbitol in type 1 (or 2) diabetics. Sorbitol is also able to inhibit • Na,K-ATPase rather strongly (remember that ouabain has a sugar • group that binds to the site for K+ on this enzyme). One study of • 1000 patients indicated an average increase in central corneal • thickness of ~5% in diabetics.

28. THE RETINA Diabetic effects on the retina are always serious since they involve the partial or complete, irreversible loss of sight. The effects involve damage to the retinal blood vessels and the condition in known as diabetic retinopathy. This damage is not unique to the retina as it also may involve blood vessels in the brain, limbs, kidneys and other parts of the body.

29. This is a fundus photograph of a retina in which the patient has an advanced stage of diabetic retino- pathy. Visible here are marked signs of blood vessel damage as seen by microaneurisms (1), aneurisms (2) and gross bleeding into the retina (3). An aneurism is a swelling of a blood vessel that may continue toward its breakage. 1 2 3

30. This picture is a close up showing better details of vessel damage. The swellings or aneurisms precede vessel breakage and loss of blood. They occur as a result of weakening of the tissues that surround the vessel including pericytes and collagen loss. Note: attempts to replace damaged vessels can be seen in “acellular” capillaries that form anastomoses (blue arrow) or outlets.

31. Here is a cross section of a typical blood vessel coming from the central retinal artery. The vessel has three components: 1) an endothelial cell that forms the inner lining and wall of the vessel lumen; 2) a pericyte(literally: a “surrounding cell”) that partially or completely encircles the endothelial cell. 3) basement membrane composed of collagen. The pericyte and the basement membrane are “support” tissues that help the blood vessel retain its integral strength. Their loss greatly weakens the vessel.

32. There are 5 biochemical hypotheses that offer explanations to the cause of blood vessel loss in diabetic retinopathy: • the osmotic effect of polyol formation (in the pericytes). •  in diacylglycerol that causes the synthesis of endothelin-1 •  in mitogen activated protein kinasethat  endothelin-1 • glycation of proteins (affecting vessel collagens) • oxidation that also  glycation of proteins

33. Hypothesis 1: The polyol pathway…Although the polyol pathway no longer seemed to be an attractive mechanism for diabetic retinopathy a few years back, new evidence suggests that it now may be so. What is lacking is an ARI (aldose reductase inhibitor) that is effective. It is quite clear that AR and the polyol pathway operate in both blood vessel endothelial cells and pericytes in the retina. It is also thought that the pathway exists in Müller cells that support the neural retinal photoreceptors. One investigator has stated that the presently available ARIs were not strong enough and when boosted would protect against cellular damage in the retina if only the well-known side effects of these agents could be prevented. So the future in prevented diabetic retinopathy may lie in the discovery of a safe inhibitor.

34. Hypothesis 2: diacylglycerol and endothelin-1….An intermediate offshoot from the E-M pathway is a 3-carbon compound esterified to fatty acids called diacyglycerol. This substance activates protein kinase C which, in turn, brings about a gene induction for the synthesis of the protein endothelin-1 (see pp. 118-119 in your text). Endothelin-1 promotes blood vessel occlusion (blockage). Since pericytes have receptors for endothelin-1, it is possible that this peptide may induce blood vessel blockage only leading to leakiness as a secondary effect. Hypothesis 3: MAP kinase and endothelin-1….This is a variation of hypotheis 2 in which mitogen stimulated protein kinase (MAP kinase) also stimulates the synthesis of endothelin-1. The stimulation of MAP kinase activity has been shown to occur in diabetes and this enzyme does bring about endothelin-1 synthesis.

35. Hypothesis 4: glycation of proteins….does occur in the diabetic state as shown previously. This hypothesis is compelling inasmuch as binding to and denaturing blood vessel collagens produces effects such as: vessel blockage; vessel leakage; vessel dilation; vessel thickening as well as apoptosis. Hypothesis 5: oxidation and glycation of proteins…This is a variation of hypothesis. It simply adds the contribution of molecular oxygen as a catalytic agent (not an enzyme!) to increase the process of forming AGEs (advanced glycation end products). This variation has been somewhat controversial without good supporting evidence being shown. The polyol pathway (hypothesis 1) and the direct glycation of collagens (hypothesis 4) seem, at present, to be the best biochemical explanations for diabetic retinopathy.

36. SUMMARY QUESTIONS FOR STUDY: • What do diabetes and galactosemia have in common to explain cataract formation & what do they not have in common? • Why are lactose intolerance and galactosemia not the same disease? • What enzyme produces the “classic” form of galactosemia? • What is a recurrent corneal erosion and what collagen type is involved with this phenomenon? • Can you explain how neuropathy might develop in diabetes? • What is important about sorbitol in corneal swelling in the diabetic state? • Why might aneurisms occur in retinopathy with diabetes and what • are aneurisms? • What anatomical components of retinal blood vessels are affected by • diabetes? • Why have ARIs not been able to cure or prevent retinopathy in • diabetes if the polyol pathway is present in cells of retinal blood vessels? • How might diacylglycerol and endothelin-1 contribute to diabetic • retinopathy?