1. Pathophysiology of Complications of Diabetes
Robina Rana
PGY - 4 Endocrinology
28th September, 2011

2. OBJECTIVES: To understand the pathophysiology of
1--Acute complications of DM due to severe
hyperglycemia( Diabetic Ketoacidosis and Hyperglycemic
Hyperosmolar non ketotic state.)
2--Chronic complications of DM due to hyperglycemia
including micro vascular and macro vascular complications.
To gain an understanding of the mechanisms that lead to glucose induced vascular damage.
To appreciate that prevention of vascular complications is possible through frequent screening , tight glycemic control and control of other risk factor for vascular disease.

3. Overview of carbohydrate metabolism showing the major pathways and end products.

5. The extracellular supply of glucose is primarily regulated by two hormones:
Insulin
Glucagon.

7. Pathophysiology of Type 1 Diabetes:
Our understanding of the pathophysiology of diabetes is evolving.
Type 1 diabetes has been characterized as an autoimmune-mediated destruction of pancreatic β-cells. The resulting deficiency in insulin also means a deficiency in the other co secreted and co located β-cell hormone, amylin.
As a result, postprandial glucose concentrations rise:
due to lack of insulin-stimulated glucose disappearance,
poorly regulated hepatic glucose production, and
increased or abnormal gastric emptying following a meal.

8. Temporal model for development of type 1 diabetes.
Individuals with a genetic predisposition are exposed to an immunologic trigger that initiates an autoimmune process, resulting in a gradual decline in beta cell mass. The downward slope of the beta cell mass varies among individuals and may not be continuous. This progressive impairment in insulin release results in diabetes when 80% of the beta cell mass is destroyed. A "honeymoon" phase may be seen in the first 1 or 2 years after the onset of diabetes and is associated with reduced insulin requirements. [Adapted from Medical Management of Type 1 Diabetes, 3rd ed, JS Skyler (ed). American Diabetes Association, Alexandria, VA, 1998.]

9. Pathophysiology of Type 2 Diabetes:
Early in the course of type 2 diabetes, postprandial β-cell action becomes abnormal, as evidenced by the loss of immediate insulin response to a meal .
Peripheral insulin resistance coupled with progressive β-cell failure and decreased availability of insulin, amylin, and GLP-1 contribute to the clinical picture of hyperglycemia in diabetes.

10. Metabolic changes during the development of type 2 diabetes mellitus
Insulin secretion and insulin sensitivity are related, and as an individual becomes more insulin resistant (by moving from point A to point B), insulin secretion increases. A failure to compensate by increasing the insulin secretion results initially in impaired glucose tolerance (IGT; point C) and ultimately in type 2 DM (point D). (Adapted from SE Kahn: J Clin Endocrinol Metab 86:4047, 2001; RN Bergman, M Ader: Trends Endocrinol Metab 11:351, 2000.)

12. Acute Complications of Diabetes:
Diabetic Ketoacidosis
Hyperosmolar hyperglycemic state
(HHS, also called non ketotic hyperglycemia)

13. Chronic Complications of Diabetes:
Microvascular disease
Nephropathy
Retinopathy
Neuropathy
peripheral
Autonomic
Macrovascular disease
Coronary artery disease
Cerebrovascular disease
Peripheral vascular disease
Associated complications
Foot ulcers
Infections

14. Epidemiology
Diabetic ketoacidosis is characteristically associated with type 1 diabetes. It also occurs in type 2 diabetes under conditions of extreme stress such as serious infection, trauma, cardiovascular or other emergencies, and, less often, as a presenting manifestation of type 2 diabetes, a disorder called ketosis-prone diabetes mellitus.

15. Pathophysiology:
The basic mechanism underlying both DKA and HHS is reduction in the net effective action of circulating insulin, with concomitant elevation of counter regulatory hormones, primarily glucagon, but also catecholamine, cortisol, and growth hormone .
The deficiency in insulin (absolute deficiency, or relative to excess counter regulatory hormones) is more severe in DKA compared to HHS. The residual insulin secretion in HHS is sufficient to minimize ketosis but does not control hyperglycemia

16. Biochemical defect in diabetes:
Loss of Insulin stimulated uptake of glucose by a hormone dependent glucose transporter  Hyperglycemia
Insulin deficiency activates hormone dependent lipase  Increased lipolysis
This leads to conversion of triglycerides to free fatty acids and glycerol
In absence of insulin , unrestrained lipolysis occurs
Fatty acids are converted to acetyl CoA which is shuttled into
1) Krebs cycle( insulin dependent)
2)ketones bodies (without insulin) including BHB and acetoacetate.

17. Hormonal alterations in DKA and HHS result in hyperglycemia by their impact on three fundamental processes in glucose metabolism
Impaired glucose utilization in peripheral tissues
Increased gluconeogenesis (both hepatic and renal)
Increased glycogenolysis

18. Insulin deficiency and/or resistance promote hepatic gluconeogenesis by two mechanisms:
increased delivery of gluconeogenetic precursors (glycerol and alanine) to the liver due to increased fat and muscle breakdown ;
increased secretion of glucagon by removal of the inhibitory effect of insulin on glucagon secretion and the glucagon gene.

20. General features of hyperglycemia-induced tissue damage.

21. Tissue damaging effects of hyperglycemia?
Damage to a particular subset of cell types:
Capillary endothelial cells in the retina,
Mesangial cells in the renal glomerulus.
Neurons and Schwann cells in peripheral nerves.

22. Most cells are able to maintain a constant intracellular glucose level when they are exposed to hyperglycemia.
In contrast cells damaged by hyperglycemia are those that cannot do this efficiently.
Thus diabetes selectively damages cells like endothelial cells and mesangial cells , whose glucose transport rate does not decline rapidly as a result of hyperglycemia, leading to high glucose inside cells.

23. This is important, because it tells us that the explanation for what causes complications must involve mechanisms going on inside these cells, rather than outside.

24. Mechanisms of Complications
Although chronic hyperglycemia is an important etiologic factor leading to complications of DM, the mechanism(s) by which it leads to such diverse cellular and organ dysfunction is unknown.
At least four prominent theories, which are not mutually exclusive, have been proposed to explain how hyperglycemia might lead to the chronic complications of DM.
An emerging hypothesis is that hyperglycemia leads to epigenetic changes in the affected cells

25. Increased flux through the polyol pathway.
The polyol pathway, focuses on the enzyme aldose reductase. Aldose reductase normally has the function of reducing toxic aldehydes in the cell to inactive alcohols. When the glucose concentration in the cell becomes too high, aldose reductase also reduces that glucose to sorbitol, which is later oxidized to fructose. In the process of reducing high intracellular glucose to sorbitol, the aldose reductase consumes the cofactor NADPH . But NADPH is also the essential cofactor for regenerating a critical intracellular antioxidant, reduced glutathione. By reducing the amount of reduced glutathione, the polyol pathway increases susceptibility to intracellular oxidative stress.

26. Hyperglycemia increases flux through the polyol pathway
Hyperglycemia increases flux through the polyol pathway. From Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820, 2001.

27. Complications due to increased sorbitol: decreased myoinositol and competition for nitric oxide synthase >>>>> diabetic cataracts nerve conduction abnormalities

28. Some of the pathogenetic mechanisms responsible for the development of microvascular complications in diabetes (diffuse neuropathy).

29. Intracellular production of AGE precursors.
Increased intracellular glucose leads to the formation of advanced glycosylation end products (AGEs), which bind to a cell surface receptor, via the nonenzymatic glycosylation of intra- and extracellular proteins.
They damage cells by three mechanism:
The first mechanism, is the modification of intracellular proteins including, most importantly, proteins involved in the regulation of gene transcription .

30. 2. The second mechanism, is that these AGE precursors can diffuse out of the cell and modify extracellular matrix molecules nearby , which changes signaling between the matrix and the cell and causes cellular dysfunction . 3.The third mechanism, is that these AGE precursors diffuse out of the cell and modify circulating proteins in the blood such as albumin. These modified circulating proteins can then bind to AGE receptors and activate them, thereby causing the production of inflammatory cytokines and growth factors, which in turn cause vascular pathology.

31. Increased production of AGE precursors and its pathologic consequences
Increased production of AGE precursors and its pathologic consequences. From Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820,

32. AGEs have been shown
to cross-link proteins (e.g., collagen, extracellular matrix proteins),
accelerate atherosclerosis,
promote glomerular dysfunction,
reduce nitric oxide synthesis,
induce endothelial dysfunction,
and alter extracellular matrix composition and structure.
The serum level of AGEs correlates with the level of glycemia, and these products accumulate as the glomerular filtration rate (GFR) declines.

33. Advanced glycation products in vascular pathology.

34. Advanced glycation products in nephropathy

35. Receptor-Mediated effects

36. Advanced glycation peptides are metabolized by the kidney.

37. Advanced glycation products are metabolized to small peptides

38. Effects of AGEs. Cdc42, Cell division cycle 42 protein; eNOS, endothelial nitric oxide synthase; ICAM-1, intercellular adhesion molecule-1; MAP, mitogen-activated protein; NAD(P)H nicotinamide dinucleotide phosphate; NO, nitric oxide; ROS, reactive oxygen s...
Effects of AGEs. Cdc42, Cell division cycle 42 protein; eNOS, endothelial nitric oxide synthase; ICAM-1, intercellular adhesion molecule-1; MAP, mitogen-activated protein; NAD(P)H nicotinamide dinucleotide phosphate; NO, nitric oxide; ROS, reactive oxygen species. [Adapted from A. Goldin, J. A. Beckman, A. M. Schmidt, and M. A. Creager: Circulation 114:597–605, 2006 (17 ).
Goh S , Cooper M E JCEM 2008;93:
©2008 by Endocrine Society

39. PKC activation.
The Third thoery is PKC activation. In this pathway, hyperglycemia inside the cell increases the synthesis of a molecule called diacylglycerol, which is a critical activating cofactor for the classic isoforms of protein kinase-C. When PKC is activated by intracellular hyperglycemia, it has a variety of effects on gene expression,. For example, the vasodilator producing endothelial nitric oxide (NO) synthase (eNOS) is decreased, while the vasoconstrictor endothelin-1 is increased. Transforming growth factor- and plasminogen activator inhibitor-1 are also increased.

40. Consequences of hyperglycemia-induced activation of PKC.

41. Increased hexosamine pathway activity.
A fourth theory proposes that hyperglycemia increases the flux through the hexosamine pathway, which generates fructose-6-phosphate, a substrate for O-linked glycosylation and proteoglycan production.
The hexosamine pathway may alter function by glycosylation of proteins such as endothelial nitric oxide synthase or by changes in gene expression of transforming growth factor  (TGF-) or plasminogen activator inhibitor-1 (PAI-1).

42. when glucose is high inside a cell, most of that glucose is metabolized through glycolysis, going first to glucose-6 phosphate, then fructose-6 phosphate, and then on through the rest of the glycolytic pathway.

43. However, some of that fructose-6-phosphate gets diverted into a signaling pathway in which an enzyme called GFAT (glutamine:fructose-6 phosphate amidotransferase) converts the fructose-6 phosphate to glucosamine-6 phosphate and finally to UDP (uridine diphosphate) N-acetyl glucosamine. The N-acetyl glucosamine gets put onto serine and threonine residues of transcription factors, just like the more familiar process of phosphorylation, and over modification by this glucosamine often results in pathologic changes in gene expression.

44. Hyperglycemia increases flux through the hexosamine pathway
Hyperglycemia increases flux through the hexosamine pathway. From Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820, 2001.

45. A UNIFIED MECHANISM
All of these different pathogenic mechanisms do reflect a single hyperglycemia-induced process and this single unifying mechanism is that hyperglycemia leads to increased production of reactive oxygen species or superoxide in the mitochondria; these compounds may activate all four of the pathways described above.

46. How does hyperglycemia increase superoxide production by the mitochondria?
Hyperglycemia leads to more glucose being oxidized in the TCA cycle, which in effect pushes more electron donors (NADH and FADH2) into the electron transport chain. As a result of this, the voltage gradient across the mitochondrial membrane increases until a critical threshold is reached. At this point, electron transfer inside complex III is blocked, causing the electrons to back up to coenzyme Q, which donates the electrons one at a time to molecular oxygen, thereby generating superoxide The mitochondrial isoform of the enzyme superoxide dismutase degrades this oxygen free radical to hydrogen peroxide, which is then converted to H2O and O2 by other enzymes.

47. Hyperglycemia-induced production of superoxide by the mitochondrial electron transport chain.

48. Mitochondrial overproduction of superoxide activates four major pathways of hyperglycemic damage by inhibiting GAPDH. From Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820, 2001.

49. schematic summary showing the elements of the unified mechanism of hyperglycemia-induced cellular damage:

50. When intracellular hyperglycemia develops in target cells of diabetic complications, it causes increased mitochondrial production of ROS. The ROS cause strand breaks in nuclear DNA, which activate PARP. PARP then modifies GAPDH, thereby reducing its activity. Finally, decreased GAPDH activity activates the polyol pathway, increases intracellular AGE formation, activates PKC and subsequently NFB, and activates hexosamine pathway flux.

51. The unifying mechanism of hyperglycemia induced cellular damage.

52. Pathways of micro vascular complications initiated by hyperglycemia
Pathways of micro vascular complications initiated by hyperglycemia. AGEs, advanced glycation end products; DAG, diacylglycerol; PKC, protein kinase C.

53. Diabetic macro vascular disease?
In contrast to diabetic microvascular disease, data from the UKPDS have shown that hyperglycemia is not the major determinant of diabetic macrovascular disease. Consequence of insulin resistance is increased free fatty acid (FFA) flux from adipocytes into arterial endothelial cells. In macrovascular, but not in microvascular endothelial cells, this increased flux results in increased FFA oxidation by the mitochondria.

54. Since both –oxidation of fatty acids and oxidation of FFA-derived acetyl CoA by the TCA cycle generate the same electron donors (NADH and FADH2) generated by glucose oxidation, increased FFA oxidation causes mitochondrial overproduction of ROS by exactly the same mechanism described above for hyperglycemia.
And, as with hyperglycemia, this FFA-induced increase in ROS activates the same damaging pathways: AGEs, PKC, the hexosamine pathway (GlcNAc).

55. Insulin resistance causes mitochondrial overproduction of ROS in macrovascular endothelial cells by increasing FFA flux and oxidation. From Hofmann S, Brownlee M: Biochemistry and molecular cell biology of diabetic complications: a unifying mechanism. In Diabetes Mellitus: A Fundamental and Clinical Text. 3rd ed. LeRoith D, Taylor SI, Olefsky JM, Eds. Philadelphia, Lippincott Williams & Wilkins, p. 1441–1457, 2004.

56. Although hyperglycemia serves as the initial trigger for complications of diabetes, it is still unknown whether the same pathophysiologic processes are operative in all complications or whether some pathways predominate in certain organs.

57. Glycemic control and vascular complications in DM
Hyperglycemia is an important risk factor for the development of micro vascular disease in patients with type 2 diabetes, as it is in patients with type 1 diabetes. This has been shown in several observational studies .
In addition, improving glycemic control improves micro vascular outcomes, as illustrated by many randomized trials.

58. DCCT/EDIC-
The importance of tight glycemic control for protection against micro vascular and cardiovascular disease in diabetes was established in the DCCT/EDIC study for type 1 diabetes .

59. UKPDS -
Although the role of glycemic control on micro vascular disease in type 2 diabetes was documented in the United Kingdom Prospective Diabetes Study (UKPDS), its role in reducing cardiovascular risk has not been established as clearly for type 2 diabetes.

60. Kumamoto study —
 In a trial of 110 patients with type 2 diabetes from Japan, showed ;
intensive therapy was beneficial .

61. ADVANCE trial —
 In the ADVANCE trial, 5571 type 2 diabetes patients receiving intensive therapy to lower A1C (mean A1C 6.5 percent) had a reduction in the incidence of nephropathy, defined as development of macro albuminuria, doubling of the serum creatinine to at least 2.26 mg/dL (200 micromol/L), the need for renal-replacement therapy, or death due to renal disease (4.1 versus 5.2 percent, hazard ratio [HR] 0.79, 95% CI ) compared with 5569 patients receiving standard therapy (A1C 7.3 percent) . There was no significant effect of glycemic control on the incidence of retinopathy.

62. Veteran's Affairs Diabetes Trial —
In the Veteran's Affairs Diabetes Trial (VADT), 892 veterans with long-standing type 2 diabetes receiving intensive therapy (A1C 6.9 percent) did not have a reduction in retinopathy or major nephropathy outcomes, which were predefined secondary endpoints, compared with 899 veterans receiving standard therapy (A1C 8.4 percent) .

63. The lack of benefit of intensive control in VADT may be secondary to the duration of diabetes in subjects participating in VADT (mean 11.5 years versus newly diagnosed in UKPDS) and the time required to show benefit (delayed benefit may require longer follow-up).
In addition, aggressive treatment of hypertension and hyperlipidemia in all VADT participants may have contributed to the inability to show a microvascular benefit of intensive glucose control.

64. ACCORD trial —
 In the ACCORD trial , 10,250 patients with long-standing type 2 diabetes were randomly assigned to intensive or standard glycemic control. After a median follow-up of 3.7 years,
Intensive therapy was stopped due to a higher number of total and cardiovascular deaths in subjects assigned to intensive therapy (median A1C 6.4 percent) compared with the standard treatment group (median A1C 7.5 percent).
Patients in the intensive therapy arm were transitioned to standard therapy, and all patients were followed for microvascular outcomes, predefined secondary endpoints, until completion of the study (five years)

65. Micro vascular summary:
The results of the UKPDS, Kumamoto, ADVANCE and ACCORD trials are consistent with those of the DCCT for patients with type 1 diabetes, taking into account the relative differences in A1C achieved between treatment groups and the differences in study duration (or exposure): intensive therapy improves the outcome of micro vascular disease.
The results of the post-trial monitoring phase of the UKPDS show that a sustained period of glycemic control in newly diagnosed patients with type 2 diabetes has lasting benefit in reducing micro vascular disease.

66. Despite these differences, all three trials consistently show that over the time period studied (3.5 to 6 years), near-normal glycemic control (A1C 6.4 to 6.9 percent) does not reduce cardiovascular events in patients with longstanding diabetes.
However, in patients with newly diagnosed type 2 diabetes, a goal A1C of ≤7.0 percent is reasonable and supported by the findings of the UKPDS follow-up study. Whether a benefit of stricter control (A1C near-normal) in newly diagnosed patients will eventually be demonstrated is uncertain.

67. Macro vascular summary:
Although the difference in mortality findings between ACCORD and ADVANCE/VADT and the difference in cardiovascular outcomes between ACCORD/ADVANCE/VADT and the meta-analyses are unexplained, there are some differences among the trials that could account for the discrepancies, including choice of pharmacologic therapy, baseline glycemic control, and frequency of hypoglycemia.

68. In spite of evidence that aggressive risk factor reduction lowers the risk of both micro- and macro vascular complications in patients with diabetes, the vast majority of patients do not achieve recommended goals for A1C, blood pressure control, and management of dyslipidemia.