New Prespectives for Type 2 Diabetes Management

LIST OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

I. INTRODUCTION

II. AIM OF THE WORK

III. MATERIALS AND METHODS

IV. RESULTS

V. DISCUSSION

VI. CONCLUSIONS AND RECOMMENDATIONS

VII. SUMMARY

VIII. REFERENCES

List of Tables

(1) Effect of STZ-induced type 2 diabetes on fasting serum glucose, fasting serum insulin and HOMA-IR in male albino rats

(2) Effect of STZ-induced type 2 diabetes on lipid profile in male albino rats

(3) Effect of STZ-induced type 2 diabetes on oxidative stress parameters in male albino rats

(4) Effect of STZ-induced type 2 diabetes on inflammatory parameters in male albino rats

(5) Effect of treatment with the studied drugs for 2 weeks on fasting serum glucose in male albino rats (mg/dl)

(6) Effect of treatment with the studied drugs for 2 weeks on fasting serum insulin in male albino rats (ng/ml)

(7) Effect of treatment with the studied drugs for 2 weeks on HOMA-IR in male albino rats

(8) Effect of treatment with the studied drugs for 2 weeks on serum triglycerides in male albino rats (mg/dl)

(9) Effect of treatment with the studied drugs for 2 weeks on serum total cholesterol in male albino rats (mg/dl)

(10) Effect of treatment with the studied drugs for 2 weeks on serum HDL-C in male albino rats (mg/dl)

(11) Effect of treatment with the studied drugs for 2 weeks on serum LDL-C in male albino rats (mg/dl)

(12) Effect of treatment with the studied drugs for 2 weeks on serum free fatty acids in male albino rats (mmol/L)

(13) Effect of treatment with the studied drugs for 2 weeks on non-HDL-cholesterol in male albino rats (mg/dl)

(14) Effect of treatment with the studied drugs for 2 weeks on triglycerides to HDL-cholesterol ratio in male albino rats

(15) Effect of treatment with the studied drugs for 2 weeks on hepatic malondialdehyde in male albino rats (nmol/gm wet tissue)

(16) Effect of treatment with the studied drugs for 2 weeks on hepatic reduced glutathione in male albino rats (µg/mg protein)

(17) Effect of treatment with the studied drugs for 2 weeks on serum monocyte chemoattractant protein-1 in male albino rats (pg/ml)

(18) Effect of treatment with the studied drugs for 2 weeks on serum C-reactive protein in male albino rats (mg/L)

(19) Effect of treatment with the studied drugs for 2 weeks on serum nitric oxide in male albino rats (nmol/ml)

List of FIGURES

(1) Model for the effects of adipocytes on pancreatic β-cell function/mass and insulin sensitivity in the pathogenesis of type 2 diabetes

(2) Mitochondrial overproduction of superoxide activates the major pathways of hyperglycemic damage by inhibiting glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

(3) Development of type 2 diabetes

(4) The cellular origins of reactive oxygen species, their targets, and antioxidant systems.

(5) Schematic of the effects of chronic oxidative stress on the insulin signaling pathway

(6) Structure of GSH (γ-glutamylcysteinyl glycine), where the N-terminal glutamate and cysteine are linked by the γ-carboxyl group of glutamate

(7) Chemical structure of L-cysteine

(8) The transsulfuration pathway in animals.

(9) Sources and actions of cysteine and glutathione (GSH)

(10) The role of serine kinase activation in oxidative stress-induced insulin resistance and the protective effect of some antioxidants by preserving the intracellular redox balance

(11) Chemical structure of biguanides

(12) Structure of human proinsulin and some commercially available insulin analogs.

(13) Model of control of insulin release from the pancreatic β-cell by glucose and by sulfonylurea drugs

(14) Schematic diagram of the insulin receptor heterodimer in the activated state.

(15) Standard curve of insulin

(16) Standard curve of Monocyte chemoattractant protein-1 (MCP-1)

(17) Standard curve of nitric oxide

(18) Standard curve of MDA

(19) Standard curve of reduced glutathione

(20) Standard curve of Protein

(21) Effect of STZ-induced type 2 diabetes on fasting serum glucose in male albino rats

(22) Effect of STZ-induced type 2 diabetes on fasting serum insulin in male albino rats

(23) Effect of STZ-induced type 2 diabetes on HOMA-IR in male albino rats

(24-a) Effect of STZ-induced type 2 diabetes on serum triglycerides in male albino rats

(24-b) Effect of STZ-induced type 2 diabetes on serum total cholesterol in male albino rats

(24-c) Effect of STZ-induced type 2 diabetes on serum HDL-C in male albino rats

(24-d) Effect of STZ-induced type 2 diabetes on serum LDL-C in male albino rats

(24-e) Effect of STZ-induced type 2 diabetes on serum free fatty acids in male albino rats

(24-f) Effect of STZ-induced type 2 diabetes on non- HDL-C in male albino rats

(24-g) Effect of STZ-induced type 2 diabetes on TGs/HDL ratio in male albino rats

(25) Effect of STZ-induced type 2 diabetes on hepatic malondialdehyde in male albino rats

(26) Effect of STZ-induced type 2 diabetes on hepatic reduced glutathione in male albino rats

(27) Effect of STZ-induced type 2 diabetes on serum monocyte chemoattractant protein-1 in male albino rats

(28) Effect of STZ-induced type 2 diabetes on serum C-reactive protein in male albino rats

(29) Effect of STZ-induced type 2 diabetes on serum nitric oxide in male albino rats

(30) Effect of treatment with the studied drugs for 2 weeks on fasting serum glucose in male albino rats

(31) Effect of treatment with the studied drugs for 2 weeks on fasting serum insulin in male albino rats

(32) Effect of treatment with the studied drugs for 2 weeks on HOMA-IR in male albino rats

(33-a) Effect of treatment with the studied drugs for 2 weeks on serum triglycerides in male albino rats

(33-b) Effect of treatment with the studied drugs for 2 weeks on serum total cholesterol in male albino rats

(33-c) Effect of treatment with the studied drugs for 2 weeks on serum high density lipoprotein cholesterol in male albino rats

(33-d) Effect of treatment with the studied drugs for 2 weeks on serum low density lipoprotein cholesterol in male albino rats

(33-e) Effect of treatment with the studied drugs for 2 weeks on serum free fatty acids in male albino rats

(33-f) Effect of treatment with the studied drugs for 2 weeks on non-HDL-cholesterol in male albino rats

(33-g) Effect of treatment with the studied drugs for 2 weeks on triglycerides to HDL-cholesterol ratio in male albino rats

(34) Effect of treatment with the studied drugs for 2 weeks on hepatic malondialdehyde in male albino rats

(35) Effect of treatment with the studied drugs for 2 weeks on hepatic reduced glutathione in male albino rats

(36) Effect of treatment with the studied drugs for 2 weeks on serum monocyte chemoattractant protein-1 in male albino rats

(37) Effect of treatment with the studied drugs for 2 weeks on serum C-reactive protein in male albino rats

(38) Effect of treatment with the studied drugs for 2 weeks on serum nitric oxide in male albino rats

(39) Histopathological evaluation of pancreatic sections stained with hematoxylin and eosin (H&E) stain (X 10).

(40) Comparison of mean percentage change in biochemical metabolic, oxidative stress and inflammatory parameters between untreated and treated (metformin, L-cysteine and their combination) experimentally induced type 2 diabetic adult male rats

(41) Comparison of mean percentage change in lipid profile between untreated and treated (metformin, L-cysteine and their combination) experimentally induced type 2 diabetic adult male rats

>LIST OF ABBREVIATIONS

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BACKGROUND OF THE STUDY

Diabetes mellitus (DM) is a chronic multisystem disorder with biochemical consequences and serious complications that affect many organs. There are complex interactions between genetic, epigenetic, environmental and behavioural factors that contribute to the development of diabetes. Non-pharmacological and pharmacological interventions have been used for diabetic management. Over the past few years, research has started to focus on the use of novel adjuvant drugs as antioxidants and anti-inflammatory drugs for better management, as it was revealed that both oxidative stress and inflammation play a critical role in the disease pathogenesis.

Metformin is a widely used oral antidiabetic agent for the management of type 2 diabetes. Its primary mode of action appears to be through improvement of insulin sensitivity and suppression of hepatic gluconeogenesis and glycogenolysis. Moreover, it affects glucose transport system, increases glucose utilization and delays its absorption from the intestine. It also shows beneficial effects on diabetes, as weight reduction and improvements in lipid profile, inflammation and endothelial function.

L-cysteine is a semi-essential sulfur containing amino acid. One important function of L-cysteine is that it is a precursor of glutathione, which is pivotal for the detoxification of cellular oxidative stress. Dietary intake of cysteine-rich proteins lowers the oxidative stress and insulin resistance. It improves glycemic control, shows an anti-inflammatory effect and implies a protective effect on pancreatic β-cells.

Taking the above mentioned data in consideration, it seems that combined therapy of metformin and an antioxidant like L-cysteine may be of value in treatment of the diabetic state and amelioration of the oxidative stress and inflammation associated with diabetes mellitus.

INTRODUCTION

Diabetes mellitus is the most common endocrine metabolic disorder, affecting about 170 million people worldwide (1). It represents a group of diseases with complex heterogeneous etiology, characterized by chronic hyperglycemia with carbohydrate, fat and protein metabolic abnormalities(2), which are due to insulin deficiency and/or insulin resistance (3). These abnormalities result in the impairment of uptake and storage of glucose and reduced glucose utilization for energy purposes. Defects in glucose metabolizing machinery and consistent efforts of the physiological system to correct the imbalance in glucose metabolism place an over-exertion on the endocrine system. Continuing deterioration of endocrine control exacerbates the metabolic disturbances and leads primarily to hyperglycemia(4), then proceeds to the development of long-term complications, such as microangiopathy; nephropathy, neuropathy and retinopathy. The basis of these complications is a subject of great debate and research. Hyperglycemia and metabolic derangement are accused as the main causes of these long-standing changes in various organs. Hyperglycemia may also lead to increased generation of free radicals and reduced antioxidant defense system (3).

Epidemiology of diabetes mellitus

Diabetes mellitus is a common growing disease, which is considered epidemic by WHO. Its incidence in adults and adolescents have been alarmingly rising in developed countries with estimate for an increase of 60% in the adult population above 30 years of age in 2025, with a higher prevalence in the 45 to 64 years-old adults (5). These increases are expected because of population ageing and urbanization. According to the WHO, undiagnosed diabetes in Egypt will be about 8.8 million by the year 2025(6).

Diabetes mellitus classification

The current classification includes four main categories (7):

I- Type 1 diabetes, either type 1A (immune-mediated, e.g. latent autoimmune diabetes in adults [LADA]) or type 1 B (idiopathic)

II- Type 2 diabetes

III- Other specific types

1. Genetic defects of β-cell function (maturity onset diabetes of the young [MODY]). These defects may be in genes of hepatic nuclear factor (HNF-1α or HNF-4α) or insulin promoter factor-1 (IPF-1).
2. Genetic defects in insulin action (Type A insulin resistance, lipoatrophic diabetes).
3. Diseases of the exocrine pancreas (pancreatitis, neoplasia, cystic fibrosis, hemochromatosis).
4. Endocrinopathies (acromegaly, Cushing's syndrome, glucagonoma, pheochromocytoma, hyperthyroidism).
5. Drug or chemical induced (vacor, streptozotocin, alloxan, glucocorticoids, thyroid hormone, diazoxide, thiazide diuretics, minoxidil, oral contraceptives, L-dopa).
6. Infections (congenital rubella, cytomegalovirus).
7. Uncommon forms of immune-mediated diabetes (“Stiff-man” syndrome, anti-insulin receptor antibodies).
8. Other genetic syndromes sometimes associated with diabetes (Down syndrome, Klinefelter syndrome, Turner syndrome).

IV-Gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is defined as any abnormal carbohydrate intolerance that begins or is first recognized during pregnancy(8). It is associated with an increased risk of perinatal mortality and congenital abnormalities, which is further increased by impaired glycemic control (9). It occurs in approximately 7% of all pregnancies and if occurred once, it is likely to occur in subsequent pregnancies. Up to 70% of women with GDM have a potential risk of developing type 2 diabetes mellitus. The risk factors for developing gestational diabetes are similar to those for type 2 diabetes, including family history, age, obesity and ethnicity (10). It is known that pregnancy is a diabetogenic state, characterized by impaired insulin sensitivity, particularly in the second and third trimester. This is due to changes in some hormones such as human placental lactogen, progesterone, prolactin and cortisol that antagonize the effects of insulin and decrease phosphorylation of insulin receptor substrate-1 (IRS-1), triggering a state of insulin resistance. Logically, the pancreas should compensate for this demand by increasing insulin secretion. However in GDM, there is deterioration of beta cell function, particularly the first phase insulin secretion (8).

An intermediate group of individuals with impaired fasting glucose and/or impaired glucose tolerance was classified as “pre-diabetics”. Their progression to diabetes is common, particularly when non-pharmacological interventions, such as lifestyle changes are not provided (7).

I-Type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) is an organ-specific progressive cellular-mediated autoimmune disease characterized by a defect in insulin production, as a result of selective and massive destruction of islet β-cells (80–90%). It accounts for only about 5–10% of all cases of diabetes; however, its incidence continues to increase worldwide. The progression of the autoimmune process is generally slow and may take several years before the onset of the clinical diabetes (11). Markers of the immune β-cell destruction, including circulating insulin autoantibodies (IAAs), islet-cell autoantibodies (ICAs) and glutamic acid decarboxylase autoantibodies (GADA), are present in 90% of patients at the time of diagnosis (7).

Two forms are identified:

- Type 1A DM, which results from a cell-mediated autoimmune attack on β-cells and has a strong genetic component, inherited through the human leukocyte antigen (HLA) complex, mainly HLA-DR3 and HLA-DR4 (12), but the factors that trigger onset of clinical disease remain largely unknown.
- Type 1B DM (idiopathic) is a far less frequent form, has no known cause, and occurs mostly in individuals of Asian or African descent(7). This form of diabetes lacks evidence for β-cell autoimmunity and is not HLA associated. While the disease is often manifested by severe insulinopenia and/or ketoacidosis, β-cell function often recovers, rendering almost normal glucose levels (13).

Starting generally at a young age, T1DM is also referred to as ‘juvenile diabetes’. Indeed, it affects children and young adults in particular and generally occurs before the age of 40, with incidence peaks at 2, 4-6 and 10-14 years (11). However, it can also occur at any age, even as late as in the eighth and ninth decades of life. The slow rate of β-cell destruction in adults may mask the presentation, making it difficult to distinguish from type 2 diabetes. This type of diabetes is known as “Latent Autoimmune Diabetes in Adults” (13).

Patients with type 1 diabetes are severely insulin deficient and are dependent on insulin replacement therapy for their survival (13).

II- Type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder of polygenic nature, that is characterized by defects in both insulin action and insulin secretion (14). T2DM affects nowadays more than 150 million people worldwide and is projected to increase to 439 million worldwide in 2030(15). By the end of the 20th century, its incidence has increased dramatically in children and adolescents, as a result of the rise in childhood obesity and is now continuing to rise, changing the demographics of the disease in this group (16).

Genetic, epigenetic and environmental factors have been implicated in type 2 diabetes mellitus pathogenesis with increasing evidences that epigenetic factors play a key role in the complex interplay between them(17). Epigenetic mechanisms are commonly associated to gene silencing and transcriptional regulation of genes (18). The epigenetic control of gene expression is based on modulation of chromatin structure and accessibility to transcription factors, which is achieved by multiple mechanisms. These mechanisms involve methylation–demethylation of cytidine–guanosine sequences in the promoter regions, acetylation–deacetylation of lysine residues of core histones in the nucleosome and presence of microRNA molecules, which bind to their complementary sequences in the 3′ end of mRNA and reduce the rate of protein synthesis(19). Actions of major pathological mediators of diabetes and its complications such as hyperglycemia, oxidative stress and inflammation can lead to the dysregulation of these epigenetic mechanisms (17).

Insulin resistance in peripheral tissues, such as muscle and fat, is often the earliest recognizable feature of T2DM, results in a compensatory hyperinsulinemia that promotes further weight gain. This occurs until the β-cells can no longer compensate for the increased insulin resistance, then β-cell failure and hyperglycemia ensue (20). It is also associated with co-morbidities, such as hypertension, hyperlipidemia and cardiovascular diseases, which taken together comprise the ‘Metabolic Syndrome’ (20).

Similar to adults, obesity in children appears to be a major risk factor for type 2 diabetes (21). Many studies show a strong family history among affected youth, with 45-80% having at least one parent with diabetes and 74-100% having a first- or second-degree relative with type 2 diabetes (22). Until now, type 2 diabetes was typically regarded as a disease of the middle-aged and elderly. While it is still true that this age group maintains a higher risk than the younger adults do, evidence is accumulating that onset in children and adolescents is increasingly common. Onset of diabetes in childhood or adolescence, around the time of puberty, heralds many years of disease and increases the risk of occurrence of the full range of both micro- and macrovascular complications (23).

Although a mother could transmit genetic susceptibility to her offspring, it is more likely that maternal diabetes increases the risk of diabetes in children by altering the intrauterine environment, which can impair the normal β-cell development and function as well as the insulin sensitivity of skeletal muscle (24). Moreover, malnutrition during fetal or early life and low birth weight appear to be associated with an increased risk of adulthood insulin resistance, glucose intolerance, T2DM, dyslipidemia and hypertension (25).

Normal glucose homeostasis

Maintenance of serum glucose concentrations within a normal physiological range (fasting blood glucose level 70-110 mg/dl), is primarily accomplished by two pancreatic hormones; insulin, secreted by the β-cells and glucagon, secreted by α-cells (26). Derangements of glucagon or insulin regulation can result in hyperglycemia or hypoglycemia, respectively.

In the postabsorptive state, the majority of total body glucose disposal takes place in insulin-independent tissues (27). Approximately 50% of all glucose utilization occurs in the brain and 25% of glucose uptake occurs in the splanchnic area (liver and the gastrointestinal tissue). The remaining 25% of glucose metabolism takes place in insulin-dependent tissues, primarily muscle with only a small amount being metabolized by adipocytes (28). Approximately 85% of endogenous glucose production is derived from the liver and the remaining amount is produced by the kidney. Glycogenolysis and gluconeogenesis contribute equally to the basal rate of hepatic glucose production (27).

In the postprandial state, the maintenance of whole-body glucose homeostasis is dependent upon a normal insulin secretory response and normal tissue sensitivity to the effects of hyperinsulinemia and hyperglycemia to augment glucose disposal. This occurs by three tightly coupled mechanisms: (i) suppression of endogenous glucose production and increase glycogen synthesis; (ii) stimulation of glucose uptake by the splanchnic tissues; and (iii) stimulation of glucose uptake by peripheral tissues, primarily muscle (27).

The route of glucose administration also plays an important role in the overall glucose homeostasis. Oral glucose ingestion has a potentiating effect on insulin secretion that the insulin concentrations in the circulation increase very rapidly (by at least two to threefold) after oral glucose, when compared to a similar intravenous bolus of glucose. This potentiating effect of oral glucose administration is known as “the incretin effect” and is related to the release of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (also called gastric inhibitory polypeptide) (GIP) from the gastrointestinal tissues (29).

Glucose homeostasis in type 2 diabetes mellitus

Type 2 diabetic individuals are characterized by defects in insulin secretion; insulin resistance involving muscle, liver and the adipocytes; and abnormalities in splanchnic glucose uptake (28).

Insulin secretion in insulin resistant, non-diabetic individuals is increased in proportion to the severity of the insulin resistance and glucose tolerance remains normal. Thus, their pancreas is able to "read" the severity of insulin resistance and adjust its secretion of insulin. However, the progression to type 2 diabetes with mild fasting hyperglycemia (120-140 mg/dl) is heralded by an inability of the β-cell to maintain its previously high rate of insulin secretion in response to a glucose challenge, without any further or only minimal deterioration in tissue sensitivity to insulin (27).

The relationship between the fasting plasma glucose concentration and the fasting plasma insulin concentration resembles an inverted U or horseshoe. As the fasting plasma glucose concentration rises from 80 to 140 mg/dl, the fasting plasma insulin concentration increases progressively, peaking at a value that is 2-2.5 folds greater than in normal weight, non-diabetic, age-matched controls. The progressive rise in fasting plasma insulin level can be viewed as an adaptive response of the pancreas to offset the progressive deterioration in glucose homeostasis. However, when the fasting plasma glucose concentration exceeds 140 mg/dl, the beta cell is unable to maintain its elevated rate of insulin secretion and the fasting insulin concentration declines precipitously. This decrease in fasting insulin level has important physiologic implications, since at this point; hepatic glucose production begins to rise, which is correlated with the severity of fasting and postprandial hyperglycemia (27).

Moreover, the largest part of the impairment in insulin-mediated glucose uptake is accounted for a defect in muscle glucose disposal. Thus in the basal state, the liver represents a major site of insulin resistance (30); however in the postprandial state, both decreased muscle glucose uptake and impaired suppression of hepatic glucose production contribute to the insulin resistance, together with defect in insulin secretion, are the causes of postprandial hyperglycemia. It should be noted that brain glucose uptake occurs at the same rate during absorptive and postabsorptive periods and is not altered in type 2 diabetes (28).

Pathophysiology of type 2 diabetes mellitus

Insulin resistance:

Insulin resistance (IR) is the impaired sensitivity and attenuated response to insulin in its main target organs, adipose tissue, liver, and muscle, leading to compensatory hyperinsulinemia (31).

In adipose tissue, insulin decreases lipolysis, thereby reducing FFAs efflux from the adipocytes. However, intra-abdominal fat is metabolically distinct from subcutaneous fat, as it is more lipolytically active and less sensitive to the antilipolytic effects of insulin. In liver, insulin inhibits gluconeogenesis by reducing key enzyme activities. While in skeletal muscle, insulin predominantly induces glucose uptake by stimulating the translocation of the GLUT4 glucose transporter to the plasma membrane and promotes glycogen synthesis (32).

Insulin resistance leads to increased lipolysis of the stored triacylglycerol molecules with subsequent increase circulating FFAs concentrations and ectopic fat accumulation. This results in increases in the flux of FFAs from fat to liver and periphery. Excess delivery of FFAs stimulates liver glucose and triglycerides production (33), impedes insulin mediated glucose uptake and decreases glycogen synthesis in skeletal muscle (34), as well as impairs vascular reactivity and induces inflammation(35). This is shown in Figure (1).

At the molecular level, impaired insulin signaling results from reduced receptor expression or mutations or post-translational modifications of the insulin receptor itself or any of its downstream effector molecules. These reduce tyrosine-specific protein kinase activity or its ability to phosphorylate substrate proteins (IRS) (36) resulting in phosphatidylinositol-3-kinase (PI3K)/Akt pathway impairment (37).

To date, several methods for evaluating insulin resistance in humans have been reported such as fasting serum insulin levels, homeostasis model assessment of insulin resistance (HOMA-IR) and insulin tolerance test (38). Because abnormalities in insulin action are poorly detected by a single determination of either glucose or insulin levels, the insulin resistance is commonly evaluated by HOMA-IR, which is likely to be the most simple and repeatable index (38, 39).

Glucotoxicity:

Insulin resistance results in chronic fasting and postprandial hyperglycemia. Chronic hyperglycemia may deplete insulin secretory granules from β-cells, leaving less insulin ready for release in response to a new glucose stimulus (40). This has led to the concept of glucose toxicity, which implies the development of irreversible damage to cellular components of insulin production over time. Hyperglycemia stimulates the production of large amounts of reactive oxygen species (ROS) in β-cells. Due to ROS interference, loss of pancreas duodenum homeobox-1 (PDX-1) has been proposed as an important mechanism leading to β-cell dysfunction. PDX-1 is a necessary transcription factor for insulin gene expression and glucose-induced insulin secretion, besides being a critical regulator of β-cell survival. Additionally, ROS are known to enhance NF-κB activity, which potentially induces β-cell apoptosis (41).

Lipotoxicity:

Chronically elevated free fatty acids (FFAs) level results from the resistance to the antilipolytic effect of insulin. It is known that FFAs acutely stimulate insulin secretion, but chronically impair insulin secretion, induce further hepatic and muscle insulin resistance, stimulate gluconeogenesis and cause a decrease β-cell function and mass, an effect referred to as β-cell lipotoxicity (42).

In the presence of glucose, fatty acid oxidation in β-cells is inhibited and accumulation of long-chain acyl coenzyme A occurs. This mechanism has been proposed to be an integral part of the normal insulin secretory process. However, its excessive accumulation can diminish the insulin secretory process by opening β-cell potassium channels. Another mechanism might involve apoptosis of β-cells, possibly via generation of nitric oxide through inducible nitric oxide synthase (iNOS) activation which results in great production of toxic peroxynitrite (ONOO-) (43).

Thus, glucolipotoxicity may play an important role in the pathogenesis of hyperglycemia and dyslipidemia associated with type 2 diabetes (44).

Dyslipidemia:

Diabetic dyslipidemia is typically defined by its characteristic lipid ‘triad’ profile, known as atherogenic dyslipidemia, which is usually an increase in plasma triglycerides, a decrease in high-density lipoprotein cholesterol and a concomitant increase in small dense oxidized low-density lipoproteins (45, 46). These lipid abnormalities may be a more important risk factor for atherosclerosis and cardiovascular diseases than hyperglycemia (46).

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Figure ( 1 ): Model for the effects of adipocytes on pancreatic β-cell function/mass and insulin sensitivity in the pathogenesis of type 2 diabetes (47) .

In diabetic patients, there is typically a preponderance of smaller, denser, oxidized LDL particles, which may increase atherogenicity and cardiovascular risk, even if the absolute concentration of LDL cholesterol is not elevated (45).

Non-HDL cholesterol (non-HDL-C) is a new measure that reflects the combined lipid profile change. It encompasses all cholesterol present in the potentially atherogenic lipoprotein particles (VLDL, remnants, IDL and LDL). Non-HDL-C has been shown to correlate with coronary artery disease severity and progression, as well as predicts cardiovascular morbidity and mortality in patients with diabetes (48).

Another simple tool, Triglycerides to HDL-cholesterol ratio (TGs: HDL-C) has been proposed as an atherogenic index, that has proven to be a highly significant predictor of myocardial infarction, even stronger than total cholesterol to HDL-C ratio and LDL-C to HDL-C ratio (49). Moreover, a significant negative relationship between TGs: HDL-C ratio and insulin sensitivity was observed. Thus a TGs: HDL-C ratio > 3.5 provides a simple mean of identifying insulin resistant, dyslipidemic patients who are at increased risk of cardiovascular diseases(50).

The precise pathogenesis of diabetic dyslipidemia is not fully known; nevertheless, a large body of evidence suggests that insulin resistance has a central role in the development of this condition as a result of the increased influx of free fatty acids from insulin-resistant fat cells into the liver, in the presence of adequate glycogen stores(51).

Diabetic complications and their pathogenesis

Hyperosmolar hyperglycemic non-ketotic state

It is one of the major acute complications, which is a life-threatening condition, commonly occurs in elderly patients with type 2 diabetes. There is almost always a precipitating factor which include; the use of some drugs, acute situations and chronic diseases. Abnormal thirst sensation and limited access to water also facilitate development of this syndrome. It is associated with four major clinical features, which are severe hyperglycemia (blood glucose more than 600 mg/dl), absent or slight ketosis, plasma hyperosmolarity and profound dehydration. Treatment of this state should be started immediately with the determination and correction of the precipitating event and lifesaving measures, while the other clinical manifestations should be corrected with the use of appropriate fluids and insulin (52).

However, chronic complications can be divided into microvascular; affecting eyes, kidneys and nerves and macrovascular; affecting the coronary, cerebral and peripheral vascular systems (53).

Microvascular complications

In fact, microvascular complications can begin in developing at least 7 years before the clinical diagnosis of type 2 diabetes. Conversely; type 1 patients may not develop signs of microvascular complications until 10 years after diagnosis of diabetes (54).

Nephropathy: Diabetic nephropathy is a frequent complication of type 1 and type 2 diabetes mellitus, characterized by excessive urinary albumin excretion, hypertension and progressive renal insufficiency. The natural history of diabetic nephropathy has 5 stages; which include hyperfiltration with normal renal function; histological changes without clinically evident disease; incipient diabetic nephropathy or microalbuminuria; overt diabetic nephropathy (macroalbuminuria and reduced renal function); and renal failure requiring dialysis (end stage renal disease) (55).

Neuropathy: Diabetic peripheral neuropathy is one of the most prevalent and complicated conditions to manage among diabetic patients. Diabetes is the major contributing reason for non-traumatic lower extremity amputations (more than 60% of cases). Ischemia occurs because of compromised vasculature that fails to deliver oxygen and nutrients to nerve fibers. This results in damage to myelin sheath covering and insulating nerve. The most common form involves the somatic nervous system; however, the autonomic nervous system may be affected in some patients. Sensorimotor neuropathy is characterized by symptoms; such as burning, tingling sensations and allodynia. Autonomic neuropathy can cause gastroparesis, sexual dysfunction and bladder incontinence (54).

Retinopathy: Diabetic retinopathy is the most frequent cause of new cases of blindness among adults aged 20-74 years (56). Non-proliferative retinopathy produces blood vessel changes within the retina, which include weakened blood vessel walls, leakage of fluids and loss of circulation. It generally does not interfere with vision (54). However, if left untreated, it can progress to proliferative retinopathy, that is very serious and severe. It occurs when new blood vessels branch out or proliferate in and around the retina (56). It can cause bleeding into the fluid-filled center of the eye or swelling of the retina, leading to blindness. The duration of diabetes and the degree of hyperglycemia are probably the strongest predictors for development and progression of retinopathy (54).

Macrovascular complications

The hallmark of diabetic macrovascular disease is the accelerated atherosclerosis; involving the aorta and the large and medium-sized arteries, which is a leading cause of morbidity and mortality in diabetes (54). Accelerated atherosclerosis caused by accumulation of lipoproteins within the vessel wall, resulting in the increased formation of fibrous plaques(53). Hyperglycemia also affects endothelial function, resulting in increased permeability, altered release of vasoactive substances, increased production of procoagulation proteins and decreased production of fibrinolytic factors (53). All these changes result in atherosclerotic heart disease, myocardial infarction and sudden death; peripheral vascular disease and cerebrovascular disease, including cerebral hemorrhage, infarction and stroke.

Hyperglycemia causes tissue damage through four major mechanisms. Several evidences indicate that all these mechanisms are activated by a single upstream event, which is the mitochondrial overproduction of reactive oxygen species (57), Figure (2).

Increased polyol pathway flux

The polyol pathway is based on a family of aldo-keto reductase enzymes, which can use as substrates a wide variety of carbonyl compounds and reduce them by NADPH to their respective sugar alcohols (polyols). Glucose is converted to sorbitol by the enzyme aldose reductase, which is then oxidized to fructose by the enzyme sorbitol dehydrogenase, using NAD+ as a cofactor. Aldose reductase is found in tissues such as nerve, retina, lens, glomerulus and vascular cells. In many of these tissues, glucose uptake is mediated by insulin-independent GLUTs; intracellular glucose concentrations, therefore, rise in parallel with hyperglycemia (57).

Several mechanisms include sorbitol-induced osmotic stress, increased cytosolic NADH/NAD+ and decreased cytosolic NADPH have been proposed to explain tissue damage resulted from this pathway (58). The most cited is an increase in redox stress, caused by the consumption of NADPH, a cofactor required to regenerate reduced glutathione (GSH), which is an important scavenger of ROS. This could induce or exacerbate intracellular oxidative stress (57).

Increased intracellular advanced glycation endproducts (AGEs) formation and increased expression of the receptor for AGEs (RAGE)

AGEs are formed by the non-enzymatic reaction of glucose and other glycating compounds derived both from glucose and fatty acids with proteins (59). Intracellular production of AGE precursors can damage cells by altering protein functions and binding of plasma proteins modified by AGE precursors to RAGE on cells such as macrophages and vascular endothelial cells. RAGE binding induces the production of ROS, which in turn activates the pleiotropic transcription factor nuclear factor (NF-κB), causing multiple pathological changes in gene expression (60). These effects induce procoagulatory changes and increase the adhesion of inflammatory cells to the endothelium. In addition, this binding appears to mediate, in part, the increased vascular permeability induced by diabetes, probably through the induction of VEGF (57).

Increased protein kinase C (PKC) activation

PKC activation results primarily from enhanced de-novo synthesis of diacylglycerol (DAG) from glucose via triose phosphate. Evidence suggests that the enhanced activity of PKC isoforms could also result from the interaction between AGEs and their cell-surface receptors (57). PKC activation implicated in many processes; such as increased vascular permeability, angiogenesis, blood flow abnormalities, capillary and vascular occlusion, which are involved in the pathology of diabetic complications (58).

Increased hexosamine pathway flux

Hyperglycemia and elevated free fatty acids also appear to contribute to the pathogenesis of diabetic complications by increasing the flux of glucose and fructose-6-phophate into the hexosamine pathway, leading to increases in the transcription of some key genes and alteration in protein functions such as eNOS inhibition (57).

Mitochondrial superoxide overproduction

It has now been established that all of the different pathogenic mechanisms described above stem from a single hyperglycemia-induced process, namely overproduction of superoxide by the mitochondrial electron-transport chain that can damage cells in numerous ways. It is hypothesized that excess ROS inhibits GAPDH (glyceraldehyde-3-phosphate dehydrogenase), a glycolytic key enzyme promoting shunting of upstream glucose metabolites into the aforementioned pathways (57). This overproduction of ROS can be prevented by manganese superoxide dismutase (61).

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Figure ( 2 ): Mitochondrial overproduction of superoxide activates the major pathways of hyperglycemic damage by inhibiting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (62).

Obesity, inflammation and insulin resistance

Although genetic predisposition to insulin resistance exists, it is widely accepted that the increasingly sedentary lifestyle; such as consumption of a high caloric diet and lack of exercise, have increased the global prevalence of not only insulin resistance and diabetes, but also of obesity (63). Between 60% and 90% of cases of type 2 diabetes now appear to be related to obesity (64). The close association of these two common metabolic disorders has been referred to as “diabesity” (65). Adipocytes are not merely a site for storage of energy in the form of triglycerides, but also a source of many adipokines (63) that have effects on many peripheral tissues, including skeletal muscles and liver. As body weight increases, there is expansion of the adipose tissue mass, particularly visceral intra-abdominal adipose tissue, resulting in, not only excessive free fatty acids release, but also altered release of these adipokines (66). Increased release of various inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), IL-6, MCP-1 and resistin; mainly from visceral fat and leptin; mainly from subcutaneous fat, together with decreased release of adiponectin contribute to the whole body insulin resistance (67). Figure (3) shows how inflammation contributes to develop insulin resistance and type 2 diabetes mellitus.

The inflammation is triggered in the adipose tissue by macrophages, which form ring-like structures surrounding dead adipocytes. As adipose tissue expands during the development of obesity, certain regions become hypoperfused, leading to adipocyte microhypoxia and cell death. Adipocyte hypoxia and death trigger a series of proinflammatory program, which in turn recruit new macrophages. Another proposed mechanism is the activation of inflammatory pathway by oxidative stress. Hyperglycemia and high fat diet have been shown to increase ROS production, via multiple pathways; such as NADPH oxidase activation, which in turn activates nuclear factor-κB, triggering inflammatory response in adipose tissue (68).

TNF-α, resistin, IL-6 and other cytokines appear to participate in the induction and maintenance of the chronic low-grade inflammatory state; which is one of the hallmarks of obesity and type 2 diabetes (69). IL-6 may interfere with insulin signaling, inhibit lipoprotein lipase activity and increase concentrations of non-esterified fatty acids (NEFA), contributing to dyslipidemia and insulin resistance (70). In addition, IL-6 stimulates the secretion of further proinflammatory cytokines; such as IL-1 and increases the hepatic production of CRP, thus explaining its increase in the metabolic syndrome and diabetes(71).

C-reactive protein (CRP), monocyte chemoattractant protein-1 (MCP-1) and other chemokines have essential roles in the recruitment and activation of macrophages in the adipose tissue and in the initiation of inflammation (69, 72). CRP activation of monocytes increases the expression of Ccr2, the receptor for MCP-1(72). Overexpression of MCP-1 causes inhibition of Akt and tyrosine phosphorylation in liver and skeletal muscle, which contributes to insulin resistance (73, 74). This demonstrates a clear association between increased levels of MCP-1 and CRP with the decreased insulin sensitivity and increased vascular inflammation (75), explaining the increased risk of atherosclerosis, cardiovascular disease and stroke in diabetic patients (76, 77).

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Figure ( 3 ): Development of type 2 diabetes; insulin resistance that precedes the development of hyperglycemia is associated with obesity and is induced by adipokines, FFAs, and chronic inflammation in adipose tissue. Pancreatic β-cells compensate for insulin resistance by hypersecretion of insulin. However, at some point, β-cell compensation is followed by β-cell failure and diabetes ensues (63).

This state of proatherogenesis and low-grade inflammation is known to cause induction of inducible nitric oxide synthase (iNOS), increasing nitric oxide production (78). Nitric oxide (NO) is a free radical, known to act as a biological messenger in mammals. It has a dual role as a mediator of physiological and pathophysiological processes.

In pancreatic islets, excess NO is produced on exposure to cytokines, which mediates β-cell injury and leads to diabetes mellitus. Nitric oxide can also combine with oxygen to produce potent cellular killers, such as the highly toxic hydroxyl radical (OH˙) and peroxynitrite (ONOO-). In diabetes mellitus, there is increased breakdown of NO by superoxide, resulting in the excessive formation of peroxynitrite, a potent oxidant that can attack many types of biological molecules. High levels of peroxynitrite cause initation of lipid peroxidation, sulfhydryl oxidation, nitration of some amino acids, direct DNA damage and oxidation of antioxidants (3).

Oxidative stress in diabetes mellitus

Oxidative stress refers to a situation of a serious imbalance between free radical-generating and radical-scavenging systems; i.e. increased free radical production or reduced activity of antioxidant defenses or both, leading to potential tissue damage (79). There is currently great interest in the potential contribution of reactive oxygen species (ROS) in pathogenesis of diabetes and more importantly in the development of secondary complications of diabetes (80).

Free radical species include a variety of highly reactive molecules, such as ROS and reactive nitrogen species (RNS). ROS include free radicals such as superoxide (O2•-), hydroxyl (OH•), peroxyl (RO2•), hydroperoxyl (HRO2•-), as well as non-radical species such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl). RNS include free radicals like nitric oxide (NO•) and nitrogen dioxide (NO2•-), as well as non-radicals such as peroxynitrite (ONOO-), nitrous oxide (HNO2) and alkyl peroxynitrates (RONOO) (81). Production of one ROS or RNS may lead to the production of others through radical chain reactions (82). Of these reactive molecules; O2•-, NO• and ONOO- are the most widely studied species, as they play important roles in diabetic complications (83).

To avoid free radical overproduction, antioxidants are synthesized to neutralize free radicals. Antioxidants include a manifold of enzymes, such as superoxide dismutase (SOD), catalase, glutathione peroxidase and glutathione reductase, as well as many non-enzymatic antioxidants as vitamin A, C and E (84). This is shown in Figure (4).

Free radicals, at physiological levels, play a key role in defense mechanisms as seen in phagocytosis and neutrophil function. They are also involved in gene transcription and, to some extent, acts as signaling molecules. However, excess generation of free radicals in oxidative stress has pathological consequences, including damage to nucleic acid, proteins and lipids, causing tissue injury and cell death (83).

Oxidative damage to DNA, lipids and proteins

1- Nucleic acid:

The hydroxyl radical is known to react with all components of the DNA molecule, damaging both the purine and pyrimidine bases and the deoxyribose backbone, causing base degeneration, single strand breakage and cross-linking to proteins. The most extensively studied DNA lesion is the formation of 8-OH-Guanine. Permanent modification of genetic material, resulting from these oxidative damage incidents, represents the first step involved in mutagenesis, carcinogenesis and ageing (85, 86).

2- Proteins:

Collectively, ROS can lead to oxidation of the side chain of amino acids residues of proteins, particularly methionine and cysteine residues, forming protein-protein cross-linkages and oxidation of the protein backbone (87), resulting in protein fragmentation, denaturation, inactivation, altered electrical charge and increased susceptibility to proteolysis (88). The concentration of carbonyl groups is a good measure of ROS-mediated protein oxidation (89).

3- Membrane lipids:

ROS attack polyunsaturated fatty acids (PUFAs) of phospholipids in the cell membranes, which are extremely sensitive to oxidation because of double and single bonds arrangement (90). The removal of a hydrogen atom leaves behind an unpaired electron on the carbon atom to which it was originally attached. The resulting carbon-centered lipid radical can have several fates, but the most likely one is to undergo molecular rearrangement, followed by reaction with O2 to give a peroxyl radical, which are capable of abstracting hydrogen from adjacent fatty acid side chains and so propagating the chain reaction of lipid peroxidation. Hence, a single initiation event can result in conversion of hundreds of fatty acid side chains into lipid hydroperoxides (91). Further decomposition of these lipid hydroperoxides produces toxic aldehydes; in particular 4-hydroxynonenal and malondialdehyde (92).

The occurrence of lipid peroxidation in biological membranes causes impairment of membrane functioning, changes in fluidity, inactivation of membrane-bound receptors and enzymes, and increased non-specific permeability to ions (93). Thus, lipid peroxidation in-vivo has been implicated as the underlying mechanisms in numerous disorders and diseases, such as cardiovascular diseases, atherosclerosis, liver cirrhosis, cancer, neurological disorders, diabetes mellitus, rheumatoid arthritis and aging (89).

Malondialdehyde (MDA) is a major highly toxic by-product formed by PUFAs peroxidation. MDA can react both irreversibly and reversibly with proteins, DNA and phospholipids, resulting in profound mutagenic and carcinogenic effects (92, 94). The determination of plasma, urine or other tissue MDA concentrations using thiobarbituric acid (TBA reaction) continues to be widely used as a marker of oxidative stress, as its level correlates with the extent of lipid peroxidation (95).

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Figure ( 4 ): The cellular origins of reactive oxygen species, their targets and antioxidant systems (68, 83).

Sources of oxidative stress in diabetes

Multiple sources of oxidative stress in diabetes including enzymatic, non-enzymatic and mitochondrial pathways have been reported (81).

Enzymatic sources of augmented generation of reactive species in diabetes include NOS, NAD(P)H oxidase and xanthine oxidase . If NOS lacks its substrate L-arginine or one of its cofactors, NOS may produce O2•- instead of NO• and this is referred to as the uncoupled state of NOS. NAD(P)H oxidase is a membrane associated enzyme that consists of five subunits and is a major source of O2•- production (81). There is plausible evidence that protein kinase C (PKC), which is stimulated in diabetes via multiple mechanisms, activates NAD(P)H oxidase (82).

Non-enzymatic sources of oxidative stress originate from hyperglycemia, which can directly increase ROS generation. Glucose can undergo auto-oxidation and generate OH• radicals. In addition, glucose reacts with proteins in a non-enzymatic manner, leading to the development of Amadori products followed by formation of advanced glycation endproducts (AGEs). ROS is generated at multiple steps during this process (96). Once AGEs are formed, they bind to various receptors termed RAGE and this step is also generating ROS (97). Moreover, cellular hyperglycemia in diabetes leads to the depletion of NADPH, through the polyol pathway, resulting in enhanced production of O2•− (98).

The mitochondrial respiratory chain is another source of non-enzymatic generation of reactive species. During the oxidative phosphorylation process, electrons are transferred from electron carriers NADH and FADH2, through four complexes in the inner mitochondrial membrane, to oxygen, generating ATP and O2• −, which is immediately eliminated by natural defense (83). However, in the diabetic cells, more glucose is oxidized by Krebs cycle, which pushes more NADH and FADH2 into the electron transport chain (ETC) thereby overwhelming complex III of ETC, where the transfer of electrons is blocked. Thus, the generated electrons are directly donated to molecular oxygen, one at a time, generating excessive superoxide (57). Therefore in diabetes, electron transfer and oxidative phosphorylation are uncoupled, resulting in excessive O2•− formation and inefficient ATP synthesis (99).

Antioxidants

Reactive oxygen species can be eliminated by a number of antioxidant defense mechanisms, which involve both enzymatic and non-enzymatic strategies. They work in synergy with each other and against different types of free radicals (100). Hyperglycemia not only engenders free radicals, but also impairs the endogenous antioxidant defense system and causes inflammation in many ways in diabetes mellitus (101), Figure (5). Decreases in the activities of SOD, catalase and glutathione peroxidase; decreased levels of glutathione and elevated concentrations of thiobarbituric acid reactants are consistently observed in diabetic patients and in experimentally-induced diabetes (100).

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Figure (5): Schematic of the effects of chronic oxidative stress on the insulin signaling pathway (102)

I- Enzymatic antioxidants

- Superoxide dismutase (SOD)

Isoforms of SOD are variously located within the cell. Cu/Zn-SOD is found in both the cytoplasm and the nucleus. Mn-SOD is confined to the mitochondria, but can be released into the extracellular space (100). SOD converts superoxide anion radicals produced in the body to hydrogen peroxide, which is then detoxified to water either by catalase or by glutathione peroxidase in the lysosomes and mitochondria, respectively(96), thereby reducing the likelihood of superoxide anion interacting with nitric oxide to form reactive peroxynitrite (100). However, H2O2 can also be converted to the highly reactive OH• radical in the presence of transition elements like iron and copper (103).

II- Non-enzymatic antioxidants

1- Vitamins

Vitamins A, C and E are diet-derived and detoxify free radicals directly. They also interact in recycling processes to generate their reduced forms. α-tocopherol is reconstituted, when ascorbic acid recycles the tocopherol radical generating dihydroascorbic acid, which is recycled by glutathione (100).

Vitamin E, a fat soluble vitamin, reacts directly with peroxyl and superoxide radicals to protect membranes from lipid peroxidation (100). It exists in eight different forms, of which α-tocopherol is the most active form in humans. Hydroxyl radical reacts with tocopherol forming a stabilized phenolic radical, which is reduced back to the phenol by ascorbate and NAD(P)H dependent reductase enzymes (96). The deficiency of vitamin E is concurrent with increased peroxides and aldehydes in many tissues. However, there have been conflicting reports about vitamin E levels in diabetic animals and human subjects that its plasma and/or tissue levels are reported to be unaltered, increased or decreased in diabetes (100).

Vitamin C, ascorbic acid, is an important potent water soluble antioxidant vitamin in human plasma, acting as an electron donor; it is capable of scavenging oxygen-derived free radicals and sparing other endogenous antioxidants from consumption (104). It can increase NO production in endothelial cells by stabilizing NOS cofactor tetrahydrobiopterin (BH4)(96). Vitamin C itself is oxidized to dehydroascorbate, which is considered as a marker of oxidative stress, as in smoking and diabetes mellitus (105). Plasma and tissue levels of vitamin C are 40–50% lower in diabetic compared with non-diabetic subjects (106).

2- Coenzyme Q10 (CoQ10)

It is an endogenously synthesized lipid soluble antioxidant that acts as an electron carrier in the complex II of the mitochondrial electron transport chain and in higher concentrations, it scavenges O2•- and improves endothelial dysfunction in diabetes (83, 96).

3- α-Lipoic acid

It is an antioxidant, which can exert beneficial effects in both aqueous and lipid environments. α-lipoic acid is reduced to another active compound dihydrolipoate, which is able to regenerate other antioxidants, such as vitamin C, vitamin E and reduced glutathione through redox cycling (83, 96).

4- Trace elements

Selenium, an essential trace element, is involved in the complex defense system against oxidative stress through selenium-dependent glutathione peroxidases and other selenoproteins (107). It has insulin-mimetic properties on glucose metabolism both in-vitro and in-vivo, by stimulating the tyrosine kinases involved in the insulin signaling cascade(108). Within the context of diabetes mellitus, controversially data on selenium levels in biological fluids can be found. Lower, similar and even higher selenium levels were reported in diabetic patients with respect to healthy subjects (109).

Zinc, magnesium and chromium are of special interest. Severe Zn deficiency is not frequent but concerns have been raised about Zn levels in diabetic patients. Some studies have reported Zn deficiency in type 2 diabetes, others failed to find significant differences with healthy subjects (110). Low magnesium levels have been associated with increased severity of type 2 diabetes, whereas controversy exists about the importance of hypomagnesaemia in pre-diabetic states(110). Previous studies also reported that diabetic patients have a significantly lower plasma chromium levels with higher urinary levels than in healthy subjects. This combination of abnormalities suggests a chronic renal loss of chromium (111).

Vanadium compounds are one of the most studied substances for the long-term treatment of diabetes. Vanadium exhibits insulin-mimetic effects in-vitro and in the streptozotocin diabetic rat with some insulin-enhancing effects (112).

5- Glutathione

Glutathione (γ-glutamyl-L-cysteinylglycine, GSH), Figure (6), is a small intracellular ubiquitous tripeptide, which is a sulfhydryl (SH) antioxidant, antitoxin and enzyme cofactor (113), present in both prokaryotes and eukaryotes (114). Being water soluble, it is found mainly in the cell cytosol and other aqueous phases of the living system (113).

Glutathione antioxidant system predominates among other antioxidants systems due to its very high reduction potential and high intracellular concentrations compared to other antioxidants in tissues. Glutathione is found almost exclusively in its thiol-reduced active form (GSH), comprises 90% of the total low molecular weight thiol in the body (115). GSH often attains millimolar levels inside cells, especially highly concentrated in the liver and in lens, spleen, kidney, erythrocytes and leukocytes, however its plasma concentration is in micromolar range (116).

Glutathione is an essential cofactor for antioxidant enzymes, namely the GSH peroxidases, which serve to detoxify hydrogen peroxide and other peroxides generated in water phase as well as the cell membranes and other lipophilic cell phases by reacting them with GSH, which then becomes in the oxidized form (GSSG). The recycling of GSSG to GSH is accomplished mainly by the enzyme glutathione reductase using the coenzyme NADPH as its source of electrons. Therefore NADPH, coming mainly from the pentose phosphate shunt, is the predominant source of GSH reducing power (117). Moreover, GSH is an essential component of the glyoxalase enzyme system, which is responsible for catabolism of the highly reactive aldehydes; methylglyoxal and glyoxal. It can also bind to these aldehydes, causing them to be excreted in bile and urine (115). These effects have particular implications for preventive health, as lipid peroxidation has been found to contribute to the development of many chronic diseases in humans.

The ratio of reduced to oxidized glutathione (GSH/GSSG) within cells is often used as a measure of cellular toxicity or vice versa as a predictor of the antioxidative capacity and redox state of the cells (114). GSH in the body is synthesized mostly de-novo, with cysteine being the limiting amino acid, so increasing cysteine supply is necessary to raise GSH synthesis and concentration. GSH may be a good reservoir for cysteine, as its concentration in tissues is 5-7 times higher than free cysteine (118).

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Figure ( 6 ): Structure of GSH (γ-glutamylcysteinyl glycine), where the N-terminal glutamate and cysteine are linked by the γ-carboxyl group of glutamate (119).

GSH makes major contributions to the recycling of other antioxidants that have become oxidized such as α-tocopherol, vitamin C and perhaps also the carotenoids (117). Moreover, GSH is important in the synthesis and repair of DNA, as it is required in the conversion of ribonucleotides to deoxyribonucleotides (120).

A major function of GSH is the detoxification of xenobiotics and/or their metabolites. It is also involved in maintaining the essential thiol status of many important enzymes and proteins (117). It participates in some cellular functions as amino acid translocation across the cell membrane (121) and folding of newly synthesized proteins (122). In addition, GSH is essential for the proliferation, growth, differentiation and activation of immune cells(117) and is implicated in the modulation of cell death (cellular apoptosis and necrosis) (123).

Some oxidative stressors are known for their ability to deplete GSH. These include smoking, alcohol intake, some over the counter drugs (as acetaminophen), household chemicals, strenuous aerobic exercise, dietary deficiency of methionine (an essential amino acid and GSH precursor), ionizing radiation, tissue injury, surgery, trauma, bacterial or viral infections (as HIV-1) and environmental toxins (117).

GSH reduction has been associated with the pathogenesis of a variety of diseases; therefore, systemic GSH status could serve as an index of general health.

Glutathione in liver diseases

GSH depletion is involved in liver injury and enhanced morbidity related to liver hypofunction. Studies had been demonstrated a decrease in plasma and liver GSH, increase in GSSG and a significant decrease in cysteine present in cirrhotic patients, chronic alcoholic and non-alcoholic liver disease (fatty liver, acute and chronic hepatitis); as compared with the healthy subjects (117).

Glutathione in immunity and HIV disease

Adequate GSH is essential for mounting successful immune responses when the host is immunologically challenged. Healthy humans with relatively low lymphocyte GSH were found to have significantly lower CD4 counts (117). It was postulated that GSH deficiency could lead to the progression of immune dysfunction, weight loss, cachexia and wasting syndrome, which are known AIDS stigmas. GSH depletion is also seen in many autoimmune diseases as Crohn's disease, an inflammatory immunomediated disorder, in which low GSH, elevated GSSG levels and altered GSH enzymes were found in the affected ileal zones (114).

Glutathione in diabetes mellitus

Low blood thiol status and reduced systemic GSH content were reported in diabetic and glucose intolerant patients as a result of insulin deficiency. It was reported that chronic hyperglycemia resulted in enhanced apoptosis in human endothelial cells, which was attenuated by insulin due to its ability to induce glutamate cysteine ligase expression (119). Platelets from diabetics have lower GSH levels and make excess thromboxane (TxA2), thus having a lowered threshold for aggregation. This may contribute to the increased atherosclerosis seen in the diabetic population(117).

Furthermore, glutathione deficiency is associated with aging and many other diseases as neurodegenerative diseases, including Parkinson's disease, schizophrenia and Alzheimer’s disease; atherosclerosis and cardiovascular diseases; human pancreatic inflammatory diseases and metal storage diseases as Wilson’s disease (117, 124).

Strategies for repleting cellular glutathione

In light of the copious evidence supporting the importance of GSH for homeostasis, and for resistance to toxic attack, as well as the contribution of its deficiency in many diseases, a number of researchers had been stimulated to find new potential approaches and methods for maintaining or restoring GSH levels (125). Optimizing GSH would likely augment antioxidant defenses and stabilize or raise the cell’s threshold for susceptibility to toxic attack (117).

- Oral glutathione

Oral GSH was reported to replete GSH in subjects with depleted GSH but not healthy ones. Intact GSH can be absorbed slowly by intestinal lumen enterocytes and epithelial cells, such as lung alveolar cells; thus intact GSH can be also delivered directly into the lungs as an aerosol (117). Circulating GSH is safe and soluble in plasma. It reacts only slowly with oxygen and is less susceptible to auto-oxidative degradation. However, currently, the use of GSH as a therapeutic agent is limited by its unfavorable pharmacokinetic properties. GSH has a short half-life in human plasma and difficulty in crossing cell membranes, so administration of high doses is necessary to reach a therapeutic value (125), which will not be a particularly cost-effective way to accomplish GSH repletion.

- L-cysteine

It is a sulfur containing semi-essential amino acid, as humans can synthesize it from the essential amino acid methionine only to a limited and generally not sufficient extent (126). Its chemical structure is illustrated in Figure (7). One important function of L-cysteine is being a precursor that limits the synthesis of glutathione. It also serves as a very important precursor for synthesis of proteins, coenzyme A and inorganic sulphate (127).

Cysteine is catabolized in the gastrointestinal tract and plasma (128), so it is relatively unstable in the blood. When substituted into the diet in place of the total protein allowance, it can replete GSH (117).

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Figure ( 7 ): Chemical structure of L-cysteine (115)

Biosynthesis

In animals, L-cysteine is synthesized from L-methionine and L-serine via trans-sulfurtion reaction (127). The sulfur is derived from methionine, which is converted to homocysteine through the intermediate S-adenosylmethionine. Cystathionine β-synthase then combines homocysteine and serine to form the asymmetrical thioether, cystathionine. The enzyme cystathionine γ-lyase converts the cystathionine into cysteine and α-ketobutyrate (129). This is shown in Figure (8).

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Figure ( 8 ): The transsulfuration pathway in animals. The first three reactions involve methyl group transfer via S-adenosylmethionine (129).

Biological functions of L-cysteine

The chemical structure of cysteine contains a free sulfhydryl group, which is the reactive entity that contributes to many of cysteine’s biological activity; serving as a nucleophile with susceptibility to be oxidized to the disulfide derivative cystine (130).

As a moderately powerful redox pair, cysteine and its disulfide partner cystine have an important physiological function as antioxidants. Cysteine's antioxidant properties are typically expressed in the glutathione, where the free SH group of cysteine within glutathione confers its functional properties. Cysteine and glutathione form a major part of the endogenous thiol pool that reacts with the vasoregulatory molecule NO to form nitrosothiol, which acts as NO-carrier molecules, stabilizing this normally volatile molecule. S-nitrosothiols have potent relaxant activity, antiaggregatory and anti-inflammatory functions. They have greater half-lives than free NO and are more resistant than free NO to degradation by superoxide. In this way, nitrosothiol increases the bioavailability of NO and potentiates its effects (115). All actions of cysteine and GSH are shown in Figure (9).

Cysteine is a component of many structural and functional proteins. It is able to stabilize protein structures by forming disulfide covalent cross-links, which add stability to the three-dimensional structures of protein, increase the rigidity of proteins, affect their susceptibility to denaturation and provide proteolytic resistance (115, 131). The precise location of cysteine within a protein also plays a direct role in the protein’s function. For example, cysteine is found at the active site of several enzymes, including eNOS; regulating its catalytic activity (115).

Proteins containing cysteine, such as metallothionein, can bind to heavy metals tightly because of the high affinity of thiol group to these metals; thus cobalt, cupper, inorganic arsenic and selenium toxicities can be ameliorated by oral cysteine ingestion(132). L-cysteine has been proposed as a preventative or antidote for some of the negative effects of alcohol, including liver damage and hangover. It counteracts the poisonous effects of acetaldehyde; the major by-product of alcohol metabolism, by supporting its conversion into the relatively harmless acetic acid (133). Aside from its oxidation to cystine, cysteine participates in numerous post-translational modifications(134).

Cysteine and insulin resistance

An early study demonstrated that cysteine has an insulin-like action, promoting the entry of glucose into adipose cells, mediated by its free SH group. Cysteine has been subsequently shown to increase the levels of GLUT3 and GLUT4, with a marked enhancement of glucose uptake, in mouse muscle and human neuroblastoma cells (135). Moreover, Cysteine may improve glucose metabolism by preventing oxidative or nitrosative inhibition of the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and glucose-6- phosphate dehydrogenase (115).

In cultured adipocytes, it was demonstrated that cysteine supplementation reverses the increased intracellular oxidative stress, after AGE-RAGE interaction, which causes a decrease in glucose uptake (136) and also prevents the methylglyoxal induced decrease in IRS-1 tyrosine phosphorylation and PI3K activity that impair insulin signaling (137). This is illustrated in Figure (10).

It was also reported that cysteine analogues potentiate the glucose-induced insulin release in pancreatic islets isolated from female Wistar rats(138). Dietary intake of whey protein and α-lactoalbumin (cysteine-rich proteins) lowers the oxidative stress and insulin resistance induced by sucrose in rats (139). Other studies have reported that N-acetylcysteine supplementation reduces fructose-induced insulin resistance in rats (140) and also improves insulin sensitivity in women with polycystic ovaries (141).

Other effects of L-cysteine

1- L-cysteine administration prevents liver fibrosis by direct inhibition of activated hepatic stellate cells proliferation and transformation (142). It also shows a cytoprotective effect against carbon tetrachloride (CCl4)-induced hepatotoxicity by reversal of CCl4 induced lactate dehydrogenase release and decreased cellular thiols, mainly glutathione (143).
2- Previous clinical studies suggest that the acquired immunodeficiency syndrome (AIDS) may be the consequence of a virus-induced cysteine deficiency. HIV-infected persons were found to have abnormally high TNF-α and IL-2 receptor alpha-chain. All the corresponding genes are associated with NF-κB, whose transcription is negatively regulated by cysteine or cysteine derivatives, thus they may be considered as adjuvant therapy for the treatment of patients with HIV-1 infection (144, 145).

Side effects of L-cysteine

Gastrointestinal problems as indigestion, flatulence, diarrhea, nausea and vomiting are the main side effects of L-cysteine. Allergic reactions, include itching and facial swelling, are another possible side effects. Copious amount of water should be taken with cysteine to prevent cystine renal stones formation. It was showed by in-vitro studies that cysteine mimics many of the chemical properties of homocysteine, which is known to increase the risk of the cardiovascular diseases (146).

N-acetylcysteine (NAC)

N-acetyl-L-cysteine is a cysteine precursor that is rapidly absorbed and converted to circulating cysteine by deacetylation. It is used as an antioxidant and as a mucolytic due to its ability to break disulphide bonds in the mucous. It has liver protecting effects, so it is a well-established antidote for acetaminophen overdose (147). It also has antimutagenic and anticarcinogenic properties. In addition, NAC can prevent apoptosis and promote cell survival, a concept useful for treating certain degenerative diseases (148).

NAC can scavenge ROS and increase depleted glutathione levels. Activation of redox-sensitive NF-κB in response to a variety of signals (IL-1, TNF-α and ROS) can be also inhibited by NAC. NAC can interfere with cell adhesion, smooth muscle cell proliferation, stability of rupture-prone atherosclerotic plaques in the cardiovascular system, reduce lung inflammation and prolong survival of transplants (148).

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Figure ( 9 ): Sources and actions of cysteine and glutathione (GSH) (115).

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Figure ( 10 ): The role of serine kinase activation in oxidative stress-induced insulin resistance and the protective effect of some antioxidants by preserving the intracellular redox balance(149).

Management of diabetes mellitus

I- Non-pharmacological management of diabetes

Lifestyle modifications are the cornerstone of management of diabetes mellitus and include the prescription of a healthy diet, regular exercise, management of stress and avoidance of tobacco (150).

1-Diet

The aims of dietary management are to achieve and maintain ideal body weight, euglycemia and desirable lipid profile, prevent and postpone complications related to diabetes and provide optimal nutrition during pregnancy, lactation, growth, old age and associated conditions, such as hypertension and catabolic illnesses (151). However, there is no single description for diet composition that can achieve these goals in all patients. Thus, the dietary recommendations should be individualized according to the person’s ethnicity, cultural and family background, personal preferences and associated co-morbid conditions(150). Diet, that contains 60% carbohydrates, high dietary fiber, low to moderate dietary fat and moderate high biological value proteins as well as vitamins and minerals; especially chromium, vitamin E and C, is considered proper for management of diabetic patients (152).

2- Physical activity

Exercise program should be individualized according to patient’s capacity and disabilities. Diabetic patient must wear appropriate footwear. It should also be noted that poorly controlled patients may develop hyperglycemia during exercise, whereas patients treated with insulin and insulin secretagogues could develop hypoglycemia (153).

The best form of exercise recommended to diabetic is a stepwise increase of aerobic exercises. There are several benefits from a regular exercise schedule. These include reduction of hypertension and weight, increase in bone density, improvement in insulin sensitivity, cardiovascular function and lipid profile (reduces serum triglycerides and increases HDL-C), as well as improvement in the sense of physical and mental well-being and the overall quality of life (152).

3- Stress management

Diagnosis of diabetes mellitus is a stressful situation in life of an individual and appropriate management; requires an approach that includes behavioural modification to develop positive attitude and healthy life style. A satisfactory treatment plan should include special attention to person with diabetes, quality of life, coping skills, optimal family support and a healthy workplace environment. Appropriate support and counseling is an essential component of the management at the time of diagnosis and throughout life(150).

II- Pharmacological management of type 2 diabetes mellitus

A- Antidiabetic agents

Even when non-pharmacological measures are successfully implemented, the progressive natural history of the disease dictates that the majority of patients will later require pharmacologic therapy, and this should be introduced promptly if the glycemic target is not met or not maintained. Preserving β-cell function and mass are important considerations in maintaining long-term glycemic control. If β-cell function deteriorates beyond the capacity of oral agents to provide adequate glycemic control, then the introduction of insulin should not be delayed(154).

Terminology within the field of antidiabetic agents may simplify the usage of the different agents. Hypoglycemic agents have the capacity to lower blood glucose below normal level to the extent of frank hypoglycemia (e.g. sulfonylureas). Antihyperglycemic agents (euglycemic agents) can reduce hyperglycemia, but when acting alone they do not have the capability to lower blood glucose below normoglycemia to the extent of frank hypoglycemia (e.g. metformin, thiazolidinediones, gliptins, α-glucosidase inhibitors) (154).

They are classified into:

- Oral antidiabetic agents:
- Insulin sensitizers:
- Biguanides, including metformin
- Thiazolidinediones or glitazones, including rosiglitazone and pioglitazone
- Insulin secretagogues:
- Sulfonylureas, including gliclazide, glipizide, glimepiride and glibenclamide
- Meglitinides (non-sulfonylurea secretagogues) including nateglinide and repaglinide
- Alpha-glucosidase inhibitors including acarbose, miglitol and voglibose
- Novel treatments: (oral and non-insulin parenteral agents)
- Gliptins, including sitagliptin and vildagliptin
- Glucagon-like peptide-1 receptor agonist, including exenatide and liraglutide
- Amylin and amylin analogs, including pramlintide
- Rimonabant
- New experimental agents

1. Insulin sensitizers

1.1. Biguanides

The history of biguanides stems from a guanidine-rich herb Galega officinalis (goat’s rue or French lilac) that was used as a traditional treatment for diabetes in Europe because of its glucose lowering effect (154, 155) . Its structural formula is illustrated in Figure (11).

Several guanidine derivatives were adopted for the treatment of diabetes in the 1920s. These agents all disappeared as insulin became available, but three biguanides – metformin, phenformin and buformin – were introduced in the late 1950s (155). However, phenformin and buformin were withdrawn in many countries in the late 1970s because of a high incidence of lactic acidosis (156). Metformin remained and was introduced into the USA in 1995 and since then it became most widely prescribed first line antidiabetic agent worldwide (157).

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Figure ( 11 ): Chemical structure of biguanides (158).

Pharmacological effects of metformin

1- Antihyperglycemic effect

Metformin exerts a range of actions that counter insulin resistance and decrease hyperglycemia by reducing fasting and postprandial blood glucose(159). The glucose-lowering efficacy of metformin requires a presence of at least some insulin because metformin does not mimic or activate the genomic effects of insulin. The precise mechanisms through which metformin exerts its glucose lowering effects are not entirely understood. However, its primary mode of action appears to be increasing hepatic insulin sensitivity, resulting in decreased hepatic glucose output through suppression of gluconeogenesis and glycogenolysis. Metformin may also modestly augment glucose uptake in peripheral tissues, increase fatty acid oxidation and increase glucose metabolism in the splanchnic bed. Metformin’s molecular effects appear to be at least in part mediated by adenosine monophosphate-activated protein kinase (AMPK), but it is unclear if this pathway represents the drug’s specific or unique target(160). AMPK activation determines a wide variety of physiological effects, including increased fatty acid oxidation and enhanced glucose uptake by skeletal muscle by increasing translocation of GLUT1 and insulin-sensitive glucose transporters, GLUT4, into the cell membrane (161). Administration of metformin to obese subjects was also found to increase levels of active GLP-1 after a glucose load, this phenomenon appears to occur through mechanisms other than DPP-4 inhibition; and may instead be due to direct stimulation of GLP-1 secretion or a reduction in DPP-4 secretion (160). Interestingly, these incretin-sensitizing effects of metformin appear to be mediated by PPAR-α dependent pathway as opposed to the more commonly described AMPK activation pathway (162). Importantly, the likelihood of hypoglycemia induced by metformin monotherapy is quite low, as the drug does not exert its effects through an increase in insulin secretion (160).

A new insight on the mechanism of action of metformin is its ability to decrease plasma glucose through the release of β-endorphin from adrenal gland, which activates peripheral opoid μ1 receptors. β-endorphin acts as a positive regulator in glucose utilization and a negative modulator in hepatic gluconeogenesis in the insulin-deficient state. These actions are mediated by the amelioration of GLUT4 gene expression and the attenuation of raised hepatic phosphenolpyruvate carboxykinase (PEPCK) gene expression, a rate-controlling enzyme of gluconeogenesis (163).

Metformin also has cardioprotective benefits and offers some protection against vascular complications; independently of its antihyperglycemic effect (164). It was reported that metformin is associated with a decrease in myocardial infarction due to its effect on various atherothrombotic risk markers and factors, including reduced carotid intima-media thickness, increased fibrinolysis and reduced concentrations of the anti-thrombolytic factor; plasminogen activator inhibitor-1 (PAI-1) (154).

2-Anti-inflammatory effect

Metformin also offers some protection against vascular inflammation and complications; independently of its antihyperglycemic effect (165). This may be mediated through the reduction of thrombotic factor and inflammatory markers (166).

3-Antioxidative effect

Metformin may also exert antioxidative effects, as it prevents hyperglycemia-induced PKC activation and protects against high glucose-induced oxidative stress through a mitochondrial permeability transition dependent pathway that is involved in cell death(167). This may be in relation to metformin’s ability to inhibit non-toxically complex I in the mitochondrial respiratory chain (165).

Metformin is also able to react in-vitro with OH• radical. However, it is not a very good scavenger of ROS at molecular level. Thus, it seems that metformin exerts its in-vivo antioxidant activity by different pathways other than the simple free radical scavenging action. These pathways include increasing the antioxidant enzyme activities, decreasing the markers of lipid peroxidation (168) and inhibiting the formation of advanced glycation end products by its ability to react directly with, and neutralize, highly reactive α-dicarbonyl intermediates involved in AGEs formation, such as methylglyoxal (164). Metformin can also increase the activity of glyoxalase, an enzyme which deactivates methylglyoxal to D-lactate (164).

Pharmacokinetics of metformin

Metformin is an orally administered medication, which is 50%–60% bioavailable. Administration with food may decrease its absorption, the clinical significance of which is unknown. The drug is minimally protein-bound, and has few known drug interactions other than that known to occur with cimetidine, which increases metformin levels in plasma by up to 40%. Metformin is not metabolized prior to its complete excretion in the urine via glomerular filtration and tubular secretion. The drug has an elimination half-life of approximately 6 hours. Decreases in renal function will decrease clearance of the medication. It is generally dosed 2–3 times daily, but is available in an extended release preparation, which may be administered once a day. 85% of the maximal glucose-lowering effect is seen at a daily dose of 500 mg 3 times daily, while the most effective glucose lowering occurs with a total daily dose of 2000 mg (160).

Indications

Because metformin does not cause weight gain, it is often preferred for overweight and obese people with T2DM. It can be introduced in insulin-resistant states before the development of hyperglycemia (163). Metformin can resume ovulation in women with anovulatory polycystic ovarian syndrome (PCOS), which is an unlicensed application of the drug in the absence of diabetes (154).

Adverse effects and contraindications

The main tolerability issue with metformin is abdominal discomfort and other gastrointestinal adverse effects, including diarrhea, nausea, vomiting, flatulence, stomach upset and metallic taste in approximately 30% of patients (169). Anorexia and stomach fullness are likely part of the reason for weight loss, noted with metformin. These effects are often transient and can be ameliorated by taking the drug with meals and using a small initial dose, which is then gradually titrated slowly until target level of blood glucose control is attained or using extended-release preparations of metformin(170); however, around 5% of patients cannot tolerate the drug at any dose (171). It can reduce gastrointestinal absorption of vitamin B12, which rarely causes frank anemia (172).

The most serious adverse event associated with metformin is lactic acidosis that is typically characterized by a raised blood lactate concentration, decreased arterial pH and/or bicarbonate concentration with an increased anion gap. It is rare, but about half of cases are fatal (154). The true likelihood of lactic acidosis, occurring as a result of metformin accumulation, is unclear. However, given these concerns, the drug is contraindicated in the setting of renal dysfunction or in those at risk for lactic acidosis, such as individuals with kidney hypoperfusion due to hypotension or septicemia, congestive heart failure, chronic cardiopulmonary dysfunction, significant hepatic dysfunction or alcohol abuse. Renal function must be assessed prior to and periodically during metformin therapy, particularly in the elderly (160).

Presenting symptoms of lactic acidosis are generally non-specific (flu-like symptoms), but often include hyperventilation, malaise and abdominal discomfort. Treatment should be commenced promptly; bicarbonate remains the usual therapy. Hemodialysis to remove excess metformin can be helpful, and may assist restoration of fluid and electrolyte imbalance occurred during treatment with high dose intravenous bicarbonate (154).

Metformin also should be temporarily stopped when using intravenous radiographic contrast media or during surgery with general anaesthesia (154).

1.2. Thiazolidinediones (TZDs)

TZDs are pharmacological ligands for the nuclear receptor peroxisome proliferator- activated receptor- γ (PPAR-γ), which is highly expressed in adipose tissue and to a lesser extent in muscle, pancreatic β-cells, vascular endothelium and macrophages (173). Therefore, thiazolidinediones can affect responsive genes at these locations, giving rise to “pleiotropic effects” (174). Many of these genes participate in lipid and carbohydrate metabolism.

Troglitazone was the first thiazolidinedione to enter routine clinical use; however, it was associated with fatal cases of idiosyncratic hepatotoxicity and was withdrawn in 2000(175). Two other thiazolidinediones; rosiglitazone and pioglitazone were then introduced, which did not show hepatotoxicity, indicating that troglitazone’s hepatotoxicity has presumably a compound specific phenomenon (176). However, rosiglitazone was withdrawn in 2010 from market, as the clinical investigations revealed its implication in cardiovascular side effects (177).

Mode of action

TZDs stimulate PPAR-γ, promoting differentiation of pre-adipocytes into mature adipocytes (178); these new small adipocytes are particularly sensitive to insulin and show increased uptake of fatty acids with increased lipogenesis (179). This, in turn, reduces circulating free fatty acids, facilitating glucose utilization and restricting fatty acid availability as a source for hepatic gluconeogenesis. By reducing circulating fatty acids, ectopic lipid deposition in muscle and liver is reduced, which further contributes to improvements of glucose metabolism. TZDs also increase glucose uptake into adipose tissue and skeletal muscle via increased availability of GLUT4 glucose transporters (154).

Pharmacokinetics

Absorption of rosiglitazone and pioglitazone is rapid and almost complete, with peak concentrations at 1-2 hours, but slightly delayed when taken with food. Both drugs are metabolized extensively by the liver and are almost completely bound to plasma proteins; but their concentrations are not sufficient to interfere with other protein-bound drugs (154).

Indications

TZDs are indicated as monotherapy in T2DM, associated with no risk for hypoglycemia development. They are often used to gain additive efficacy in combination with other antidiabetic drugs, particularly metformin in overweight patients(180). Interestingly, because of the effects of thiazolidinediones on hepatic fat metabolism, these drugs might even be useful for the treatment of non-alcoholic steatohepatitis (181).

Adverse effects and contraindications

Fluid retention, leading to weight gain, anemia and development of heart failure as well as increased incidence of bone fractures are the major adverse effects of TZDs(153, 182).

Their use is contraindicated in patients with evidence of heart failure or pre-existing liver disease (183) and they should be used with caution in patients with osteoporosis and pre-existing macular edema (184). A debate on the risk of tumor development upon stimulation of PPAR-γ in colonic cells has been reported; thus, familial polyposis coli is a contraindication to TZDs on the theoretical grounds (183).

2. Insulin secretagogues

2.1. Sulfonylureas

Since their introduction in the 1950s, sulfonylureas (SUs) have been used extensively as insulin secretagogues for the treatment of T2DM. Sulfonylureas were developed as structural variants of sulfonamides, after the latter were reported to cause hypoglycemia. Early sulfonylureas such as carbutamide, tolbutamide and chlorpropamide are often referred to as “first generation”. These have been largely superceded by the more potent, probably safer “second generation” sulfonylureas, notably glibenclamide (glyburide), gliclazide, glipizide; and then followed by glimepiride, which is considered “third generation” of SUs (183).

Mode of action

Sulfonylureas act directly on the β-cells of the islets of Langerhans to stimulate insulin secretion. They enter β-cell and bind to the cytosolic surface of the sulfonylurea receptor 1 (SUR1), which forms part of voltage dependent K+ ATP channels, leading to its closure and reducing the efflux of potassium, enabling membrane depolarization, which in turn opens adjacent voltage-dependent L-type calcium channels, increasing calcium influx and causing release of insulin (185). They are ineffective in totally insulin-deficient patients, requiring about 30% of normal β-cells function for successful therapy(186). SUs don’t increase insulin formation but stimulate the release of stored insulin in response to glucose concentrations, which are below the normal threshold for glucose-stimulated insulin release (approximately 5 mmol/L), thus they are capable of causing hypoglycemia in normal and diabetic subjects (183).

Sulfonylureas appear to enhance insulin-stimulated glucose utilization in liver, muscle and adipose tissue through increasing insulin receptor number and enhancing the post-receptor complex enzyme reactions mediated by insulin (187). They are capable of suppressing hepatic glucose production and potentiating adipose tissue glucose transport and lipogenesis, as well as skeletal muscle glucose uptake and glycogen synthesis (154). It has been advocated that sulfonylurea drugs have extrapancreatic effects, in addition to their insulin secretory effect on pancreatic β-cells, as they effectively improve peripheral insulin resistance through activation of peroxisome proliferator-activated receptor-γ (PPAR-γ like activity) (188).

Pharmacokinetics

Sulfonylureas vary considerably in their pharmacokinetic properties, which in turn affects their clinical suitability for different patients. Longer acting sulfonylureas can be given once daily, but carry greater risk of hypoglycemia, especially those with active metabolites. Sulfonylureas are highly bound to plasma proteins, which can lead to interactions with other protein-bound drugs, such as salicylates, NSAIDs, sulfonamides and warfarin, increasing the risk of hypoglycemia (154).

Other drug interactions include:

- Interactions that increase glucose lowering effect of SUs; as with some antifungals and MAOIs by reducing hepatic metabolism and with probencid by decreasing excretion.
- Interactions that decrease glucose lowering effect of SUs; as with rifampicin and other microsomal enzyme inducers.

Indications

Sulfonylureas are widely used as monotherapy and in combination with metformin, a thiazolidinedione or an α-glucosidase inhibitor (189). These combinations afford an additive glucose-lowering efficacy, at least initially, but increase the risk of hypoglycemia.

Adverse effects and contraindications

Weight gain, reflects the anabolic effects of increased plasma insulin concentrations. Hypoglycemia is the most common and potentially most serious adverse effect of sulfonylurea therapy. Very occasionally, sulfonylureas produce sensitivity reactions (183).

2.2. Meglitinides (short-acting prandial insulin releasers)

Nowadays, postprandial hyperglycemia is widely recognized as a central feature of early diabetes and impaired glucose tolerance (IGT). It is caused primarily by the impairment of first phase insulin secretion and its correction is important for long-term glycemic control (190). Meglitinide analogs, known as non-sulfonylurea secretagogues, were evaluated as potential antidiabetic agents after an observation in the 1980s that meglitinide, the non-sulfonylurea moiety of glibenclamide, could stimulate insulin secretion similar to sulfonylureas. Repaglinide, which was the first approved member of this group, and nateglinide, were introduced as “prandial insulin releasers” (191).

Mode of action

Prandial insulin releasers act similar to SUs. However, they activate a different potassium channel in the pancreatic β-cell, leading to membrane depolarization and insulin release (192). By generating a prompt increase of insulin to coincide with meal digestion; these agents help to restore partially the first phase glucose-induced insulin response that is lost in T2DM. Specifically targeting postprandial hyperglycemia might also address the vascular risk attributed to prandial glucose excursions and reduce the risk of interprandial hypoglycemia as less insulin is secreted several hours after meal (154).

Pharmacokinetics

The pharmacokinetic properties of these compounds favored a rapid but short-lived insulin secretory effect that suited administration with meals to promote prandial insulin release. Repaglinide is almost completely and rapidly absorbed with peak plasma concentrations after about 1 hour. It is quickly metabolized in the liver to inactive metabolites and rapidly eliminated in the bile with a terminal elimination half-life of 1 to 1.7 hours (193). Taken about 15 minutes before a meal; repaglinide produces a prompt insulin response, which lasts about 3 hours, coinciding with the duration of meal digestion(183). Repaglinide may be more suitable than nateglinide in patients with moderate renal insufficiency, where metformin and some SUs are contraindicated (183, 192).

Indications

They are theoretically safer in older adults; particularly if other agents are contraindicated because of their short half-life and lower risk of hypoglycemia; however, the need for multiple daily dosages may be a disincentive. They can be used in patients who have an allergy to SUs medication (192). Prandial insulin releasers can be used as monotherapy in patients inadequately controlled by non-pharmacological measures or as add-ons to metformin or TZDs to produce a synergistic effect (154).

Adverse effects

Fewer and less severe hypoglycemic episodes occur with meglitinides than with sulfonylureas. They have a similar risk for weight gain as SUs (192). Sensitivity reactions are uncommon (183).

3. α-Glucosidase inhibitors

Acarbose, the first α-glucosidase inhibitor, was introduced in the early 1990s, followed by two further agents, miglitol and voglibose (194).

Mode of action

Their mechanism of action is unique. This is the sole dug class not targeted at a specific pathophysiological defect of type 2 DM. They competitively inhibit the activity of α-glucosidase enzymes in the brush border of enterocytes lining the intestinal villi, preventing the enzymes from cleaving disaccharides and oligosaccharides into monosaccharides, the final steps of carbohydrate digestion, delaying glucose absorption. By moving glucose absorption more distally along the intestinal tract, α-glucosidase inhibitors may alter the release of glucose-dependent intestinal hormones, GIP and GLP-1, which probably reduce postprandial insulin concentrations concurrently with the attenuated rise in postprandial glucose levels (183). Thus, α-glucosidase inhibitors can effectively reduce postprandial glucose excursions and improves glycemic control (195).

Pharmacokinetics

Acarbose is degraded by amylases in the small intestine and by intestinal bacteria; less than 2% of the unchanged drug is absorbed along with some of the intestinal degradation products. Absorbed material is mostly eliminated in the urine within 24 hours(183, 194). Miglitol is almost completely absorbed and eliminated unchanged in the urine and faeces (194).

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