GLUCOSE HOMEOSTASIS:

Glucose homeostasis is maintained by the highly coordinated interaction of three physiologic processes: insulin secretion, tissue glucose uptake and hepatic glucose production. By this way, the body tries to keep a constant supply of glucose for cells by maintaining a constant glucose concentration in the blood. Normal glucose homeostasis represents the balance between intake (glucose absorption from the gut), tissue utilization (glycolysis, pentose phosphate pathway, tricarboxylic acid cycle, glycogen synthesis) and endogenous production (glycogenolysis and gluconeogenesis) [1]. The most important metabolic fuels are glucose and fatty acids.

Glucose is preferentially used by brain and muscles. To ensure the continuous provision of glucose to the brain and other tissues, metabolic fuels are stored for use in time of need. Glucose homeostasis is controlled primarily by the anabolic hormone insulin and also by some insulin-like growth factors [2]. Several catabolic hormones (glucagon, catecholamines, cortisol, and growth hormone) oppose the action of insulin and are known as anti-insulin or counter-regulatory hormones [3].

1.1. Role of insulin and glucagon:

Insulin is a 51 amino-acid protein hormone [4] secreted by the ȕ-cell of the pancreatic islets in response to the increase in plasma glucose following a meal. It decreases the plasma glucose concentration by promoting i) the uptake of glucose into tissues, ii) intracellular glucose metabolism and iii) glycogen synthesis [5-7]. Insulin is secreted by ȕ-cells, which constitute approximately 70% of all islet cells. Insulin acts on three main target tissues: the liver, muscle, and adipose tissue. Its action occurs via the ȕ-subunit of a membrane receptor. The binding of insulin to this membrane

receptor triggers serial reactions which affect glycogen synthesis and glucose transport [8].

Glucagon is a small, single chain, 29 amino-acid peptide secreted by the pancreatic Į-cells. Overall, glucagon focuses energy metabolism on the endogenous production

of glucose. Its main effect is the mobilization of the fuel reserves for maintenance of the blood glucose level between meals. Glucagon inhibits glucose-utilizing pathways and the storage of metabolic fuels. It acts rapidly on the liver to stimulate glycogenolysis and to inhibit glycogen synthesis, glycolysis, and lipogenesis. As with insulin, the first stage of glucagon action corresponds to its binding to a specific membrane receptor. The signalling pathway involves GTP binding to a G-protein complex and induces activation of a second messenger and phosphorylation of regulatory enzymes for carbohydrate and lipid metabolism [9]. The fine balance between insulin and glucagon action is a key factor in the control of fuel metabolism.

1.2. Role of glucose transporters:

Glucose is not able to cross the cell plasma membrane alone. It needs the presence of integral membrane carrier proteins called glucose transporters (Glut) [10]. Different isoforms of Glut have been highlighted. Each of the Glut isoforms has a specific tissue distribution. Glut1 to Glut 4 isoforms are strongly involved in the control of glucose homeostasis. Glut 1 is the first glucose transporter that has been characterized [11]. It is predominantly expressed in erythrocytes and endothelial cells. One of the important roles of Glut 1 is to allow the passage of glucose through the blood brain barrier [12]. Glut 2 is strongly expressed in pancreatic ȕ cells and in the liver. In case of an increase of plasma glucose concentration, Glut 2 allows glucose to enter in pancreatic ȕ-cells. This entry of glucose induces the release of

insulin by ȕ-cells. In liver Glut 2 allows shutting off glucose output [13]. Glut 3 is the main glucose transporter present in neurons. One of the principal characteristics of Glut 3 is its very high affinity for glucose, which probably ensures a continuous and adequate brain glucose supply under conditions of starvation [14]. Glut4 is present in tissues that exhibit insulin-stimulated glucose transport such as skeletal muscles, cardiac muscles or adipose tissue. In these tissues insulin allows an increase of glucose intake by inducing the translocation of Glut4-containing intracellular vesicles to the plasma membrane [15].

1.3. Role of liver:

The liver is the central organ of the glucose production control via insulin action, which stimulates both glycolysis and glycogen synthesis. On the one hand, insulin acts after a meal on the hepatocytes to stimulate the action of several enzymes, including glycogen synthase, to synthesized glycogen. On the other hand, it is also able to transforms non-carbohydrate compounds into glucose during gluconeogenesis [16, 17], allowing a continuous supply of glucose over long periods.

This way completes the short-term supply of endogenous glucose provided by the mobilization of glycogen after the decreasing of the glucose content of the extracellular fluid. Gluconeogenesis uses non-sugar carbon substrates, such as pyruvate, lactate (originate from anaerobic glycolysis), glycerol (from adipose tissue triglycerides) and glucogenic amino-acids. Insulin has also various effects in the liver.

It suppresses lipolysis and promotes the lipogenesis by the synthesis of long-chain fatty acids. These lipids are then packaged into very-low-density lipoproteins (VLDL), and secreted into the blood [18].

1.4. Role of adipose tissue:

As the caloric value of fats (9Kcal/g) [19] is higher than that of carbohydrates (4 Kcal/g) [20], long-chain fatty acids are the ideal storage fuel. Two main processes occur in adipocytes: lipogenesis and lipolysis. Lipogenesis enables the storage of triglycerides from glucose and circulating lipoproteins thanks to the lipoprotein-lipase.

Insulin has a key role in lipogenesis: it is necessary for glucose penetration in adipocytes and promotes glucose transformation in fatty acids. It also helps synthesis of glycerol phosphate from glucose to form triglycerides [21]. Lipolyse-activated triglycerides could also release fatty acids and glycerol in blood by lipolysis, which will be especially used by cardiac muscles [22, 23]. Fatty acid could be synthesized from glucose and pyruvate in adipocytes too. Triglycerides undergo lipolysis by lipases and are broken down into glycerol and fatty acids. Once released into the blood, the relatively hydrophobic free fatty acids bind to serum albumin for transport to tissues that require energy. The glycerol also enters the bloodstream and is absorbed by the liver or kidney, where it is converted to glycerol 3-phosphate by the enzyme glycerol kinase. Hepatic glycerol phosphate is mostly converted into glyceraldehyde 3-phosphate to rejoin the glycolysis and gluconeogenesis pathway [18].

1.5. Role of Muscles:

The rate of glucose entry into muscles can be modified significantly by the circulating concentration of insulin, which stimulates a variety of metabolic pathways in the muscle cells. In fact, insulin increases glucose transport, glucose metabolism, and glycogen synthesis. Insulin also increases cellular uptake of amino acids and stimulates protein synthesis. Muscle can use both glucose and fatty acids as energy sources. During intensive exercise, glucose is the preferred fuel. Fatty acids are the

main energy source at rest and during prolonged exercise. Muscle handles carbohydrate quite differently to the liver. In contrast to the liver, it cannot release glucose into the circulation, as it does not have glucose-6-phosphatase. Therefore, muscle uses glycogen for its own needs of energy and contributes to endogenous glucose production by releasing lactate, a product of anaerobic glycolysis, which is transported to the liver (for gluconeogenesis) [24] .

1.6. Role of the brain:

The brain is also a player in glucose homeostasis. In fact, in recent years, the idea that glucose metabolism throughout the body is coordinated by the brain has gained growing support [25]. Because the brain derives its metabolic energy almost exclusively from glucose, glucose sensing systems are located in the central nervous system and are able to control glucose homeostasis and energy storage [26, 27]. For insulin-stimulated glucose metabolism to occur in the brain, insulin receptors, and insulin-sensitive glucose transporters are required. Insulin receptors have been demonstrated throughout the human brain, with particularly high concentrations in the hypothalamus, cerebellum and cortex. Neuronal systems sense and respond to input from hormones, and reply by adaptive changes in energy intake, energy expenditure and hepatic glucose production. Findings showed that insulin has a direct effect on potassium ATP channels in the hypothalamus [28]. However, even if the role of the brain in metabolic regulation is clearly integrated now, exact mechanisms remain confusing.

2. GLUCOTOXICITY:

In the case of glucose homeostasis deficiency, blood glucose concentration will chronically increase. In contrast to physiological glucose concentration, chronic supraphysiological glucose concentration negatively affect a large number of organs and tissues, such as pancreas, eyes, liver, muscles, adipose tissues, brain, heart, kidneys and nerves. Glucose toxicity is the main cause of diabetic complications, which are often observed only several years after the development of the illness [29].

However, chronic hyperglycaemia can also increase the rate of development of early diabetic state by affecting the secretion capacity of pancreatic cells, which in turn increase blood glucose concentration. This vicious circle finally leads to the total incapacity of ȕ-cells to secrete insulin [30, 31].

2.1. Mechanism of glucotoxicity:

2.1.1. Oxidative stress:

Metabolic reactions continuously produce reactive oxygen species (ROS), such as superoxides (O2^-), hydroxyl radicals (OH^-), peroxyl radicals (ROO^-) or nitric oxide (NO). However, several antioxidant enzymes (SOD, Glutathione,…) allow maintaining low levels of ROS. Oxidative stress corresponds to the overproduction of ROS that can induce damages of cellular components, such as lipids, proteins or DNA [32]. Oxidative stress is strongly supposed to be a key event in diabetic complications [33]. Indeed, it has been shown that generation of ROS is increased in type 1 and type 2 diabetes, since in diabetic patients, the amount of the oxidative stress marker 8-hydroxydeoxyguanosine is 5 times higher compared to healthy patients [34]. Generation of ROS in diabetes seems to be directly linked with chronic hyperglycaemia. Indeed, hyperglycaemia is supposed to generate ROS through

different pathways, such as impairment of the red-ox balance, increase of advanced glycation products, activation of protein kinase C, or mitochondrial overproduction of superoxides [35]. The susceptibility of each tissue to oxidative stress can vary depending on the amount of expressed antioxidant enzymes. Pancreatic islets seem to be very sensitive to oxidative stress, since it expressed very low amounts of antioxidant enzymes [36]. Comparison of pancreatic islets between type 2 diabetic patients and healthy patients has shown a significant increase of oxidative stress markers in diabetic patients, which correlates with the level of glucose-induced insulin secretion impairment. Moreover, the use of antioxidant drugs allowed an improvement of insulin secretion capacity, as well as insulin mRNA expression [37].

Oxidative stress is also strongly suspected to be involved in chronic hyperglycaemia-induced insulin resistance, in different tissues [38]. Indeed, it has been shown that incubation of primary adipocyte cells with chronic high glucose concentration was able to induce oxidative stress [39]. Moreover, oxidative stress was also shown to induce insulin resistance in the 3T3-L1 adipocyte cell line, by inhibiting the translocation of Glut4 to the plasma membrane [40]. Hyperglycaemia-induced oxidative stress is also supposed to negatively act on liver and muscle cells. Indeed, chronic hyperglycaemia is known to induce oxidative stress in hepatocytes which can lead to liver cell damages [41]. It is also known that oxidative stress can induce insulin resistance in intact rat muscles [42] and that chronic hyperglycaemia is able to provoke oxidative stress in the L6 muscle cell line [43]. However, no direct link between chronic hyperglycaemia and insulin resistance has been shown so far in this tissue. Hyperglycaemia-induced oxidative stress is also involved in the development of macrovascular and microvascular diabetic complications [44]. Retina is highly sensitive to oxidative stress, since it has the highest oxygen uptake and glucose

oxidation than any other tissue [45]. Studies of diabetic rat retina and retinal cells incubated with high glucose concentration have shown an elevated concentration of superoxides [46, 47]. Animal models have shown that oxidative stress is not only involved in the development of retinopathy but also in the persistence of the pathology after normalization of glucose concentration, which is probably caused by the persistence of ROS [48]. Oxidative stress is also strongly suspected to be involved in the development of diabetic neuropathy [49]. Several studies have shown the capacity of antioxidant enzymes to prevent or reverse the toxic effect of chronic hyperglycaemia in the nerves [50, 51]. Moreover, the presence of high concentrations of the mitochondrial oxidative stress markers has been demonstrated in urine and kidneys of hyperglycaemic and obese ZSF1 rats [52, 53].

2.1.2. ER-stress:

Endoplasmic reticulum (ER) is an important organelle of the cell since it allows correct folding and post-translational modifications of proteins. Dysfunctions of the ER machinery lead to proteotoxicity in the ER, which is called ER-stress. In order to survive in ER-stress conditions, cells activate different protective mechanisms, which are called ER-stress response [54, 55]. Activation of the ER-stress response during diabetes has been demonstrated by several studies [56-59]. This observation has been confirmed at the cellular level since incubation of rat pancreatic islets with high glucose concentration has shown a strong activation of several ER-chaperone proteins, such as Bip and Grp94, which is a characteristic feature of ER-stress response activation [58]. Chronic hyperglycaemia also induces the activation of 2 other proteins involved in the ER-stress response: CHOP and GADD34 [60]. These 2 proteins are known to promote apoptosis in response to ER-stress activation [61-63].

In ȕ-cells, activation of hyperglycaemia-induced ER-stress has been postulated as being one possible mechanism leading to ȕ-cell death [64]. Reticulum stress activation is also involved in the apparition of insulin resistance in peripheral tissues.

Drug induction of ER-stress in liver cells has shown an inhibition of the insulin signalling pathway [65]. In a similar manner, ER-stress induction in adipocytes is also supposed to induce insulin resistance [66, 67]. However, so far, no direct link between chronic hyperglycaemia-induced ER-stress and insulin resistance in liver and adipose tissue has been demonstrated. Concerning skeletal muscles, it is known that chronic hyperglycaemia induces insulin resistance in this tissue [68], even if the mechanism triggering this resistance is still unknown.

2.1.3. Protein glycation:

Another consequence of prolonged exposition of organisms to hyperglycaemia conditions derived from the non-enzymatic reaction between glucose and proteins.

Protein glycation is a highly-selective process that occurs between reducing carbohydrates with either terminal or Į-amino groups in lysines or guanidine groups in arginine residues. The mechanism of the glycation reaction based on the Maillard reaction can be described at three stages as shown in Fig. 1.

The relevance of this glucotoxicity consequence is based on the alteration of the structure, biological function and turnover of proteins when glycation takes place. In this sense, two different levels can be distinguished in this post-translational modification (PTM) according to the Maillard reaction (see Fig. 1): (1) that occurring at the initial and intermediate stages in which proteins are still recognizable as glycoconjugates of original proteins and (2) that taking place as a consequence of a

longer exposition to glucose, in which the structure of the protein is seriously altered by formation of Advanced Glycation End-products (AGEs).

The first level of protein glycation is kinetically slow in the human body [69, 70].

However, these modifications are accelerated under pathophysiologic conditions being strongly associated with hyperglycaemia conditions. For this reason, glycation has often been related to chronic complications of diabetes mellitus, renal failure and degenerative changes occurring in the course of aging [71-73]. Lapolla et al. have evaluated the glycation level of ApoA-I, which can be a diagnostic tool to assess the metabolic state of diabetic and/or nephropathic patients. Glycation of ApoA-I due to glyco-oxidation stress involves a change in the functionality of the protein which might affect cholesterol transport efficiency [74]. Calvo et al. also evaluated the glycation of HDL in type 1 and 2 diabetic patients as compared to that on control healthy subjects. Glycated ApoA-I was isolated from diabetic patients in order to compare its lipid binding properties to those of ApoA-I from healthy subjects.

Fluorescent measurements evidenced that ApoA-I glycation involves a decrease in the stability of the lipid–apolipoprotein interaction and also in the apolipoprotein self-association [75-77].

Also during diabetes, haemoglobin, transferrin and other proteins present in blood can suffer modifications in their structure due to reaction with glucose. For instance, glycohaemoglobin is frequently used as an indicator for long-term (4−6 weeks) control in patients with diabetes mellitus [78]. The AGEs formation is thought to play a role in age-related chronic diseases such as atherosclerosis [79], amyloidosis, actinic elastosis or neurodegenerative disorders such as Alzheimer [80], Parkinson [81], Pick and Lewy body diseases [82]. Some authors have suggested that the most serious damaging effects are caused by AGEs and not by initial glycation products

[83]. Advanced glycation end-products form protein crosslinks, which can alter their physicochemical properties [84]. These modifications have also been suggested to lead to alterations in the binding of glycated human albumin serum to drugs such as phenytoin, salicylate or ibuprofen [85-87]. Changes in structure and function of structural proteins, such as collagen, fibronectin, tubulin or actin have been ascribed to AGEs formation as well. In addition, protein-AGEs adducts seem to inactivate metabolic enzymes [69]. Another critical property of AGEs is their ability to activate transduction receptors of the immunoglobulin family. Ramasamy et al. hypothesised that AGEs impart a potent impact on tissue stimulating processes linked to inflammation and further consequences due to interaction with these receptors [88].

Protein–AGEs adducts may be repaired by enzymatic mechanisms or removed by macrophages, lysosome-containing cells or proteolytic enzymes [89, 90]. The degradation of AGE-modified proteins leads to the production of AGE-peptides, which are only partially and selectively excreted by the kidneys. In cases of renal failure, this excretion is impaired with subsequent accumulation of AGE–peptides in plasma. Thus, AGE peptides exhibit the same toxic activity as AGEs–proteins and, for this reason, high AGE-peptide concentration can be considered as a marker of the presence of AGE-modified proteins [91, 92].

2.2. Glucotoxicity and pancreatic islet cells:

Many studies have been performed to better understand the effect of glucotoxicity on pancreatic islet cells, which are involved in the control of insulin secretion. They have shown that prolonged exposure of pancreatic ȕ-cell lines, such as INS-1, HIT-15 or ȕTC6 to high glucose concentration, strongly decrease their insulin secretion capacity [93]. Several proteins involved in the regulation of insulin secretion have

been shown to be down-regulated in response to long-term hyperglycaemia [31]. In addition, chronic hypoglycaemia also induces a decrease of insulin mRNA expression due to the degradation of the insulin gene promoter but also of the PDX-1 and MafA transcription factors, which are essential for insulin gene expression [94].

Oxidative stress seems to be linked to this decrease of insulin mRNA expression since the concomitant use of antioxidant compounds and chronic high glucose concentration does not induce the decrease of insulin mRNA expression [95]. The toxic effect of glucose on insulin secretion and insulin gene expression can be reversed by cell incubation with low glucose concentration. However, after a certain time of cell incubation with high glucose concentration, the toxic effect of glucose becomes irreversible [96]. The final step of ȕ-cell glucotoxicity corresponds to the induction of apoptosis. Several studies have shown the induction of apoptosis in ȕ-cell lines after long-term ȕ-cell incubation with high glucose concentration [97].

Apoptosis induction was also demonstrated in human and Psammomys obesus pancreatic islets incubated with high glucose concentration (11.2mM) compared to

Apoptosis induction was also demonstrated in human and Psammomys obesus pancreatic islets incubated with high glucose concentration (11.2mM) compared to