METABOLISM

Diabetes mellitus, a common metabolic disorder resulting from defects in

insulin secretion or action or both and is characterized by an increase in

insulin resistance in conjunction with the inability of pancreatic beta cells

to secrete sufficient insulin to compensate  (American Diabetes

Association, 2005; Centre for Disease Control and Prevention 2005).

Mortality and morbidity in T2DM are due to occurrence of microvascular

complications such as diabetic nephropathy, neuropathy and retinopathy

along with macrovascular complications such as accelerated

atherosclerosis causing ischemic heart and cerebrovascular disease

( Morrish NJ, et al. 2001).

Types of Diabetes mellitus

Two major forms of diabetes occur—Type 1 diabetes mellitus (T1DM) and

Type-2 diabetes mellitus (T2DM). T1DM occurs when the insulin-

producing -cells in the pancreas are destroyed, typically by anβ

autoimmune, T cell-mediated mechanism, resulting in the production of

insufficient amounts of insulin (Wang TT, et al. 1998). T2DM is caused by a

resistance to insulin combined with a failure to produce sufficient insulin.

T2DM is commonly linked to obesity, which can cause insulin resistance

(Henegar JR, et al. 2001; Chagnac A, et al. 2003). Despite the different

pathogenic mechanisms of T1DM and T2DM, they share common

symptoms including glucose intolerance, hyperglycemia, hyperlipidemia

and similar complications. A pivotal role for reactive oxygen species (ROS)

13

METABOLISM

has been proposed in both the development of insulin resistance and in

the pathogenesis of both micro- and macro-vasculature complications

(Sharma K, et al. 1999; Zanatta CM, et al. 2008; Brosius FC, et al. 2008).

Etiology and Pathophysiology of Type-2 Diabetes

Etiology

Diabetes mellitus has reached epidemic proportions and affects more than

170 million individuals worldwide. Global estimates for the year 2010

predict a further growth of almost 50%, with the greatest increases in the

developing countries of Africa, Asia, and South America (Zimmet P, et al.

2001). In more developed societies, the prevalence of diabetes mellitus has

reached about 6% (King H, et al. 1995) and, even more alarmingly, among

obese white adolescents 4% had diabetes and 25% had abnormal glucose

tolerance (Sinha R, et al. 2002) Some 90% of diabetic individuals have

type-2 (non-insulin-dependent) diabetes mellitus, and within this category

no more than 10% can be accounted for by monogenic forms such as

maturity onset diabetes of the young (Fajans SS, et al.

2001) and mitochondrial diabetes (Maassen JA, et al. 2004) or late onset a

utoim-mune diabetes of the adult, which is actually a late-onset type-1

diabetes (Pozzilli P, et al. 2001). Thus, most diabetes in the world is

accounted for by “common” type-2 diabetes, which has a multifactorial

pathogenesis caused by alterations in several gene products.

14

METABOLISM

The medical and socioeconomic burden of the disease is caused by

the associated complications, (Wei M, et al. 1998; UK Prospective Diabetes

Study (UKPDS), Lancet 1998) which impose enormous strains on health-

care systems. The incremental costs of patients with type-2 diabetes arise

not only when the diagnosis is established but at least 8 years earlier

(Nichols GA, et al. 2000). The devastating complications of diabetes

mellitus are mostly macro vascular and micro vascular diseases as a

consequence of accelerated atherogenesis. Cardiovascular morbidity in

patients with type-2 diabetes is two to four times greater than that of non-

diabetic people (Zimmet P, et al. Nature 2001).

Pathophysiology of Type-2 Diabetes

To understand the cellular and molecular mechanisms responsible for

type-2 diabetes it is necessary to conceptualise the framework within

which glycemia is controlled. Insulin is the key hormone for regulation of

blood glucose and, generally, normoglycemia is maintained by the

balanced interplay between insulin action and insulin secretion.

Importantly, the normal pancreatic β-cell can adapt to changes in insulin

action i.e. a decrease in insulin action is accompanied by upregulation of

insulin secretion (and vice versa). Figure: 1 illustrates the curve linear

relation between normal β-cell function and insulin sensitivity (Bergman

RN, 1989). Deviation from this hyperbola, such as in the patients with

impaired glucose tolerance and type-2 diabetes in fig: 1, occurs when β-

15

METABOLISM

cell function is inadequately low for a specific degree of insulin sensitivity.

Thus, β-cell dysfunction is a critical component in the pathogenesis of

type-2 diabetes (Weyer C, et al. 1999).

However,not only deviation from but also progression along the hyp

erbola affects glycemia. When insulin action decreases (as with increasing

obesity) the system usually compensates by increasing β-cell function.

However, at the same time, concentrations of blood glucose at fasting and

2 hrs, after glucose load will increase mildly (Stumvoll M, et al. 2003). This

increase may well be small, but over time becomes damaging because of

glucose toxicity and in it a cause for β-cell dysfunction. Thus, even with

(theoretically) unlimited β-cell reserve, insulin resistance paves the way

for hyperglycemia and type-2 diabetes (Michael S, et al. 2005).

Insulin resistance

Insulin resistance is said to be present when the biological effects of

insulin are less than expected for both glucose disposal in skeletal muscle

and suppression of endogenous glucose production primarily in the liver

(Dinneen S, et al. 1992). In the fasting state, however, muscle accounts for

only a small proportion of glucose disposal (less than 20%) whereas

endogenous glucose production is responsible for all the glucose entering

the plasma. Endogenous glucose production is accelerated in patients with

type-2 diabetes or impaired fasting glucose (Weyer C, et al. 1999; Meyer

C, et al. 1998). Because this increase occurs in the presence of

hyperinsulinaemia, at least in the early and intermediate disease stages,

16

METABOLISM

hepatic insulin resistance is the driving force of hyperglycemia of type-2

diabetes (Stumvoll M, et al. 2005).

Figure 1: Hyperbolic relation between cell function and insulin sensitivity

In people with normal glucose tolerance (NGT) a quasi-hyperbolic relation exists between cell

function and insulin sensitivity. With deviation from his hyperbola, deterioration of glucose

tolerance (impaired glucose tolerance [IGT] and type2 diabetes [T2DM]) occurs.

Insulin resistance is strongly associated with obesity and physical

inactivity, and several mechanisms mediating this interaction have been

identified. A number of circulating hormones, cytokines, and metabolic

fuels, such as non-esterified (free) fatty acids (NEFA) originate in the

adipocytes and modulate insulin action. An increased mass of stored

triglyceride, especially in visceral or deep subcutaneous adipose depots,

leads to large adipocytes that are themselves resistant to the ability of

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METABOLISM

insulin to suppress lipolysis. This results in increased release and

circulating levels of NEFA and glycerol, both of which aggravate insulin

resistance in skeletal muscle and liver (Boden G. 1997). Excessive fat

storage not only in adipocytes but “ectopically” in non-adipose cells also

has an important role (Danforth E Jr. 2000). For example, increased

intramyocellular lipids are associated with skeletal muscle insulin

resistance under some circumstances. The coupling between intrahepatic

lipids and hepatic insulin resistance seems to be even tighter (Seppala-

Lindroos A, et al. 2002; Bajaj M, et al. 2003).

The current understanding of the Pathophysiology of Type-2

diabetes suggests that complex interactions exist between multiple

pathways. These include abnormalities in glucose transport mechanisms,

increased activity of specific intracellular metabolic pathways, activation

of protein kinase C isoforms, formation of reactive oxygen species (ROS),

increased production of advanced glycation end products (AGEs) and,

altered activity of a variety of growth factors and cytokines (Liu Y, et al.

2005).

ROLE OF INSULIN RESISTANCE IN TYPE-2 DIABETES

Even if insulin exerts numerous different effects, so far insulin sensitivity

has been considered mainly in the context of glucose metabolism,

especially at the liver and muscle sites (Scheen AJ, et al. 1992). The

presence of insulin resistance in vivo can be evidenced during various

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METABOLISM

dynamic tests such as an oral glucose tolerance test, an intravenous

glucose tolerance test and a so-called euglycaemic hyperinsulinaemic

clamp (Scheen AJ, et al. 1995). Using the latter approach, it has been

extensively demonstrated that insulin-mediated glucose disposal

(essentially in the skeletal muscle) is largely reduced in patients with type-

2 diabetes. Furthermore, the concomitant use of isotopes showed that

hepatic glucose production is insufficiently inhibited by insulin, thus

demonstrating the presence of both muscular and hepatic insulin

resistance. Despite tremendous advances in molecular biology and the

continued identification of increasingly more molecules involved in the

insulin signaling cascade, the molecular mechanism (or mechanisms) that

underlines the development of insulin resistance in subjects prone to

develop type-2 diabetes still remains elusive (Gerich J. 1998). Genetic

mutations associated with insulin resistance are rare and it seems unlikely

that a single genetic alteration explains a large number of cases of insulin

resistance among type-2 diabetic patients. Rather, it is more likely that a

number of different genes may contribute, some of which may be obesity

genes. Three commonly encountered factors that influence insulin

sensitivity and are apparently not genetically determined are aging,

exercise and dietary constituents. However, even if the effects of age,

exercise and diet are considered, there is still a large between-subject

variation in insulin sensitivity that has to be related to other factors. A

major component of this residual variation may be related to obesity, but

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more importantly to differences in body fat distribution (Montague CT, et

al. 2000). A vast majority of type-2 diabetic patients are overweight, and

obesity undoubtedly plays a major role in the development of the disease

(Kahn SE. Diabetologia. 2003). While it is recognized that obesity is an

important determinant of insulin sensitivity (Scheen AJ, et al. 1995), body-

fat distribution seems to be a critical aspect (Montague CT, et al. 2000).

Several groups have made a strong case that the intra-abdominally fat

depot is the primary correlate of insulin sensitivity; while others have

proposed that the central subcutaneous fat depot is the major factor

determining a reduction in insulin sensitivity. Obese individuals with most

of their fat stored in visceral adipose depots generally suffer greater

adverse metabolic consequences than similarly overweight subjects with

fat stored predominantly in subcutaneous sites. Excess abdominal fat mass

is associated with an increased release of NEFA that may trigger a

reduction in insulin sensitivity at both the hepatic and the muscular levels.

In the liver, this results in an increased glucose output (essentially due to

enhanced gluconeogenesis), a decreased insulin extraction and an

increased VLDL production while in the skeletal muscle this results in a

reduction in glucose oxidation and glucose storage as glycogen (so-called

Randle’s effect) (Reaven GM, et al. 1995 ). Numerous insulin-resistant

obese patients have also a so-called metabolic syndrome associating

impaired glucose tolerance (or type-2 diabetes), dyslipidaemia and

arterial hypertension, all factors aggravating the risk of cardiovascular

20

METABOLISM

diseases (Scheen AJ. 1996). A fuller understanding of the biology of central

obesity will require information regarding the genetic and environmental

determinants of human fat topography and of the molecular mechanisms

linking visceral adiposity to degenerative metabolic and vascular disease.

Long-term positive energy balance may lead not only to excess triglyceride

depots in the adipose tissue, but also to ectopic triglyceride storage. The

ability of the adipocytes to function properly when engorged with lipid can

lead to lipid accumulation in other tissues, reducing their ability to

function and response normally. Liver steatosis is a common finding in

obese subjects, especially in those with intra-abdominal fat depot, and

non-alcoholic fatty liver disease is now considered as part of the metabolic

syndrome associated to insulin resistance. In addition, intramuscular

triglyceride levels are increased in obese subjects, and a close relationship

has been repeatedly reported between the degree of ectopic

intramyocellular triglyceride depot and the severity of muscular insulin

resistance (Ravussin E, et al. 2002). Interestingly, ectopic fat accumulation

in insulin sensitive tissues may be associated with insulin resistance

independent of overall obesity. However, the understanding of the causes

and mechanisms underlying fat accumulation in skeletal muscle and the

liver are limited. Identifying why some individuals store fat in insulin-

sensitive tissues, but others do not, may be of great importance for the

development of new insulin sensitizing agents. The role of counter

regulatory hormones in the resistance to insulin in patients with type-2

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METABOLISM

diabetes remains unclear. Nevertheless, plasma glucagon levels are

regularly increased in type-2 diabetic patients and this hormone could

contribute to enhance gluconeogenesis and hepatic glucose output,

especially in presence of insulin deficiency (Scheen AJ, et al. 1996).

Pharmacological attempts to decrease glucagon secretion led to

substantial reduction in plasma glucose levels, arguing for a significant

role of this hormone in the development of hyperglycemia in type -

2diabetes. Finally, a hemodynamic hypothesis of insulin resistance has

also been put forward (Scheen AJ, et al. 1996). Reduced number of muscle

capillaries and impaired insulin-induced vasodilatation (essentially in the

postprandial state) may contribute to increase the distance and to alter the

insulin diffusion process from the capillary to the muscular cells and there

by insulin action in obese patients with type-2 diabetes (Scheen AJ, et al.

1996).

Fig 2: Contribution of endocrine pancreas, liver, skeletal muscle and adipose tissue in the

pathogenesis of type-2 diabetes: emerging role of ectopic fat storage in liver, muscle and beta-

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METABOLISM

cell and of adipose tissue as an endocrine organ releasing various adipocytokins in addition to

non-esterified fatty acids (NEFA) in presence of positive energy balance and obesity.

Role of insulin deficiency in Type-2 diabetes

Beta-cell function in type-2 diabetes has been the subject of intense

investigation for several decades, and considerable progress has been

made during the recent years in the knowledge of the physiology and

Pathophysiology of insulin secretion (Polonsky KS, 1995). Recent data

demonstrated that beta-cell deficit and beta-cell apoptosis are present in

humans with type-2 diabetes (Butler AE, et al. 2003). Once hyperglycemia

exists, beta-cell dysfunction is clearly present in subjects with type-2

diabetes (Polonsky KS. 1995). Individuals with type-2 diabetes also show a

decrease in the potentiation by oral rather than parenteral glucose

loading, a phenomenon known as the “incretion effect” which is associated

to glucose-dependent insulin tropic peptide (GIP) and glucagon-like

peptide (GLP)-1 secreted by enterocytes. In addition, alterations in

pulsatile insulin release and ultradian oscillatory insulin secretion can be

observed. Finally, inefficient proinsulin processing to insulin and a

reduction in the release of islet amyloid polypeptide (IAPP, also known as

amylin) has been observed in established type-2 diabetes (Kahn SE,

Diabetologia. 2003).

Several mechanisms have been proposed to explain the -cellβ

deficiency observed in subjects prone to develop type-2 diabetes (Scheen

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METABOLISM

AJ, et al. 1996). Unfavorable metabolic environment may also play a

deleterious role, especially increased glucose levels that may induce

glucotoxicity (Yki -Jarvinen H. 1992) and a chronic increase in NEFA levels

that may induce lipotoxicity, both processes contributing to alter insulin

secretion. Interestingly, ectopic deposition triglycerides in pancreatic

islets has also been reported, a condition that may contribute to

dysfunction of the beta cell. Indeed, although the mechanism of lipotoxicity

in the beta cell remains unclear, it has been suggested that accumulation of

triglycerides increases nitric oxide, which causes oxidative damage and

apoptosis in the cells (Unger RH. 2002). Finally, defects in insulin signaling

pathways associated with insulin resistance in peripheral tissues have

recently been found to disrupt insulin secretion by pancreatic beta cells,

suggesting that insulin resistance in the beta cells may be, at least partly,

responsible for the beta-cell dysfunction and the development of type-2

diabetes (Greenberg AS, et al. 2002).

Metabolic changes in Type-2 Diabetes

Hyperglycemia is considered a major factor in the development of T2DM

and the adverse effects are recognizable through induces through four

major pathways: (i) increased polyol pathway flux (ii) increased advanced

glycation end-product formation (iii) activation of protein kinase C and

(iv) increased Hexosamine pathway flux (Setter SM, et al. 2003). An

increase in the entry of glucose into the polyol pathway, the diacylglycerol

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METABOLISM

(DAG) synthetic pathway, and the Hexosamine pathway was found in

mesangial cells cultured under high glucose conditions (Masakazu H, et al.

2003).

Polyol pathway

The polyol pathway, shown schematically in Fig: 3, focuses on the enzyme

aldose reductase. Aldose reductase normally has the function of reducing

toxic aldehydes in the cell to inactive alcohols, but when the glucose

concentration in the cell becomes too high, aldose reductase also reduces

that glucose to sorbitol, which is later oxidized to fructose. In the process

of reducing high intracellular glucose to sorbitol, the aldose reductase

consumes the cofactor NADPH (Lee AY, et al. 1999). But as shown in Fig: 3,

NADPH is also the essential cofactor for regenerating a critical intracellular

antioxidant, reduced glutathione (Engerman RL, et al. 1994).

Subsequently, sorbitol is oxidized to fructose via sorbitol dehydrogenase,

with NAD+ reduced to NADH, providing increased substrate to complex-I

of the mitochondrial respiratory chain. Since the mitochondrial

respiratory chain is thought to be a major source of excess ROS in diabetes,

provision of additional electrons for transfer to oxygen-forming

superoxide would augment mitochondrial ROS production. In addition,

since sorbitol does not cross cell membranes, its intracellular

accumulation results in osmotic stress. Osmotic stress perse increases

cellular cytosolic generation of H2O2 (Pingle SC, et al. 2004). The polyol

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pathway increases susceptibility to intracellular oxidative stress

(Brownlee M, 2005).

Fig 3: Hyperglycemia increases flux through the polyol pathway. From Brownlee M: Biochemistry

and molecular cell biology of diabetic complications. (Nature 414:813–820, 2001.)

Formation of AGE’s

Binding of advanced-glycation end products (AGE) to receptor for AGE

(RAGE) produces a cascade of cellular signaling, such as Protein kinase C

activation. Indeed, AGE-induced PKC activation stimulates collagen mRNA

expression in human mesangial cells (Kim YS, et al. 2001). Moreover,

Amadori modified glycated albumin also stimulate PKC activation,

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METABOLISM

followed by increased production of TGF- protein and type IV collagen inβ

glomerular endothelial cells, even in physiologic glucose concentrations

(Chen S, et al. 2001).The ultimate fate of most AGEs within the body is

renal clearance; they can interact with a number of renal cellular binding

sites that mediate many of their biological effects. Arguably, the most

important of these binding sites is the receptor for AGEs (RAGE), a

member of the immunoglobulin super family (Neeper M, et al. 1992).

RAGE is a multiligand pattern recognition receptor involved in the

amplification of immune and inflammatory responses primarily via

activation of nuclear factor- B and production of interleukins and tumorⱪ

necrosis factor- (Schmidt AM, et al. 1995).β

Fig: 4 (Increased production of AGE precursors and its pathologic consequences. From Brownlee

M: Biochemistry and molecular cell biology of diabetic complications Nature 414:813–820,

2001.)

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Protein kinase C pathway

In this pathway, shown schematically in Fig: 5, hyperglycemia inside the

cell increases the synthesis of a molecule called diacylglycerol, which is a

critical activating cofactor for the classic forms of protein kinase-C, -β,- ,δ

and -α (Koya D, et al. 1997; Koya D, et al. 1998) When PKC is activated by

intracellular hyperglycemia, it has a variety of effects on gene expression,

examples of which are shown in the row of open boxes in Fig: 5, In each

case, the things that are good for normal function are decreased and the

things that are bad are increased. For example, starting from the far left of

Fig: 5, the vasodilator producing endothelial nitric oxide (NO) synthase

(eNOS) is decreased, while the vasoconstrictor endothelin-1 is increased.

Transforming growth factor-β and plasminogen activator inhibitor-1 are

also increased. At the bottom of the figure the row of black boxes lists the

pathological effects that may result from the abnormalities in the open

boxes (American Diabetes Association, 2005).

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METABOLISM

Fig: 5 Consequences of hyperglycemia-induced activation of PKC.

DIABETES, VOL. 54, JUNE 2005

Hexosamine pathway

Hexosamine pathway as shown schematically in Fig: 6, when glucose is

high inside a cell, most of that glucose is metabolized through glycolysis,

going first to glucose-6 phosphate, then fructose-6 phosphate, and then on

through the rest of the glycolytic pathway. However, some of that fructose

6-phosphate gets diverted into a signaling pathway in which an enzyme

called GFAT (glutamine: fructose-6 phosphate amidotransferase) converts

the fructose-6 phosphate to glucosamine-6 phosphate and finally to UDP

(uridine diphosphate) N-acetyl glucosamine. What happens after that is

the N-acetylglucosamine gets put onto serine and threonine residues of

transcription factors, just like the more familiar process of

phosphorylation, and over modification by this glucosamine often results

in pathologic changes in gene expression (Kolm-Litty V, et al. 1998;

Sayesk PP, et al. 1996; Wells L ,et al.

2003) For example, in Fig: 6,

increased modification of the

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transcription factor Sp1 results in increased expression of transforming

growth factor-β1 and plasminogen activator inhibitor-1, both of which are

bad for diabetic blood vessels (Du XL ,et al. 2000).

Inflammation & Type-2 DM

One of the most difficult questions to answer is why obesity elicits an

inflammatory response. Why, if the ability to store excess energy has been

preserved through the course of evolution, does the body react in a

manner that is harmful to itself? We hypothesize that this reaction is tied

to the inter dependency of metabolic and immune pathways (Kathryn E, et

al. 2005).

The role of TNF and IL-6 in Type –2 DMα

The TNF- and IL-6 found in plasma are likely produced by variousα

tissues, including activated leukocytes, adipocytes, and endothelial cells.

Because the increased circulating cytokine levels found in diabetes seem to

originate from noncirculating cells, and given the prompt increase of

plasma cytokine levels after acute hyperglycemia. Several studies have

demonstrated elevated levels of IL-6 and tumor necrosis factor- (TNF-α

) among individuals both with features of the insulin resistanceα

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METABOLISM

syndrome and with clinically overt type-2 diabetes mellitus ( Katherine E,

et al. 2002).

TNF-α

TNF-α is synthesized as a 26-kDa transmembrane pro-hormone, which

undergoes proteolytic cleavage to yield a 17-kDa soluble TNF-α molecule.

Despite the differences in size and location, both forms of TNF- α are

capable of mediating biological responses and together may be

responsible for both local and systemic actions of this cytokine. To date

most (if not all) of the cellular actions of TNF- α have been attributed to the

activities of two distinct receptors: type 1 TNFR1, a 55- or 60-kDa peptide

in rodents and humans, respectively ; and a type II TNFR2, a 75- or 80-kDa

in rodents and humans, respectively . Both of these receptors are

expressed ubiquitously (albeit at different ratios) and oligomerise upon

ligand binding. The extracellular domains of these two receptors exhibit

some sequence homology, while the intracellular domains appear to be

quite dissimilar. The latter has been interpreted as an indication that they

might signal for different biological functions (Jaswinder K, et al. 1999).

TNFR1 and TNFR2 promote a complex array of post receptor

signaling events. Primarily through three major pathway: 1) an apoptotic

signaling pathway, 2) activation JNK and MAPK Pathway and 3) activating

of NFkB pathway. Both TNFR1 and TNFR2 are expressed by skeletal

muscles (Uysal KT, et al. 1998). TNF- decreased tyrosineα

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phosphorylation of IRS-1 (Hotamisligil GS, et al. 1994; del Aguila LF, et al.

1999) and increases IRS-1 serine phosphorylation. This relative increase

in serine to tyrosine phosphorylation may lead to decreased ability of IRS-

1 to engage the p85 subunits of PI3K leading to decreased insulin

metabolic signaling. TNF- has also been shown to reduce signalα

transduction at the level of protein kinase B (PKB or Akt) and insulin

stimulated glucose uptake in skeletal muscle tissue. TNF- signalingα

through TNFR1 suppresses AMPK activity via transcriptional upregulation

of protein phosphates. Activation of this phosphates in turn reduces

skeletal muscle acetyl CoA Carboxylase phosphorylation, suppresses fatty–

acid oxidation and increases intramuscular DAG accumulation, effects that

are associated with insulin resistance both in vitro and vivo ( Bouzakri K,

et al. 2007).

IL-6

Interleukin-6 is produced by many cell types (fibroblasts, endothelial cells,

monocytes), and many tissues including adipose tissue. It is now well

known that IL-6 production by adipose tissue is enhanced in obesity (Fried

SK, et al. 1998; Bastard JP, et al. 2002). It is thought that 15 to 30% of

circulating IL-6 levels derives from adipose tissue production in the

absence of an acute inflammation (Mohamed-Ali V, et al. 1997).

Accumulating evidence also indicates the IL-6 is involved in glucose

metabolism and insulin action. However the nature of this role remains

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controversial. IL-6 may exert deleterious effects in insulin action and

glucose homeostasis. For example, the circulating level of IL-6 is elevated

in various insulin- resistance states including type-2 DM (Kim HJ, et al.

2004). Recent studies have suggested that IL-6 could be involved in insulin

resistance and its complications (Bastard JP, et al. 2002). The IL-6 receptor

belongs to the cytokine class I receptor family involving JAK/STATs (Janus

kinases/signal transducers and activators of transcription) signal

transduction pathway. Janus kinase activation induces STAT

phosphorylation, dimerisation and translocation to the nucleus to regulate

target gene transcription (Ihle JN, et al. 1995). It is now clearly established

that a strong interaction occurs between cytokine and insulin signaling

pathways, and generally leads to an impaired biological effect of insulin.

Although the exact mechanisms have not yet been clearly elucidated, it

could involve tyrosine phosphates’ activation (Kroder G, et al. 1996).  Or

an interaction between suppressor of cytokine signaling (SOCS) proteins

and the insulin receptor. Whatever the mechanisms involved, it has now

been clearly demonstrated that cytokines such as TNF- and IL-6 are ableα

to decrease insulin action. Therefore the chronic increase in circulating

cytokine levels could contribute to insulin resistance (Jean-Philippe

Bastard, et al. 2006).

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Fig: 7 Adipokines expression and secretion by adipose tissue in insulin-resistant subjects.

TNF- and Glucose metabolism α

Several mechanisms have been proposed to explain how TNF- inducesα

insulin resistance and hyperglycemia in adipocytes as well as systemically

(Fig: 8). First, TNF- has ability to inhibit the insulin stimulated tyrosineα

kinase activity of the insulin receptor and IRS-1 by inducing a serine

phophorylation of IRS-1and thus converting IRS-1 into an inhibitor of the

insulin receptor tyrosine kinase in vitro (Hotamisligil, et al. 1994). Second,

TNF- stimulates lipolysis in the adipose tissue, thus increasingα  the

plasma concentration of the FFA that eventually contributes to the

development of the insulin resistant (Feingold KR, et al. 1994).

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Accordingly, hepatic glucose production increases and glucose uptake and

metabolism in the muscles decrease. In adipocytes, TNF- down regulatesα

the expression of several proteins implicated in the insulin receptor

pathway, including IRS-1, GLUT-4, PPAR- , and adiponectin (Hotamisligil,ϒ

et al. 1994; Bruun JM, et al. 2003). Moreover, TNF- regulate theα

production of leptin, which known to regulate energy homeostasis, reduce

pancreatic insulin secretion and promote hyperglycemia (Ahima RS, et al.

2000). Therefore TNF- may also contribute indirectly to a hyperglycemicα

state by inhibiting adiponectin and by stimulating leptin action on the on

the glucose metabolic pathways. In addition monocytes chemotactic

protein-1, expression and production can be stimulated by TNF- (Sica A,α

et al. 1999), increasing the recruitment of macrophages into the adipose

tissue, which will augment the inflammatory state and trigger

hyperglycemia.

35

TNF

IR

Hyperglycemia

Adiponectin Lipolysis

Pro-inflammatory

PPAR-γ

FFA

LeptinMCP-1

IRS-1

AGE

AGE+RAGE

Cellular dysfunction and damage

GLUT4 TNF-RI

METABOLISM

Figure: 8 Mechanisms used by TNF- α to exert its effects on glucose metabolic pathways. GLUT4,

glucose transporter 4; IR, insulin receptor; IRS-1, insulin receptor substrate-1; MCP-1, monocyte

chemotactic protein-1; PPAR, peroxisome proliferator-activated receptor; TNF-RI, TNF-α

receptor type I.

Role of TNF-α in lipid metabolism

A key function of adipocytes is the regulation of lipid metabolism

according to the physiological energy requirements. The three biochemical

sites of regulation are fatty acid uptake, lipogenesis and lipolysis. Each of

these can be altered in response to extracellular stimuli such as insulin,

36

METABOLISM

cortisol, catecholamine’s, growth hormone, testosterone, free fatty acids

(FFA) and cytokines (Ramsay TG 1996). There is also an increasing body

of evidence to support a role for TNF-α in modulating lipid metabolism.

First, treatment of tumor-bearing (cachectic) rodents with anti- TNF-α

antibodies protects against abnormalities in lipid metabolism (Carbo N, et

al. 1994). In obesity, the elevated levels of TNF-α may also contribute to

the elevated basal lipolysis that is a characteristic of adipocytes from obese

subjects (Ramsay TG. 1996). This is further supported by studies in which

the administration of the sTNFR-IgG chimera, (Cheung AT, et al. 1998) but

not anti-TNF-α antibody (Lopezsoriano FJ, et al. 1997), decreases serum

FFA levels of obese rodents. Moreover, TNF-α deficient mice exhibit

lower circulating FFA and triglycerides than their wild-type littermates

(Uysal KT, et al. 1997; Ventre j, et al. 1997). Finally, the exogenous

application of TNF-α can stimulate lipolysis and increase circulating FFA

levels in vivo and in vitro. Since FFA can also mediate insulin resistance

the action of TNF-α on insulin sensitivity (see ‘Role of TNF-α in mediating

insulin resistance’ below) may be potentiated by increased lipolysis

(Jaswinder K, et al. 1998). In adipocytes, fatty acids are derived

predominantly via uptake from the circulation or from intracellular

lipolysis and to a lesser extent by de novo Synthesis from glucose. Fatty

acid uptake is facilitated by the extracellular activity of lipoprotein lipase

(LPL) which varies with nutritional and endocrine status (Ramsay TG.

1996). TNF-α has been shown to inhibit LPL activity and to down-regulate

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METABOLISM

its protein expression in vitro, and in vivo (Jaswainder K, et al.

1998). However, this action in human adipocytes remains controversial 

(Kern PA. 1998). More recently, TNF-α has also been shown to decrease

the expression of FFA transporters (such as FATP and FAT) in adipose

tissue (Memon RA, et al. 1998). Together, these actions of TNF-α could

decrease FFA uptake from the circulation and contribute to the

hyperlipidemia that is observed during infections and also in obesity.

In addition to inhibiting FFA uptake, TNF-α also acts to decrease the

expression of key enzymes involved in lipogenesis, namely acetyl-Co A

carboxylase and fatty acid synthase (Doerrler W, et al. 1994). However,

this may not occur in mature adipocytes (Memon RA, et al. 1998). None the

less, a recent report suggests that TNF-α can also decrease. Acyl-

CoA synthetase (ACS) mRNA and activity in hamster adipose tissue

(Memon RA, et al. 1998).This would result in reduced re-esterification of

FFA and together with the decreased substrate availability (through

inhibition of. insulin-sensitive glucose uptake) may be the cause of

suppressed triglyceride accumulation.

The third target of TNF-α action is the lipolytic machinery of

adipocytes. While the lipolytic process is predominantly regulated by

adrenergic stimulation and mediated by a cAMP-dependent pathway, the

mechanism of TNF-α -induced lipolysis appears to be distinct and less well

understood. Despite this, biochemical and genetic studies have clearly

38

METABOLISM

shown that the lipolytic actions are mediated by TNFR1 (Lopezsoriano J, et

al. 1997; Sethi J, et al. 1998). This is consistent with earlier studies which

used human TNF- (α selective for TNFR1 in murine systems) to stimulate

lipolysis in murine cultures (Kawakami M, et al. 1987). The actions

downstream of TNFR1 may involve transcriptional regulation of key

proteins involved in lipolysis, since a chronic exposure is required to

induce lipolysis fat cell cultures (Feingold KR, et al. 1992; Hauner H, et al.

1995) This is supported by the demonstration that TNF-α -induced

lipolysis is blocked by activators of the transcription factor, PPARϒ, such

as indomethacin, thiazolidinediones and 15-dPGJ2 (Jaswainder K, et al.

1998). The rate limiting enzyme, hormone sensitive lipase (HSL) is a

candidate protein whose levels may be regulated by TNF-α. However, this

action remains controversial since studies have either shown no

regulation by TNF-α (in human cultured adipocytes), or a significant

decrease in 3T3-L1 adipocytes (Souza SC, et al. 1998). It remains to be

seen if TNF-α can modulate the activity of HSL directly or acts by up-

regulating other, as yet unidentified, modulatory proteins. Perilipin is

another protein implicated in lipolysis, although its exact function remains

unknown. It is localized at the surface of lipid droplets (Greenberg AS, et al.

1991) and down regulated by TNF-α but not by catecholamine.

Intriguingly, over-expression of perilipin in 3T3-L1 adipocytes selectively

blocks TNF-α but not catecholamine-stimulated lipolysis (Sovz SC, et al.

1998). Whilst the down regulation of key lipolytic proteins is not

39

METABOLISM

consistent with the stimulation of lipolysis by TNF-α, these studies do

highlight one problem facing mechanistic investigations on differentiated

adipocytes: since the expression of lipogenic and lipolytic proteins is

intrinsically linked to adipocytes differentiation, and TNF-α can suppress

the expression of adipogenic genes, it is difficult to identify the direct

targets of TNF-α signaling. Indeed, the observations discussed above

(particularly on cultured 3T3-LI adipocytes) may be the causal effects of

modifying the differentiation program and this may not be representative

of actions in vivo. Nonetheless, the possibility remains that the lipolytic

actions of TNF-α  are mediated not by up regulation of key lipolytic

enzymes but by the down regulation of proteins involved in the trapping of

FFA in adipocytes (e.g. ACS and perilipin). Interestingly, 2β adrenergic

receptor ( 2β -AR) expression has been reported to be up-regulated by

TNF-α in adipocytes. However, this is accompanied by a significant

decrease in 1β and 3 β adrenoceptor expression (Hardri KE, et al. 1997).

40

METABOLISM

Figure: 9 Actions of TNF- α on lipid metabolism in adipocytes. As indicated by the shaded area,

the net action of TNF- α in adipocytes is to decrease FFA uptake and triglyceride synthesis

lipogenesis. While increasing lipolysis, LPL, lipoprotein lipase; FFA, free fatty acids; FATP, fatty

acid transporter protein; aP2, adipocytes fatty acid binding protein; ACS, acyl-CoA synthetase;

HSL, hormone sensitive lipase; βAR, β3 adrenergic receptor; AC, adenylate cyclase; PKA, protein

kinase A; Glut 4, insulin sensitive glucose transporter. An asterisk indicates proteins whose

activity and/or expression is down-regulated by TNF- α but unregulated by PPAR ƴ and its

Ligands. This also includes lipogenic enzymes such as acetyl-CoA carboxylase and fatty acid

synthetase.

IL-6 and glucose metabolism:

IL-6 is a biologically active substance that is not only secreted by

adipocytes but is also released from contracting skeletal muscle

(Steensberg A, et al. 2002). There is little evidence to suggest that IL-6

causes hyperglycaemia in vivo. In fact, it is difficult to believe that IL-6

41

METABOLISM

would impair glucose uptake, given that it is upregulated in contracting

muscle when the requirement for glucose uptake is augmented

(Steensberg A, et al. 2003). There are some mechanisms have been

proposed to explain how IL-6 induces the hyperglycemia in type-2 diabetic

subjects.

IL-6 is expressed and released from adipose tissues and positively

correlate with diabetes (Fried SK, et al. 1998; Pedersen M et al, 2003;

Weiss R, et al. 2004). Diabetes is associated macrophage accumulation in

adipose tissue and these macrophages release inflammatory mediators

and molecules promoting inflammation. IL-6 is expressed in macrophages

but more so in adipocytes (Weisberg SP, et al. 2003). IL-6 expression is

thought to be stimulated in a paracrine fashion by proinflammatory

mediators released in the tissue. There is a striking paucity of human and

animal studies investigating the effect of acute and long-term IL-6

exposure on insulin signaling and glucose metabolism in adipocytes but IL-

6 infusion in a physiological concentration increases subcutaneous

adipose tissue glucose uptake in humans (Lyngso D, et al. 2002). It is of

interest to note that SOCS-3 expression is increased in adipocytes cell lines

exposed to IL-6 in vitro (Lagathu C, et al. 2003; Shi H, et al. 2004). SOCS-3

is also induced in adipocytes upon insulin stimulation and functions as a

negative regulator of insulin signaling by binding to and ubiquitinating

IRS-1 and -2, thereby targeting these important signaling intermediates for

proteosomal degradation (Emanuelli B, et al. 2000). In recent studies

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METABOLISM

(Carey AL, et al. 2004), adipose tissue IL-6 mRNA has been shown to be

elevated in diabetic humans, and the elevated IL-6 mRNA levels correlated

with reduced rates of insulin stimulated glucose disposal (Bastard JP, et al.

2002).

Skeletal muscle is the largest insulin-sensitive tissue and

contributes to 90% of the insulin stimulated glucose disposal in healthy˃

individuals (Reaven GM. 1988). Small amounts of IL-6 are released from

resting skeletal muscle in elderly but not young individuals and IL-6

stimulates its own expression in muscle cells (Carey AL et al, 2004).

During exercise, large quantities of IL-6 are released from muscle tissue

beds (Febbraio MA, et al. 2004) and co regulating glucose homeostasis

during exercise. From a physiological point of view, it seems irrational that

working muscle releases a factor that inhibits insulin signaling when the

muscle needs insulin action for aerobic glucose metabolism. Thus, it is

unclear whether IL-6 causes hyperglycemia in skeletal muscle (Kim HJ, et

al. 2004; Rieusset J, et al. 2004). However, the studies suggest that dose

and time of exposure of IL-6 may alter the effect of IL-6 on insulin

sensitivity in skeletal muscle. Interestingly, a study modeling long-term

low concentration exposure to IL-6 in individuals developing type- 2 DM

support that IL-6 causes hyperglycemia (Klover PJ, et al. 2003).

In contrast to what has been detailed for adipocytes and skeletal

muscle cells, there is far more consistency in studies investigating that IL-6

43

METABOLISM

may have a marked influence on hepatic glucose metabolism (Senn JJ, et al.

2003; Kim HJ. et al. 2004). The in vitro studies on the human

hepatocarcinoma HepG2 cell line are strikingly consistent. These studies

indicate that IL-6 signaling in hepatocytes is mainly directed via the

JAK/STAT pathway leading to STAT3 phosphorylation, SOCS3

transcription and inhibition of insulin receptor autophosphorylation and

tyrosine phosphorylation of IRS-1 and -2, decreased glycogen storage due

to decreased gluconeogenesis and increased glycogenolysis. Collectively,

these studies provide strong evidence for the ability of IL-6 to reduce

insulin sensitivity in hepatocytes by hampering insulin signaling

(Kristiansen OP, et al. 2005).

IL-6 and lipid metabolism

IL-6 is a pleuripotent cytokine that is secreted by many different cell types

and tissues including macrophases, skeletal muscle and adipose tissue

(Papanicolaou DA, et al. 2000). Circulating skeletal muscle and adipose

tissue levels of IL-6 are chronically elevated in diabetics (Kern PA, et al.

2001) and correlate with increased triglycerides and plasma free fatty acid

levels (Bastard JP, et al. 2000). IL-6 directly influence human adipocytes

metabolism by increasing the production of leptin which in turn decreasing

the activity of LPL, an enzyme that regulates uptake of circulating

triglycerides into skeletal muscle and adipocytes (Greenberg AS, et al.

1992).

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METABOLISM

In a recent study, IL-6 was shown to enhance AMP-activated protein

kinase (AMPK) in both skeletal muscle and adipose tissue (Kelly M, et al.

2004). AMPK plays a central role in the regulation of fuel metabolism in

skeletal muscle because its activation stimulates lipid oxidation in vitro

and in vivo (Van Hall G, et al. 2003; Bruce CR, et al. 2004; Petersen EW, et

al. 2005). It is well known that AMPK phosphorylate acetyl-CoA

carboxylase (ACC) resulting in inhibition of ACC activity, which in turn

leads to a decrease in malonyl CoA content, relieving inhibition of CPT-1

and increasing fatty acid oxidation (Fig: 9) (Kahn BB, et al. 2005). Whether

the IL-6 induced increase in lipid oxidation is mediated by the activation of

AMPK had not been elucidated, but it was shown by the use of an AMPK

dominant-negative–infected cell line, that IL-6 mediated phosphorylation

of ACC and subsequent palmitate oxidation is AMPK dependent (Carey AL,

et al. 2006).

IL-6 has been shown to activate SOCS proteins in liver (Senn JJ et al,

2003) and hepatic over expression of SOCS1 and -3 leads to liver

hyperglycemia (Ueki K, et al. 2004). IL-6 induces SOCS3 is much greater in

hepatic tissue. The possibility exists that the negative effects of IL-6

induced increases in SOCS3 in tissues such as skeletal muscle may be

overridden by the positive effects of activation of AMPK. While in vitro

experiments provide solid evidence that the activation of AMPK is a major

mechanism by which IL-6 exerts its metabolic effect on lipid metabolism

(Carey AL, et al. 2006).

45

METABOLISM

46

Adipose tissue and Skeletal muscle

↑Production of IL-6

Hyperglycemia

Adipocytes

Leptin

AMPK

Acetyl-CoA

Pi

ACC ACC

Pi

Malonyl-CoA

Fatty acyl

Oxidation

CPT1

+

METABOLISM

Figure: 9 Effects of IL-6 on lipid metabolism, AMPK, AMP-activated protein kinase; inorganic

phosphate; ACC, Acetyl-CoA carboxylase; CPT, Carnitine palmitoyl transferase.

47

METABOLISM

INFLAMMATORY PROCESSES AND INSULIN RESISTANCE IN TYPE-2

DIABETES

Type-2 Diabetes is characterized by an increase in insulin resistance in

conjunction with the inability of pancreatic beta cells to secret sufficient

insulin to compensate (Centre for Disease Control and Prevention, 2005).

Insulin affects cells through binding to its receptor on the surface of

insulin-responsive cells. The stimulated insulin receptor phosphorylates

itself and several substrates, including members of the insulin receptor

substrate (IRS) family, thus initiating downstream signaling events (Saltail

A.R, et al. 2002). The inhibition of signaling downstream of the insulin

receptor is a primary mechanism through which inflammatory signaling

leads to insulin resistance. Exposure of cells to TNF- or elevated levels ofα

free fatty acids stimulates inhibitory phosphorylation of serine residues of

IRS-1 (Yin M.J, et al. 1980; Hotamisligil G.S, et al. 1996). This

phosphorylation reduces both tyrosine phosphorylation of IRS-1 in

response to insulin and the ability of IRS-1 to associate with the insulin

receptor and there by inhibits downstream signaling and insulin action

(Kathryn E, et al. 2005). Recently it has become clear that inflammatory

signaling pathways can also become activated by metabolic stresses

originating from inside the cell as well as by extracellular signaling

molecules. It has been demonstrated that obesity overloads the functional

capacity of the ER and that this ER stress leads to the activation of

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METABOLISM

inflammatory signaling pathways and thus contributes to insulin

resistance (Ozcan U, et al. 2004; Ozawa K, et al. 2005). Additionally,

increased glucose metabolism can lead to a rise in mitochondrial

production of ROS. ROS production is elevated in obesity, which causes

enhanced activation of inflammatory pathways (Lin Y, et al. 2005;

Furukawa S, et al. 2004). Several serine/threonine kinases are activated by

inflammatory or stressful stimuli and contribute to inhibition of insulin

signaling, including Janus kinase, inhibitor of NF- B kinase (IKK), and PKC-κ

(Zick Y. 2003). Again, the activation of these kinases in obesityθ

highlights the overlap of metabolic and immune pathways; these are the

same kinases, particularly Inhibitor of NF- B kinase (IKK)and JNK that areκ

activated in the innate immune response by Toll-like receptor (TLR)

signaling in response to LPS, peptidoglycan, double-stranded RNA, and

other microbial products ( Medzhitov R, et al.2001). Hence it is likely that

components of TLR signaling pathways will also exhibit strong metabolic

activities. The 3 members of the JNK group of serine/threonine kinases,

JNK-1, -2, and -3, belong to the MAPK family and regulate multiple

activities in development and cell function, in large part through their

ability to control transcription by phosphorylating activator protein–1

(AP-1) proteins, including c-Jun and JunB (Davis R.J. 2000). JNK has

recently emerged as a central metabolic regulator, playing an important

role in the development of insulin resistance in obesity (Hirosumi J, et al.

2002). In response to stimuli such as ER stress, cytokines, and fatty acids,

49

METABOLISM

JNK is activated, where upon it associates with and phosphorylates IRS-1

on Ser307, impairing insulin action (Kathryn E, et al. 2005). PKC and

IKK, two other inflammatory kinases that play a large role in counteracting

insulin action, particularly in response to lipid metabolites, are IKK and

PKC- . Lipid infusion has been demonstrated to lead to a rise in levels ofθ

intracellular fatty acid metabolites, such as diacylglycerol (DAG) and fatty

acyl CoAs. This rise is correlated with activation of PKC- and increasedθ

Ser307 phosphorylation of IRS-1 (Yu C, et al. 2002). PKC- may impairθ

insulin action by activation of another serine/threonine kinase, IKK , orβ

JNK (Perseghin G, et al. 2003). Other PKC isoform have also been reported

to be activated by lipids and may also participate in inhibition of insulin

signaling (Schmitz-Peiffer C. 2002) IKK can impact on insulin signalingβ

through at least 2 pathways. First, it can directly phosphorylate IRS-1 on

serine residues (Kathryn E, et al. 2005). Second, it can phosphorylate

inhibitor of NF- B, thus activating NF- B, a transcription factor that,κ κ

among other targets, stimulates production of multiple inflammatory

mediators, including TNF- and IL-6 (α Shoelson, et al. 2003). Activation of

IKK in liver and myeloid cells appears to contribute to obesity-induced

insulin resistance, (Kathryn E, et al. 2005) IKK can impact on insulinβ

signaling through at least 2 pathways. First, it can directly phosphorylate

IRS-1 on serine residues (Yin M.J. 1998; Gao Z, et al. 2002). Second, it can

phosphorylate inhibitor of NF- B, thus activating NF- B, a transcriptionκ κ

factor that, among other targets, stimulates production of multiple

50

METABOLISM

inflammatory mediators, including TNF- and IL-6 (Shoelson S.E, et al.α

2003). Activation of IKK in liver and myeloid cells appears to contribute to

obesity-induced insulin resistance, though this pathway may not be as

important in muscle. Other pathways, In addition to serine/ threonine

kinase cascades, other pathways contribute to inflammation-induced

insulin resistance. For example, at least 3 members of the SOCS family,

SOCS1, -3, and -6, have been implicated in cytokine mediated inhibition of

insulin signaling (Kathryn E, et al. 2005). These molecules appear to

inhibit insulin signaling either by interfering with IRS-1 and IRS-2 tyrosine

phosphorylation or by targeting IRS-1 and IRS-2 for proteosomal

degradation (Rui L, et al. 2002; Ueki, K, Kondo. 2004). SOCS3 has also been

demonstrated to regulate central leptin action, and both whole body

reduction in SOCS3 expression (SOCS3+/–) and neural SOCS3 disruption

result in resistance to high-fat diet–induced obesity and insulin resistance

(Mori H, et al. 2004; Howard, J.K, et al. 2004). Inflammatory cytokine

stimulation can also lead to induction of NOS. Overproduction of nitric

oxide also appears to contribute to impairment of both muscle cell insulin

action and cell function in obesity (β Shimabukuro M, et al. 1997; Perreault

M, et al. 2001). Deletion of NOS prevents impairment of insulin signaling in

muscle caused by a high-fat diet (Perreault M, et al. 2001). Thus, induction

of SOCS proteins and NOS represent 2 additional and potentially important

mechanisms that contribute to cytokine-mediated insulin resistance. It is

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METABOLISM

likely that additional mechanisms linking inflammation with insulin

resistance remain to be uncovered. (Kathryn E, et al. 2005).

Micro and Macro vascular complication in type-2 DM

Insulin resistance and insulin deficiency give rise to a hyperglycemic state

that is a major risk factor for the development of diabetic complications.

Hyperglycemia is considered the key contributor to microvascular

complications, including retinopathy, neuropathy, and nephropathy. It is

also one of several major risk factors associated with cardiovascular

disease along with insulin resistance or deficiency (Scott JA, et al. 2004).

Diabetic Retinopathy

Diabetic retinopathy (DR) can be defined as damage to microvascular

system in the retina due to prolonged hyperglycemia that may lead to

blindness. Various complications associated with DR have been noted that

include vitreous hemorrhage, retinal detachment and glaucoma DR has

been considered as a disease of the retina which is the leading cause of

acquired blindness in working adults in which the microvasculature of the

retina gets severely damaged, the blood vessels swell and leak fluid,

growth of new vessels initiates that ultimately lead to the detachment of

the retina (Rohilla A, et al .2012). In DR The retina swells like balloon and

is recognized as the earliest stage of DR moderate non proliferative

retinopathy, in which the blood vessels nourishing the retina get blocked;

severe non proliferative retinopathy, in which the retinopathy spreads and

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METABOLISM

blood vessels get blocked in several areas in retina; and proliferative

retinopathy, in which in four types; mild non proliferative the retina sends

signals to trigger the growth of new blood vessels (Kowluru RA, et al.

2007; Aiello LM, et al. 2003).

Role of inflammatory cytokines in diabetic retinopathy

IL-6

Interleukin-6 has been well known as a pro-inflammatory cytokine.

However, there are an increasing number of reports about its anti-

inflammatory character (Sanchez RN, et al. 2003; Sappington RM, et al.

2006; Nandi D, et al. 2010). In vitro in presence of increased pressure and

injury resulting from ischemia, IL-6 inhibited apoptosis of retinal ganglion

cells (Sanchez RN, et al. 2003; Sappington RM, et al. 2006). On the other

hand M1 macrophages prevented neovascularisation within retina due to

high secretion of IL-6, IL-12 and IL-23 (Dace DS, et al. 2008).  Although,

majority of reports confirm its negative role in the onset and progression

of diabetic retinopathy (Noma H, et al. 2009; Funk M, et al, 2010; Koleva-

Georgieva DN, et al. 2011). IL-6 is produced mainly by macrophages,

monocytes, lymphocytes T and B, while in the eye IL-6 is produced by

keratocytes, Muller cells, pigmented epithelium, corneal epithelium, iris

and ciliary body (Yoshida S, et al. 2001). IL-6 secretion is activated by

hypoxia, AGEs, and PKC (protein kinase C). In children and adolescents

with diabetic retinopathy, higher levels of IL-6 were demonstrated in

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METABOLISM

serum (Zorena K, et al. 2012). A significant increase in the level of IL-6 was

found in proliferative diabetic retinopathy (PDR) patients compared to

non proliferative diabetic retinopathy (NPDR) and healthy children.

Authors recorded significant gradation in the IL-6 increase when

comparing healthy children, children with T1DM without abnormalities in

the eyes, and diabetic children with non proliferative diabetic retinopathy.

Higher IL-6 and TNF- level in diabetic children are attributed to worseα

metabolic balance and chronic inflammation (Zorena K, et al. 2007). A part

from IL-6 influence on the set and progression of diabetic retinopathy in

young patients with diabetes, higher levels of VEGF and C-reactive protein

were also found (Coulon J, at al. 2005; Zorena K, et al. 2007). In

inflammatory conditions IL-6 levels in serum may increase even 100

times, therefore IL-6 is regarded as an early and sensitive but non-specific

indicator of inflammatory process affecting the organism (Abrahamsson J,

et al. 1997). High IL-6 levels have been observed also in patients with type-

2 diabetes and retinopathy as compared to patients without retinopathy.

Also in aqueous from eyes with diabetic macular edema found to be

significantly increased IL-6 and VEGF (Zorena K, et al. 2012). When

patients were given bevacizumab, it was noted that VEGF levels dropped

below physiological levels (Oh IK, et al. 2010) demonstrated positive

correlation between the aqueous levels of IL-6 and macular thickness

indicating that IL-6 may play a central role in the development of diabetic

macular edema. Other studies concerned the increased levels of IL-6, TNF-

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METABOLISM

,α  ET-1 vW F, Se–selectin in vitreous detected  in  patients with  type-2

diabetes and PDR (Adamiec-Mroczek J, et al. 2010).  Furthermore, authors

note correlation between TNF -α, ET-1 and HbA1c suggesting that there is

close relation between metabolic equilibrium and inflammatory factors in

Type-2 DM patients. Thus, those studies support pro-inflammatory and

proangiogenic role of IL-6 (Zorena K, et al. 2012).

Tumor Necrosis Factor alpha (TNF- ) α

TNF- α is one of most important inflammatory cytokines. It is produced

primarily by monocytes and macrophages on which it exerts its endo-,

para- and autocrine actions. It stimulates cytotoxic properties of

monocytes and macrophages and simultaneously is a mediator of

cytotoxicity. Its biological effects depends strongly on quantity and

intensity of TNF- α secretion. Apart from taking part in inflammatory

processes it also plays important role in neovascularisation (Wilson &Balk

will F, 2002). TNF-α exerts versatile effects due to its ability to induce

synthesis of other cytokines functionally related to TNF-α, extracellular

matrix proteins, monocyte and fibroblast chemotaxis modulation and also

influences the expression of adhesive molecules in retinal vessels

(Doganay S, et al. 2002; Naldini A, et al. 2005). An antiangiogenic action of

TNF- α using human endothelial cells, The authors showed that incubation

of those cells with known proangiogenic factor VEGF for 24h augmented

their proliferative activity more than two fold, whereas 12h pre-incubation

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METABOLISM

abolished this effect. However, TNF- α alone revealed a weak cytotoxic

effect towards endothelial cells. Inhibition of endothelial cell proliferation

by TNF- α was associated with reduction in VEGFR-2 (KDR/Flt-1)

receptor mRNA transcription level which depends on dose and the

duration of cytokines. Low concentrations of TNF-α can trigger signaling

pathways through p55 and p75 receptor, but in high concentrations only

through p55) (Zorena K, et al. 2012). TNF-α may become a relevant

indicator of development and risk of diabetic retinopathy. Similar

observations were made in adult patients with PDR (Gustavsson C, et al.

2008; Koleva–Georgieva DN, et al. 2011). Levels of TNF-α in vitreous of

Type-2 diabetic patients with PDR were higher than those found in control

group. Furthermore a correlation between TNF-α and HbA1c is observed,

suggesting that there is a close relation between glycaemic control and

inflammatory factors in T2DM patients (Adamiec-Mroczek J, et al. 2008;

Lee JH. 2008). TNFRI and TNFRII receptors’ levels in vitreous of patients

with PDR and proliferative vitreo retinopathy were much higher than in

patients with perforation in macula (Limb G, et al. 2001).

Attempts are being made to block TNF- α actions with monoclonal

antibodies (Sfikkakis PP, et al. 2010; Giganti M, et al. 2010; Biswas NR, et

al. 2010). In randomized studies, the intravenous use of infliximab has

improved visual acuity in patients with diabetic macular edema (DME)

(Sfikakis PP, et al. 2010). Etanercept is a soluble TNF- α receptor that acts

as competitive inhibitor blocking effects of TNF-α binding to cells.

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METABOLISM

Etanercept reduced leukocyte adherence in retinal blood vessels of

diabetic rats for 1 week as compared to control. Etanercept did not reduce

retinal VEGF levels, but it inhibited blood-retinal barrier breakdown and

NF-κB activation in the diabetic retina (Joussen AM, et al. 2002; Zheng L ,

et al. 2004).

Cardio vascular disease and insulin resistance

Elevated serum levels of triglycerides and free fatty acids in association

with obesity and type-2 DM led many researchers to assume that the

status of lipotoxicity is responsible for the initiation and progression of

hepatic and peripheral insulin resistance and the deterioration of

pancreatic -cell function seen in type-2 DM (β Bergman RN, et al. 1998).

Accumulating evidence showed that increased cytokines secreted from

adipose tissue, also known as adipokines, may be responsible for initiating

a proinflammatory status that percolates the development of both insulin

resistance and endothelial dysfunction (ED; the initial stage in the

development of atherosclerosis). Cytokines are a group of

pharmacologically active low molecular weight proteins that possess

autocrine and paracrine effects and are known products of inflammatory

and immune system. Although adipose tissue is a major source of cytokine

production, it is still uncertain about the proportion of cytokines produced

by adipocytes in comparison to the total cytokines produced by all other

tissues. Among the many recognized cytokines, few have been widely

studied in adipose tissue. They include TNF- , IL-6, adiponectin,α

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METABOLISM

leptin, resistin, and plasminogen activator inhibitor-1(PAI-1) (Aldhahi W,

et al. 2003).

Role of cytokines in Insulin Resistance as a Link to Inflammation

Increased circulating cytokines and growth factors are strongly related to

inflammation, which is play an important role in the etiology of

atherosclerosis and diabetes (Ross R, 1999; Yuan M, et al. 2001).

Although little is known about the extent of involvement and interaction

between inflammation and adipose tissue in relation to the etiology and

the progression of insulin Resistance, cytokines merge as possible links

between inflammation and insulin resistance and diabetes. Levels of the

proinflammatory cytokines, IL-6 and TNF- , the most widely studiedα

cytokines in adipose tissue, were shown to be correlated with all

measures of obesity and were strongly related to insulin resistance

(Aldhahi W, et al. 2003). TNF-α is thought to play a major role in the path

physiology of insulin resistance in rodents through the phosphorylation of

the insulin receptor substrate-1 (IRS-1) protein on serine residues. This

could prevent its interaction with the insulin receptor beta subunit, and

stop the insulin signaling pathway. Although clinical studies have shown

that visceral adipose tissue is closely linked to insulin resistance, TNF-α

mRNA expression was similar in serosal adipose tissue and visceral

adipose tissue. Moreover, TNF- is weakly expressed either inα

subcutaneous or in deep human adipose tissue depots and this expression

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METABOLISM

is not always modified in obesity (Bastard JP, et al. 2006). This

corresponds with the evaluation of in vivo secretion, which showed

that TNF-α production by subcutaneous abdominal adipose tissue was

quantitatively negligible in lean and obese subjects (Mohamed-Ali V, et al.

1997). This suggests that adipose tissue is not directly implicated in the

increased circulating TNF- level observed in obesity in human. It can beα

hypothesized that other mechanisms involving a systemic effect of leptin

or of other adipokines may induce TNF- secretion by other cell typesα

such as macrophages. Nevertheless, the precise role of TNF- in humanα

obesity requires further investigation. (Bastard JP, et al. 2006).

Interleukin-6 is produced by many cell types (fibroblasts,

endothelial cells, monocytes), and many tissues including adipose tissue. It

is now well known that IL-6 production by adipose tissue is enhanced in

obesity. It is thought that 15 to 30 % of circulating IL-6 levels derives from

adipose tissue production in the absence of an acute inflammation.

Secretion is higher in VAT than in SAT. Accordingly, IL-6 mRNA expression

is higher in VAT than in SAT (Fried SK, et al. 1998; Bastard JP, et al. 2002).

However, in adipose tissue, the greater proportion of IL-6 is not produced

by mature adipocytes but rather by cells of the stroma vascular fraction

including preadipocytes, endothelial cells and monocytes- macrophages.

Interleukin-6 is multifunctional cytokine acting on many cells and tissues.

One phosphatase activation or an interaction between suppressor of

cytokine signaling (SOCS) proteins and the insulin of the main effects of IL-

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METABOLISM

6 is the induction of hepatic CRP production, which is now known to be an

independent, major risk marker of cardiovascular complications ( Bastard

JP, et al. 2006).

Interestingly, there is a strong relationship between IL-6 protein

content in adipose tissue and circulating levels of both IL-6 and CRP. In

addition, IL-6 has been recently proposed to play a central role in the link

between obesity, inflammation and coronary heart diseases (Yudkin JS, et

al. 2000). This could partly explain the relationship between central fat

depots and cardiovascular risk complications in human. Moreover, IL-6

production by adipose tissue could directly affect liver metabolism by

inducing VLDL secretion and hypertriglyceridemia, since the VAT is closely

connected to the liver by the venous portal system. Recent studies have

suggested that IL-6 could be involved in insulin resistance and its

complications (Bastard JP, et al. 2000). The IL-6 receptor belongs to the

cytokine class I receptor family involving JAK/STATs (Janus kinases/signal

transducers and activators of transcription) signal transduction

pathway. Janus kinase activation induces STAT phosphorylation,

dimerisation and translocation to the nucleus to regulate target gene

transcription (Ihle JN, et al. 1995). It is now clearly established that a

strong interaction occurs between cytokine and insulin signaling

pathways, and generally leads to an impaired biological effect of insulin.

Although the exact mechanisms have not yet been clearly elucidated, it

could involve tyrosine receptor (Mooney RA, et al. 2001; Lagathu C, et al.

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2003; Rieusset J, et al. 2004)). whatever the mechanisms involved, it has

now been clearly demonstrated that cytokines such as TNF- and IL-6 areα

able to decrease insulin action. Therefore, in addition to the aggravation of

the cardiovascular risk linked to inflammation, the chronic increase in

circulating cytokine levels could contribute to insulin resistance. (Bastard

JP, et al. 2006).

Diabetic Nephropathy

Diabetic nephropathy is one of the most common microvascular

complications of type-2 diabetes mellitus and the leading cause of end-

stage renal disease worldwide (Lopes AA, et al. 2009; Ohga S, et al.

2007).  Diabetic nephropathy is a progressive disease that takes several

years to develop. Many factors contribute to the development of diabetic

nephropathy including hyperglycemia, hypertension, and obesity, and

sedentary lifestyle, hereditary, smoking, and advancing age (Rossing P,

2006; Romero Aroca P, et al. 2010). Diabetic nephropathy is characterized

by morphological and ultrastructural changes in the kidney including

expansion of the molecular matrix and loss of the charge barrier on the

glomerular basement membrane (Hovind P, et al. 2001).The progression

from normal albuminuria to microalbuminuria is considered the initial

step in diabetic nephropathy which further progresses to

macroalbuminuria as renal function continues to deteriorate and

glomerular filtration rate (GFR) starts to decline (Parving H. 2001).

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Cytokines (TNF- and IL-6) in type-2 diabetic nephropathy α

An inflammatory cell by definition is any cell that participates in an

inflammatory response which can be resident or infiltrating cells. Classical

inflammatory cells are macrophages, lymphocytes, dendritic cells (DC),

neutrophils and eosinophils (Borst SE, 2004). Inflammation plays an

important role in many aspects of diabetes and related complications

(Jager J, et al. 2007). Systemic levels of pro-inflammatory cytokines

including tumour necrosis factor (TNF)- , interleukin (IL)-1 and IL-6 areα β

elevated in patients with both type-1 and type-2 diabetes. Increases in

these cytokines can directly promote insulin resistance (Senn JJ, et al.

2002). There is a growing appreciation of the role of inflammation in

diabetic complications (Navarro JF, et al. 2008).

Diabetic nephropathy (DN) is one of the major diabetic

complications which finally lead to end-stage renal disease. Cytokines are

polypeptides that regulate immune and inflammatory events. Circulating

levels of inflammatory cytokines such as interleukin-6 (IL-6) and tumor

necrosis factor- α (TNF-α) are increased in T2DM (Alexandraki K, et al.

2006). Tumor necrosis factor- α (TNF-α) is a proinflammatory cytokine

with a wide range of biologic effects including the stimulation for the

production of prostaglandins, platelet-activating factor, and plasminogen

activator inhibitor; chemotaxis; the induction of adhesion molecules

expression; and the synthesis of other inflammatory mediators (Mariano F,

et al. 1997; Navarro JF, et al. 2008). Renal cells are capable of synthesizing

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TNF-α, which may act in a paracrine or autocrine manner to induce a

variety of effects on different renal structures (Ohga S, et al. 2007). This

proinflammatory cytokines play a significant role in the development and

progression of several renal disorders (Noronha IL, et al, 1995), including

diabetic nephropathy (Ostendorf T, et al. 1996)

TNF- α is a Pleiotropic cytokine involved in systemic inflammation.

TNF- α induces a local inflammatory response by initiating a cascade of

cytokines and increasing vascular permeability, there by recruiting

macrophage and neutrophils to a site of infection (Sugimoto H, et al. 1999;

Pamir N, et al. 2009). TNF- α activates NFkB signaling which mediates the

transcription of various cytokines involved in cell survival and

proliferation, inflammatory responses and cell adhesion, and antiapoptotic

factors (Cao YL, et al. 2008). TNF- α being cytotoxic to glomerular,

mesangial, and epithelial cells are the factors causing renal damage in

type-2 diabetic patients (McCarthy ET, et al. 1998)

IL-6 is a 22–27kDa polypeptide secreted from activated monocytes,

macrophages, fibroblasts, adipocytes and endothelial cells in response to

various stimuli, such as TNF-α, IL-1β, bacterial endotoxins, physical

exercise and oxidative stress. IL-6 belongs to the IL-6 family of cytokines,

which are characterized by the common use of the IL-6R β receptor. IL-6

receptors and IL-6Rβ belong to the type I cytokine receptor family

(Kamimura D, et al. 2003).Elevated levels of IL-6 predict future risk of

complication development in T2DM (Carey AL, et al. 2004) and IL-6 mRNA

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was found to be elevated in insulin resistant humans as well (Bastard

2002).

Interleukin-6 (IL-6) is a pleiotropic cytokine with a key impact on

both immunoregulation and non immune events in most cell types and

tissues outside the immune system. Chronic low-grade inflammation and

activation of the innate immune system are closely involved in the

pathogenesis of diabetes and its microvascular complications.

Inflammatory cytokines, mainly IL-6, and IL-18, as well as TNF- areα

involved in the development and progression of diabetic nephropathy

(Carmen Mora-Fernandez et al, 2008). Interleukin-6 affects extracellular

matrix dynamics at mesangial and podocyte levels, stimulates mesangial

cell proliferation, increases fibronectin expression, and enhances

endothelial permeability in type-2 diabetes mellitus (Dalla Vestra M, et al.

2005).

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