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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
17
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
18
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
19
METABOLISM
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
21
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-
22
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
23
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
24
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
25
METABOLISM
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,
26
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.)
27
METABOLISM
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).
28
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
29
METABOLISM
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
30
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
31
METABOLISM
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
32
METABOLISM
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).
33
METABOLISM
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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METABOLISM
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
37
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
42
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
48
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
52
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-
54
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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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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METABOLISM
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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METABOLISM
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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