Diabetes Mellitus (DM):
Diabetes mellitus (DM) is a group of metabolic
disorders distinguished by hyperglycemia resulting from failures in insulin action,
insulin secretion, or both. Insulin is a hormone secreted by the beta cells (?-cells)
of the pancreas, which is essential for utilizing of glucose from digested food
as an energy source. The chronic hyperglycemia of DM is referred to long-term dysfunction,
damage and failure of several organs, especially the kidneys, hearts, nerves, eyes,
and blood vessels (ADA, 2014).
Various pathogenic processes are related
to the development of DM. These range from pancreatic ?-cells autoimmune destruction
with consequent insulin insufficiency and abnormalities in insulin action (ADA, 2014).
The first widely accepted classification
of DM was published by World Health Organization (WHO) in 1980. The 1980
Expert Committee proposed two major classes of diabetes mellitus, type 1
diabetes mellitus and type 2 diabetes mellitus (WHO, 1980).
Type 1 Diabetes Mellitus (T1DM):
This form of diabetes, formerly
called insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes,
results from a cellular-mediated autoimmune destruction of the ?-cells of the pancreas.
Onset most often appears in childhood, but it can also occur in adults in their
late 30s and early 40s (ADA, 2014). The mechanisms which lead to ?-cell
destruction are still not completely understood but it is more accepted that
the selective destruction of ?-cell is interposed by cytotoxic T-cells and by certain
cytokines (Graham et al., 2012).
In this type of diabetes, the rate of ?-cell destruction is widely
variable, being slow in others, mainly adults, and rapid in some individuals, mainly
infants and children (ADA, 2014). Patients with this form often become relied on insulin for survival
and are at risk for ketoacidosis disorder (Willis et al.,
Type 2 Diabetes Mellitus (T2DM):
This type of diabetes, formerly
known as non-insulin-dependent diabetes mellitus (NIDDM), is an expression used
for persons who have insulin resistance and usually have relative insulin insufficiency.
It usually exists in individuals over 40 and is called adult onset diabetes
mellitus. T2DM frequently goes not diagnosed for many years since the
hyperglycemia appears gradually and at earlier stages is often not severe
enough for the patient to notice any of the classic symptoms of diabetes (ADA, 2014).
The risk of progressing
this type of diabetes increases with obesity, age, and lack of physical activity
and it exists more frequently in individuals with hypertension or dyslipidemia and
in women with prior gestational diabetes mellitus (GDM) (ADA, 2014). T2DM also requires continuous medical care
and multifactorial risk reduction strategies to normalize blood glucose levels
and prevent or minimize acute and long-term microvascular or macrovascular complications
(DeFronzo et al., 2015).
Many T2DM individuals need
exogenous insulin in the later stages of the disease since endogenous insulin secretion
becomes insufficient to maintain acceptable levels of glycemia despite ongoing
therapy with other anti-diabetic agents (Ma et al., 2012).
Although insulin resistance
is considered the initiating event in the pathogenesis of T2DM, pancreatic ?-cell
dysfunction is an indispensable condition for the development of the disease
and hyperglycemia does not become apparent until there is severe ?-cell
dysfunction (Tripathy and Chavez, 2010). Several factors contribute to ??cell dysfunction, including ageing,
genetic abnormalities, lipotoxicity, glucotoxicity, insulin resistance
leading to ??cell stress and reactive oxygen stress (DeFronzo et al.,
T2DM and Insulin Resistance (IR):
Insulin is a
pleiotropic hormone secreted from ?-cells of pancreas and has different
functions including enhancing of nutrient transport into cells, modification of
enzymatic activity and regulation of energy homeostasis. These functions of
insulin are done across a variety of insulin target tissues; skeletal muscle, liver
and adipose tissue (Zeyda and Stulnig, 2009).
In skeletal muscle, insulin enhances glucose
uptake by stimulating translocation of the glucose transporter 4 (GLUT4) to the plasma membrane. In the liver,
insulin inhibits gluconeogenesis
by reducing key enzyme activities resulting in decreasing of hepatic
glucose production. In adipose
tissue, insulin reduces lipolysis thereby decreasing free fatty acid (FFA)
efflux from adipocytes (Zeyda and Stulnig, 2009).
These metabolic effects of insulin are mediated by a complex
insulin-signaling cascades (Figure 1.1), that is initiated when insulin
binds to its receptor on the cell membrane of target tissues, leading to phosphorylation
and activation of insulin receptor substrate (IRS) proteins that are associated
with the activation of two main signaling pathways: the Ras-mitogen-activated
protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase
(PI3K)-AKT/protein kinase B (PKB) pathway.
The phosphorylated IRS-1 activates
PI3K which converts phosphatidylinositol 4,5-bisphosphate (PIP2) to
phosphatidylinositol 3,4,5-trisphosphate (PIP3) which thereby activates various
phosphoinositide-dependent protein kinase 1(PDK1) and recruits Akt to the cell
Ultimately, these signalling events result in
the translocation of GLUT 4 to the plasma membrane, resulting in an increase in
glucose uptake. The MAPK pathways are not involved in mediating metabolic
actions of insulin but in inducing mitogenic and growth effects of insulin including
the activation of genes that are involved in cell growth, thereby promoting
inflammation and atherogenesis (Jung and Choi, 2014).
Figure (1.1) Schematic view of insulin signaling pathway in adipose tissue (Jung
and Choi, 2014).
Abbreviations: IRS-1: insulin
receptor substrate, MAPK: mitogen-activated protein kinase, PDK:
phosphoinositide-dependent protein kinase 1, PI3K: phosphatidylinositol
3-kinase, PIP2: phosphatidylinositol 4,5-bisphosphate, PIP3:
phosphatidylinositol 3,4,5-trisphosphate, PKB: protein kinase B, MEK:
signal-regulated kinase kinase.
Insulin resistance (IR),
a central feature of T2DM, is believed to be the underlying mechanism for the
metabolic syndrome (Guo, 2014). By definition, IR
is an impairment of insulin action which causes the impairment of glucose
uptake in muscle and the increase of endogenous glucose production by the liver
resulting in hyperglycemia (Kim et al., 2008). The defect
in insulin action occurs in multiple tissues, including adipocytes, liver, and
skeletal muscle. In insulin-resistant individuals, insulin-stimulated glucose
disposal is impaired in skeletal muscle due to impaired insulin signaling and
multiple intracellular defects in glucose metabolism. Similar defects in
insulin signaling in the adipocytes and liver have been reported (Lambadiari
et al., 2015).
Data of many investigators supposed
that the IR in many type 2 diabetic patients results from an increase in
visceral adiposity. It has been speculated that the direct release of FFA or
other products from visceral adipose tissue into the portal circulation may be
an important mechanism in inducing IR (Banerji et al., 1997).
Lipotoxicity and Glucotoxicity in the
pathophysiology of T2DM
elevation of plasma FFA adversely affects insulin secretion and insulin action
(lipotoxicity). Chronic exposure of pancreatic ?-cells to FFA recruits
multiple mechanisms of toxicity, including accelerated ceramide synthesis,
increased fatty acid oxidation and esterification and fatty acid-induced
apoptosis (Del Prato, 2009).
increased FFA concentrations contribute to IR in peripheral tissues. Initially,
Randle et al. were the first to suggest a primary role for elevated FFA
availability and the development of IR. They showed that there are substrate
competition between glucose and FFA. Moreover, they speculated that increase in
fat oxidation would cause an increase in the mitochondrial acetyl CoA:CoA and
NADH:NAD ratios with subsequent inactivation of pyruvate dehydrogenase (PDH).
This in turn would induce an increase in intracellular citrate levels, resulting
in inhibition of phosphofructokinase (PFK) and glucose-6-phosphate (G6P) accumulation.
As G6P inhibits hexokinase activity, this would result in intracellular accumulation
of glucose and decreased glucose uptake (Randle et al.,
Another hypothesis account for the effects
of fatty acids
in IR is shown in (Figure 1.2), this model holds that increasing intracellular fatty acid metabolites such as fatty
acyl CoA’s, diacylglycerol or ceramides activate a serine/threonine kinase cascade (possibly initiated
by protein kinase Cq (PKCq)), resulting in
phosphorylation of serine/threonine sites on insulin receptor substrates (IRS-1
and IRS-2) which in turn decreases the ability of the IRSs to activate
PI3-kinase. As a consequence, glucose transport activity and other events
downstream of insulin receptor signaling are diminished including muscle glycogen synthesis (Shulman,
2000). All are the main reasons that shed a light for the involvement of
other mechanisms in the development of IR.
Figure (1.2) Mechanism of
fatty acid-induced insulin resistance in skeletal muscle (Savage et al.,
Abbreviations: DAG: diacylglycerol, GLUT: glucose transporter, G6P: glucose
6-phosphate, GSK3: glycogen synthase kinase-3, IRS: insulin receptor substrate,
LCCoA: long-chain acyl coenzyme A; nPKCs, novel protein kinase Cs, PI 3-kinase:
phosphatidylinositol 3-kinase; PTB: phosphotyrosine binding domain, PH:
pleckstrin homology domain, SH2: src homology domain, AKT2: protein kinase B.
Chronic elevation of
plasma glucose impairs both insulin secretion and insulin action
(glucotoxicity) where it may induces IR and decreases pancreatic ?-cell
function by several different mechanisms. Hyperglycaemia in vivo as well as in
vitro repeatedly has been shown to exert a cytostatic and proapoptotic effect
on ?-cells including cytoplasmic DNA fragmentation, higher
caspase 3 (a pro-apoptotic protease) activity and greater expression of the
gene encoding the pro-apoptotic protein (Del Prato, 2009).
Multiple mechanisms have
been reported to show the hyperglycemia-induced loss of ?-cell function, but a
major contributor is alteration of intracellular energy metabolism and
oxidative stress, as well as mitochondrial dysfunction. Other pathways linked
to hyperglycemia include endoplasmic reticulum (ER) stress and hypoxia-induced
stress (Kim and Yoon, 2011). Therefore, glucotoxicity is one of the most
important mechanisms of ?-cell dysfunction and loss in diabetic patients.
T2DM and Mitochondrial dysfunction:
There is evidence that
mitochondrial dysfunction is related to T2DM and IR (Montgomery and Turner,
A free radical defined as
any chemical species that have one or more unpaired electrons and that often makes
the free radical to be very reactive and acts as an electron acceptor that steals
electrons from other molecules (Bhattacharya, 2015). Free
radicals and related molecules are generally classified as reactive oxygen
species (ROS) due to their ability to create oxidative changes inside the cell
and can be divided into two types: free radical ROS, like hydroxyl
radical ion (OH?), superoxide anion (?O2•)
and nitric oxide ion (NO?) and highly reactive non-radical ROS,
like molecular oxygen (O2) and hydrogen peroxide
(H2O2) producing radical forms of ROS (Chen et al., 2012).
Most intracellular ROS are
brought from ?O2•, whose formation is often
through NADPH oxidases (NOXs), xanthine oxidase (XO) and the mitochondrial electron-transport
chain (mETC) in endogenous biologic systems. ?O2•
is short-lived and can be converted to H2O2 either through
spontaneous dismutation or through the catalytic action of superoxide dismutase
(SOD), mitochondrial MnSOD and cytosolic CuZnSOD. H2O2 is
eventually converted to highly toxic OH? in the existence of reduced
iron (Fe2+) or copper (Cu+) through the Fenton reaction (Wen et al., 2013) (Figure 1.3).
The most characterized NOX
enzyme is Nox2 NADPH oxidase which can induce electron transfer from cytosolic NADPH
to the oxygen molecules in the phagosomal lumen, producing ?O2•.
Another enzyme, XO, can also produce ?O2•
by transferring electrons from hypoxanthine to oxygen molecules (Perevoshchikova
et al., 2013).
Moreover, mETC, composed of four protein complexes
(complexes I to IV), cytochrome c (cyto c) and coenzyme Q (CoQ), is the major source
of ROS in living cells, through which continued aerobic respiration produces ?O2•.
Great amounts of ?O2• are generated at the mitochondrial complex I when the NADH/NAD+
ratio is high or reverse electron transport happens. For H2O2,
both peroxisomes and ER luminal thiol oxidase I (EroI) are major sources
for H2O2 production (Wen et al., 2013).
Figure (1.3) Reactive
oxygen species from diverse sources (Wen et al., 2013)
ROS are by-products of
cellular metabolism and cells normally possess several mechanisms to defend
against damage produced by free radicals. Problems happen when the generation
of ROS overrides the ability of cells to defend against these species. This
imbalance between cellular generation of ROS and the ability of cells to defend
against them is referred to as oxidative stress (Chen et al., 2012).
Oxidative stress can result
from multiple sources and ROS appear to be generated in larger amounts by islets
?-cell from T2DM patients than by
those from nondiabetic individuals (Tangvarasittichai, 2015). Indeed, mitochondria
of islets ?-cell from T2DM patients have been found to show morphologic
abnormalities including a rounded rather than elliptical shape, hypertrophy and
higher density compared to mitochondria of islets ?-cell from control individuals
(Molina et al., 2009).
Accumulating evidence shows
that hyperglycemia and obesity in T2DM are associated with increased ROS genertation
(Furukawa et al., 2004). One possible explanation is that the
nutrient overload can increase the reducing equivalents, FADH2 and NADH, which
increases flux through the mETC resulting in increased ?O2•
is dismutated into H2O2 by action of SOD classes
(Figure 1.4). In addition, Non-esterified FFAs are increased in T2DM and
enter the citric acid cycle to produce acetyl-CoA to receive NADH
overproduction and cause mitochondrial superoxide over production.
important source of ROS in T2DM is ROS related to ER stress and induction of
the unfolded protein response (Figure 1.4). Proper folding of many
proteins requires the formation of inter-and/or intra-molecular disulfide bonds
involving the oxidation of cysteine residues and the release of electrons followed
by ER oxidoreductin and then O2 to form ?O2•. It is generally thought that nutrient excess
in obesity and T2DM might overload the protein folding capacity of the ER and
result in increased ROS generation (Tiganis, 2011).
Figure (1.4) ROS and type 2 diabetes mellitus (Tiganis, 2011).
If ROS are not rapidly removed,
they can injure mitochondria either by inducing DNA fragmentation, protein
crosslinking, and peroxidation of membrane phospholipids or by activating a
series of stress pathways (Chen et al., 2012).
Endoplasmic Reticulum Stress (ER):
endoplasmic reticulum (ER) is one of the important organelles and it exerts various
vital functions, including, calcium storage and post-translational
modification, folding and gathering of newly synthesized proteins. Therefore,
proper function of the ER is essential to cell survival (Schwarz and Blower, 2016).
Several events can disturb ER functions,
of disulfide bonds formation, inhibition of protein glycosylation, depletion of
calcium from the ER lumen, drop in protein transport from the ER to the Golgi,
expression of misfolded proteins, etc. Such ER dysfunction causes
proteotoxicity in the ER, collectively termed “ER stress” (Schönthal, 2012).
Unfolded Protein Response
under ER stress conditions, cells possess a self-protective mechanism against
ER stress which is termed the ER stress response or unfolded protein response
(UPR) (Demirtas et al.,
least four functionally distinct responses have been identified (Figure 1.5).
The first response includes upregulation
of the genes encoding ER chaperone proteins including binding immunoglobulin
protein (Bip), also known as glucose-regulated protein 78 (GRP78), and glucose-regulated protein 94 (GRP94), that increase activity of protein-folding
and prevent protein accumulation. These proteins have a consensus sequence in
their promoters, which is termed as the cis-acting ER stress response
element (ERSE) or the unfolded protein response element (UPRE). Three distinct
ER-membrane localized upstream components, Ire1 (inositol requiring 1), PERK (RNA-dependent protein kinase-like endoplasmic
reticulum kinase), and ATF6 (activating transcription factor 6), transmit
stress signals from the ER to nucleus in response to disorder of protein
folding in the ER (Schönthal, 2012).
The second response includes translational
alleviation to decrease new protein synthesis and to prevent further assembly
of unfolded proteins. This event happens at the level of translational
initiation via phosphorylation of eukaryotic initiation factor 2? (eIF2 ?) which regulates the binding
of the initiator Met-tRNA to the ribosome. Phosphorylation of eIF2 at ? subunit
of its Ser51 inhibits protein synthesis. PERK is the essential kinase
responsible for this phosphorylation during ER stress (Schönthal, 2012).
The third response is degradation of
misfolded proteins in the ER, which is called ER-associated degradation (ERAD).
Misfolded proteins, which are not refolded in the ER, can be detected by the ER
quality control system, retrotransported from the ER to the cytosol and
degraded by the 26S proteasome.
Figure (1.5) Schematic of the unfolded protein response (Gebremeskel and
triggers PERK activation. Activated PERK attenuates protein biosynthesis by
phosphorylating eIF2? which halts global translation, but leads to activation
of ATF4. ER stress also triggers IRE1? activation which initiates the splicing
of (X-box binding protein 1 mRNA) XBP1 mRNA, producing an active transcription
factor, spliced XBP1 (sXBP1). This leads to the expression of chaperone
proteins and proteins involved in protein degradation. In addition, ER stress
also activates ATF6 which also increases chaperone synthesis to alleviate ER
The fourth response is apoptosis that
occurs when severe and prolonged ER stress impairs the ER functions (Araki et al., 2003).There are three
known apoptosis pathways that are induced by ER stress (Figure 1.6).
The first apoptotic pathway is transcriptional activation of the
gene for CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) (Ron and Habener, 1992). Transcriptional stimulation of the CHOP gene is intermediated
by three distinct upstream components of ER stress, PERK, Ire1 and ATF6 (Harding et al., 2000).
The second is induction of the cJUN
NH2-terminal kinase (JNK) pathway. JNKs constitute a family of signal
transduction proteins that are important for controlling gene expression and
participating in deduction between survival and apoptosis in response to
stresses. Activated Ire1 triggers the
tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating
kinase 1 (ASK1) resulting in formation of the Ire1-TRAF2-ASK1 complex leading
to JNK activation (Urano et
The third pathway is induction of the
ER-localized cysteine protease, caspase-12 (Nakagawa et al., 2000). Caspase protease family includes a set of enzymes which cleave their substrates
at C-terminal aspartic acid residues. It has been reported that they exert an essential
role in the progression of apoptosis. Caspases are synthesized as precursor
proteins (procaspases) and consists of N-terminal regulatory prodomain of
various length followed by two subunits, p20 and p10. The caspase family is
broadly divided into two groups: initiator caspases (caspase-8, -9, and -12)
and effector caspases (caspase-3, -6, and -7). Initiator caspases are activated
through autoprocessing in response to apoptotic stimuli. Active initiator
caspases in turn process precursors of the effector caspases responsible for
dismantling cellular structures