Clinica Chimica Acta 375 (2007) 20 – 35

www.elsevier.com/locate/clinchim

Invited critical review

Molecular mechanisms of insulin resistance and associated diseases
Barbara Mlinar a , Janja Marc a,⁎, Andrej Janež b , Marija Pfeifer b
a
Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia
b
Department of Endocrinology and Metabolic Diseases, University Medical Centre, Ljubljana, Slovenia

Received 26 May 2006; received in revised form 7 July 2006; accepted 10 July 2006
Available online 14 July 2006

Abstract

Insulin resistance is a state in which higher than normal concentrations of insulin are required for normal response. The most common
underlying cause is central obesity, although primary insulin resistance in normal-weight individuals is also possible. Excess abdominal adipose
tissue has been shown to release increased amounts of free fatty acids which directly affect insulin signalling, diminish glucose uptake in muscle,
drive exaggerated triglyceride synthesis and induce gluconeogenesis in the liver. Other factors presumed to play the role in insulin resistance are
tumour necrosis factor α, adiponectin, leptin, IL-6 and some other adipokines. Hyperinsulinaemia which accompanies insulin resistance may be
implicated in the development of many pathological states, such as hypertension and hyperandrogenaemia. Insulin resistance underlies metabolic
syndrome and is further associated with polycystic ovary syndrome and lipodystrophies. When β-cells fail to secrete the excess insulin needed,
diabetes mellitus type 2 emerges, which is, besides coronary heart disease, the main complication of insulin resistance and associated diseases.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Diabetes mellitus type 2; Insulin resistance; Lipodystrophy; Metabolic syndrome; Obesity; Polycystic ovary syndrome

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2. Mechanisms of insulin resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.1. Dysregulation of FFA and adipokine secretion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.1.1. FFA and tumour necrosis factor α . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.1.2. Adiponectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.1.3. Leptin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.1.4. Resistin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.1.5. Other adipokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.2. Glucocorticoids and FFA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.3. Molecular mechanisms of impaired insulin signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Abbreviations: α-MSH, melanocyte-stimulating hormone; AGPAT2, 1-acylglycerol-3-phosphate-O-acyl-transferase; AgRP, agouti-related protein; ATPIII, The
National Cholesterol Education Program's Adult Treatment Panel III; BMI, body mass index; CAP, Cbl associated protein; CART, cocaine and amphetamine related
transcript; Cbl, casitas B-lineage lymphoma; DM2, diabetes mellitus type 2; ESHRE/ASRM, European Society of Human Reproduction/American Society for
Reproductive Medicine; FFA, free fatty acids; GLUT4, glucose transporter 4; GSK-3, glycogen synthase kinase 3; HDL, high density lipoprotein; HOMA,
homeostasis model assessment; IDF, International Diabetes Federation; IGF-1, insulin-like growth factor-1; IR, insulin resistance; IRS-1/4, insulin receptor substrate-
1/4; LC-CoA, long-chain-CoA(s); LH, luteinizing hormone; MAP-kinase, mitogen-activated protein kinase; Mc4R, melanocortin 4 receptor; MCH, melanin-
concentrating hormone; MCP-1, macrophage and monocyte chemoattractant protein-1; MODY, maturity-onset diabetes of the young; mTOR, mammalian target of
rapamycin; PAI-1, plasminogen activator inhibitor-1; PCOS, polycystic ovary syndrome; PEPCK, phosphoenolpyruvate carboxykinase; PI3K, phosphoinositide-3-
kinase; PIP3, phosphatidylinositol-3,4,5-triphosphate; PKB, protein kinase B; PKC, protein kinase C; POMC, pro-opiomelanocortin; PPAR-γ, peroxisome
proliferator-activated receptor γ; PYY, peptide YY3–36; SOCS-1, -3, suppressors of cytokine signalling-1, -3; TNF-α, tumour necrosis factor α; TZD,
thiazolidinediones; VLDL, very low density lipoprotein.
⁎ Corresponding author. Tel.: +386 1 4769 500; fax: +386 1 4258 031.
E-mail address: janja.marc@ffa.uni-lj.si (J. Marc).

0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.cca.2006.07.005
 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35 21

2.3.1. Normal insulin signalling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

2.3.2. Defects in insulin signalling resulting in IR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3. Obesity as the main factor in IR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.1. Pathophysiology of obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2. Categories of obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.3. Insulin and leptin resistance in obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4. IR associated diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4.1. Metabolic syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4.1.1. Definition of metabolic syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4.1.2. Clinical manifestations of metabolic syndrome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.2. Inherited and acquired lipodystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.3. Diabetes mellitus type 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.3.1. Detrimental effect of FFA on β-cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.3.2. Susceptibility genes for DM2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.4. Polycystic ovary syndrome and IR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

1. Introduction Alternative measures of IR are fasting plasma insulin concen-

Insulin resistance (IR) is a common pathologic state in which
target cells fail to respond to ordinary levels of circulating insulin.
It results in inability of insulin to provide normal glucose and
lipid homeostasis. Hence, higher than normal concentrations of
insulin are needed in order to maintain normoglycaemia [1,2].
Compensatory hyperinsulinaemia due to enhanced β-cell secre-
tion is therefore an obligate accompanying feature in IR. Never-
theless, the main characteristics of IR are disinhibited lipolysis in
adipose tissue, impaired uptake of glucose by muscle and
disinhibited gluconeogenesis. Clinical markers of IR are visceral
obesity, acantosis nigricans [3], acne, hirsutism [4], and hepatic
steatosis [1]. IR is associated with a number of diseases including
obesity, metabolic syndrome, type 2 diabetes mellitus (DM2),
lipodystrophies, polycystic ovary syndrome and chronic infec-
tion. The overall prevalence of IR is reported to be 10–25% [5].
The gold standard for evaluating IR is hyperinsulinaemic
euglycaemic clamp: insulin is infused at a constant rate and
glucose is held at basal levels by glucose infusion. The rate of
the latter is a measure of insulin mediated glucose disposal [6].
tration [3], homeostasis model assessment (HOMA) index [7],
quantitative insulin sensitivity check index (QUICKI) [8] and
McAuley index [9], which are presented in Fig. 1.
The aetiology of IR includes genetic factors resulting in syn-
dromic forms of IR, and environmental factors: food intake,
reduced physical activity, aging, smoking or administration of
drugs, including thiazide diuretics, beta adrenergic antagonists,
glucocorticoids, which can cause or contribute to IR [3]. The most
important factor is obesity which is usually of combined poly-
genetic and environmental origin [3,10]. In IR, visceral adipose
tissue is resistant to the antilipolytic effect of insulin and conse-
quently releases excessive amounts of free fatty acids (FFA). This
is a driving force in the development of IR. In addition, various
hormones and cytokines from adipose tissues (adipokines) also
contribute to IR. Conversely, restricted calorie intake, weight
reduction and physical activity improve insulin sensitivity [3,4,11].
The present paper is an overview of currently known mecha-
nisms of IR which include influence of various factors on
metabolism and insulin prereceptor, receptor and postreceptor
events.

Fig. 1. Models for IR assessment: HOMA [7], QUICKI [8] and McAuley [9] index.
22 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35
2. Mechanisms of insulin resistance
2.1. Dysregulation of FFA and adipokine secretion
It is well established that increased availability and utilization
of FFA play a critical role in the development of IR [12].
Numerous studies have also found associations between IR and
increased tumour necrosis factor α (TNF-α), interleukin 6 (IL-6),
macrophages and monocyte chemoattractant protein-1 (MCP-1),
plasminogen activator inhibitor-1 (PAI-1), adipsin, and decreased
adiponectin [13].
2.1.1. FFA and tumour necrosis factor α
IR is the accompanying feature in the majority of obese
patients. Adipose tissue releases large amounts of TNF-α, which
is at least partly responsible for development of IR in obesity [14].
On the other hand, weight loss is well known to increase insulin
sensitivity, which is thought to be mediated in part by decreased
adipose TNF-α release [15]. TNF-α is the main autocrine/
paracrine factor that triggers secretion of free fatty acids (FFA)
from adipose tissue into the circulation [14]. However, it remains
unclear which factors actually trigger adipokine release from
adipose tissue.
TNF-α mediates repression of many genes responsible for
glucose and FFA uptake and storage. For 3T3-L1 adipocytes it
has been shown that TNF-α dependent gene up- and down-
regulation occurs by obligatory activation of nuclear transcrip-
tion factor κB [16]. As a result of TNF-α action, enhanced
lipolysis occurs with release of FFA and cytokines.
TNF-α and FFA impair insulin signalling in insulin responsive
tissues, especially in muscle. The released FFA, according to the
lipid supply hypothesis (Randle hypothesis), act as the predominant
substrate in intermediary metabolism. Increased NADH/NAD+
and acetyl-CoA/CoA ratios could be the reason for decreased
glucose uptake, which means IR [17,18]. Visceral adipose tissue
secretes FFA directly to the portal blood flow, thereby exerting
potent effects on the liver rather than subcutaneous adipose tissue
releasing its products to the peripheral circulation [1].
In liver, high levels of FFA provide abundant substrate for
triglyceride and VLDL-synthesis, and for gluconeogenesis. TNF-
α represses genes for glucose uptake and beta-oxidation [19]. De
novo cholesterol synthesis occurs, followed by TNF-α upregula-
tion of corresponding genes [13]. FFA also trigger fibrinogen and
PAI-1 synthesis in the liver [20]. FFA draining from visceral
adipose tissue to portal circulation and further to the liver decrease
hepatic insulin clearance, thereby contributing to hyperinsulinae-
mia [18]. Excess triglycerides accumulate in the liver [1].
In muscle, high FFA concentrations favour beta-oxidation,
which diminishes glucose uptake and oxidation [21]. But beta-
oxidation is inadequate to clear FFA effectively from the circu-
lation, and even more so in the absence of physical activity [22].
Glycogen synthesis in muscle is inhibited. Muscle is the main
glucose disposer (80–90%) and diminished uptake contributes
greatly to hyperglycaemia [14,23]. Excess FFA are stored as
triglyceride droplets in muscles [1,11,22].
In adipose tissue, FFA inhibit lipoprotein lipase activity,
which is otherwise stimulated by insulin, and thereby render
clearance of FFA from circulation impossible [24]. Overall,
lipids released from adipocytes as FFA are transported as tri-
glycerides by VLDL and shifted to non-adipose tissues [25].
In β-cells, long exposure to high FFA concentrations impairs
insulin secretion, as described below at DM2.
TNF-α has been shown to downregulate the genes for
adiponectin, glucose transporter 4 (GLUT4), insulin receptor
substrate-1 (IRS-1), CCAAT/enhancer binding protein α (C/EBP-
α), peroxisome proliferator-activated receptor γ (PPAR-γ), and
perilipin in adipocytes [16,19]. TNF-α upregulates many genes
expressed in adipose tissue that are responsible for inflammation,
immune response and energy balance (vascular cell adhesion
molecule-1 (VCAM-1), plasminogen activator inhibitor-1 (PAI-1),
IL-6, IL-1β, angiotensinogen, resistin, leptin) [19].
2.1.2. Adiponectin
Adiponectin is strongly inversely correlated to IR in lipo-
dystrophy and obesity, and to inflammatory states [26,27]. It is
secreted exclusively by adipocytes [26]. Adiponectin improves
insulin sensitivity by various mechanisms. In liver, it induces
fatty acid oxidation, decreases lipid synthesis, decreases uptake
of FFA and represses gluconeogenesis by enzyme downregula-
tion [13,28,29]. In muscle, adiponectin favours glucose and FFA
oxidation. These effects are partly due to the activation of AMP-
kinase [26]. Thereby, adiponectin decreases plasma FFA and
glucose levels [13,28]. FFA catabolism is stimulated either
directly or through stimulation of PPAR-γ nuclear receptors [30].
Adiponectin levels increase with weight loss [31] and treatment
with thiazolidinediones (TZD) [32], which are PPAR-γ agonists.
Synthesis of adiponectin is impaired in states of calorie excess,
which might be associated with leptin resistance or deficiency.
Insulin and insulin-like growth factor (IGF-1) stimulate adipo-
nectin synthesis [28].
Adiponectin exerts effects on gene transcription also through
inhibition of nuclear transcription factor κB, the latter being
activated on the other hand by TNF-α [33]. Adiponectin de-
creases the expression of adhesion molecules on blood vessel
wall, inhibits chemotaxis of macrophages and their conversion to
foam cells, proliferation of smooth muscle cells and inflamma-
tory events in atherogenesis, which are promoted by IL-6, PAI-1,
etc. [13]. Additionally, adiponectin suppresses secretion of TNF-
α [34].
2.1.3. Leptin
Leptin has been shown to be implicated in providing normal
insulin sensitivity [35,36]. It is a hormone secreted predomi-
nantly by adipose tissue and is a signal of sufficiency of energy.
It decreases food intake and increases energy expenditure,
thereby indirectly promoting insulin sensitivity [37]. Leptin
effects are mediated by its action on the hypothalamus and
directly on target tissues (muscle, gonads, β-cells, liver) [38].
In normal conditions of weight maintenance, leptin con-
centrations are positively correlated with total body fat mass. In
short term food deprivation, serum leptin levels decrease and
the opposite is true for short term overfeeding [28].
Clinical states with diminished adipose mass (lipodystro-
phies) are characterized by reduced plasma leptin concentrations
 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35
and IR. Leptin administration significantly improves insulin
sensitivity in these conditions [35]. Moreover, a mouse model
with suppressed adipose tissue development resulted in
hyperphagic mice with severe insulin resistant diabetes [39].
The suggested mechanism causing IR was leptin and adipo-
nectin deficiency due to the absence of adipose tissue. Thus, the
administration of these hormones together reversed IR [36].
These two hormones therefore appear to be critical for normal
insulin sensitivity.
Leptin is an essential fertility factor, since decreased leptin
concentrations following food-deprivation have been shown to be
responsible for suppressing the hypothalamic-pituitary-gonadal
axis [40]. An important leptin effect is sympathetic stimulation,
achieved by eliciting leptin-responsive neurons in the hypothal-
amus [13]. This could in part explain the development of
hypertension in visceral obesity and non-obese states. In ac-
cordance with this, sympathetic activity in non-obese, normoten-
sive men correlates well with protein-bound leptin concentrations
[41].
2.1.4. Resistin
Resistin is secreted to a much greater extent by visceral than
by subcutaneous adipose tissue [13]. In rodent obesity, serum
resistin levels are increased, and some experiments have proved
resistin as a factor contributing to IR [42], although some other
studies failed to confirm these results [13,43]. Human resistin
shows only 64% homology with rodent resistin [44] and there is
growing evidence that expression of resistin in human adipose
tissue and its plasma concentration are not associated with IR or
obesity in humans [28,45]. Moreover, it is argued whether
resistin is secreted by adipocytes or yet unidentified stromal
cells and whether circulating resistin has any association with
adipose tissue [46]. Its function in humans is unknown.
2.1.5. Other adipokines
Other adipokines, MCP-1, PAI-1, IL-6 and IL-10, have been
studied in association with IR.
MCP-1 is suggested to be directly implicated in IR as it was
shown to impair insulin stimulated glucose uptake and insulin
receptor Tyr phosphorylation in cultured adipocytes [47]. MCP-1
is secreted by adipocytes; it attracts macrophages to adipose
tissue and promotes their IL-1 and TNF-α release [13]. It also
inhibits adipocyte growth and differentiation. MCP-1 promotes
atherosclerosis by attracting macrophages and favouring their
accumulation in vessel walls [48].
PAI-1 is more highly expressed in visceral than in sub-
cutaneous adipose tissue [49]. It is strongly associated with
visceral obesity, IR and metabolic syndrome. PAI-1 is suggested
to contribute to obesity and IR and has a causal role in the
further development of cardiovascular disease based on its
prothrombotic activity [13]. PAI-1 plasma levels decrease after
weight loss [13] and after therapy with insulin sensitizing sub-
stances [50].
Studies with IL-6 have suggested its strong implication in IR,
since plasma concentrations and adipose tissue expression, and
polymorphisms of IL-6, correlate well with obesity and IR. IL-6
was shown to interfere with insulin signalling [51], to inhibit
23
adipogenesis and secretion of adiponectin [13]. It also induces
IR in the liver and adipocytes [52].
IL-6 has opposite effects in the central nervous system,
suggesting that it favours energy expenditure. Administration of
IL-6 to IL-6 gene knockout rodents reversed obesity [53].
Similarly to adiponectin, IL-10 is a factor promoting insulin
sensitivity: in obesity without the metabolic syndrome IL-10
plasma levels were increased, but in the metabolic syndrome its
levels were decreased [54].
2.2. Glucocorticoids and FFA
Glucocorticoids are well known insulin antagonists and thereby
could induce IR. Glucocorticoids enhance liver gluconeogenesis
and glucose release. Furthermore, they impair peripheral glucose
uptake by various mechanisms. One of them is increased
availability of FFA which diminishes glucose oxidation and
hence its uptake to peripheral tissues. There are at least three
possible ways in which glucocorticoids could promote lipolysis:
(1) glucocorticoids increase noradrenalin conversion to
adrenalin (by phenyl-ethanolamine N-methyltransferase
in skeletal muscles), which acts on hormone-sensitive
lipase in adipose tissue and increases lipolysis [55];
(2) they could directly upregulate hormone-sensitive lipase
[56];
(3) they could inhibit lipoprotein lipase and thus disable
uptake of FFA in adipose tissue [57].
The next mechanism of aggravated glucose uptake is
inhibited translocation of GLUT4 which was seen in skeletal
muscle in the presence of glucocorticoids [58]. Glucocorticoids
were, additionally, shown to inhibit vasodilation (induced by
insulin via nitrogen oxide) and less glucose was then delivered
to the target tissues [59]. These effects of glucocorticoids are
potentiated by increased FFA levels, which enhance glucocor-
ticoid binding to their receptor [60]. All of these mechanisms
contribute to or exaggerate IR.
2.3. Molecular mechanisms of impaired insulin signalling
2.3.1. Normal insulin signalling
The insulin receptor is a heterotetramer, expressed on liver,
adipose and skeletal muscle cells. Binding of insulin triggers
oligomerization and receptor autophosphorylation on tyrosine
residues, and tyrosine phosphorylation of insulin receptor
substrates IRS-1, -2, -3, -4, IRS5/DOK4, IRS6/DOK5. This
phosphorylation provides the basis for the subsequent associ-
ation with downstream signal proteins which diverge into three
different pathways: the phosphoinositide-3-kinase (PI3K)
pathway, the CAP/Cbl/TC10 pathway and the mitogen-
activated protein kinase (MAP-kinase) dependent pathway
[61,62] (Fig. 2). PI3K interacts with phosphorylated tyrosines
on IRS molecules, resulting in phosphorylation of phosphatidyl
inositol-4,5-diphosphate (PIP2) to phosphatidyl inositol-3,4,5-
triphosphate (PIP3). The latter, as a second messenger, activates
phosphoinositide-dependent kinase 1 (PDK-1), which further
24 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35

Fig. 2. Insulin signal transduction [61,62]. After autophosphorylation of insulin receptor Tyr, Tyr phosphorylation in insulin receptor substrates 1 to 4 (IRS-1 to 4) takes
place and subsequently three different pathways transduce the signal: the PI3K-dependent pathway mediating metabolic responses, including glucose/lipid/protein
metabolism and insulin-stimulated glucose uptake, the CAP/Cbl pathway which is additionally required for glucose transporter-4 (GLUT4) translocation, and the
mitogen-activated protein kinase (MAP kinase) pathway that results in cell proliferation and differentiation. Solid line: stimulation, dashed line: inhibition. IRS —
insulin receptor substrate; PI3K — phosphoinositide-3-kinase; PDK-1 — phosphoinositide-dependent kinase 1; PKB — protein kinase B; aPKC — atypical protein
kinases C; mTOR — mammalian target of rapamycin; p70S6K — p70 ribosomal S6 kinase; PEPCK — phosphoenolpyruvate carboxykinase; GSK3 — glycogen
synthase kinase 3.

phosphorylates protein kinase B (PKB) and atypical protein
kinases C (aPKC) [63]. PKB phosphorylates, and thereby
inactivates, glycogen synthase kinase 3 (GSK-3) which, at the
final stage, results in glycogen synthesis. PKB also mediates
upregulation of the fatty acid synthase gene, downregulation of
the phosphoenolpyruvate carboxykinase (PEPCK) gene and
affects the expression of several other genes, together with
translocation of the main glucose transporter GLUT4 to the
plasma membrane [61,62]. PKB leads additionally to activation
of protein synthesis which is mediated by mammalian target of
rapamycin (mTOR) [64].
For glucose uptake to be fully manifested, the adapter protein
CAP in the second pathway recruits proto-oncogene Cbl to the
phosphorylated insulin receptor which finally results in
reinforced GLUT4 translocation [61]. The third pathway after
IRS-activation includes GRB2, SHP2 and several other proteins
which lead to MAP-kinase activation, cellular proliferation, and
differentiation via gene transcription regulation [62,65].
2.3.2. Defects in insulin signalling resulting in IR
Impairment of insulin action involves inactivation of insulin
during its circulation in blood, and defects at the receptor and
postreceptor levels. These defects are caused by genetic or
environmental factors. A minority of insulin resistant cases is
characterized by a single genetic or acquired trait. Anti-insulin
autoantibodies have been found in diabetes mellitus type 1 [66].
On the other hand, more than 60 mutations have been identified
in the insulin receptor gene. Among them, type A IR is
 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35
associated with a heterozygous mutation state which is
responsible for decreased Tyr phosphorylation of the β-subunit
after insulin binding [67]. In Rabson-Mendenhall syndrome and
leprechaunism (Donohue syndrome), insulin receptor gene
mutations are presumed to impair insulin binding to the receptor
[67]. IR may be due to abnormal production of anti-insulin
receptor antibodies (type B IR) [68]. PPAR-γ mutations which
are not associated with lipodystrophy are also reported to cause
IR [69].
Increased degradation of insulin receptor has been reported
[70]. It has also been suggested that serine/threonine phosphor-
ylation by various PKCs could be an additional factor in regulating
insulin receptor activity [71]. The decreased Tyr phosphorylation
state of IRS-1 that was found in hyperinsulinaemic ob/ob mice
could be a consequence of decreased insulin receptor activity [72].
2.3.2.1. Defects in insulin postreceptor signalling. Recent
studies focus mainly on dysregulation at the insulin-postreceptor
level. An essential factor contributing to IR is increased Ser/Thr
phosphorylation of IRS-1, which could be the consequence of
increased TNF-α concentration and of hyperinsulinaemia which
is secondary to IR. PKCs, PI3K-downstream kinases, MAP-
kinases (extracellular signal-regulated kinase-1 (Erk) and -2) are
thought to be responsible for Ser/Thr phosphorylation, which is
followed by enhanced proteosomal degradation of IRS-1.
Decreased IRS-1 protein content was reported in humans, other
animals and cultured cells with IR [61]. It has to be emphasized
that a certain basal Ser/Thr phosphorylation state of IRS-1 is
necessary for successful Tyr phosphorylation and insulin sig-
nalling. It therefore remains to be elucidated to what degree and
on which Ser/Thr sites IRS-1 must be phosphorylated to display
its diverse effects.
Besides mediating IRS-1 degradation, hyperinsulinaemia
might affect the concentration of IRS-2, and provoke some
downstream changes in insulin signalling [61].
TNF-α, IL-6 and insulin induce another important group of
IR factors — suppressors of cytokine signalling, SOCS-1 and
-3, which have at least three different mechanisms of action:
they compete with IRS-1 for association with insulin receptor,
they inhibit Janus kinase, involved in insulin signalling, and
they augment proteosomal IRS-1 degradation [73]. TNF-α also
decreases expression of GLUT4 [19].
Hyperglycaemia was shown to severely decrease PKB activity,
although some other proximal components of insulin signalling
(insulin receptor, IRS-1, IRS-2, PI3K) were activated [61].
FFA in high plasma concentrations were shown, by trans-
formation to diacylglycerol and acyl-CoA, to increase serine
phosphorylation on IRS-1 by PKC-θ in rats. In another ex-
periment, diminished activities of IRS-1, IRS-2 and of PI3K,
PKB-α, PKC-λ, PKC-ζ and GSK in rat muscle were demon-
strated as a consequence of increased FFA concentrations [61].
In transgenic mice, FFA increased activity of Munc18c protein,
a negative regulator of GLUT4 translocation to plasma mem-
brane [74]. Nevertheless, unsaturated FFA allow normal insulin
sensitivity in some target tissues [61].
Glycated proteins, emerging as a consequence of hyperglycae-
mia, were shown to diminish intracellular PI3K, PKB, and GSK-3
25
activity, thus possibly contributing to IR. Similarly, glucosamine
(UDP-N-acetyl glucosamine), produced from glucose and being
the main substrate for cellular glycosylation, enhances glycosyla-
tion of IRS-1, thereby decreasing its activity, and glycosylation of
glycogen-synthase, which reduces its insulin responsiveness [61].
On the other hand, development of transgenic animal models
and reports on naturally occurring mutations in humans
demonstrate that genetically predisposed dysfunction of certain
signalling proteins also results in IR in vivo. This was shown for
proteins IRS-1, -2, aPKC, PKBβ, glycogen synthase, and Foxo1
protein. Foxo-proteins are transcription factors that upregulate
the genes for gluconeogenesis enzymes in liver and are inhibited
by PKB phosphorylation in normal insulin signalling [75].
3. Obesity as the main factor in IR
3.1. Pathophysiology of obesity
All persons have their own genetically determined weight set-
point, and body weight is hence tightly regulated by an energetic
homeostatic mechanism, as seen in Fig. 3 [10,76,77]. Adipocytes
secrete leptin and β-cells secrete insulin, both in proportion to
body-fat content. The two hormones enter the brain. They bind to
their central receptors on the hypothalamic neurons, exerting
effects to reduce body weight. Hypothalamic neurons express
peptides and their receptors which could be categorized either as
orexigenic (neuropeptide Y, agouti-related protein (AgRP), mela-
nin-concentrating hormone (MCH), orexins A and B) or as ano-
rexigenic (melanocortins (i.e. melanocyte-stimulating hormone,
α-MSH), cocaine and amphetamine related transcript (CART)). In
leptin or insulin abundance the anorexigenic pathway prevails,
with increased energy expenditure, increased thermogenesis and
diminished food intake. Decreased leptin and insulin serum con-
centrations lead to activation of the orexigenic pathway, resulting
in low metabolic rate and enhanced appetite [10].
Leptin and insulin mediate long-term body-mass regulation.
However, they are also active as short-term signals that affect
single meal to be initiated and terminated. In addition, there are
some other briefly acting hormones/factors which accompany
food intake: ghrelin, motilin, neuromedin U, neurotensin [78],
cholecystokinin, peptide YY3–36 (PYY) [77] and glucagon-like
peptide-1 [79], all secreted by the gastrointestinal tract, and vagal
afferent signalling [77]. Ghrelin is secreted by the stomach
fundus and increases the sense of hunger and stimulates gastric
emptying [78]. PYY is secreted by the small intestine and colon,
signals satiety and inhibits gut motility [77]. Ghrelin stimulates
neuropeptide Y and AgRP neurons, while PYY inhibits the same
neurons in animals [77]. Recently, a peptide derived from the
same prohormone as ghrelin was discovered. Named obestatin, it
opposes the effects of ghrelin [80].
The leptin-melanocortin anorexigenic signalling pathway in
particular appears to be highly conserved among species, and
mutations in genes coding for components of this pathway –
leptin, leptin receptor, pro-opiomelanocortin (POMC), prohor-
mone-convertase 1 (PC1), and melanocortin 4 receptor (Mc4R) –
cause rare forms of morbid monogenic obesity in humans and
lead to some naturally occurring murine models of obesity (ob,
26 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35

Fig. 3. Energetic homeostasis. Neuropeptide Y (NPY)/agouti related protein (AgRP) neurones and pro-opiomelanocortin (POMC)/cocaine and amphetamine related
transcript (CART) neurones in the arcuate nucleus in the hypothalamus receive signals from the periphery. They relay orexigenic and anorexigenic signals to the
downstream neurons which provide the balance between food intake and energy expenditure [10,76,77]. Solid line: stimulation; dashed line: inhibition. PYY —
peptide YY3–36; Y1R and Y2R — subtypes of neuropeptide Y receptor; α-MSH — α-melanocyte stimulating hormone derived by cleavage of POMC; Mc4R —
melanocortin 4 receptor; GHSR — growth hormone secretagogue receptor (receptor for ghrelin); InsR — insulin receptor; MCH — melanin concentrating hormone;
TRH — thyrotropin-releasing hormone; CRH — corticotropin-releasing hormone.

db, Ay and mg) [10]. In contrast, knockouts in genes for
orexigenic pathways in mice fail to produce lean phenotypes,
which demonstrates the extremely powerful mutual effects of
anabolism and weight gain system components [77].
Today's high incidence of obesity can be explained by a
»thrifty genotype« hypothesis: over a period of time, alleles were
selected that favoured weight gain and fat storage in order to
provide enough nutrients for times of food deprivation. In
today's constant food availability and decreased need of phys-
ical activity, such genotypes cause pandemia of obesity [81].
The potential of the body to maintain body weight precisely
is reflected by the failure of interventions aimed at body
weight reduction. Calorie-restricted diets result in compensa-
tory increase in ghrelin levels, thereby stimulating eating [77].
Reduction of body weight results in decreased leptin levels,
which again stimulates weight gain [77]. Surgical removal of
adipose tissue results in restoration of fat in new locations, and
stimulation of thermogenesis by adrenergic β3-agonists elicits
a compensatory central nerve system response [10]. Only
extreme intervention in mice by gross overexpression of
uncoupling protein-3 (UCP-3), which is a signal protein in
thermogenesis, was successful in overriding central adaptive
mechanisms, and mice remained lean [82]. In humans, gastric
bypass surgery is successful, as it results in decreased ghrelin
 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35 27

plasma concentration and increased PYY, which suppresses
hunger and maintains reduced body weight [77].
Besides monogenic forms of obesity there are at least 20 rare
syndromes with obvious genetic bases which appear to be more
complex as they predispose to more dysfunctions (mental
retardation, multiple signs of hypothalamic disorder and other
congenital abnormalities) [76].
The common human obesity is thought to be an oligogenic
state and its expression is modulated by multiple modifier genes
and by environmental factors — food intake, physical activity,
and smoking [10]. The genetic basis of obesity is estimated to be
40–80% [76]. At least 204 putative gene loci associated with
obesity have been identified, and those that have been confirmed
by multiple studies are presented in Table 1 [83].
3.2. Categories of obesity
The state of nutrition is best described by the body mass
index (BMI). BMI is calculated by dividing weight in kilograms
by the square of height in metres. BMI, with some exceptions,
correlates well with the amount of total body fat. According to
the BMI, the following categories of excessive body mass or
nutrition have been proposed [84]:
BMI 25–30 kg/m2 overweight
BMI 30–40 kg/m2 obesity
BMI 40–50 kg/m2 morbid obesity
BMI N 50 kg/m2 extreme obesity
In the United States, 26% of adults are reported to be obese
and approximately 60% of adults are overweight [85].
Subcutaneous obesity can be measured by hip circumference,
whereas visceral obesity is described by waist circumference.
Plasma leptin concentrations are increased in obese indivi-
duals and exogenous administration of leptin has no effect on
body weight [13]. The phenomenon has been explained as leptin
resistance or desensitization [28]. In addition, soluble leptin
receptor concentrations, and hence the fraction of bound leptin,
are low in obesity. This feature is associated, independently, with
abdominal obesity and with IR. Soluble leptin receptors are
thought to be important for transport to or over the blood–brain
barrier, and it is the saturation of this transport or impairment of
leptin receptor signal transduction that may be the cause of leptin
resistance. Leptin concentrations in CSF of obese patients are
only modestly increased [28].
A certain amount of adipose tissue is necessary for normal
insulin sensitivity. This was demonstrated by animal models in
which suppression of adipose tissue development resulted in
severe IR. Thus, adipose tissue provides two hormones essential
for normal insulin sensitivity: adiponectin and leptin [36,39].
4. IR associated diseases
IR is an integrative feature of metabolic syndrome, the co-
existence of risk factors for cardiovascular diseases and DM2. A
group of lipodystrophies constitute a special syndromic form of
IR, characterized by disrupted distribution of adipose tissue.
Further diseases arising from IR include DM2 and polycystic
ovary syndrome.
4.1. Metabolic syndrome

3.3. Insulin and leptin resistance in obesity 4.1.1. Definition of metabolic syndrome
The clustering of hypertension, hyperglycaemia and gout was
Visceral obesity is thought to be the main factor causing IR. first recognized in the 1920s [1]. In 1988, Reaven identified
Visceral obesity predisposes to the development of hypertension, Syndrome X originating from IR. In 2004, The National Cho-
DM2, cardiovascular disease and certain types of cancer [76]. lesterol Education Program's Adult Treatment Panel III (ATPIII)
Increased risk exists already in the overweight group. Besides
total body fat mass, distribution of fat predisposes to eventual
metabolic complications. Visceral and subcutaneous adipose Table 1
tissue differ in their endocrine activities. Specific receptors, such A list of genes associated with obesity confirmed by 5 or more studies [83]
as angiotensin II receptors type-1 (AT1), β1-, β2-, β3-adrenergic Gene name (accord. to HUGO Protein name
receptors, glucocorticoid, and androgen receptors, are present to nomenclature committee)
a larger degree in visceral adipocytes where they promote ADIPOQ adiponectin
lipolysis [13,86,87]. On the other hand, antilipolytic insulin ADRA2A adrenergic receptor α-2A
receptors, α-2A adrenergic receptors, and estrogen receptors are ADRA2B adrenergic receptor α-2B
predominantly expressed in subcutaneous adipocytes [86,87]. ADRB1 adrenergic receptor β-1
ADRB2 adrenergic receptor β-2
Additionally, visceral adipose tissue secretes its products into the
ADRB3 adrenergic receptor β-3
portal circulation which brings released FFA directly to the liver DRD2 dopamine receptor D2
where they promote gluconeogenesis, VLDL synthesis, decrease LEP leptin
glucose uptake and cause overall IR. LEPR leptin receptor
Visceral adipose tissue is characterized by a relatively higher NR3C1 nuclear receptor subfamily 3,
group C, member 1
secretion of IL-1 and PAI-1, while leptin and adiponectin
PPARG PPAR-γ
secretion is greater from subcutaneous adipose tissue [13]. This UCP1 uncoupling protein 1
accounts for the relationship between visceral obesity and UCP2 uncoupling protein 2
inflammatory/thrombotic events and explains the effectiveness UCP3 uncoupling protein 3
of TZD in improving insulin sensitivity by redistribution of TNF TNF-α
LIPE hormone sensitive lipase
lipids to subcutaneous adipose tissue [88].
28 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35

Table 2
Diagnostic criteria for metabolic syndrome according to ATPIII [11] and IDF [89]
ATPIII IDF
Clinical feature Defining level Clinical feature Defining level
Central obesity–waist Men N102 cm central obesity–waist Europid men ≥94 cm (or specific values for other ethnic groups)
circumference Women N88 cm circumference Europid women ≥80 cm
plus any 2 of the following:
Triglycerides ≥1.7 mmol/L triglycerides ≥1,7 mmol/L or specific treatment
HDL-cholesterol Men ≤1.03 mmol/L HDL-cholesterol Men ≤1.03 mmol/L or specific treatment
Women ≤1.29 mmol/L Women ≤1.29 mmol/L
Blood pressure ≥130/≥ 85 mm Hg Blood pressure ≥130/≥85 mm Hg or treatment of
previously diagnosed hypertension
Fasting plasma glucose ≥6.1 mmol/L Fasting plasma glucose ≥5.6 mmol/L or previously diagnosed DM2

defined the metabolic syndrome by the criteria listed in Table 2,
which were further modified by the International Diabetes
Federation (IDF) in spring 2005 (Table 2). According to ATPIII,
patients having at least 3 of the 5 characteristics – abdominal
obesity, elevated triglycerides, decreased HDL-cholesterol, in-
creased blood pressure or increased fasting plasma glucose – can
be diagnosed as having the metabolic syndrome [11]. IDF de-
clares similar criteria fairly consistent with ATPIII [89]. Slight
differences include central obesity as the major feature and
fulfillment of two of the other four manifestations. The borderline
glucose concentration is lower in IDF criteria, with a strong
recommendation for OGTT when exceeded. The waist circum-
ference criteria are more strict by IDF and different for different
ethnic groups.
The main features of the metabolic syndrome are abdominal
obesity, IR, atherogenic dyslipidaemia, hypertension, pro-
inflammatory and prothrombotic states [11]. These features
are strong risk factors for DM2 and cardiovascular disease, with
additional possible complications including cholesterol gall-
stones, sleep apnoea, polycystic ovary syndrome in women,
hypogonadism in men, fatty liver and some types of cancer [11].
The prevalence of metabolic syndrome is age, sex and ethnicity
dependent, as seen in Table 3 [1].
Three possible aetiologies for the metabolic syndrome were
postulated. The first is visceral obesity, which was found to be
responsible for the excessive release of free fatty acids, cytokines
and other pro-inflammatory products, which are implicated in the
development of IR, hypertension and dyslipidemia [11].
IR as the second putative cause of metabolic syndrome raises
the question as to whether it is possible to separate obesity from
IR. Indeed, IR exists to various degrees in all categories of BMI,
suggesting the contribution of an independent, inheritable factor
to at least some extent. In some populations (South Asians), IR
exists in mildly overweight people and this is said to be primary
IR. In this case IR can be classified as a specific aetiological factor
for metabolic syndrome. Hyperinsulinaemia, as a consequence of
IR, increases VLDL synthesis and secretion from the liver and
causes hypertension. IR of muscle can cause hyperglycaemia,
exaggerated by gluconeogenesis in insulin-resistant liver [11].
The third aetiology is thought to include independent factors:
immunological, vascular, hepatic, etc., which are influenced by
the specific genetic background of an individual, and by
environmental factors [11].
4.1.2. Clinical manifestations of metabolic syndrome
4.1.2.1. Plasma glucose and FFA levels. In early stages of
metabolic syndrome normal glucose tolerance and only moder-
ately elevated plasma FFA are the consequences of insulin hyper-
secretion [11]. At suboptimal insulin concentrations, impaired
fasting glucose (IFG) or impaired glucose tolerance (IGT)
emerge. The latter is demonstrated by oral glucose tolerance
test (OGTT) glucose values above normal, but not sufficiently
elevated to define DM2 [90]. When plasma insulin concentrations
decline as a result of β-cell degeneration, FFA levels increase
considerably with accompanying hyperglycaemia, and the
condition of IR advances to DM2 [90].
4.1.2.2. Atherogenic dyslipidaemia. Hypertriglyceridaemia
occurs as a consequence of increased liver VLDL synthesis.
Plasma HDL is decreased, with a greater proportion of small
dense HDL as a consequence of cholesteryl ester depletion and
triglyceride increase. Similarly, the composition of LDL is shifted
towards the predominance of small dense LDL. The latter is more
atherogenic than ordinary LDL [1].
4.1.2.3. Hypertension. In healthy subjects, insulin acts on
blood vessels, causing vasodilation via endothelium-derived NO
[91]. Insulin also stimulates sodium reabsorption in the kidney. In
the insulin resistant state the vasodilation can be lost but the renal
effect is maintained and even increased [1]. Both contribute to
hypertension, which is further exacerbated by the vasoconstric-
tion favoured by increased FFA plasma levels. Moreover, insulin
stimulates the sympathetic nervous system, which might also be
preserved in the state of IR [92].
Nevertheless, it appears that events linked directly to insulin
play only a moderate role in the development of hypertension
[1]. Additionally, leptin might have some contribution to
Table 3
The prevalence of metabolic syndrome [1]
Population Prevalence (age interval)
USA 7% (20–29) 44% (60–69) 42% (≥ 70)
France b5.6% (30–39) 17.5% (60–64) –
Iran b10%(20–29) 38% (60–69) men; –
67% (60–69) women
 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35 29

hypertension, as plasma concentrations of protein bound leptin
in normotensive men correlate with sympathetic nerve activity
[41]. On the other hand, it has been proposed that obesity might
be associated with mild chronic inflammatory state which leads
to IR and endothelial dysfunction. The latter could cause
hypertension independently of IR [91].
4.1.2.4. Other clinical features. The pro-inflammatory state is
characterized by a rise in plasma C-reactive protein, and the
prothrombotic state by elevation of PAI-1 and fibrinogen [11].
Additional manifestations of the metabolic syndrome are fatty
liver and steatohepatitis, resulting from increased liver VLDL
production in the state of IR, hyperuricaemia, increased
homocysteine and vascular abnormalities with microalbuminuria
[1]. Hyperuricaemia is particularly interesting as it is most likely a
consequence of hyperinsulinaemia. The kidney, which maintains
normal sensitivity to insulin, adapts to high insulin concentrations
with decreased uric acid secretion, thereby elevating the plasma
level of the latter [90].
Another important clinical marker of IR is acanthosis nigricans
[3], which can be seen as hyperpigmented skin changes based on
epidermal hyperplasia. The feature is thought to be related to the
hyperinsulinaemic state, as insulin was shown to accelerate
epidermal cell growth in culture [93].
4.2. Inherited and acquired lipodystrophies
As already mentioned, an excess or deficiency of adipose
tissue is associated with IR [10]. In addition, IR accompanies a
group of disorders, characterized by selective loss of adipose
tissue, named lipodystrophies. They present with dyslipidae-
mia, hepatic steatosis and acanthosis nigricans [94]. The group
of disorders includes both inherited and acquired syndromes.
All forms of inherited lipodystrophies are rare, with congenital
generalized lipodystrophy being the most prevalent and
occurring in 1 in 10 million people [94].
Congenital generalized (Berardinelli-Seip) lipodystrophy is
associated with two possible gene defects, in the AGPAT2 (1-
acylglycerol-3-phosphate-O-acyl-transferase 2) gene–type 1
[85] and in the seipin gene–type 2 [95]. While AGPAT2 is
involved in triglyceride synthesis in adipocytes, the role of
seipin is not known, but its expression in the central nervous
system indicates a possible central nervous system defect in this
type of lipodystrophy. Patients of both types present with
generalized lack of adipose tissue from birth and other clinical
features listed in Table 4. Patients with type 2 congenital
generalized lipodystrophy exhibit mental retardation [4,94].
Familial partial lipodystrophy exists as Koebberling or type
1 and Dunningan or type 2, which are characterized by loss of
fat from arms and legs but normal or excess of facial, neck and
intra-abdominal fat [4,94]. Type 2 is associated with a defect in
LMNA gene coding for lamin A and C by alternative splicing.
These proteins play a role in nuclear envelope integrity and
interact with chromatin and various nuclear proteins [96].
Another variety of familial partial lipodystrophy is PPARG
gene associated lipodystrophy. PPAR-γ receptor activity is
required for adipocyte differentiation [94].
The most common form of lipodystrophy by far is acquired
lipodystrophy in HIV-infected patients which develops in
approximately 40% of patients receiving therapy with protease
inhibitors for more than 1 year [97]. These drugs were shown to
disturb development of subcutaneous adipose tissue and are
associated with decreased levels of mRNA for two transcription
factors: sterol-response elements binding protein 1c (SREBP1c)

Table 4
Types of lipodystrophy with underlying mechanisms and clinical features
Type of lipodystrophy
Congenital generalized
lipodystrophy (Berardinelli-Seip)
Familial partial lipodystrophy
(face-sparing)
Familial partial lipodystrophy
Acquired lipodystrophy in
HIV-infected patients
Acquired partial lipodystrophy
(Barraquer-Simons syndrome)
Acquired general lipodystrophy
AGPAT2 — gene for 1-acylglycerol-3-phosphate-O-acyl-transferase 2; LMNA — gene for lamin A and C; PPARG — gene for peroxisome proliferator-activated
receptor γ.
Gene defect/underlying mechanism Clinical features
type 1 AGPAT2 • generalized lack of subcutaneous, intra-abdominal,
intrathoracic adipose tissue
• accelerated appetite in childhood
• skeletal and cardial muscle hypertrophy
• hypertriglyceridemia eventually leading to acute pancreatitis
• acanthosis nigricans
type 2 seipin • the same as type 1
• mental retardation
type 1 ? • identified in women
(Koebberling) • absence of limb fat and excess of facial, neck and
intra-abdominal fat from childhood
type 2 LMNA • normal fat distribution in childhood; loss of limb fat
(Dunningan) and accumulation of facial, neck and intra-abdominal fat in
puberty or later
PPAR-γ PPARG • loss of limb and face fat
associated
protease inhibitors • loss of subcutaneous fat from face, arms and legs, and
excess fat in the neck, upper back and in the trunk
autoimmune adipocyte lysis • fat loss from face, neck, arms and excess fat on hips and legs
immune mechanism (panniculitis)/ • generalized loss of fat in childhoood and puberty
autoimmune mechanism/idiopathic • acanthosis nigricans, hepatic steatosis
30 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35
and PPAR-γ [98]. Lipodystrophy develops more frequently in
older HIV-infected patients and in longer duration of anti-
retroviral therapy. This type of lipodystrophy presents with
isolated peripheral adipose atrophy or isolated fat accumulation
or as a mixed syndrome [99].
Acquired partial lipodystrophy (Barraquer-Simons syn-
drome) is associated with presence of polyclonal IgG named
C3 nephritic factor, which favours complement activation and
lysis of adipocytes expressing factor D [100].
Acquired general lipodystrophy is in 50% of cases preceded
by subcutaneous inflammatory lesions (panniculitis) or auto-
immune diseases and is associated with fat loss from large areas
of the body [94].
4.3. Diabetes mellitus type 2
DM2 is a complication of IR. Diabetes mellitus is estimated
to affect 6% of the population and DM2 accounts for 90–95%
of diabetes cases [101]. DM2 represents a strong risk factor for
cardiovascular disease. There is much evidence for a powerful
genetic component in the aetiology of DM2. For example, a
person with a first degree relative with DM2 is at 3.5 times
greater risk to develop DM2 [102]. The common insulin resis-
tance (e.g. in the metabolic syndrome) is usually accompanied
by hyperinsulinaemia as a result of relative hyperglycaemia to
override diminished insulin action. When β-cells fail to secrete
excessive amounts of insulin, DM2 develops [22]. Increased
susceptibility to β-cell failure has been postulated as the
predominant risk factor for DM2 and intensive investigation of
susceptibility genes (eventually affecting β-cell secretion) is
under way [103]. On the other hand, mechanisms involving
lipid oversupply of β-cells have arisen from aetiology studies,
pointing, at least in part to environmental factors. Combining
both suggestions, the excess of FFA could affect individuals
with genetically predisposed increased sensitivity of β-cell
signal molecules, e.g. protein kinases [101].
4.3.1. Detrimental effect of FFA on β-cells
FFA act as incretins, that is, augment glucose stimulated
insulin secretion, which is important under physiological con-
ditions. However, FFA alone are not secretagogues. A two-arm
signalling pathway is proposed to trigger secretion of insulin. The
first arm is associated with acetyl-CoA mediated ATP/ADP ratio
increase, which closes ATP-sensitive K+ channels, thereby depo-
larizing the cell. The consequence is the prolonged open-time of
voltage-dependent Ca-channels. Finally the elevated intracellular
Ca2+ concentration modulates kinases and other signalling pro-
teins managing insulin secretion. The activity of the second arm
is associated with pyruvate synthesis from glucose, followed by
oxaloacetate and citrate production which is responsible for the
subsequent malonyl-CoA synthesis. The latter is a switch
repressing β-oxidation and stimulating synthesis of long-chain-
CoA (LC-CoA) and complex lipids — diacylglycerols and
phosphatidate. FFA are implicated by being a direct substrate for
LC-CoA synthesis. Complex lipids and LC-CoA are presumed to
activate PKC or modify the acylation state of proteins acting on
ion-channels and the exocytosis of granules containing insulin.
The exact targets of LC-CoA and complex lipids action are not
fully known, but the list of potential molecules being bound or
acylated by LC-CoA includes PKC, Ca-ATPase, carnitine-
palmitoyl transferase 1, hormone-sensitive lipase, voltage
dependent Ca-channels and others. Thus, LC-CoA appears to
be implicated distally in stimulus-secretion coupling in β-cells
[101].
Short term exposure to high FFA plasma concentrations has
been proved to augment glucose-stimulated insulin secretion,
while long-term oversupply with FFA increases basal insulin
secretion and exacerbates glucose-dependent secretion. Both
were shown in vitro [104] and in vivo [105,106]. A few mecha-
nisms have been proposed for the dual response of β-cells, one
being that there could be enzymes with various sensitivities to
exposure time and concentration of LC-CoA, as some enzymes
were shown to be stimulated and others inhibited by high
concentrations of FFA [101]. In addition, hyperglycaemia results
in early enhanced sensitivity to glucose and subsequently
diminished insulin secretion [107]. Both phenomena are named
with the common name glucolipotoxicity. In conclusion, chron-
ically elevated FFA inhibit insulin biosynthesis and secretion,
decrease the expression of homeodomain transcription factor
(PDX-1), glucose transporter GLUT2 and acetyl-CoA carboxyl-
ase, and increase the expression of carnitin-palmitoyltransferase-1
[101]. Additionally, mitogenesis of β-cells is reported to be
inhibited by FFA via activation of various PKC isoforms [108].
4.3.2. Susceptibility genes for DM2
Affected genes in monogenic forms of diabetes, insulin
resistance and lipodystrophy provide an excellent base for the
search for susceptibility genes for polygenic multifactorial
DM2, although the latter can be distinguished from monogenic
Mendelian diseases. On certain genetic backgrounds, with par-
ticular gene interaction – epistasis – and with a particular
environment, the gene defects otherwise associated with
monogenic disorders could contribute to DM2 [22].
Maturity-onset diabetes of the young (MODY) is a mono-
genic form of diabetes and exists in 6 forms arising from 6
affected MODY genes. Three of them, HNF4A, TCF1 (or
HNF1A) and GCK genes encode for two transcriptional factors
and glucokinase in the β-cells, respectively, and were proved to
be involved in DM2 [109–111].
From the genes responsible for the monogenic form of
insulin resistance, the gene for insulin with class III variable
number tandem repeat alleles [112] has been associated with
multifactorial DM2 as well. Additionally, the AGPAT2 gene
underlying Berardinelli-Seip congenital lipodystrophy and
the Mt-tRNA Leu(UUR) mitochondrial gene, otherwise
associated with maternally inherited diabetes and deafness
(MIDD) syndrome, have been shown to be implicated in
DM2 [103].
Search for susceptibility genes among the genes dealing with
insulin secretion and action gave positive results for the already
mentioned PPARG gene with another polymorphism Pro12Ala
(different from that implicated in monogenic IR [113]) and the
KCNJ11 gene, encoding for protein contained in KATP channels
[114]. Suggestive, but not yet definite, evidence for implication
 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35
in DM2 concerns IRS-1, GLUT2 and PGC1A (co-activator of
PPAR-γ) genes [103].
The only proved DM2 susceptibility gene found through
genome wide-scan methods is CAPN10 which encodes for
calpain-10 from the family of calcium-activated neutral pro-
teases expressed in β-cells [115]. This gene was identified after
strong linkage disequilibrium found with DM2, which was
followed by positional cloning [116].
When studying the importance of particular susceptibility
genes it must be borne in mind that their individual effects are
modest. The listed susceptibility genes do contribute to the basis
for future diagnosis, prognosis and therapy, but for now, the
mutual influences of various gene loci and interactions of genes
with dietary and other lifestyle factors remain to be precisely
determined and quantitated [22,103].
4.4. Polycystic ovary syndrome and IR
Polycystic ovary syndrome (PCOS) is a heterogeneous group
of disorders with metabolic and endocrine defects, which affects
5–10% of women of reproductive age [117,118]. In PCOS
patients, 50–70% present with IR [117,119]. Diagnostic criteria
for PCOS according to the Rotterdam European Society of
Human Reproduction/American Society for Reproductive Med-
icine (ESHRE/ASRM) sponsored PCOS Consensus Workshop
Group include at least two of three clinical features — oligo- or
an-ovulation, clinical or biochemical signs of hyperandrogenism
and polycystic ovaries on ultrasound examination in the absence
of other possible aetiologies [119]. Excess androgens primarily
originate from the ovary [93]. Additional frequent manifestations
in PCOS, beside IR, include clustering of metabolic syndrome
features: obesity, dyslipidaemia, hypertension, impaired glucose
tolerance, and elevated luteinizing hormone/follicle stimulating
hormone (LH/FSH) ratio [117,119]. Increased risk for DM2
[120,121], cardiovascular disease [122,123] and endometrial
cancer [124] have been observed in PCOS patients.
IR and its attendant hyperinsulinaemia have been suggested to
play a key role in the aetiology of PCOS, as weight loss and
insulin sensitizing drugs improve hyperandrogenaemia and
restore ovulation [117], and fasting insulin levels correlate well
with androgen levels [125]. Ovarian steroidogenesis is essentially
LH-dependent. Nevertheless, some studies have shown a potent
synergistic effect of insulin on ovarian steroidogenesis, in spite of
peripheral IR [117].
Defects in insulin signalling in insulin resistant PCOS patients
were shown to be tissue specific, there being reports of a 30%
decrease in insulin receptor autophosphorylation in adipocytes,
but none in fibroblasts [126], increased Ser phosphorylation in
fibroblasts of 50% PCOS patients [127], lower IRS-1-associated
PI3K activity in muscle cells [128], but normal activity in fibro-
blasts of PCOS women [129].
Despite IR, evident in the glucose uptake pathway in insulin
resistant patients, pathways mediating proliferation were shown
to be intact, at least in PCOS fibroblasts [129]. It was initially
proposed that insulin could stimulate ovarian androgen biosyn-
thesis via IGF-1 receptors or hybrid insulin/IGF-1 receptors.
With the use of antibodies to insulin receptor, insulin was proved
31
to stimulate steroidogenesis in theca cells from PCOS patients via
its own receptors [130]. This was confirmed in normal theca cells
and, further, a PI3K inhibitor was shown to prevent this induction
[128]. Thus, it was postulated that, distally to PI3K, a divergence
into 2 pathways may take place, one regulating glucose transport
and another regulating androgen biosynthesis [128]. Some
mechanisms should exist in insulin resistant PCOS patients to
confer greater activity to the pathway regulating steroidogenesis
while the pathway for glucose transport is defective.
There is no evidence that insulin can regulate androgen synthesis
through the MAP-kinase pathway. Nevertheless, this pathway
(MEK/Erk) was shown, in PCOS theca cells, to be suppressed with
concomitantly increased P450c17 (17α-hydroxylase/C17–20 lyase)
activity. The latter is the key enzyme in ovarian androgen bio-
synthesis. Indeed, the MAP-kinase suppression and steroidogenesis
augmentation were independent of insulin concentration [131].
Another proposed mechanism of increased androgen biosyn-
thesis is abnormally increased Ser phosphorylation of P450c17,
which increases the enzyme activity [132]. Thus, the same
mechanism could be responsible for diminished glucose uptake
and hyperandrogenism.
Insulin receptors have been identified in human pituitary [133],
leading to the suggestion that insulin could stimulate LH secretion
and thereby augment LH action on ovarian steroidogenesis.
Additionally, hyperinsulinaemia suppresses sex-hormone
binding globulin synthesis in the liver and accounts for the
increased bioavailability of androgens [134].
However, a considerable number of women with IR do not
develop PCOS [135] and there are reports of lean PCOS patients
without IR [81,136]. A study on sisters of PCOS patients demon-
strated that, on the same genetic basis, hyperandrogenaemia and/
or anovulation developed mostly in those sisters who were obese
[121]. Thus, an intrinsic ovarian defect(s) of possibly genetic
origin might be present but IR is needed to reveal or exaggerate
the PCOS [135]. In support of this hypothesis, theca cells from
PCOS patients uniformly show increased activities of certain
steroidogenic enzymes and increased contents of dehydro-
epiandrosterone, progesterone, 17 α-hydroxyprogesterone, and
androstenedione [137]. Intrinsic ovarian defect(s) or extra-ovarian
stimuli without IR and/or hyperinsulinaemia could by themselves
be sufficient to drive aetiopathogenesis in some PCOS patients.
Familial clustering encouraged the search for susceptibility
genes for PCOS and many were found. This accords with the
heterogeneity of phenotypes and both suggest that PCOS is a
polygenic trait influenced by environmental factors [138]. The
candidate gene list consists of approximately 40 genes being
involved in androgen biosynthesis and action (e.g. CYP17,
SHBG, CYP11A), in insulin resistance (gene for insulin receptor,
IRS-1), genes encoding inflammatory cytokines (TNFRSF1B)
and others [138].
5. Conclusion
IR is a common feature in obesity and possibly can lead to
further disorders — metabolic syndrome, DM2 and PCOS.
Lipodystrophies on the other hand are associated with decreased
adipose mass but are, again, characterized by insulin resistance.
32 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35
Various studies have shown that adipose tissue seriously affects
whole body insulin sensitivity by secreting adipokines impli-
cated in glucose and lipid metabolism.
According to some reports, IR is an inherited feature [11,139]
but, on the other hand, arising from obesity which at least partly
depends on lifestyle. Hyperinsulinaemia, which accompanies IR,
is thought to play an important role in the pathogenesis of some
syndromes. Despite the finding that insulin hypersecretion is a
consequence of IR [1,2], there are studies – showing examples of
PCOS patients with normal insulin sensitivity but insulin
hypersecretion – indicating that IR and insulin hypersecretion
could be two distinguishable features [136]. Additionally, basal
insulin levels are also affected by (defective) hepatic insulin
clearance [136]. On the other hand, besides IR and hyperinsu-
linaemia, some special impairments, e.g. vascular or ovarian
defects, could finally contribute to the full development of IR-
related diseases. A combination of genetic and environmental
factors should therefore be considered and the aetiological
mechanisms of these diseases need to be studied further. This is
particularly important in the case of metabolic syndrome, as there
is still some doubt about whether it exists or whether it is merely
the random co-existence of cardiovascular risk factors without
any common origin [140]. On the other hand, studies with
powerful statistical evidence of uniformity of metabolic syn-
drome have been recently published [141].
Future study of the molecular mechanisms of IR-associated
diseases would help in the management of their frequent and
serious complications, DM2 and coronary heart disease.
References
[1] Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet
2005;365:1415–28.
[2] Wang CC, Goalstone ML, Draznin B. Molecular mechanisms of insulin
resistance that impact cardiovascular biology. Diabetes 2004;53:
2735–40.
[3] Granberry MC, Fonseca VA. Insulin resistance syndrome: options for
treatment. South Med J 1999;92:2–15.
[4] Greenfield JR, Campbell LV. Insulin resistance and obesity. Clin
Dermatol 2004;22:289–95.
[5] Ferrannini E, Natali A, Bell P, Cavallo-Perin P, Lalic N, Mingrone G.
Insulin resistance and hypersecretion in obesity. European Group for the
Study of Insulin Resistance (EGIR). J Clin Invest 1997;100:1166–73.
[6] DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method
for quantifying insulin secretion and resistance. Am J Physiol 1979;237:
E214–23.
[7] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner
RC. Homeostasis model assessment: insulin resistance and beta-cell
function from fasting plasma glucose and insulin concentrations in man.
Diabetologia 1985;28:412–9.
[8] Katz A, Nambi SS, Mather K, et al. Quantitative insulin sensitivity check
index: a simple, accurate method for assessing insulin sensitivity in
humans. J Clin Endocrinol Metab 2000;85:2402–10.
[9] McAuley KA, Williams SM, Mann JI, et al. Diagnosing insulin resistance
in the general population. Diabetes Care 2001;24:460–4.
[10] Cummings DE, Schwartz MW. Genetics and pathophysiology of human
obesity. Annu Rev Med 2003;54:453–71.
[11] Grundy SM, Brewer Jr HB, Cleeman JI, Smith Jr SC, Lenfant C.
Definition of metabolic syndrome: Report of the National Heart, Lung,
and Blood Institute/American Heart Association conference on scientific
issues related to definition. Circulation 2004;109:433–8.
[12] DeFronzo RA. Pathogenesis of type 2 diabetes mellitus. Med Clin North
Am 2004;88:787–835 [ix].
[13] Kershaw EE, Flier JS. Adipose tissue as an endocrine organ. J Clin
Endocrinol Metab 2004;89:2548–56.
[14] Ruan H, Lodish HF. Insulin resistance in adipose tissue: direct and
indirect effects of tumor necrosis factor-alpha. Cytokine Growth Factor
Rev 2003;14:447–55.
[15] Kern PA, Saghizadeh M, Ong JM, Bosch RJ, Deem R, Simsolo RB. The
expression of tumor necrosis factor in human adipose tissue. Regulation by
obesity, weight loss, and relationship to lipoprotein lipase. J Clin Invest
1995;95:2111–9.
[16] Ruan H, Hacohen N, Golub TR, Van Parijs L, Lodish HF. Tumor necrosis
factor-alpha suppresses adipocyte-specific genes and activates expression
of preadipocyte genes in 3T3-L1 adipocytes: nuclear factor-kappaB
activation by TNF-alpha is obligatory. Diabetes 2002;51:1319–36.
[17] Randle PJ, Garland PB, Newsholme EA, Hales CN. The glucose fatty
acid cycle in obesity and maturity onset diabetes mellitus. Ann N Y Acad
Sci 1965;131:324–33.
[18] Lewis GF, Carpentier A, Adeli K, Giacca A. Disordered fat storage and
mobilization in the pathogenesis of insulin resistance and type 2 diabetes.
Endocr Rev 2002;23:201–29.
[19] Ruan H, Miles PD, Ladd CM, et al. Profiling gene transcription in vivo
reveals adipose tissue as an immediate target of tumor necrosis factor-
alpha: implications for insulin resistance. Diabetes 2002;51: 3176–88.
[20] Aubert H, Frere C, Aillaud MF, Morange PE, Juhan-Vague I, Alessi MC.
Weak and non-independent association between plasma TAFI antigen
levels and the insulin resistance syndrome. J Thromb Haemost
2003;1:791–7.
[21] Boden G, Chen X, Ruiz J, White JV, Rossetti L. Mechanisms of fatty acid-
induced inhibition of glucose uptake. J Clin Invest 1994;93: 2438–46.
[22] Jenkins AB, Campbell LV. The genetics and pathophysiology of diabetes
mellitus type II. J Inherit Metab Dis 2004;27:331–47.
[23] DeFronzo RA, Gunnarsson R, Bjorkman O, Olsson M, Wahren J. Effects
of insulin on peripheral and splanchnic glucose metabolism in noninsulin-
dependent (type II) diabetes mellitus. J Clin Invest 1985;76:149–55.
[24] Saxena U, Witte LD, Goldberg IJ. Release of endothelial cell lipoprotein
lipase by plasma lipoproteins and free fatty acids. J Biol Chem 1989;264:
4349–55.
[25] Sharma AM, Chetty VT. Obesity, hypertension and insulin resistance.
Acta Diabetol 2005;42(Suppl 1):S3–8.
[26] Chandran M, Phillips SA, Ciaraldi T, Henry RR. Adiponectin: more than
just another fat cell hormone? Diabetes Care 2003;26:2442–50.
[27] Kinlaw WB, Marsh B. Adiponectin and HIV-lipodystrophy: taking
HAART. Endocrinology 2004;145:484–6.
[28] Meier U, Gressner AM. Endocrine regulation of energy metabolism:
review of pathobiochemical and clinical chemical aspects of leptin, ghrelin,
adiponectin, and resistin. Clin Chem 2004;50:1511–25.
[29] Combs TP, Berg AH, Obici S, Scherer PE, Rossetti L. Endogenous
glucose production is inhibited by the adipose-derived protein Acrp30.
J Clin Invest 2001;108:1875–81.
[30] Stefan N, Vozarova B, Funahashi T, et al. Plasma adiponectin levels are not
associated with fat oxidation in humans. Obes Res 2002;10: 1016–20.
[31] Yang WS, Lee WJ, Funahashi T, et al. Weight reduction increases plasma
levels of an adipose-derived anti-inflammatory protein, adiponectin.
J Clin Endocrinol Metab 2001;86:3815–9.
[32] Satoh N, Ogawa Y, Usui T, et al. Antiatherogenic effect of pioglitazone in
type 2 diabetic patients irrespective of the responsiveness to its anti-
diabetic effect. Diabetes Care 2003;26:2493–9.
[33] Sonnenberg GE, Krakower GR, Kissebah AH. A novel pathway to the
manifestations of metabolic syndrome. Obes Res 2004;12:180–6.
[34] Aldhahi W, Hamdy O. Adipokines, inflammation, and the endothelium in
diabetes. Curr Diab Rep 2003;3:293–8.
[35] Oral EA, Simha V, Ruiz E, et al. Leptin-replacement therapy for
lipodystrophy. N Engl J Med 2002;346:570–8.
[36] Yamauchi T, Kamon J, Waki H, et al. The fat-derived hormone adiponectin
reverses insulin resistance associated with both lipoatrophy and obesity.
Nat Med 2001;7:941–6.
[37] Webber J. Energy balance in obesity. Proc Nutr Soc 2003;62:539–43.
 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35
[38] Bjorbaek C, Kahn BB. Leptin signaling in the central nervous system and
the periphery. Recent Prog Horm Res 2004;59:305–31.
[39] Shimomura I, Hammer RE, Richardson JA, et al. Insulin resistance and
diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in
adipose tissue: model for congenital generalized lipodystrophy. Genes
Dev 1998;12:3182–94.
[40] Chehab FF, Qiu J, Mounzih K, Ewart-Toland A, Ogus S. Leptin and
reproduction. Nutr Rev 2002;60:S39-46; discussion S68-84, 85-37.
[41] Tank J, Jordan J, Diedrich A, et al. Bound leptin and sympathetic outflow
in nonobese men. J Clin Endocrinol Metab 2003;88:4955–9.
[42] Steppan CM, Bailey ST, Bhat S, et al. The hormone resistin links obesity
to diabetes. Nature 2001;409:307–12.
[43] Way JM, Gorgun CZ, Tong Q, et al. Adipose tissue resistin expression is
severely suppressed in obesity and stimulated by peroxisome proliferator-
activated receptor gamma agonists. J Biol Chem 2001;276:25651–3.
[44] Banerjee RR, Lazar MA. Resistin: molecular history and prognosis.
J Mol Med 2003;81:218–26.
[45] Utzschneider KM, Carr DB, Tong J, et al. Resistin is not associated with
insulin sensitivity or the metabolic syndrome in humans. Diabetologia
2005;48:2330–3.
[46] Arner P. Resistin: yet another adipokine tells us that men are not mice.
Diabetologia 2005;48:2203–5.
[47] Sartipy P, Loskutoff DJ. Monocyte chemoattractant protein 1 in obesity
and insulin resistance. Proc Natl Acad Sci U S A 2003;100:7265–70.
[48] Takahashi K, Mizuarai S, Araki H, et al. Adiposity elevates plasma
MCP-1 levels leading to the increased CD11b-positive monocytes in
mice. J Biol Chem 2003;278:46654–60.
[49] Fain JN, Madan AK, Hiler ML, Cheema P, Bahouth SW. Comparison of
the release of adipokines by adipose tissue, adipose tissue matrix, and
adipocytes from visceral and subcutaneous abdominal adipose tissues of
obese humans. Endocrinology 2004;145:2273–82.
[50] Vestergaard H, Lund S, Pedersen O. Rosiglitazone treatment of patients
with extreme insulin resistance and diabetes mellitus due to insulin receptor
mutations has no effects on glucose and lipid metabolism. J Intern Med
2001;250:406–14.
[51] Senn JJ, Klover PJ, Nowak IA, et al. Suppressor of cytokine signaling-3
(SOCS-3), a potential mediator of interleukin-6-dependent insulin
resistance in hepatocytes. J Biol Chem 2003;278:13740–6.
[52] Korc M. Update on diabetes mellitus. Dis Markers 2004;20:161–5.
[53] De Benedetti F, Alonzi T, Moretta A, et al. Interleukin 6 causes growth
impairment in transgenic mice through a decrease in insulin-like growth
factor-I. A model for stunted growth in children with chronic inflammation.
J Clin Invest 1997;99:643–50.
[54] Esposito K, Pontillo A, Giugliano F, et al. Association of low interleukin-10
levels with the metabolic syndrome in obese women. J Clin Endocrinol
Metab 2003;88:1055–8.
[55] Kennedy B, Elayan H, Ziegler MG. Glucocorticoid induction of
epinephrine synthesizing enzyme in rat skeletal muscle and insulin
resistance. J Clin Invest 1993;92:303–7.
[56] Slavin BG, Ong JM, Kern PA. Hormonal regulation of hormone-sensitive
lipase activity and mRNA levels in isolated rat adipocytes. J Lipid Res
1994;35:1535–41.
[57] Ong JM, Simsolo RB, Saffari B, Kern PA. The regulation of lipoprotein
lipase gene expression by dexamethasone in isolated rat adipocytes.
Endocrinology 1992;130:2310–6.
[58] Coderre L, Vallega GA, Pilch PF, Chipkin SR. In vivo effects of
dexamethasone and sucrose on glucose transport (GLUT-4) protein tissue
distribution. Am J Physiol 1996;271:E643–8.
[59] Andrews RC, Walker BR. Glucocorticoids and insulin resistance: old
hormones, new targets. Clin Sci (Lond) 1999;96:513–23.
[60] Haourigui M, Vallette G, Martin ME, Sumida C, Benassayag C, Nunez EA.
In vivo effect of free fatty acids on the specific binding of glucocorticoster-
oids to corticosteroid binding globulin and liver receptors in immature rats.
Steroids 1994;59:46–54.
[61] Pirola L, Johnston AM, Van Obberghen E. Modulation of insulin action.
Diabetologia 2004;47:170–84.
[62] Saltiel AR, Kahn CR. Insulin signalling and the regulation of glucose and
lipid metabolism. Nature 2001;414:799–806.
33
[63] Brazil DP, Hemmings BA. Ten years of protein kinase B signalling: a
hard Akt to follow. Trends Biochem Sci 2001;26:657–64.
[64] Raught B, Gingras AC, Sonenberg N. The target of rapamycin (TOR)
proteins. Proc Natl Acad Sci U S A 2001;98:7037–44.
[65] Bevan P. Insulin signalling. J Cell Sci 2001;114:1429–30.
[66] Sharma K, Khosla PK, Tiwari HK, Sharma RK, Bajaj JS. Anti-insulin
antibodies and retinopathy in juvenile onset type-1 diabetes. Indian
J Ophthalmol 1991;39:174–5.
[67] McIntyre EA, Walker M. Genetics of type 2 diabetes and insulin resistance:
knowledge from human studies. Clin Endocrinol (Oxf) 2002;57:303–11.
[68] O'Rahilly S. Insights into obesity and insulin resistance from the study of
extreme human phenotypes. Eur J Endocrinol 2002;147:435–41.
[69] Barroso I, Gurnell M, Crowley VE, et al. Dominant negative mutations in
human PPARgamma associated with severe insulin resistance, diabetes
mellitus and hypertension. Nature 1999;402:880–3.
[70] Taylor SI, Samuels B, Roth J, et al. Decreased insulin binding in cultured
lymphocytes from two patients with extreme insulin resistance. J Clin
Endocrinol Metab 1982;54:919–30.
[71] Bossenmaier B, Strack V, Stoyanov B, et al. Serine residues 1177/78/82
of the insulin receptor are required for substrate phosphorylation but not
autophosphorylation. Diabetes 2000;49:889–95.
[72] Saad MJ, Araki E, Miralpeix M, Rothenberg PL, White MF, Kahn
CR. Regulation of insulin receptor substrate-1 in liver and muscle
of animal models of insulin resistance. J Clin Invest 1992;90:
1839–49.
[73] Kile BT, Schulman BA, Alexander WS, Nicola NA, Martin HM, Hilton DJ.
The SOCS box: a tale of destruction and degradation. Trends Biochem Sci
2002;27:235–41.
[74] Schlaepfer IR, Pulawa LK, Ferreira LD, James DE, Capell WH, Eckel
RH. Increased expression of the SNARE accessory protein Munc18c in
lipid-mediated insulin resistance. J Lipid Res 2003;44:1174–81.
[75] Schinner S, Scherbaum WA, Bornstein SR, Barthel A. Molecular mecha-
nisms of insulin resistance. Diabet Med 2005;22:674–82.
[76] Bell CG, Walley AJ, Froguel P. The genetics of human obesity. Nat Rev
Genet 2005;6:221–34.
[77] Korner J, Leibel RL. To eat or not to eat- how the gut talks to the brain. N
Engl J Med 2003;349:926–8.
[78] Nogueiras R, Tschop M. Biomedicine. Separation of conjoined hormones
yields appetite rivals. Science 2005;310:985–6.
[79] King PJ. The hypothalamus and obesity. Curr Drug Targets 2005;6: 225–40.
[80] Zhang JV, Ren PG, Avsian-Kretchmer O, et al. Obestatin, a peptide
encoded by the ghrelin gene, opposes ghrelin's effects on food intake.
Science 2005;310:996–9.
[81] Neel JV. Diabetes mellitus: a “thrifty” genotype rendered detrimental by
“progress”? Am J Hum Genet 1962;14:353–62.
[82] Clapham JC, Arch JR, Chapman H, et al. Mice overexpressing human
uncoupling protein-3 in skeletal muscle are hyperphagic and lean. Nature
2000;406:415–8.
[83] Perusse L, Rankinen T, Zuberi A, et al. The human obesity gene map: the
2004 update. Obes Res 2005;13:381–490.
[84] Pfeifer M. Morbid obesity. In: Omejc M, Repše S, editors. Workshop and
Symposium Gastric Surgery-Standards and Novelties Proceedings of the
symposium Klinični oddelek za abdominalno kirurgijo Kirurška klinika
Klinični center Ljubljana: Ljubljana; 2005. p. 11–21.
[85] Friedrich MJ. Epidemic of obesity expands its spread to developing
countries. Jama 2002;287:1382–6.
[86] Vohl MC, Sladek R, Robitaille J, et al. A survey of genes differentially
expressed in subcutaneous and visceral adipose tissue in men. Obes Res
2004;12:1217–22.
[87] Arner P. Differences in lipolysis between human subcutaneous and
omental adipose tissues. Ann Med 1995;27:435–8.
[88] Yki-Jarvinen H. Thiazolidinediones. N Engl J Med 2004;351:1106–18.
[89] International Diabetes Federation. The IDF consensus worldwide definition
of the metabolic syndrome; 2001.
[90] Reaven G. Syndrome X. In: DeGroot L, Jameson JL, editors. Endo-
crinology. Philadelphia: WB Saunders; 2001. p. 954–8.
[91] Abdu TA, Elhadd T, Pfeifer M, Clayton RN. Endothelial dysfunction in
endocrine disease. Trends Endocrinol Metab 2001;12:257–65.
34 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35
[92] Anderson EA, Hoffman RP, Balon TW, Sinkey CA, Mark AL. Hyper-
insulinemia produces both sympathetic neural activation and vasodilation
in normal humans. J Clin Invest 1991;87:2246–52.
[93] Doi SA, Towers PA, Scott CJ, Al-Shoumer KA. PCOS: an ovarian
disorder that leads to dysregulation in the hypothalamic-pituitary-adrenal
axis? Eur J Obstet Gynecol Reprod Biol 2005;118:4–16.
[94] Garg A. Acquired and inherited lipodystrophies. N Engl J Med 2004;350:
1220–34.
[95] Magre J, Delepine M, Khallouf E, et al. Identification of the gene altered
in Berardinelli-Seip congenital lipodystrophy on chromosome 11q13. Nat
Genet 2001;28:365–70.
[96] Burke B, Stewart CL. Life at the edge: the nuclear envelope and human
disease. Nat Rev Mol Cell Biol 2002;3:575–85.
[97] Chen D, Misra A, Garg A. Clinical review 153: Lipodystrophy in human
immunodeficiency virus-infected patients. J Clin Endocrinol Metab
2002;87: 4845–56.
[98] Bastard JP, Caron M, Vidal H, et al. Association between altered
expression of adipogenic factor SREBP1 in lipoatrophic adipose tissue
from HIV-1-infected patients and abnormal adipocyte differentiation and
insulin resistance. Lancet 2002;359:1026–31.
[99] Tomazic J, Silic A, Karner P, et al. Lipodystrophy and metabolic
abnormalities in Slovenian HIV-infected patients. Wien Klin Wochenschr
2004;116:755–9.
[100] Mathieson PW, Wurzner R, Oliveria DB, Lachmann PJ, Peters DK.
Complement-mediated adipocyte lysis by nephritic factor sera. J Exp
Med 1993;177:1827–31.
[101] Yaney GC, Corkey BE. Fatty acid metabolism and insulin secretion in
pancreatic beta cells. Diabetologia 2003;46:1297–312.
[102] Rich SS. Mapping genes in diabetes. Genetic epidemiological perspec-
tive. Diabetes 1990;39:1315–9.
[103] McCarthy MI. Progress in defining the molecular basis of type 2 diabetes
mellitus through susceptibility-gene identification. Hum Mol Genet
2004;13(Spec No 1):R33–41.
[104] Opara EC, Garfinkel M, Hubbard VS, Burch WM, Akwari OE. Effect of
fatty acids on insulin release: role of chain length and degree of
unsaturation. Am J Physiol 1994;266:E635–9.
[105] Paolisso G, Gambardella A, Amato L, et al. Opposite effects of short-and
long-term fatty acid infusion on insulin secretion in healthy subjects.
Diabetologia 1995;38:1295–9.
[106] Carpentier A, Mittelman SD, Lamarche B, Bergman RN, Giacca A,
Lewis GF. Acute enhancement of insulin secretion by FFA in humans is
lost with prolonged FFA elevation. Am J Physiol 1999;276:E1055–66.
[107] Roche E, Farfari S, Witters LA, et al. Long-term exposure of beta-INS
cells to high glucose concentrations increases anaplerosis, lipogenesis,
and lipogenic gene expression. Diabetes 1998;47:1086–94.
[108] Wrede CE, Dickson LM, Lingohr MK, Briaud I, Rhodes CJ. Fatty acid
and phorbol ester-mediated interference of mitogenic signaling via novel
protein kinase C isoforms in pancreatic beta-cells (INS-1). J Mol
Endocrinol 2003;30:271–86.
[109] Mohlke KL, Silander K, Peck EC, et al. Common non-coding SNPs near
the hepatocyte nuclear factor-4 alpha gene are associated with type 2
diabetes. Am J Hum Genet 2003;73:210 [abstract].
[110] Triggs-Raine BL, Kirkpatrick RD, Kelly SL, et al. HNF-1alpha G319S, a
transactivation-deficient mutant, is associated with altered dynamics of
diabetes onset in an Oji-Cree community. Proc Natl Acad Sci U S A
2002;99:4614–9.
[111] Weedon MN, Shields B, Knight B, et al. A common variant in the
glucokinase beta-cell gene promoter increases fasting glucose levels and is
associated with altered birth weight. Diabet Med 2003;20:A44 [abstract].
[112] Huxtable SJ, Saker PJ, Haddad L, et al. Analysis of parent–offspring trios
provides evidence for linkage and association between the insulin gene and
type 2 diabetes mediated exclusively through paternally transmitted class III
variable number tandem repeat alleles. Diabetes 2000;49: 126–30.
[113] Altshuler D, Hirschhorn JN, Klannemark M, et al. The common
PPARgamma Pro12Ala polymorphism is associated with decreased risk
of type 2 diabetes. Nat Genet 2000;26:76–80.
[114] Gloyn AL, Weedon MN, Owen KR, et al. Large-scale association studies
of variants in genes encoding the pancreatic beta-cell KATP channel
subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) confirm that the KCNJ11
E23K variant is associated with type 2 diabetes. Diabetes 2003;52:
568–72.
[115] Weedon MN, Schwarz PE, Horikawa Y, et al. Meta-analysis and a large
association study confirm a role for calpain-10 variation in type 2 diabetes
susceptibility. Am J Hum Genet 2003;73:1208–12.
[116] Hanis CL, Boerwinkle E, Chakraborty R, et al. A genome-wide search for
human non-insulin-dependent (type 2) diabetes genes reveals a major
susceptibility locus on chromosome 2. Nat Genet 1996;13:161–6.
[117] Guzick DS. Polycystic ovary syndrome. Obstet Gynecol 2004;103:
181–93.
[118] Norman RJ, Wu R, Stankiewicz MT. 4: Polycystic ovary syndrome. Med
J Aust 2004;180:132–7.
[119] Revised 2003 consensus on diagnostic criteria and long-term health risks
related to polycystic ovary syndrome. Fertil Steril 2004;81:19–25.
[120] Ehrmann DA, Kasza K, Azziz R, Legro RS, Ghazzi MN. Effects of race
and family history of type 2 diabetes on metabolic status of women with
polycystic ovary syndrome. J Clin Endocrinol Metab 2005;90:66–71.
[121] Legro RS, Driscoll D, Strauss III JF, Fox J, Dunaif A. Evidence for a
genetic basis for hyperandrogenemia in polycystic ovary syndrome. Proc
Natl Acad Sci U S A 1998;95:14956–60.
[122] Cussons AJ, Stuckey BG, Watts GF. Cardiovascular disease in the
polycystic ovary syndrome: new insights and perspectives. Atheroscle-
rosis 2006;185:227–39.
[123] Dahlgren E, Janson PO, Johansson S, Lapidus L, Oden A. Polycystic
ovary syndrome and risk for myocardial infarction. Evaluated from a risk
factor model based on a prospective population study of women. Acta
Obstet Gynecol Scand 1992;71:599–604.
[124] Hardiman P, Pillay OC, Atiomo W. Polycystic ovary syndrome and
endometrial carcinoma. Lancet 2003;361:1810–2.
[125] Lobo RA, Granger LR, Paul WL, Goebelsmann U, Mishell Jr DR.
Psychological stress and increases in urinary norepinephrine metabolites,
platelet serotonin, and adrenal androgens in women with polycystic ovary
syndrome. Am J Obstet Gynecol 1983;145:496–503.
[126] Ciaraldi TP, el-Roeiy A, Madar Z, Reichart D, Olefsky JM, Yen SS.
Cellular mechanisms of insulin resistance in polycystic ovarian
syndrome. J Clin Endocrinol Metab 1992;75:577–83.
[127] Dunaif A, Xia J, Book CB, Schenker E, Tang Z. Excessive insulin
receptor serine phosphorylation in cultured fibroblasts and in skeletal
muscle. A potential mechanism for insulin resistance in the polycystic
ovary syndrome. J Clin Invest 1995;96:801–10.
[128] Munir I, Yen HW, Geller DH, et al. Insulin augmentation of 17alpha-
hydroxylase activity is mediated by phosphatidyl inositol 3-kinase but not
extracellular signal-regulated kinase-1/2 in human ovarian theca cells.
Endocrinology 2004;145:175–83.
[129] Book CB, Dunaif A. Selective insulin resistance in the polycystic ovary
syndrome. J Clin Endocrinol Metab 1999;84:3110–6.
[130] Nestler JE, Jakubowicz DJ, de Vargas AF, Brik C, Quintero N, Medina F.
Insulin stimulates testosterone biosynthesis by human thecal cells from
women with polycystic ovary syndrome by activating its own receptor
and using inositolglycan mediators as the signal transduction system.
J Clin Endocrinol Metab 1998;83:2001–5.
[131] Nelson-Degrave VL, Wickenheisser JK, Hendricks KL, et al. lterations in
mitogen-activated protein kinase kinase and extracellular regulated kinase
signaling in theca cells contribute to excessive androgen production in
polycystic ovary syndrome. Mol Endocrinol 2005;19: 379–90.
[132] Zhang LH, Rodriguez H, Ohno S, Miller WL. Serine phosphorylation of
human P450c17 increases 17,20-lyase activity: implications for adre-
narche and the polycystic ovary syndrome. Proc Natl Acad Sci U S A
1995;92:10619–23.
[133] Unger JW, Livingston JN, Moss AM. Insulin receptors in the central
nervous system: localization, signalling mechanisms and functional
aspects. Prog Neurobiol 1991;36:343–62.
[134] Nestler JE. Role of hyperinsulinemia in the pathogenesis of the polycystic
ovary syndrome, and its clinical implications. Semin Reprod Endocrinol
1997;15:111–22.
[135] Ben-Shlomo I. The polycystic ovary syndrome: what does insulin
resistance have to do with it? Reprod Biomed Online 2003;6:36–42.
 B. Mlinar et al. / Clinica Chimica Acta 375 (2007) 20–35
[136] Vrbikova J, Cibula D, Dvorakova K, et al. Insulin sensitivity in women with
polycystic ovary syndrome. J Clin Endocrinol Metab 2004;89: 2942–5.
[137] Strauss III JF. Some new thoughts on the pathophysiology and genetics of
polycystic ovary syndrome. Ann N Y Acad Sci 2003;997:42–8.
[138] Escobar-Morreale HF, Luque-Ramirez M, San Millan JL. The molecular-
genetic basis of functional hyperandrogenism and the polycystic ovary
syndrome. Endocr Rev 2005;26:251–82.
[139] Diamanti-Kandarakis E, Alexandraki K, Bergiele A, Kandarakis H,
Mastorakos G, Aessopos A. Presence of metabolic risk factors in non-
35
obese PCOS sisters: evidence of heritability of insulin resistance. J Endocrinol
Invest 2004;27:931–6.
[140] Kahn R, Buse J, Ferrannini E, Stern M. The metabolic syndrome: time for
a critical appraisal. Joint statement from the American Diabetes
Association and the European Association for the Study of Diabetes.
Diabetologia 2005;48:1684–99.
[141] Pladevall M, Singal B, Williams LK, et al. A single factor underlies the
metabolic syndrome: a confirmatory factor analysis. Diabetes Care
2006;29:113–22.