Chapter 5 Pro-Inflammatory Cytokines , Lipid Metabolism and Inflammation in Gestational Diabetes Mellitus as Cause of Insulin Resistance

Gestational diabetes mellitus (GDM) is a glucose intolerance of varying severity with onset or first recognition, during pregnancy that complicates 2–4% of pregnancies (Ben-Haroush et al 2004, American Diabetes Association 2005, NICE Guidelines 2008). Both patients with GDM, and their offspring, have greater risk of developing type 2 diabetes later in life (Damn 1998). There is a close relationship between GDM and prediabetes state in addition to the risk of future deterioration in insulin resistance and ultimate development of overt type 2 diabetes mellitus (Kjos et al 1999). Diabetes in pregnancy is increasing and therefore it is important to raise awareness of the associated health risks to the mother, the growing fetus, and the future child. Perinatal mortality and morbidity is increased in diabetic pregnancies through increased stillbirths and congenital malformation rates (Canadian Diabetes association 2003, HAPO 2008, RCOG SAC 2011). These are mainly the result of early fetal exposure to maternal hyperglycaemia. In the mother, pregnancy may lead to worsening or development of diabetic complications such as retinopathy, nephropathy, and hypoglycaemia (Ali and Dornhorst 2011). Although glycaemic control is important in reducing microvascular complications due to diabetes in pregnancy, it has not reduced the rate of congenital anomalies, macrosomia and other adverse outcomes (Canadian Diabetes Association 2003). This may be as a result of our lack of understanding of the epidemiology and pathogenesis of GDM (Omu et al. 2010), especially the role of inflammation, cytokines and lipid metabolism.


Introduction
Gestational diabetes mellitus (GDM) is a glucose intolerance of varying severity with onset or first recognition, during pregnancy that complicates 2-4% of pregnancies (Ben-Haroush et al 2004, American Diabetes Association 2005, NICE Guidelines 2008). Both patients with GDM, and their offspring, have greater risk of developing type 2 diabetes later in life (Damn 1998). There is a close relationship between GDM and prediabetes state in addition to the risk of future deterioration in insulin resistance and ultimate development of overt type 2 diabetes mellitus (Kjos et al 1999). Diabetes in pregnancy is increasing and therefore it is important to raise awareness of the associated health risks to the mother, the growing fetus, and the future child. Perinatal mortality and morbidity is increased in diabetic pregnancies through increased stillbirths and congenital malformation rates (Canadian Diabetes association 2003, HAPO 2008, RCOG SAC 2011). These are mainly the result of early fetal exposure to maternal hyperglycaemia. In the mother, pregnancy may lead to worsening or development of diabetic complications such as retinopathy, nephropathy, and hypoglycaemia (Ali and Dornhorst 2011). Although glycaemic control is important in reducing microvascular complications due to diabetes in pregnancy, it has not reduced the rate of congenital anomalies, macrosomia and other adverse outcomes (Canadian Diabetes Association 2003). This may be as a result of our lack of understanding of the epidemiology and pathogenesis of GDM (Omu et al. 2010), especially the role of inflammation, cytokines and lipid metabolism.
b. Elucidate the effect of lipid metabolism and lipid peroxidation, oxidative stress on antioxidant gene expression and other inflammatory cytokines.
c. Investigate the role of risk factors including obesity and adipokines like adiponectin, leptin and tumor necrosis factor alpha, acute phase proteins like C-reactive protein (C-RP), IL-6 and plasminogen activation inhibitor -1 (PAI-1) and proinflammatory Cytokines and the mechanisms involved in the pathogenesis.
d. Highlight the role of intervention strategy in the prevention of progression of GDM to type 2 Diabetes Mellitus and alteration of Maternal effects of GDM

Epidemiology of gestational diabetes mellitus
Gestational diabetes has been recognised as a heterogenous disorder of glucose intolerance (Kjos et  There is an urgent need to develop and unify appropriate diabetic diagnostic and prevention strategies and address potentially modifiable risk factors such as obesity.

Diagnostic criteria of gestational diabetes mellitus (GDM)
Historically, the diagnosis of gestational diabetes mellitus (GDM), like diabetes mellitus in general, has been by measuring the fasting plasma glucose level and performing an oral glucose tolerance test (OGGT) with the threshold as shown in Table 1. In 2010, the American Diabetic Association added haemoglobin A1c (Hb A1c) as a diagnostic tool for individual with type 2 diabetes mellitus with a threshold fixed at 6.5 % for diagnosis. It was however, not recommended for use in GDM. For diagnosis of GDM, 2 values in each diagnostic group must be met or exceeded (ADA 2011).

Gestational Diabetes -Causes, Diagnosis and Treatment
According to the NICE guidelines No 63 (2008), screening for GDM using fasting plasma glucose, random blood glucose, glucose challenge test and urinalysis for glucose, should not be recommended. Instead a 2 hour 75 g OGTT should be done at 24-28 weeks of gestation.

Non-genetic factors
Ethnicity, old age, family history, obesity and high fat diet and sedentary lifestyle, represent some important non-genetic identifiable predisposing factors for GDM, and in the absence of risk factors, there is low incidence of GDM. Ethnicity has been proven to be an independent risk factor for GDM (Dooley et al 1991, Weerakiet et al. 2004) which varies in prevalence in direct proportion to the prevalence of Type 2 diabetes in a given population or ethnic group. Women with an early diagnosis of GDM, in the first half of pregnancy, represent a high-risk subgroup, with an increased incidence of obstetric complications, recurrent GDM in subsequent pregnancies, and future development of

Genetics of GDM
GDM is considered to result from interaction between genetic and environmental risk factors. Women with mutations in MODY (Maturity onset diabetes of the young) genes often present with GDM. Genetic predisposition to GDM has been suggested given the occurrence of the disease within family members. GDM is reported to be often present in women with mutation of MODY gene mutations (Lapolla et al 1996, Ferber et al. 1999

Lipid and lipoproteins
Hyperlipidemia is a common comorbidity among patients with diabetes mellitus (

Effects of lipid peroxidation
The markers of lipid oxidation, especially malondialdehyde (MDA) increased with hyperglycaemia (Davi et al. 2005). Lipid peroxidation is a crucial process generated naturally in the body, mainly by the effect of several reactive oxygen species such as hydroxyl radical, hydrogen peroxide and superoxide. These reactive oxygen species readily attack the polyun-saturated fatty acids of the fatty acid membrane, initiating a self-propagating chain reaction (Hanachi et al. 2009). The destruction of membrane lipids and the end-products of such lipid peroxidation reactions cause cell damage. Enzymatic (catalase, superoxide dismutase) and nonenzymatic (vitamins A and E) natural antioxidant defense mechanisms exist; however, these mechanisms may be overcome, causing lipid peroxidation to take place. Lipid peroxidation has been implicated in disease states such as atherosclerosis, asthma, Parkinson's disease, kidney damage and preeclampsia (Mylonas and Kouretas 1999, Veskoukis et al 2012)

Effect of lipid metabolism on neonatal outcome
The 8-Isoprostane is a product of lipid peroxidation that can be used as a measure of free radical exposure or injury. Periventricular-intraventricular hemorrhage, necrotizing enterocolitis, chronic lung disease and retinopathy of prematurity have been referred to as oxygen radical diseases (ORD) because they are thought to be related to excess oxidant stress relative to antioxidant defenses in premature infants (Weinberger et al 2006). Umbilical cord venous, but not arterial, 8-isoprostane levels are associated with mortality and the development of one or more of the ORD. In a study, serum triglyceride, total cholesterol, and LDL-c concentrations were higher in the SGA neonates than in AGA neonates, whereas high-density lipoprotein cholesterol concentrations were similar, suggesting a limited ability to clear intravenous lipids in SGA infants (Arends et al 2005). These findings are in agreement with many previous studies in adults and children that show that low birth weight was significantly associated with a less favorable lipid profile (Mortaz et al 2001). Hyperinsulinemia is known to enhance hepatic verylow-density lipoprotein synthesis, which may contribute to increased plasma triglycerides and LDL-c levels. Resistance to the action of insulin on lipoprotein lipase in peripheral tissues may also contribute to elevated triglyceride and LDL-c levels. (Barker et al 1993).

Adipose tissue as an endocrine organ
The discovery of leptin in the mid-1990s has focused attention on the role of proteins secreted by adipose tissue (Wang et al 2004). Leptin has profound effects on appetite and energy balance, and is also involved in the regulation of neuroendocrine and immune function.

Immunology of gestational diabetes
Pregnancy represents a distinct immunologic state in the fetus that acts as an allograft to the mother, needing protection against potential rejection. There is some evidence that inflammation and activated innate immunity is associated with the pathogenesis of Type 2 Diabetes (Pickup 2004), but this needs comfirmation, especially in GDM.

Humoral reactivity in pregnancy
The placental HLA-G proteins facilitate semiallogeneic pregnancy by inhibiting maternal immune responses to foreign (paternal) antigens via their actions on immune cells is now well established, and the postulate that the recombinant counterparts of these proteins may be used as powerful tools for accommodating the fetus and prevent immune rejection (Hunt et. al 2005). Humoral immune-reactivity does not change much during pregnancy, with the exception of lowered immunoglobulin G concentration at late phase, probably explained by placental transport. Regarding cellular immunity, the reduction, elevation, and lack of variation in the number of different lymphocytic populations, have been reported (Mahmoud et al 2005(Mahmoud et al , 2006).

Cellular immune response and gestational diabetes
There is a significant increase in the absolute number of total and activated (CD3+HLA-DR+) T lymphocytes and a significant increase in the absolute number and percentage of suppressor/ cytotoxic T lymphocytes (CD8) and NK lymphocytes (CD57) in GDM patients compared with normal pregnant controls (Mahmoud et al.2006). Concerning frequency for HLA A, B, C, DR antigens in the GDM population, only Cw 7 was found to be significantly increased and A 10 significantly decreased in comparison with controls (Lapolla et al 1996). When compared with healthy pregnant women, both GDM cohorts showed higher percentages CD4+CD25+ (P < 0.05), CD4+CD45RO+ (P < 0.05) and CD4+CD29+ (Mahmoud et al 2005).

The placenta is a target of cytokines: maternal and fetal influences
There is a robust cytokine network in the placenta with diverse pathogenesis and effects on the development of the fetus.

The role of adipokines and other inflammatory markers
Research has recently focused on a group of substances produced mainly by adipose tissue called adipokines, this group includes, among others, adiponectin, leptin, Retinol- . Increased levels of inflammatory agents during and after pregnancy have been reported in patients with GDM, while increased body fat has been strongly associated with inflammation and adipocyte necrosis, hypoxia, and release of chemokines which cause macrophages to infiltrate adipose tissue. Macrophages secrete cytokines which activate the subsequent secretion of inflammation mediating agents, specifically interleukin-6 (IL-6) and Creactive protein (CRP) (Festa et al 2000). Other molecules such as Plasminogen Activator Inhibitor 1 (PAI-1) and sialic acid lead to dysregulations of metabolism, hyperglycemia, insulin resistance, and diabetes. These are a group of substances that are produced mainly in the adipose tissue. The group includes leptin, adiponectin, tumor necrosis factor alpha (TNF-α), retinol-binding protein-4 (RBP-4), resistin, visfatin, and apelin. These molecules are involved in a wide range of physiological processes including lipid metabolism, atherosclerosis, blood pressure regulation, insulin sensitivity, and angiogenesis, while they also influence immunity and inflammation. Their levels in pathologic states appear increased, with the exception of adiponectin which shows decreased levels (Vrachnis et al 2012).

Adiponectin
Since

Leptin
Leptin is a 16-kDa protein hormone that is known to play a key role in the regulation of energy intake and energy expenditure and in a number of physiological processes including regulation of endocrine function, inflammation, immune response, reproduction and angiogenesis.

Association between leptin and TNF-alpha and insulin resistance
Placental leptin mRNA production is upregulated by tumour necrosis factor (TNF) α and interleukin (IL)-6. Most studies have found increased leptin concentrations in GDM. Moreover, hyperleptinaemia in early pregnancy appears to be predictive of an increased risk to develop GDM later in pregnancy independent of maternal adiposity The discovery that some of these adipokines as key players in the regulation of insulin action suggests possible novel interactions between the placenta and adipose tissue in understanding pregnancy-induced insulin resistance, which is evident in gestational diabetes mellitus (GDM) (Winzer et al 2004).

Inflammatory mediators in diabetes mellitus
Gestational diabetes mellitus is characterized by an amplification of the low-grade inflammation already existing in normal pregnancy (Retnakaran et al 2010). This hypothesis is supported by increased circulating concentrations of inflammatory molecules like TNFα and IL-6 in GDM pregnancies. TNFα is one of the candidate molecules responsible for causing insulin resistance. Comparison of the placental gene expression profile between normal and diabetic pregnancies indicates that increased leptin synthesis in GDM is associated with a higher production of proinflammatory cytokines, e.g. IL-6 and TNFα causing a chronic inflammatory environment that enhances leptin production (

Link between inflammation and insulin resistance
In 1876 Ebstein asserted that sodium salicylate could make the symptoms of diabetes mellitus totally disappear. Similarly, in 1901 Williamson found that "sodium salicylate had a definite influence in greatly diminishing the sugar excretion" (

Mechanisms of insulin resistance by pro-inflammatory cytokines
The JNK (also referred to as SAPK) and p38 MAPKs are members of the complex superfamily of MAP serine/threonine protein kinases. This superfamily also includes the ERKs (Lewis et al 1998). In contrast to ERKs (also referred to as MAPKs), which are typically activated by mitogens, JNK/SAPK and p38 MAPK are known as stress-activated kinases. This can be attributed to the fact that the activities of these enzymes are stimulated by a variety of exogenous and endogenous stress-inducing stimuli including hyperglycemia, ROS, oxidative stress, osmotic stress, proinflammatory cytokines, heat shock, and UV irradiation (Tibbles et al 1999). Many of the more typical proinflammatory stimuli simultaneously activate JNK and IKKβ pathways, including cytokines and TLRs (Seger and Krebs 1995). Concordantly, genetic or chemical inhibition of either JNK or IKKβ/NF-κB can improve insulin resistance. The several mechanisms have been postulated to explain how obesity activates JNK and NF-κB. These can be separated into receptor (Lowes et al 2002) and nonreceptor pathways (Tamura et al 2002). Proinflammatory cytokines such as TNF-α and IL-1β activate JNK and IKKβ/NF-κB through classical receptor-mediated mechanisms that have been well characterized (Shen et al 2001, Tournier et al 2001. JNK and IKKβ/NF-κB are also activated by pattern recognition receptors, defined as surface proteins that recognize foreign substances. These include the Toll-like receptors (TLRs) and the receptor for advanced glycation end products (RAGE). Many TLR ligands are microbial products, including LPS and lipopeptides (Tamura et al 2002).

Transcription versus phosphorylation in the pathogenesis of insulin resistance
JNK is a stress kinase that normally phosphorylates the c-Jun component of the AP-1 transcription factor, but to date there are no known links between this well-established transcriptional pathway and JNK-induced insulin resistance. JNK has been shown to promote insulin resistance through the phosphorylation of serine residues (Shen et al 2001, Tournier et al 2001. Insulin receptor signaling that normally occurs through a tyrosine kinase cascade is inhibited by counterregulatory serine/threonine phosphorylations.

IKK β signaling pathway and insulin resistance
Unlike JNK, IKKβ does not phosphorylate IRS-1 to cause insulin resistance but causes insulin resistance through transcriptional activation of NF-κB. Increased lipid deposition in adipocytes leads to the production of proinflammatory cytokines, including TNF-α, IL-6, IL-1β, and resistin, which further activate JNK and NF-κB pathways through a feed-forward mechanism (Hou et al 2008). In addition to the cytokines, there is upregulated expression of transcriptions factors, receptors, and other relevant proteins including chemokines that recruit monocytes and stimulate their differentiation into macrophages.
Cytokines and chemokines produced locally include MCP-1 and macrophage inflammatory protein-1α (MIP-1α), MIP-1β, MIP-2, and MIP-3α. T cell activation leads to expression of IFNγ and lymphotoxin; macrophages, endothelial cells, and SMCs produce TNF-α; and together these stimulate the local production of IL-6 in the atheroma

Oxidative stress and activation of JNK and NF-κB pathways
In addition to proinflammatory cytokine and pattern recognition receptors, cellular stresses activate JNK and NF-κB, including ROS and ER stress. Elevated glucose cause oxidative stress through ( Muoio et al. 2008). Systemic markers of oxidative stress increase with adiposity, consistent with a role for ROS in the development of obesity-induced insulin resistance, (Ozdemir et al 2005). One potential mechanism is through the activation of NADPH oxidase by lipid accumulation in the adipo-cyte, which increases ROS production. This mechanism was shown to increase the production of TNF-α, IL-6, and MCP-1, and decrease the production of adiponectin (Barbour et al 2007). Consistent with this, the antioxidant N-acetyl cysteine can reduce ROS and improve insulin resistance in a hyperglycemia-induced model (Pieper et al 1997, Ozkilic et al 2006. Lipid accumulation also activates the unfolded protein response to increase ER stress in fat and liver. ER stress has been shown to activate JNK and subsequently, lead to serine phosphorylation of insulin receptor substrate-1 (IRS-1), but as with all of the stimuli, ER stress similarly activates NF-κβ (Evans et al 2002)

Health implications of cytokines, lipid metabolism, inflammation and GDM
The triad of cytokines, lipid metabolism and inflammation are hooked together by a biological thread of oxidative stress and pathogenetic end-point of insulin resistance.
Oxidative stress inhibits expression of Pax 3, a gene that is essential for neural tube closure, and possibly congenital cardiac anomalies which have been associated with uncontrollable diabetes in pregnancy before and in early pregnancy (Chang et al 2003). The association between GDM and macrosomia is real, with a secondary effect of increased cesarean section and increased risk of postpartum genital infection and development of overt type 2 diabetes mellitus. The markers of inflammation, dyslipidemia, oxidative stress and endothelial dysfunction may provide additional information about a patient's risk of developing cardiovascular disease and hypertension. This may provide new attractive targets for drug development.

Intervention strategies
GDM offers an important opportunity for the development for testing and the implementation of clinical strategies in diabetic prevention (Volpe et al 2007). The main objective should be to improve insulin sensitivity and prevent diabetes mellitus.

Lifestyle modification
Lifestyle modifications have been shown to be successful in decreasing the progression to T2DM in several populations, including American, Finnish and Asian, so it seems rational to consider similar interventions in women with a history of GDM (An Empowered Based Diabetic Prevention 2011). The ACOG (2003), RCOG Guidelines (2011), NICE Guidelines (2009) and the ADA (2005) all recommend that women at increased risk for T2DM should be counseled about the benefits of diet, exercise, and weight reduction and/or maintenance in an effort to prevent the development of T2DM as part of preconception care.

Breast-feeding
Breast-feeding is associated with reduced blood glucose levels and a reduced incidence of T2DM among both women with a history of GDM and women in the general population. Lactation has also been associated with postpartum weight loss, reduced long-term obesity risk, and a lower prevalence of the metabolic syndrome (O'Reilly et al 2003).

Pharmacologic interventions
Insulin is the drug to use, especially in GDM. Drugs with anti-inflammatory and vascular effects have future potential of being used in interventions aimed at reducing the enormous cardiovascular burden associated with Type 2 diabetes (Ziegler 2005). Use of sodium salicylate (Aspirin) (Hostamistigil et al 1993, Karasik et al 1993 has the concerns with high dose and possible side-effect of peptic ulceration. The use of antioxidant E and C reduces embryopathy in animal model (Cederberg ) and in human with use of N-acetyl Cysteine has beneficial effects (Ozkilic et al 2006).

Concluding remarks
Recent advances in the understanding of carbohydrate metabolism during pregnancy, suggest that preventive measures should be aimed at improving insulin sensitivity in women with strong risk factors of developing GDM. The mechanisms involved in the pathogenesis of insulin resistance and Gestational Diabetes Mellitus are summarized in Figure 1. Further research is needed to elucidate the mechanisms and consequences of alterations in lipid metabolism during pregnancy (Marseille-Tremblay et al 2008). Inflammation-induced insulin resistance is certainly increasing in parallel with the epidemic of obesity. Strategies for reducing this trend should be part of the Public Health initiatives.

Future directions
There is need for genetic studies especially from the Human Genome to identify those with candidate genes for diabetes and epigenetic factors that may affect gene expression and predisposition to inflammation. It should be possible to directly target inflammation with pharmacological interventions to treat and/or prevent insulin resistance and T2D and modulate risk for CVD and other metabolic conditions. In addition to anti-inflammatory drugs such as NF-κβ inhibitors and IL-1 receptor antagonists already known to improve inflammatory and glycemic parameters, should have utility to block the prolonged exposure to inflammatory danger signals may further enhance the metabolic and cardiovascular outcome of obese patients. Early recognition and management of women predisposed to develop T2DM is crucial in the development of primary health care strategies, modification of lifestyle, and dietary habits significantly to prevent or delay of insulin resistance and development of glucose intolerance.