**5. Mechanisms of action involved in development of T2DM**

In addition to the classical pathway modulated by EDCs (as interaction with aryl hydrocar‐ bon receptor (AhR) or nuclear hormone receptors, in particular estrogens, androgens and thyroid receptors), it was observed that EDCs exhibit the capacity to modulate signalling pathways involved in energy regulation, in general, and glucose homeostasis in particu‐ lar. EDCs can decrease insulin sensitivity, impair β-cell insulin production, impair cellu‐ lar insulin action or alter the intermediary metabolism. All these mechanisms contribute to the pathogenesis of T2DM. Experimental data revealed that one EDC acts on different levels and receptors, therefore the ultimate effect on insulin action may be the result of all pathways involved (Table 2).


Legend: (+) activate; (-) inhibit; ↑ increase; ↓ decrease; ER-estrogenic receptors; ERK1/2 extracellular regulated kin‐ ase1/2; GLUT4 – glucose transporter type4; ER-GPR30-membrane G protein related estrogen-receptor; CREB-cAMP re‐ sponse element-binding protein; [Ca2+]i - intracellular calcium ion levels; IRS – insulin receptor substrate 1; PI3 phosphatidylinositol-4,5-bisphosphate 3-kinase; PPARγ - peroxisome proliferator-activated receptor gamma; TNF-α tumor necrosis factor-α; IL-6 interleukin-6; PDI - protein disulfide isomerase; IR – insulin receptor; AhR -aryl hydrocarbon receptor; MCP-1- monocyte chemoattractantprotein-1;PEPCK- phosphoenolpyruvate carboxykinase; MAPK1/2 - mitogen-activated protein kinase 1/2; CaMK2 - Ca2+/calmodulin-dependent kinase II

**Table 2.** Example of EDC that acts on multiple pathways

Interestingly, some EDCs such as TCDD [58], PCBs [59], inorganic arsenic [60] or cadmium [61] that modulate β-cell function, may also play a role in type 1 diabetes mellitus, as a result of β-cell destruction or dysfunction as well as promotion of β-cell death.

The following mechanisms of action can explain the development of T2DM: (1) activation of aryl hydrocarbon receptor (AhR) or interaction with estrogenic receptors (ERs); (2) β-cell dysfunction and impairment of insulin secretion; (3) impairment of cellular insulin action and (4) alteration of the intermediary metabolism.

#### **5.1. Activation of AhR or interaction with ERs**

EDCs can exhibit their metabolic effects through the classical pathways, such as the activation of AhR or the interaction with ERs. It is well known that AhRs are involved in the glucose homeostasis [50], therefore activation of AhR or its heterodimerization partner, called ARNT (AhR nuclear translocator) by EDCs could interfere with glucose uptake. Gunton et al. [62] revealed that abnormal ARNT expression causes impaired insulin release in human islets, but it is unclear if this effect is a cause or a consequence of T2DM.

PCBs [63] or PBDE [64] reduce primary hepatocyte glycogen levels and impair gluconeogen‐ esis due to a specific down regulation of phosphoenolpyruvate carboxykinase (PEPCK) expression, a central regulator of gluconeogenesis. The alteration of PEPCK expression was proportional to activation of the AhR, suggesting a direct correlation between AhR activation and perturbation in intermediary metabolism.

PCBs (especially PCB-77) also impaired glucose homeostasis through another AhR-depend‐ ent mechanism, associated with an adipose-specific increase in TNF-α (tumor necrosis factor-α) expression and interleukin-6 (IL-6) levels [65, 66]. In addition to its effects on TNFα and IL-6, PCB-77 also increases expression of MCP-1 (monocyte chemoattractant protein-1), an adipocyte-secreted molecule with inflammatory function that contributes to global insulin sensitivity [67].

The implication of estrogenic receptors (especially ERα) in the pancreatic β-cell insulin content is confirmed by BPA studies. At physiologically relevant doses, BPA increases pancreatic βcell insulin content, the effect being mediated by ERα activation via extracellular regulated kinase1/2 (ERK1/2) [53]. ERα is also implicated in β-cell survival [68], regulates the glucose transporter (GLUT4) in skeletal muscle [69] and insulin sensitivity in the liver [70]

Also the non-classical membrane G protein related estrogen-receptor (ER-GPR30), expressed in pancreatic islets is involved in the effects of estrogens on glucose metabolism, its deficiency inducing hyperglycemia, impaired glucose tolerance, and elevated blood pressure [71]. GPR30 activation by several EDCs, e.g. BPA could partly contribute to the increase of insulin after BPA exposure [72]

### **5.2. Beta-cell dysfunction and impairment of insulin secretion**

**Compound Mechanisms of action Primary effects BPA** (+)ERα via ERK1/2 ↑ β-cell insulin content

> ↑ Akt phosphorylation ↑ PI3-kinase activity

(-) PDI

**Table 2.** Example of EDC that acts on multiple pathways

(4) alteration of the intermediary metabolism.

**5.1. Activation of AhR or interaction with ERs**

it is unclear if this effect is a cause or a consequence of T2DM.

(+) MAPK1/2 (+) CaMK2

**PCBs**

**(especially PCB-77)**

226 Treatment of Type 2 Diabetes

Potent antagonist of PPARγ

MAPK1/2 - mitogen-activated protein kinase 1/2; CaMK2 - Ca2+/calmodulin-dependent kinase II

β-cell destruction or dysfunction as well as promotion of β-cell death.

↓ GLUT4

↓ IRS activity

↓ PEPCK

↑ [Ca]i

↓ IR phosphorylation

(-) IRS-1 phosphorylation

↑ TNF-α, IL-6; ↓ adiponectin release

(+) ER-GPR30 ↑ β-cell insulin content

(+) AhR ↑ TNF-α, IL-6; ↑ MCP-1

(-)Akt phosphorilation Insulin resistance ↑ oxidative stress ↑ β-cell death Legend: (+) activate; (-) inhibit; ↑ increase; ↓ decrease; ER-estrogenic receptors; ERK1/2 extracellular regulated kin‐ ase1/2; GLUT4 – glucose transporter type4; ER-GPR30-membrane G protein related estrogen-receptor; CREB-cAMP re‐ sponse element-binding protein; [Ca2+]i - intracellular calcium ion levels; IRS – insulin receptor substrate 1; PI3 phosphatidylinositol-4,5-bisphosphate 3-kinase; PPARγ - peroxisome proliferator-activated receptor gamma; TNF-α tumor necrosis factor-α; IL-6 interleukin-6; PDI - protein disulfide isomerase; IR – insulin receptor; AhR -aryl hydrocarbon receptor; MCP-1- monocyte chemoattractantprotein-1;PEPCK- phosphoenolpyruvate carboxykinase;

Interestingly, some EDCs such as TCDD [58], PCBs [59], inorganic arsenic [60] or cadmium [61] that modulate β-cell function, may also play a role in type 1 diabetes mellitus, as a result of

The following mechanisms of action can explain the development of T2DM: (1) activation of aryl hydrocarbon receptor (AhR) or interaction with estrogenic receptors (ERs); (2) β-cell dysfunction and impairment of insulin secretion; (3) impairment of cellular insulin action and

EDCs can exhibit their metabolic effects through the classical pathways, such as the activation of AhR or the interaction with ERs. It is well known that AhRs are involved in the glucose homeostasis [50], therefore activation of AhR or its heterodimerization partner, called ARNT (AhR nuclear translocator) by EDCs could interfere with glucose uptake. Gunton et al. [62] revealed that abnormal ARNT expression causes impaired insulin release in human islets, but

PCBs [63] or PBDE [64] reduce primary hepatocyte glycogen levels and impair gluconeogen‐ esis due to a specific down regulation of phosphoenolpyruvate carboxykinase (PEPCK) expression, a central regulator of gluconeogenesis. The alteration of PEPCK expression was

↑ CREB phosphorylation ↑ [Ca2+]i

Taking into account their reduced capacity to fight against chronic oxidative stress and the lack of detoxification mechanisms, β-cells are the perfect target for EDCs that disrupt their structure and function or promote death.

OxidativestressisthemechanismimplicatedinT2DMinducedbyexposuretoinorganicarsenic. At relatively low concentrations arsenic-induced oxidative stress produces impairment of glucose-stimulated insulin secretion [73], while exposure to high concentrations results in irreversible damage (including oxidative damage) to β-cells followed by apoptosis or ne‐ crosis [74]. Actually, the mechanism behind arsenic-induced oxidative stress is more com‐ plex. Chronic exposure to relative low concentration of arsenite (1–2µM)producedan adaptive response, activating the transcription factor NF-E2–related factor 2 (Nrf2). Even if Nrf2 is generally considered a protective cellular component that induces antioxidant / detoxifica‐ tion enzymes [73], in this case Nrf2 activation thatdiminishes the reactive oxygen species (ROS) have a negative impact on insulin secretion. In normal cells, ROS signals produced during glucose metabolism increase the insulin secretion [75], thefore arsenic Nrf2-mediated re‐ sponse appears to play an important role in reduced glucose-stimulated insulin secretion. Inorganic arsenic also promotes β-cell apoptosis via induction of endoplasmic reticulum stress, but this mechanism is poorly studied and necessitates further investigations [76].

Regarding the interference and impairment of insulin secretion, different examples can be provided, especially taking into account that insulin secretion is a calcium-dependent process. On isolated pancreatic β-cells BPA at low concentration (10-9 M) increases the phosphorylation of CREB (transcription factor cyclic adenosine monophosphate-response element-binding protein) via an alternative mechanism, involving a non-classical membrane estrogen receptor [77], which provokes the closure of K+ /ATP channels. As a result the plasma membrane depolarizes, opening the L-type voltage-dependent calcium channels and increasing intracel‐ lular calcium ion levels [Ca2+]i and triggering insulin secretion [78].

Also abnormal levels of [Ca2+]i and the impairment of insulin secretion were observed on isolated islet cells exposed to TBT and are associated with the disruption of protein-kinase A activity [79].

PCB treatment of RINm5F cells resulted in a rapid increase of [Ca2+]i as a result of Ca2+/ calmodulin-dependent kinase II (CaMK2) and mitogen-activated protein kinase 1 and 2 (MAPK 1 and 2) activation [80]. In addition, RINm5F cells exposed to inorganic arsenic (III) exhibited a reduction of insulin secretion as a result of decreased calcium-dependent calpain-10 activity, a pathway that triggers insulin exocytosis [81]. Arsenic also reduces the β-cell line proliferation in a dose-dependent manner, as an indirect consequence of the decrease in insulin secretion.

### **5.3. Impairment of cellular insulin action**

Taking into account that insulin signalling mechanisms are described in detail elsewhere [82], we present only a short analysis of the insulin signalling cascade in order to provide some insights into how EDCs might modulate insulin action.

Insulin acts on target cells and stimulates glucose uptake via membrane –bound tertrametric insulin receptor (IR) with tyrosine kinase activity. Binding to extracellular α-subunits of IR leads to activation of tyrosine kinase. Once the tyrosine kinase of IR is activated, it promotes autophosphorylation of the β subunit, where phosphorylation of three tyrosine residues (Tyr-1158, Tyr-1162, and Tyr-1163) is required for amplification of the kinase activity. Then tyrosine kinase phosphorylates the insulin receptor substrate proteins (IRS 1 and 2) and phosphotyrosine residues on IRS proteins become targets for the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase).

The activated PI3-kinase generates higher levels of phosphotidylinositides, such as phospha‐ tidyl-inositol-3,4-bisphosphate (PIP2) and phosphatidyl-inositol-3,4,5-trisphosphate (PIP3), which bind to the phosphoinositidedependent kinase-1 (PDK1). PDK1 can directly phosphor‐ ylate all protein kinase C (PKCs).

Downstream from PI3-kinase, activation of Akt (protein kinase B) produces its effects, including those on gene transcription as well as glucose uptake through the translocation of facilitative glucose transporter 4 (GLUT4) to the cell membrane.

Each step in this signaling cascade is a potential target for EDCs. EDCs interact and impair the cellular insulin effect acting at different levels: on IRS, PI3-kinase, Akt, PDK or PKC or through associated mechanisms. Some examples are included in figure 1.

of CREB (transcription factor cyclic adenosine monophosphate-response element-binding protein) via an alternative mechanism, involving a non-classical membrane estrogen receptor

depolarizes, opening the L-type voltage-dependent calcium channels and increasing intracel‐

Also abnormal levels of [Ca2+]i and the impairment of insulin secretion were observed on isolated islet cells exposed to TBT and are associated with the disruption of protein-kinase A

PCB treatment of RINm5F cells resulted in a rapid increase of [Ca2+]i as a result of Ca2+/ calmodulin-dependent kinase II (CaMK2) and mitogen-activated protein kinase 1 and 2 (MAPK 1 and 2) activation [80]. In addition, RINm5F cells exposed to inorganic arsenic (III) exhibited a reduction of insulin secretion as a result of decreased calcium-dependent calpain-10 activity, a pathway that triggers insulin exocytosis [81]. Arsenic also reduces the β-cell line proliferation in a dose-dependent manner, as an indirect consequence of the decrease in insulin

Taking into account that insulin signalling mechanisms are described in detail elsewhere [82], we present only a short analysis of the insulin signalling cascade in order to provide some

Insulin acts on target cells and stimulates glucose uptake via membrane –bound tertrametric insulin receptor (IR) with tyrosine kinase activity. Binding to extracellular α-subunits of IR leads to activation of tyrosine kinase. Once the tyrosine kinase of IR is activated, it promotes autophosphorylation of the β subunit, where phosphorylation of three tyrosine residues (Tyr-1158, Tyr-1162, and Tyr-1163) is required for amplification of the kinase activity. Then tyrosine kinase phosphorylates the insulin receptor substrate proteins (IRS 1 and 2) and phosphotyrosine residues on IRS proteins become targets for the p85 regulatory subunit of

The activated PI3-kinase generates higher levels of phosphotidylinositides, such as phospha‐ tidyl-inositol-3,4-bisphosphate (PIP2) and phosphatidyl-inositol-3,4,5-trisphosphate (PIP3), which bind to the phosphoinositidedependent kinase-1 (PDK1). PDK1 can directly phosphor‐

Downstream from PI3-kinase, activation of Akt (protein kinase B) produces its effects, including those on gene transcription as well as glucose uptake through the translocation of

Each step in this signaling cascade is a potential target for EDCs. EDCs interact and impair the cellular insulin effect acting at different levels: on IRS, PI3-kinase, Akt, PDK or PKC or through

lular calcium ion levels [Ca2+]i and triggering insulin secretion [78].

/ATP channels. As a result the plasma membrane

[77], which provokes the closure of K+

**5.3. Impairment of cellular insulin action**

phosphatidylinositol 3-kinase (PI3-kinase).

ylate all protein kinase C (PKCs).

insights into how EDCs might modulate insulin action.

facilitative glucose transporter 4 (GLUT4) to the cell membrane.

associated mechanisms. Some examples are included in figure 1.

activity [79].

228 Treatment of Type 2 Diabetes

secretion.

**Figure 1.** A schematic illustration of EDCs interference on insulin signaling pathways. Arrows represent an activation process; X represent an inhibition process

For example, TCDD, arsenic or PCB alter IRS activity (especially IRS-1 phosphorylation) through different mechanisms: TCDD increasing MAPK (mitogen-activated protein kinase) activity and JNK (c-Jun N-terminal kinase) activity [83], arsenic decreasing p70-S6-kinase activity [84] and PCBs increasing CaMK2 and MAPK 1 and 2 activity [80].

Other EDCs act on insulin-stimulated Akt phosphorylation. Akt phosphorylation is attenuated by PCB-77 [65] or BPA [55]. Arsenic (III) exposure was also associated with suppression of AkT phosphorylation and glucose uptake in 3T3-L1 adipocytes, causing an insulin resistant phenotype [85,86].

BPA acts not only on Akt phosphorylation, but also stimulates tyrosine phosphorylation via PI3-kinase, the global effect being the impairment of IRS activity [87].

Additional studies have demonstrated that TCDD [83], BPA [88] or DEHP [89] are modulating the insulin signalling cascade by down-regulation of the insulin receptors or acting on plasma membrane GLUT4 level and antagonizing insulin action [90].

Also, cadmium induces impaired glucose tolerance by down-regulating GLUT4 expression in adipocytes [91].

Inorganic arsenic (III) inhibits PDK-1 activity, thus suppressing PDK-1-catalyzed phosphory‐ lation of PKB/Akt and p-PKB/Akt–mediated translocation of GLUT4 transporters to the plasma membrane [85,92].

#### **5.4. Alteration of the intermediary metabolism**

In addition to direct effects on the insulin signalling cascade, EDCs alter the intermediary metabolism, mainly the gluconeogenesis. TCDD [63], or PCBs [93] have been shown to downregulate the expression of phosphoenolpyruvate carboxykinase (PEPCK), reducing its activity and inducing hypoglicemia. In the case of PCBs, the suppression of hepatic PEPCK expression was proportional to activation of the AhR, suggesting a direct correlation between AhR activation and perturbation of the intermediary metabolism.

Alternative mechanisms are implicated in the development of T2DM, such as inflammation or oxidative stress. For example, PCB-77 has been shown to promote expression of IL-6 and TNF-α, leading to impaired insulin signalling in endothelial cells [64]. In addition to its effects on TNF-α and IL-6, PCB-77 also increases expression of MCP-1, adipocyte-secreted molecule that contributes to global insulin sensitivity [66].

BPA augments secretion of IL-6 and TNF-α, but simultaneously inhibits the release of adiponectin in human adipose tissue explants [94]. The suppression of adiponectin release could promote insulin resistance and increase the risk of developing the metabolic syndrome. The same outcome is expected based on elevated IL-6 levels.

We should highlight the strong correlation between increased TNFα production and insulin resistance [95]. TNFα affects insulin resistance by downregulating the glucose transporter, interfering with IR phosphorylation and signaling, and by inhibiting transcription factors that affect insulin sensitivity.

Some EDCs are acting on other nuclear receptors involved in fat metabolism and regulation of glucose uptake, like PPARs (peroxisome proliferator-activated receptors), especially on PPARγ which are involved in the regulation of adipocyte differentiation, production of adipokines or insulin responsiveness [96]. By antagonizing PPARγ, EDCs significantly inhibit the release of adiponectin that has insulin-sensitizing effects, as it enhances inhibition of hepatic glucose output as well as glucose uptake and utilization in fat and muscle tissues. So, adiponectin levels are correlated with insulin sensitivity, therefore supressing its biological effects affects glucose homeostasis.

For example, BPA at 0.1 and 1 nM doses is a potent antagonist of PPARγ, which suppresses adiponectin release in human adipose tissue explants [97]. In the same time, BPA influences adiponectin level via another mechanism that implies binding to protein disulfide isomerase (PDI), a critical player in the retention of adiponectin in cells [98]

Interestingly, *in vitro* it was observed that ERβ can act as a negative regulator of PPARγ, decreasing ligand-induced PPARγ and PPARγ induced adipogenesis [99], therefore it is obvious that PPARγ function is affected by EDCs directly interacting with the receptor, but also by EDCs that modulate ERβ activity. Also, TCDD inhibits adipogenesis through a suppression of PPARγ [100].

Other EDCs such as phthalates (DEHP) act as potent agonists of PPARα or PPARγ. In rodent models, PPARα appears to mediate high-dose DEHP-induced body weight loss [101], but these effects can not be extrapolated to humans, taking into account that the levels required to activate human PPARα are almost three times higher than the concentrations required to activate mouse PPARα, and the maximum-fold induction is less for human PPARα than for mouse PPARα [102].

In conclusion, the investigation of insulin signaling pathways may explain how EDCs modulate insulin action, especially in the case of exposure to singular compound; however, in the context of accidental or occupational exposures, humans are exposed to mixtures of compounds and this complicates understanding the global biological effects. For example, if different compounds are acting through the same pathway, but at different points, co-exposure is likely to have additive or synergistic effects that promote the development of insulin resistance and T2DM. Moreover, points of pathway convergence (e.g., IRS) might be the perfect target of drug intervention to treat environmentally-mediated diabetes.
