**6. Evidence of T2DM in animal models after EDC exposure**

There are enough published data in animal models that investigated the correlation between EDCs exposure and T2DM. This correlation between exposure in the animal models and alterations in glucose homeostasis, including hyperglycemia and glucose intolerance is crucial, taking into account that epidemiological studies fail to establish a causality.

A number of examples are given below.

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

Also, cadmium induces impaired glucose tolerance by down-regulating GLUT4 expression in

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

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

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

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.

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

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

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

membrane GLUT4 level and antagonizing insulin action [90].

activation and perturbation of the intermediary metabolism.

The same outcome is expected based on elevated IL-6 levels.

**5.4. Alteration of the intermediary metabolism**

that contributes to global insulin sensitivity [66].

adipocytes [91].

230 Treatment of Type 2 Diabetes

plasma membrane [85,92].

affect insulin sensitivity.

effects affects glucose homeostasis.

Repeated low dose of TCDD (500 ng/kg bw), administered orally, reduced glucokinase gene expression, predicting a rise in blood glucose levels on C57BL/6 mice [103], while in diabetic rats, a higher dose of 12.8 µg/kg bw TCDD had significantly reduced serum glucose levels by day 8 of treatment [51]. Moreover, a single but high dose of TCDD (116 µg/kg bw i.p.) impaired insulin-stimulated glucose uptake in C57BL/6 and DBA/2J mice [52].

Administration of 75 µg/kg bw DEHP for 14 days reduced insulin levels and raised serum glucose levels in exposed female Wistar Kyoto rats [104]. Almost similar results were obtained on male rats treated for a longer period (21 days) with diet supplemented with 2% (w/w) of DEHP [105].

Male mice treated orally with 0.5-50 µg/kg bw TBT for 45 days demonstrated hepatic steatosis, hyperinsulinemia, hyperleptinemia and a reduction in hepatic adiponectin levels, in a dosedependent fashion, confirming PPARγ stimulation observed *in vitro* [106].

Similar results were obtained in adult Sprague-Dawley rats exposed for 28 days to crude salmon oil containing POPs [107]. The animals developed insulin resistance syndrome, abdominal obesity and hepatosteatosis, the contribution of POPs to insulin resistance being confirmed also by the same authors in cultured adipocytes. These findings are important since POPs are accumulating in the lipid fraction of fish, and fish consumption represents the main source of POP exposure to humans.

Also coplanar PCBs (e.g. PCB-77 and PCB-126), at dosage of 50 mg/kg orally, impaired glucose homeostasis in lean C57BL/6 mice and mitigate beneficial effects of weight loss on glucose homeostasis in obese mice [66], while inorganic arsenic (III) administered in the drinking water for 20 weeks, at doses of 25 or 50 ppm As/kg bw/day, impaired glucose tolerance in C57BL/6 mice in a dose-dependent manner [108].

In the animal studies mentioned before, not just blood glucose levels were investigated, but other markers of insulin regulation, such as HOMA-IR, pancreatic production of NO, SOD and CAT activity, in order to reflect the magnitude of the global disturbance. While most studies have demonstrated perturbations in insulin action, some of them have shown improved glucose tolerance or even hypoglycaemia. Acute exposure of adult male mice to high dosage of BPA (100 µg/kg bw/day) produced a rapid hyperinsulinemia based on significant increase in β-cell insulin content, as a direct result of BPA estrogenic properties [109], while sustained exposure to lower dosage (10 µg /kg bw/day) impaired glucose tolerance and reduced the hypoglycaemic effect of insulin, through a compensatory peripheral insulin resistance [54]. These results are easily correlated with non-monotonic dose response curve exhibited *in vitro* by BPA.

Taking into account that EDCs alter glucose homeostasis and endocrine pancreatic function not just in adult animals but also during pregnancy or in offspring, these effects were also investigated in pregnant animals. For example, prenatal exposure to high dosage of diisobutyl phthalate (600 mg/kg bw/day) from gestation Day 7 to Day 21 reduced plasma leptin and insulin levels in male and female offspring, complementary to sexual distrurbance [110]. Maternal glucose intolerance was observed in pregnant mice exposed to inorganic arsenic (V) at dosage of 9.6 mg/kg bw, this explaining the neural tube defects induced by arsenate [111].

In conclusion, all these examples regarding *in vivo* effects in animal models are highly suggestive, taking into account that the experimental exposure is very close or even similar with environmental human exposure. However, data on co-exposure are lacking, therefore new studies should focus on this issue, in order to reveal possible additive, synergistic or antagonistic effects exhibited by the mixtures.
