Although the necessity of the PI3K/AKT pathway in insulin signal transduction is documented , it has not attained sufficient in vivo and in vitro evidence to identify the underlying mechanisms of the pathway and contradictory findings have been reported through knockout and RNAi studies –.
It is now commonly accepted that metabolic regulation relies on three types of control which involves; 1) Allosteric control of a key enzyme activity that triggers a metabolic pathway by binding to the activator, (mostly its substrate). 2) Posttranslational modifications such as phosphorylation, acetylation, glycosylation and proteolytic cleavage, which may affect the protein stability and/or equilibrium between active and inactive enzyme. In these kinds of control, subsequent changes in protein-protein interaction may participate in generating the active/non-active enzymatic complex. 3) Transcriptional regulation such as DNA methylation, which affects the gene expression level of key enzymes and is considered as a longer time regulation scale. Most metabolic regulations rely on a collaboration of these various mechanisms. As the insulin signalling starts at the cell membrane and subsequent events occur via phosphorylation cascades, which mainly happen through the PI3K/AKT pathway, it is probable that a part of the insulin function results from posttranslational modifications of numerous transcription factors , –.
Insulin resistance in Type II diabetes has been characterized by several defects in the insulin signalling cascade , –. These events are related to short-term post-translational regulation of specific protein functions and long-term transcriptional regulation of key genes of insulin signaling pathway . This hypothesis is supported by findings of altered expression of genes encoding metabolic enzymes in Type II diabetic patients .
In this study, there was no alteration in insulin signalling at the level of IRS1 and PTEN expression in diabetic participants despite the presence of reduced PI3K, AKT2 and GLUT4 expression levels and increased PDK1 expression. These findings were in agreement with previous studies ,  and suggesting that reduced expression levels of these genes may induce insulin resistance . A contradiction in the results obtained from different investigations , – indicates several possible mechanisms of transcriptional regulation of the PI3K/AKT pathway. For instance, in the present study, no significant difference in gene expression of IRS1 and PTEN in diabetic participants suggesting that defects in insulin signalling via IRS1 and PTEN are unlikely to be the primary cause. Another possibility is that these genes exert their main role in the PI3K/AKT pathway, which, beyond a very narrow range of their changes, the homeostasis of the pathway will disappear. Thus, the insulin signalling is very sensitive to the alteration of these components. Otherwise, it should be considered that insignificantly higher level of IRS1 in this study, might be due to the collaboration of various mechanisms including signal amplification as a compensatory mechanism and convergence of other signalling pathways. However, the role of negative feedback loops cannot be neglected, as control of insulin signalling can be achieved by autoregulation whereby downstream elements inhibit upstream components , . For example, AKT negatively regulates PTEN and prevents dephosphorylation of IRS1 by PTEN . Therefore, it can be concluded that higher gene expression level of IRS1 could be due to the higher amount of PTEN expression as well as lower AKT2 expression as a compensatory mechanism. Alternatively, signals from other pathways can inhibit insulin signalling. The IR and the IRS are targets for such feedback control mechanisms. Phosphorylation of IRS on Serine residues could be a key step in these feedback control processes –. Most of the Serine/Threonine kinases that are stimulated by insulin, are downstream effectors of IRS and serve as negative modulators of its action. The blockage of these kinases by the PI3K pathway inhibitors, indicates that these kinases are downstream of PI3K as potential IRS kinases . Also, insulin resistance inducers such as cellular stress, free fatty acids and tumor necrosis factor-α use similar mechanisms which activate some IRS kinases and inhibit their function by phosphorylation of Serine residues , . Serine phosphorylation is considered as a short-term inhibitory mechanism, while regulation of IRS expression might promote long-term insulin resistance. Also, it should be considered that as PTEN antagonizes PI3K, it may cause the activation of a feedback loop involving IRS1 by upregulating signalling through PI3K .
Insulin induces PI3K-mediated activation of PDK1 and produces PIP3 that regulates AKT activity and its plasma membrane translocation. Interaction between PDK1 and PKC may be required for insulin-induced phosphorylation of AKT . Moreover, PDK1 activates PKC which stimulates gluconeogenesis and contributes to insulin resistance through activation of pyruvate carboxylase . Though, this study did not examine the gene expression level of PKC, contribution of endogenous glucose production along with impaired PI3K/AKT pathway is proposed in insulin resistance as a result of higher gene expression level of PDK1in diabetics.
It has been revealed that insulin's signal being mediated by protein phosphatases such as PTEN and SHIP. Knockout and RNAi studies can induce diabetes by up-regulating PTEN. These phosphatases which have different biological functions in vivo, can induce insulin resistance through attenuating the PI3K/AKT pathway . Overexpression of PTEN decreases insulin-stimulated PI3K/AKT pathway, GLUT4 translocation and glucose uptake into the cells , . Microinjection of anti-PTEN antibody increases insulin-stimulated GLUT4 translocation to the cell membrane and glucose uptake . Therefore, PTEN reduces insulin sensitivity , as it is increased by inhibition of PTEN –. Although numerous phosphatases could be considered to be significant players in insulin signal transduction, only PTEN has been considered in this study. Changes in the abundance of PTPases and their collaboration or interaction may be involved in the pathogenesis of insulin resistance. Therefore, further ex vivo studies are required to assess the underlying mechanisms of PTEN function as well as other phosphatases and differentiate their roles, interaction and collaboration in antagonizing PI3K/AKT pathway and induction of diabetes. Understanding of mechanisms underlying the regulation of PTEN is important to identify its roles in diabetes. Regulation of PTEN is controlled at three steps; transcriptional regulation, post-translational mechanisms and membrane recruitment , , . Initially it was assumed that PTEN expression is constitutively until numerous transcription factors have been observed that binding directly to PTEN promoter and regulating its expression , , . Localization of PTEN plays an important role in the regulation of its activity in order to dephosphorylate PIP3 back to PIP2 at the cell membrane , , . Since, PTEN acts as the main antagonist of the PI3K/AKT signalling pathway by converting PIP3 into PIP2 , directly reversing the effects of PI3K and deactivating/dephosphorylating AKT through a decrease in PIP3 levels , . Reduced concentration of cellular PIP3 has been reported in Type II diabetic participants . Hence, PTEN inactivation leads to PIP3 accumulation and consequently to hyper-activation of AKT, which leads to decreased serum glucose level . Therefore, the intracellular concentration of PIP3 and PIP2 is regulated by the PI3K/PTEN equilibrium and dysregulation of PI3K/AKT pathway or no equilibrium between the PI3K and PTEN concentration might be implicated in Type II diabetes .
The findings of the present study showed reduced expression level of PI3K, AKT2 and GLUT4 in diabetic participants compared to non-diabetics, confirming previous studies , . Nevertheless, there was no significant difference in gene expression level of PTEN and IRS1 in diabetic participants that it was in consistent with the findings of some studies –. This may lead one to the hypothesis that localization of PTEN plays an important role in the regulation of its activity –. It means that the main role of PTEN in regulation of insulin function is performed by dephosphorylating the active form (insulin-stimulated) of insulin receptor and by modulating post-receptor signalling through antagonizing PI3K/AKT pathway –. These findings indicates that PTEN’s transmembrane function is probably more imperative than its intracellular function in insulin signal attenuation.
From another aspect it can be concluded that, although the PTEN level was higher in diabetic participants than in non-diabetics, the difference was not enough to be statistically significant while it was enough to affect GLUT4 expression. It means that insulin sensitivity is impaired by reduced expression of components that amplify the insulin signalling such as PI3K and AKT –. Even though presence of bistable response has not been proved in insulin signalling pathway and we are waiting for more verifications of this property, there are indications that this pathway includes the required components to exhibit bistable behaviour –. Bistability can be generated due to the non-linearity in positive feedback loops or double negative feedback loops . The non-linearity is due to the ultrasensitive response that is usually obtained through enzymatic cascades . Bistable systems display hysteresis, which means that the signalling system switches between two separate steady states without resting in a transitional state and the required amount of stimulatory input for transition from one state to another is completely different from that required for the reverse transition . The insulin signalling pathway includes multiple feedback loops , such as phosphorylated/activated AKT phosphorylates and negatively regulates PTEN. This phosphorylation impairs the function of PTEN to dephosphorylate IR and IRS1  and reveals a positive feedback loop. In other words, AKT inhibits PTEN as a signal attenuation, hence, it inhibits dephosphorylating of IR and IRS. This phenomenon consists of a double negative feedback loop (phosphorylated AKT negatively regulates PTEN that in turn dephosphorylates AKT). In except of this positive feedback loops considered in PI3K/AKT pathway, it is also known that many feedback loops have not been entirely characterized . Thus, this pathway has the potential to convert stimulatory inputs into bistable responses. Therefore, we cannot rule out the hypothesis that bistablity might exist in insulin-induced glucose absorption due to the ultrasensitivity of GLUT4 expression level in response to the PTEN expression at this study. Our findings indicate that the PI3K/AKT pathway losses bistability beyond a very narrow range of PTEN expression levels in addition to impaired insulin sensitivity by reduced expression of components that amplify the insulin signalling such as PI3K and AKT. These results are in accord with the literature on the existence of bistability in insulin signal transduction –. Consequently, PTEN/PI3K could be a phosphatase-kinase couple that controls the transition of the signalling molecule between two phosphorylation states.
Since, the regulation of insulin function is performed by the balance between phosphorylation and dephosphorylation of the PI3K/AKT pathway components and PTEN has been identified as a negative regulator of this pathway, it has generated great interest in new therapeutic approaches. Nevertheless, this study reveals that insulin resistance is caused through reduced PI3K/AKT2/GLUT4 signalling and not through alteration in PTEN expression. Furthermore, significant positive correlation between PTEN expression level and duration of diabetes indicates that PTEN expression increases by the years of having diabetes. As the time is a proposed new classification system for diabetes , PTEN may not be the cause of the reduced expression of PI3K/AKT pathway in diabetics while it can be the effect of that.
According to the several studies , vitamin D is required for normal insulin function. A number of studies ,  revealed that vitamin D level is positively correlated with insulin sensitivity and lower risk of impaired glucose tolerance and T2DM. The modulatory action of vitamin D in insulin receptor gene expression and insulin secretion may point to its role in the pathogenesis and development of T2DM . Vitamin D deficiency causes reduced insulin secretion in rats and humans, whereas its replenishment increases glucose tolerance through improvements in β-cell function . In addition, certain allelic variations in the vitamin D–binding protein (DBP) and vitamin D receptor (VDR) might affect glucose tolerance and insulin secretion ,  thus contributing to the occurrence of T2DM. Furthermore, vitamin D has been reported to contribute to normalization of extracellular calcium which determines the normal intracellular calcium pool. Increased intracellular calcium impairs phosphorylation of insulin receptors, leading to decreased GLUT4 activity and impaired insulin signal transduction , . Also, it has been documented that vitamin D deficiency and obesity in adult C57BL/6 mice entailed hyperinsulinemia and impaired expression level of the PI3K/AKT pathway components which caused impaired glucose homeostasis and insulin resistance . Furthermore, it has been demonstrated that vitamin D-induced activation of PI3K/AKT pathway is through PTEN down regulation and AKT up regulation . Insulin controls glucose and lipid metabolism through the PI3K/AKT pathway and PTEN is a negative regulator of this pathway, hence, down-regulation of PTEN enhances the metabolic effects of insulin ,  and reverses insulin resistance , .
Nevertheless, it remains to be elucidated whether alterations in insulin signalling gene expression in T2DM are influenced by the regulatory transcriptional properties of vitamin D. Since the active form of vitamin D, 1,25-dehydroxyvitamin D3, influences expression of various genes , , the relationship between serum vitamin D concentration and gene expression level of insulin signal transduction components were assessed.
In this study, there was no significant correlation between serum vitamin D concentration and gene expression level of GOIs in either group of participants. Data presented in current report is not in agreement with previous study on vitamin D-induced activation of PI3K/AKT pathway by down regulation of PTEN in mice . Also, in our previous in vitro study, vitamins D increased the expression level of IR, PI3K and GLUT4 and phosphorylation level of AKT which caused increased glucose uptake on insulin-resistant model of neuronal cells . The reason of contradiction between present study and our previous in vitro study  could be due to the non-linearity relationships, as the Pearson correlation test shows a linear correlation while insulin signal transduction is a cascade with amplifying properties –. Also, it should be considered that Pearson correlation test does not reveal the cause and effect relationships. Furthermore, in this study, only five diabetic participants were vitamin D deficient, thus, it was impossible to compare gene expression level of GOIs based on vitamin D status.