1. The role of PTP1B in mediating insulin resistance caused by reduced Zip7 activity.
Ingrid Wise
School of Applied and Biomedical Sciences
Honours supervisor: Assoc. Prof. Mark Myers

2. The prevalence of type 2 diabetes in Australia has risen dramatically in the past decade, from 2.4% in 1995 to 7.5% in 2010, and is correlated with shifts in lifestyle habits, leading to obesity.
Understanding the pathogenesis of type 2 diabetes is complicated by several factors. Patients typically present with a combination of varying degrees of insulin resistance and relative insulin deficiency, and each of the clinical features can arise through genetic or environmental influences, making it difficult to determine the exact cause in an individual patient.
Insulin resistance is a physiological condition in which cells fail to respond to the normal actions of the insulin hormone. Despite some production, the cells in the body become resistant to insulin and are unable to use it as effectively, leading to hyperglycemia, or an increase in blood glucose. All of these factors combine to cause type 2 diabetes.

3. Insulin Resistance in T2DM
Insulin resistance occurs when there is a defect in insulin signalling.
PTP1B overactivity is evident in insulin resistant tissue.
Insulin activates and causes a conformational change of the insulin receptor. This triggers numerous signalling events which finally result in end products such as regulation of glycogen synthesis, fatty acid synthesis, protein synthesis and mitogenesis.
Insulin resistance occurs when there is a defect in the insulin signalling pathway****. This causes reduced insulin receptor activation, and reduced translocation of Glut4 (which is the main glucose transporter) and as a consequence, reduced glucose uptake into the cell.
Protein tyrosine phosphatase 1B **** is a primary negative regulator of insulin signalling. It acts by dephosphorylating the insulin receptor, ceasing the insulin signal.
Autopsy analysis of individuals with diabetes revealed that there was increased activity of PTP1B in insulin resistant tissue.

4. Regulation of PTP1B by zinc
Zinc transporter Zip7 releases intracellular zinc.
Free intracellular zinc inhibits PTP1B activity.
It is understood that the efficacy of the insulin signalling pathway is dependent on the suppression of PTP1B activity****.
Zinc is a known inhibitor of PTP1B****. Consequentially, increasing intracellular concentrations of zinc increase insulin signalling efficacy.
There are 14 zip zinc transporters which are responsible for increasing cytosolic concentrations of zinc. Of these Zip7*** was selected for manipulation, due to its location on the endoplasmic reticulum and the golgi apparatus. This positioning puts Zip7 directly responsible for the control of intracellular zinc flux.

5. Hypothesis
Reduced expression of Zip7 reduces cytosolic free zinc, which in turn decreases PTP1B inhibition.
Therefore, our hypothesis was…..

6. Is normal insulin signalling restored? Or is there no effect?
Research questions
siRNA treatment to silence Zip7 mRNA – confirm decreased insulin signalling.
siRNA treatment to silence PTP1B – confirm increased insulin signalling.
siRNA treatment to simultaneously silence Zip7 & PTP1B
Is normal insulin signalling restored? Or is there no effect?
The current research project aims to clarify the role of PTP1B in mediating insulin resistance due to reduced Zip7 activity.
Firstly this will be done by silencing the mRNA expression of Zip7 to confirm decreased insulin signalling.
Secondly, the mRNA expression of PTP1B will be silenced to confirm increased insulin signalling.
Lastly, a simultaneous silence of both PTP1B and Zip7 will be conducted, which has various outcome possibilities. Normal insulin signalling may be restored, similar to PTP1B silencing alone. This result would be consistent with Zip7 induced insulin resistance being mediated through PTP1B activity. A second outcome may be that the simultaneous silencing will have no effect, similar to siRNA-Zip7 alone. This would be consistent with Zip7 mediated insulin resistance operating through a mechanism other than PTP1B inhibition.

7. siRNA silencing of Zip7 & PTP1B
Zip7 expression
So onto some results:
We used short interfering RNA to silence the mRNA expression of Zip & PTP1B which binds to and deactivates target gene expression.
As you can see we achieved a successful reduction of Zip7 & PTP1B expression**** x4 in both the individual and simultaneously silenced groups.
PTP1B expression

8. Silencing of Zip7 & PTP1B reduces pAkt
siRNA treatment resulted in a reduction of pAkt.
pAkt is reduced further when Zip7 & PTP1B are silenced simultaneously.
Activation of Akt mediates the metabolic effects of insulin, for example the modulation of glucose uptake and glycogen synthesis. The successful silencing treatment resulted in reduced phosphorylation (or activation) of Akt. Furthermore, the simultaneous silencing had an additive effect on the reduction of pAkt.
The reduction of pAkt in the Zip7 silenced group is expected. However it is possible that the unexpected reduction seen in the PTP1B silenced group is a result of cellular compensation. The reduced PTP1B allowed too much Akt activation, so the cell somehow reduced this anther way.
Total Akt
pAkt/total Akt

9. Silencing of PTP1B reduces IR & IRS-1 mRNA expression
IR expression
siRNA treatment resulted in reduced IR & IRS-1 expression.
Possibly caused by cellular compensation to excessive increases in pAkt following PTP1B silencing.
The insulin receptor and the insulin receptor substrate are responsible for the activation of downstream signalling molecules that result in the activation of Akt
In this study, the mRNA expression of the IR and IRS1 was reduced in the cell groups which had PTP1B silenced. It is possible that this is also due to cellular compensation.
The silencing of PTP1B may have reduced the expression of the IR & IRS1 to reduce the subsequent activation of Akt.
IRS-1 expression

10. Silencing Zip7 & PTP1B reduces of Glut4 & Gbe-1 expression
Glut4 expression
siRNA treatment resulted in a reduction of GLUT4 & Gbe-1 expression.
Suggestive of reduction in glucose metabolism and glycogen synthesis.
Additionally the silencing treatment resulted in reduced expression of Glut4 (which you remember is the main glucose transporter) and Gbe-1 (which assists with the formation of glycogen molecules). This is suggestive of a loss of insulin mediated glucose uptake and glycogen storage, and is expected to be a consequence of the reduction in mRNA of the IR and IRS-1.
Gbe-1 expression

11. Silencing Zip7 & PTP1B increases MT-1 & MT-2 expression
siRNA treatment resulted in increased MT-1 & MT-2 expression.
Suggestive of a alternative pathway involvement or a possible increase in oxidative stress.
In exploring alternative explanations for the reduced Akt phosphorylation in cells with silenced PTP1B, MT-1 and MT-2 were examined.
MTs mainly act in homeostasis and detoxification of toxic metals, but also have a role in scavenging of free radicals (Vergani et al., 2005).
This study revealed that the MT expression was increased when Zip7 and PTP1B expression was reduced. The greatest change in MT expression was observed in the siRNA-PTP1B and simultaneously silenced group, indicating that PTP1B may affect pathways other than those involved with glucose metabolism. Such as those involved oxidative stress.
MT-2 expression

12. Silencing PTP1B reduces UCP2 mRNA expression
siRNA treatment resulted in decreased UCP2 expression.
Suggestive of a alternative pathway involvement or a possible increase in oxidative stress.
Findings from whole mouse PTP1B -/- knockout studies suggest that the total energy dissipation in the knockout model was increased. Adaptive thermogenesis is often mediated by enhanced uncoupling of electron transport from oxidative phosphorylation (Ricquier et al., 1985).
Uncoupling can be represented by an increase in expression of UCPs in skeletal muscle and brown fat (Boss, Hagen & Lowell, 2000). However in this study we observed a decrease in UCP2 expression in PTP1B silenced cells.
This decrease in UCPs may also be due to cellular compensatory mechanisms. The decrease in insulin signalling and glucose uptake, represented by reduced pAkt and Glut4 expression respectively, may require an increase in metabolic efficiency within the cell, in which case a decrease in UCP2 expression is expected.
UCP2 expression

13. Conclusions
Reduction of PTP1B resulted in alterations in numerous components involved in the insulin signalling pathway.
Simultaneous silencing of Zip7 & PTP1B had an additive effect. Insulin signalling was not restored.
Silenced mRNA expression of Zip7 confirmed decreased insulin signalling, demonstrated by the reduction in pAkt.
However, the silencing of PTP1B mRNA did not increase insulin signalling.
Furthermore, the simultaneous silencing did not restored normal insulin signalling, nor did it have zero effect. It had an additive effect.
The Zip7 mediated insulin resistance was not reversed when PTP1B was silenced. Instead, the decreased in PTP1B caused a decreased in various key mechanisms responsible for the activation of Akt. This in turn reduced the expression of Glut4 and Gbe1, decreasing glucose uptake and glycogen synthesis respectively.
Lastly, the decrease in glucose uptake resulted in reduced expression of UCP2 indicating that the cell may have adapted, thereby enhancing the efficiency of glucose utilisation.

14. References
Haase, H. & Maret, W. (2003). Intracellular zinc fluctuations modulate protein tyrosine phosphatase activity in insulin/ insulin-like growth factor- signaling. Experimental Cell Research, 291(2),
Haase, H. & Maret, W. (2005). Fluctuations of cellular, available zinc modulate insulin signaling via inhibition of protein tyrosine phosphatases. Journal of Trace Elements in Medicine and Biology, 19(1),
Myer, S., Nield, A. & Myers, M. (2012). Zinc Transporters, Mechanisms of Action and Therapeutic Utility: Implications for Type 2 Diabetes Mellitus. Journal of Nutrition and Metabolism, 2012, 1-13.
Myers, S. A., Nield, A., Chew, G. & Myers, M. (2013). The zinc transporter, Slc39a7 (Zip7) is implicated in glycaemic control in skeletal muscle cells. PLoS ONE, 8(11), 1-15.

15. ANY QUESTIONS?  8.0 FUTURE RESEARCH 8.1 Live cell zinc imaging
Our findings of impaired insulin signalling in skeletal muscle following siRNA-Zip7 treatment is consistent with current literature (Myers et al., 2013). However the reduced phosphorylation of Akt in both the siRNA-PTP1B and the simultaneously silenced group require further investigation. Zinc has a critical role in numerous cellular processes, however low nanomolar concentrations of free zinc can be cytotoxic (Pizzorno, 2014). Zinc homeostasis is a delicate balance that is not well understood. Measurement of free zinc concentration possibly using Flurozin3, a fluorescent zinc probe, to determine the fluctuations in intracellular free zinc may help determine relationship between the expressions of crucial insulin signalling elements and modulation of the activity of PTP1B.
8.2 Glycogen synthesis
A key feature of T2DM is impairment of insulin stimulated glycogen synthesis in skeletal muscle. A glycogen synthesis assay will help determine if lowered Gbe-1 mRNA expression translates to a functional difference.
8.3 Glucose assay
Glucose is a primary source of energy and a critical component in the production of proteins and in lipid metabolism. Decreased UCP2 mRNA in siRNA-PTP1B treated cells is suggestive of a requirement for increased efficacy in the utilisation of glucose. A decrease in the cellular concentration of glucose would support this hypothesis.
8.4 ROS analysis
It is possible that there is involvement of reactive oxygen species (ROS), which requires further analysis via ROS detection. Accumulation of ROS, which are highly reactive molecules which, in excessive amounts, can lead to protein and DNA oxidation, protein cross linking and cell death, are highly cell type specific (Uy, McGlashan & Shaikh, 2011). Further investigation is required to validate the involvement of ROS in the C2C12 cell line when Zip7 and PTP1B are silenced.
8.5 Detection of LAR activity
LAR, is a receptor-like PTP with wide tissue distribution which is suggested to have a role similar to PTP1B in negatively regulating IR signalling (Mooney & LeVea, 2003). We were unable to obtain antibodies to the PTP LAR to determine any compensatory activity of LAR following PTP1B silencing.