Paper: Reduction of Low-Molecular-Weight Protein Tyrosine Phosphatase Expression Improves Hyperglycemia and Insulin Sensitivity in Obese Mice
Summary:
The purpose of this experiment was to investigate the role of Low-Molecular-Weight Protein Tyrosine Phosphatase (LMW-PTP) in glucose metabolism and insulin action. More specifically, the researchers were attempting to determine if reducing the expression of LMW-PTP in vitro and in vivo would enhance insulin signaling activity and reduce glucose levels. The basis for this experiment was built around the findings of recent studies which suggest that LMW-PTP may have a role as a negative regulator of insulin-mediated mitogenic and metabolic signaling. For instance, LMW-PTP also known as acid phosphatase locus 1 (ACP1) is an 18 kDa cytosolic enzyme with catalytically active isoforms (A and B). It is widely expressed in various tissues in mammals, however, it is most highly expressed in the liver and brain and least expressed in skeletal muscle tissue. In any event, LMW-PTP dephosphorylates tyrosine phosphorylated proteins which is how it is thought to affect the insulin pathway.
For example, insulin initiates a physiological response by binding to its membrane bou
nd receptor which causes autophosphorylation of the beta subunit and in turn activates the receptor. This results in the phophorylation of the receptors two major downstream substrates, IRS-1 and IRS-2. These substrates then phophorylate a number of other enzymes within the target cell which induce the appropriate biological response such as the phophorylation and inactivation of glycogen synthase kinase. The inactivation of this enzyme results in the activation of glycogen synthase which increases the utilization of glucose for glycogen synthesis. Thus, the end result is the reduction of blood glucose levels in the bloodstream.
The way in which LMW-PTP actually affects this pathway is it dephosphorylates the tyrosine residues on the beta subunit of the insulin receptor. This decreases the receptors ability to respond to insulin as the activation of the receptor depends on the phosphorylation of these tyrosine residues. In this way, LMW-PTP reduces insulin sensitivity which makes it a possible contributor to both hyperglycemia and diabetes. In fact, recent studies have shown that LMW-PTP is associated with dyslipidemia and hyperglycemia in human subjects, but its exact role in regulating insulin levels in vivo is still unknown. In any event, in knowing how LMW-PTP operates itself, the researchers of this experiment investigated the effects of reducing its expression in vitro and in vivo to see how it affects the actions of insulin. Their hypothesis for the experiment was that reducing the expression of LMW-PTP should increase insulin sensitivity.
To perform this experiment, they used a specific antisense oligonucleotide (ASO) to suppress the expression of LMW-PTP in cultured mouse hepatocytes as well as the liver and fat of diet-induced obese (DIO) and leptin-deficient obese (ob/ob) mice. They did this by selecting and isolating a LMW-PTP ASO. They then isolated mouse hepatocytes and transfected them with the LMW-PTP ASO. At the same time, they also transfected the DIO and ob/ob mice with the same LMW-PTP ASO. After weeks of treatment (varying lengths of time for each), the cells for all three trials were analyzed using a variety of tests to determine the effects that the ASO treatment had on insulin sensitivity and plasma glucose levels. In doing so, it was discovered that the reduction of LMW-PTP expression with ASO in all three cell types (isolated hepatocytes, DIO mice, and ob/ob mice) resulted in increased phosphorylation and activ
ity of key insulin signaling intermediates including the insulin receptor (beta subunit), P13-kinase, and Akt in response to insulin stimulation. In this way, the ASO-treated DIO and ob/ob mice both showed enhanced insulin sensitivity as the plasma insulin and glucose levels were lowered in both animals. Similarly, there was improved glucose and insulin tolerance observed in the DIO mice, however, the treatment did not decrease body weight or increase metabolic rate. Therefore, it was concluded that LMW-PTP is a key negative regulator of insulin action and that reducing its expression improves insulin sensitivity. In turn, the action of this enzyme holds potential as a novel target for the treatment of insulin resistance and type 2 diabetes.
Overall the authors show that:
1) The reduction of LMW-PTP expression with ASO in all three cell types (isolated hepatocytes, DIO mice, and ob/ob mice) results in increased phosphorylation and activity of key insulin signaling intermediates including the insulin receptor (beta subunit), P13-kinase, and Akt in response to insulin stimulation.
2) LMW-PTP is a key negative regulator of insulin action.
3) The reduction of LMW-PTP expression enhances insulin signaling in mouse hepatocytes.
4) The reduction of LMW-PTP expression improves insulin sensitivity in DIO mice with no change in body weight.
5) The reduction of LMW-PTP expression improves insulin sensitivity in ob/ob mice with no change in body weight.
6) The reduction of LMW-PTP expression improves insulin action in both liver and fat cells of both mice.
7) The plasma insulin and glucose levels were lowered in both types of mice.
8) There was improved glucose and insulin tolerance observed in the DIO mice.
Critique of the paper:
- The paper is very well written, however, it seemed to be dragged out and there was a lot of repetition.
- The figures were clear and appropriate, however, I did not like their layout. They were all jumbled together at the end of the paper and the captions for each figure were not on the same page as the figure itself which was inconvenient when analyzing the figures.
- The paper itself was also very clear and easy to understand and the key points were presented in an appropriate order.
- I found the experimental methods section to be a bit exhaustive and it got confusing at times. For example, it mentions that C57BL/6J-Lep mice were purchased for this experiment which made no sense to me. As far as I could gather, I believe they were the ob/ob mice. I think a small explanation of what these mice were would have been appropriate.
- It was also unclear how the antisense oligonucleotide reduced the expression of the LMW-PTP gene. I feel that they could have explained that a bit better.
- Other than that, all of the conclusions seemed to support the results and the researchers of the experiment accomplished what they set out to do which was to determine the role of LMW-PTP in insulin signaling and glucose metabolism.
- The data was presented in a very organized manner and the explanations of the results were very detailed.
- The results also followed their expectations very closely and they successfully accomplished the goal of the experiment which was to prove that reducing LMW-PTP expression in vivo and in vitro would increase insulin sensitivity.
- Overall the paper was very good and I enjoyed learning about this topic.
Possible future experiments:
1) Question: Does treating normal mice (mice that have normal blood glucose levels) with LMW-PTP ASO injections reduce their plasma blood glucose concentrations to unhealthy levels?
Methods: Treat normal mice with daily injections of a LMW-PTP ASO as performed
in this experiment to suppress LMW-PTP expression. The blood glucose concentrations of the mice will then have to be monitored for several weeks to determine if they are lowered below normal levels. One will also have to check for any negative impacts that this treatment may be having on the mice.
Results: If the blood glucose levels are lowered significantly in the mice, causing negative impacts such as kidney problems or death then it can be concluded that LMW-PTP ASO injections can reduce blood glucose concentrations to unhealthy levels. Therefore, this would have to be taken into account when considering LMW-PTP ASO injections as a possible treatment for type 2 diabetes.
2) Question: Can suppressing LMW-PTP expression be used as a valid treatment for type 2 diabetes?
Methods: Treat mice that have type 2 diabetes with daily injections of a LMW-PTP ASO as performed in this experiment to suppress LMW-PTP expression. One can then monitor glucose levels in the mice over a long-term period to determine if blood glucose concentrations return to normal levels in response to the injections. The mice will also have to be analyzed for any significant side effects of the treatment that would negatively impact the mice in anyway. Similarly, such an experiment would also have to be performed numerous times with consistent results in order to be considered valid.
Results: If reducing LMW-PTP expression in the diabetic mice reduces glucose to normal levels without any side effects then this technique will be considered a successful means for treating type 2 diabetes in mice. Thus, this treatment can then be considered to treat type 2 diabetes in humans.
Summary:
The purpose of this experiment was to investigate the role of Low-Molecular-Weight Protein Tyrosine Phosphatase (LMW-PTP) in glucose metabolism and insulin action. More specifically, the researchers were attempting to determine if reducing the expression of LMW-PTP in vitro and in vivo would enhance insulin signaling activity and reduce glucose levels. The basis for this experiment was built around the findings of recent studies which suggest that LMW-PTP may have a role as a negative regulator of insulin-mediated mitogenic and metabolic signaling. For instance, LMW-PTP also known as acid phosphatase locus 1 (ACP1) is an 18 kDa cytosolic enzyme with catalytically active isoforms (A and B). It is widely expressed in various tissues in mammals, however, it is most highly expressed in the liver and brain and least expressed in skeletal muscle tissue. In any event, LMW-PTP dephosphorylates tyrosine phosphorylated proteins which is how it is thought to affect the insulin pathway.
For example, insulin initiates a physiological response by binding to its membrane bou
nd receptor which causes autophosphorylation of the beta subunit and in turn activates the receptor. This results in the phophorylation of the receptors two major downstream substrates, IRS-1 and IRS-2. These substrates then phophorylate a number of other enzymes within the target cell which induce the appropriate biological response such as the phophorylation and inactivation of glycogen synthase kinase. The inactivation of this enzyme results in the activation of glycogen synthase which increases the utilization of glucose for glycogen synthesis. Thus, the end result is the reduction of blood glucose levels in the bloodstream.The way in which LMW-PTP actually affects this pathway is it dephosphorylates the tyrosine residues on the beta subunit of the insulin receptor. This decreases the receptors ability to respond to insulin as the activation of the receptor depends on the phosphorylation of these tyrosine residues. In this way, LMW-PTP reduces insulin sensitivity which makes it a possible contributor to both hyperglycemia and diabetes. In fact, recent studies have shown that LMW-PTP is associated with dyslipidemia and hyperglycemia in human subjects, but its exact role in regulating insulin levels in vivo is still unknown. In any event, in knowing how LMW-PTP operates itself, the researchers of this experiment investigated the effects of reducing its expression in vitro and in vivo to see how it affects the actions of insulin. Their hypothesis for the experiment was that reducing the expression of LMW-PTP should increase insulin sensitivity.
To perform this experiment, they used a specific antisense oligonucleotide (ASO) to suppress the expression of LMW-PTP in cultured mouse hepatocytes as well as the liver and fat of diet-induced obese (DIO) and leptin-deficient obese (ob/ob) mice. They did this by selecting and isolating a LMW-PTP ASO. They then isolated mouse hepatocytes and transfected them with the LMW-PTP ASO. At the same time, they also transfected the DIO and ob/ob mice with the same LMW-PTP ASO. After weeks of treatment (varying lengths of time for each), the cells for all three trials were analyzed using a variety of tests to determine the effects that the ASO treatment had on insulin sensitivity and plasma glucose levels. In doing so, it was discovered that the reduction of LMW-PTP expression with ASO in all three cell types (isolated hepatocytes, DIO mice, and ob/ob mice) resulted in increased phosphorylation and activ
ity of key insulin signaling intermediates including the insulin receptor (beta subunit), P13-kinase, and Akt in response to insulin stimulation. In this way, the ASO-treated DIO and ob/ob mice both showed enhanced insulin sensitivity as the plasma insulin and glucose levels were lowered in both animals. Similarly, there was improved glucose and insulin tolerance observed in the DIO mice, however, the treatment did not decrease body weight or increase metabolic rate. Therefore, it was concluded that LMW-PTP is a key negative regulator of insulin action and that reducing its expression improves insulin sensitivity. In turn, the action of this enzyme holds potential as a novel target for the treatment of insulin resistance and type 2 diabetes.Overall the authors show that:
1) The reduction of LMW-PTP expression with ASO in all three cell types (isolated hepatocytes, DIO mice, and ob/ob mice) results in increased phosphorylation and activity of key insulin signaling intermediates including the insulin receptor (beta subunit), P13-kinase, and Akt in response to insulin stimulation.
2) LMW-PTP is a key negative regulator of insulin action.
3) The reduction of LMW-PTP expression enhances insulin signaling in mouse hepatocytes.
4) The reduction of LMW-PTP expression improves insulin sensitivity in DIO mice with no change in body weight.
5) The reduction of LMW-PTP expression improves insulin sensitivity in ob/ob mice with no change in body weight.
6) The reduction of LMW-PTP expression improves insulin action in both liver and fat cells of both mice.
7) The plasma insulin and glucose levels were lowered in both types of mice.
8) There was improved glucose and insulin tolerance observed in the DIO mice.
Critique of the paper:
- The paper is very well written, however, it seemed to be dragged out and there was a lot of repetition.
- The figures were clear and appropriate, however, I did not like their layout. They were all jumbled together at the end of the paper and the captions for each figure were not on the same page as the figure itself which was inconvenient when analyzing the figures.
- The paper itself was also very clear and easy to understand and the key points were presented in an appropriate order.
- I found the experimental methods section to be a bit exhaustive and it got confusing at times. For example, it mentions that C57BL/6J-Lep mice were purchased for this experiment which made no sense to me. As far as I could gather, I believe they were the ob/ob mice. I think a small explanation of what these mice were would have been appropriate.
- It was also unclear how the antisense oligonucleotide reduced the expression of the LMW-PTP gene. I feel that they could have explained that a bit better.
- Other than that, all of the conclusions seemed to support the results and the researchers of the experiment accomplished what they set out to do which was to determine the role of LMW-PTP in insulin signaling and glucose metabolism.
- The data was presented in a very organized manner and the explanations of the results were very detailed.
- The results also followed their expectations very closely and they successfully accomplished the goal of the experiment which was to prove that reducing LMW-PTP expression in vivo and in vitro would increase insulin sensitivity.
- Overall the paper was very good and I enjoyed learning about this topic.
Possible future experiments:
1) Question: Does treating normal mice (mice that have normal blood glucose levels) with LMW-PTP ASO injections reduce their plasma blood glucose concentrations to unhealthy levels?
Methods: Treat normal mice with daily injections of a LMW-PTP ASO as performed
in this experiment to suppress LMW-PTP expression. The blood glucose concentrations of the mice will then have to be monitored for several weeks to determine if they are lowered below normal levels. One will also have to check for any negative impacts that this treatment may be having on the mice.Results: If the blood glucose levels are lowered significantly in the mice, causing negative impacts such as kidney problems or death then it can be concluded that LMW-PTP ASO injections can reduce blood glucose concentrations to unhealthy levels. Therefore, this would have to be taken into account when considering LMW-PTP ASO injections as a possible treatment for type 2 diabetes.
2) Question: Can suppressing LMW-PTP expression be used as a valid treatment for type 2 diabetes?
Methods: Treat mice that have type 2 diabetes with daily injections of a LMW-PTP ASO as performed in this experiment to suppress LMW-PTP expression. One can then monitor glucose levels in the mice over a long-term period to determine if blood glucose concentrations return to normal levels in response to the injections. The mice will also have to be analyzed for any significant side effects of the treatment that would negatively impact the mice in anyway. Similarly, such an experiment would also have to be performed numerous times with consistent results in order to be considered valid.
Results: If reducing LMW-PTP expression in the diabetic mice reduces glucose to normal levels without any side effects then this technique will be considered a successful means for treating type 2 diabetes in mice. Thus, this treatment can then be considered to treat type 2 diabetes in humans.
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