Wednesday, August 19, 2015

Unraveling the Mechanisms of Insulin Resistance

Insulin Signalling Defect

It’s well understood that insulin resistance typically precedes the development of type 2 diabetes by many years.  Not only is insulin resistance a key risk factor for type 2 diabetes, but it’s also a risk factor for many other chronic diseases, so understanding what causes insulin resistance and effectively dealing with it can solve many problems at once

Perhaps the dominant idea of what causes insulin resistance is that it’s due to a defect or inhibition in the insulin signalling pathway.  You might be familiar that one of the actions of insulin is to increase muscle glucose uptake by causing the translocation of a glucose transporter called GLUT4 to the cell membrane so that glucose can pass from the bloodstream to the muscle cell and either be used or stored.  The nitty gritty of physiology is complex, for insulin cause the translocation of GLUT4 to the cell membrane it must first activate a complex signalling pathway shown below.  In summary [1] [2]:

·         Insulin binds to insulin receptor (IR)
·         The insulin receptor tyrosine phosphorylates the insulin receptor substrate (IRS)
·         The tyrosine phosphorylated IRS associates with PI3K
·         PI3K activates Akt
·         Akt inhibits AS160 (Akt substrate 160) and GSK3 (glycogen synthase kinase 3)
o   Inhibition of AS160 increases the activity of certain enzymes that lead to the translocation of GLUT4 to the cell membrane
o   Inhibition of GSK3 promotes the activity of glycogen synthase, leading to increased glycogen synthesis

[2]

Several human studies have examined the activation of the insulin signalling pathway in muscle during a euglycemic-hyperinsulinemic clamp.  The euglycemic-hyperinsulinemic clamp is a technique whereby insulin is infused to high levels and glucose is also infused to maintain euglycemia (normal glucose levels, usually 5.0 mmol/l).  The euglycemic-hyperinsulinemic clamp is not a method that replicates physiological conditions*, but is the gold standard for measuring insulin resistance.  These studies have consistently found a defect in insulin-stimulated IRS-1 association with PI3K among people with type 2 diabetes, but are less consistent with other parts of the insulin signalling pathway [3] [4] [5] [6] [7] [8].  In addition, a defect in IRS-1 association with PI3K has also been found in women with gestational diabetes compared to pregnant women during an oral glucose tolerance test [9]

However, there are some issues with this mechanism:

·         While studies have consistently found that there is a defect in IRS-1 tyrosine phosphorylation/IRS-1 association with PI3K among individuals with IR/T2D during a euglycemic-hyperinsulinemic clamp, this has not been tested under more physiological conditions such as an oral glucose tolerance test or a standardised meal
·         While studies have consistently found that there is a defect in IRS-1 tyrosine phosphorylation/IRS-1 association with PI3K among individuals with IR/T2D, there is inconsistent evidence that this translates into impaired insulin signalling downstream of IRS-1/PI3K, with some studies finding that there is impaired insulin-stimulated Akt phosphorylation [3] [5] [10] and others finding that it is normal [7] [8] [11]
·         Even if insulin-stimulated Akt activation was impaired, very little Akt activation is needed for maximal GLUT4 translocation, therefore a defect in insulin-stimulated Akt activation of 50% may not be relevant in postprandial conditions and offset by higher fasting insulin in basal conditions [10]
·         Only a few of those insulin signalling studies have found that activation of the insulin signalling pathway is not reduced under basal conditions [4], whereas many find it to be normal or even slightly elevated [5] [7] [8] [10] [11]
·         Current glucose lowering drugs do not target any of the proteins in the insulin signalling pathway.  Thiazolidinediones promote fat storage, which lowers plasma NEFA, thereby increasing glucose oxidation.  Biguanides (such as metformin) reduce gluconeogenesis by inhibiting complex I of the mitochondrial electron transport chain [12] [13], rather than by AMPK dependent mechanisms [12] [14] [15].  And sulfonylureas reduce glucose levels by increasing insulin secretion
·         Growth hormone impairs glucose tolerance and insulin sensitivity without inhibiting the insulin signalling pathway [16] [17], suggesting that insulin resistance can develop through alternative mechanisms

* There are many differences between a meal or OGTT and the euglycemic-hyperinsulinemic clamp (see below)


Meal or OGTT
Clamp
Glucose levels
Transiently high
Basal (~5 mmol/l)
Glucose delivery
Portal vein
Systemic circulation
Portal signal (very high glucose in portal vein)
Yes
No
Insulin levels
Transiently high
Chronically very high
Insulin delivery
Portal vein and systemic circulation
Systemic circulation

The unusual concentrations and delivery of both glucose and insulin into the system means that tissues respond differently to the clamp compared with a meal or an OGTT.  You may have heard that skeletal muscle is responsible for ~70-80% of insulin stimulated glucose uptake.  This is correct in relation to the euglycemic-hyperinsulinemic clamp, but is the not case following a meal or an OGTT, where muscle and liver each take up roughly a third of the ingested glucose [18].  The clamp doesn’t result in the high levels of glucose and insulin levels in the portal vein that enhance liver glucose uptake and the concentrations of glucose and insulin play to the strengths of skeletal muscle due to differences in the main glucose transporters and glucokinase enzymes in each tissues.  Regardless the extrapolation that skeletal muscle takes up 70-80% of glucose from a meal doesn’t make sense when you consider that the brain uses a lot of glucose and liver needs to store glycogen to supply it continuously.  Anyway, something you don’t need to know but may find interesting

Inflammation and Insulin Resistance

Whether insulin resistance is cause by a defect in the insulin signalling pathway is a relevant question because it will influence how insulin resistance is treated.  The good thing with that particular diagram is that it also shows how other things affect and/or are effected by the insulin signalling pathway, as well as showing the pathways responsible for the anabolic effects of insulin.  For example, note that TNF-α, JNK, inflammatory cytokines, SOCS proteins and PTP1B are shown to inhibit the insulin signalling pathway.  The inflammatory cytokines do this by serine phosphorylating IRS-1, which inhibits tyrosine phosphorylation, thereby inhibiting the ability of IRS-1 to associate with PI3K [2].  As such, one of the prime candidates for this defect or inhibition of the insulin signalling pathway is chronic inflammation

An inflammatory cause of insulin resistance and type 2 diabetes is supported by several observations:

·         Elevated markers of inflammation precede and can predict the development of type 2 diabetes [19] [20] [21] [22]
·         Anti-TNFa drugs for rheumatoid arthritis is inversely associated with development of T2D [23] [24]
·         Gene knockouts of some inflammatory cytokines protect against diet induced insulin resistance in rodents [25]

While inflammation seems to be an attractive mechanism for insulin resistance, it’s unfortunately not so simple:

·         An IL-1R antagonist (anakinra) lowers fasting glucose, but does so by improving β-cell function, rather than by improving insulin sensitivity* [26]
·         Salicylates (such as aspirin) also lower fasting glucose, but do so by either increasing insulin secretion [27] or by reducing insulin clearance [28]
·         Inhibiting TNF-a does not improve insulin sensitivity, fasting glucose or glucose tolerance in humans [29] [30] (does improves insulin sensitivity in rodents [31])

* If inflammation is involved in the development of type 2 diabetes, it’s probably not because inflammation causes insulin resistance, but rather than inflammation, in particular IL-1, impairs β-cell function and/or promotes the destruction of β-cells [32].  There are properties of the pancreatic β-cells that make some particularly vulnerable to IL-1, and therefore a ‘weak link’.  Pancreatic islet cells express the highest density of IL-1 receptors among all body tissues, and the pancreatic β-cells seem to be particularly sensitive to IL-1 [32].  In addition there are also some unique aspects of β-cell metabolism and their vulnerability to oxidative stress that also makes them a ‘weak link’ [33]

Measuring Metabolism

Metabolomics and lipidomics are methods to simultaneously measure the concentrations of many different metabolites and lipids in biological tissues or fluids (such as plasma, saliva and urine).  These methods are often used in research to try to develop non-invasive biomarkers of disease, to better predict who is at risk.  For example, some common metabolites and lipids associated with insulin resistance and/or type 2 diabetes include:

·         Higher levels of most amino acids and fatty acids [34] [35] [36] [37]
·         Lower levels of glycine [34] [36] [37] [38] [39]
·         Higher levels of α-hydroxybutyrate (a by-product of elevated glutathione synthesis that is indicative of oxidative stress) [36] [39]

An advantage of metabolomics and lipidomics is that you can measure lots of metabolites and lipids at once.  As such there are many other metabolites and lipids I could mention such as carnitines, bile acids and glycerophosphocholine species (and other obscure lipids).  But a limitation with an approach like this is that it can be difficult to determine cause and effect.  For example, elevated levels of certain amino acids and fatty acids in plasma have consistently been found to be associated with the development of type 2 diabetes.  But do these amino acids and fatty acids promote diabetes, or are they merely elevated due to the insulin resistance that develops long before type 2 diabetes is diagnosed

A few studies have also examined how plasma metabolites and/or lipids change following an OGTT [35] [40] [41] [42] [43] or a high fat meal (P:F:C = 12:59:30) [44].  And fewer still have compared how individuals with insulin resistance and/or type 2 diabetes [35] [40] [41] [42].  To my knowledge, no studies measured tissue metabolites and lipids under fasting conditions or following a meal or OGTT, or whether they are altered in individuals with insulin resistance and/or type 2 diabetes.  Studying this could improve our understanding of metabolism and the causes of insulin resistance and type 2 diabetes as changes and alterations in tissue metabolites and lipids more closely reflects metabolism than changes in plasma metabolites and lipids.

For example, a proposed mechanism whereby insulin resistance impairs glucose uptake by tissues is that insulin resistance ultimately impairs GLUT4 translocation, which is related to the idea that insulin resistance is caused by a defect in the insulin signalling pathway.  This mechanism would predict that individuals with insulin resistance would have lower intracellular glucose compared with healthy controls and either lower or normal glucose-6-phosphate (the first step of glycolysis).  This has been demonstrated by a research group using the euglycemic-hyperinsulinemic clamp technique and nuclear magnetic resonance imaging (NMR) to measure metabolite concentrations in skeletal muscle [44], but has not been replicated.  An alternative proposed mechanism is that impaired glucose uptake is due to impaired glucose metabolism.  This mechanism would predict that individuals with insulin resistance have either normal or elevated intracellular glucose levels, but a lower concentration of glucose-6-phosphate due to reduced activity of the enzyme that catalyses the first step of glycolysis, which is seen in mice on a HFD [45]

My PhD Project

So the aim of the first study for my PhD is to characterise the insulin signalling and metabolic responses to a meal in skeletal muscle and adipose tissue in healthy individuals.  And the aim of the second study is to investigate whether these responses are altered in individuals with insulin resistance and/or type 2 diabetes.  (The third project will likely be on cancer metabolism)

We are recruiting for the first study at the moment and I’ve copied the content from our flyer below.  Feel free to ask any questions and I can send though more detailed information about the study if you’re interested.  If you’re eligible and interested to participate (or know someone that would be) we would really appreciate it.  Just please note we are at Deakin University's Burwood Campus in Melbourne, Australia:



Healthy males and females (18-40 years old), with a BMI of 23-30, are required for a study examining the physiological responses to a meal.

You will be screened prior to involvement to ensure eligibility.  Participation would involve 1 visit to our laboratory for approximately 4 hours, which will include:

·         Eating a standardised breakfast consisting of an egg, mozzarella cheese and a glucose drink
·         A total of 3 small muscle and fat biopsies that will be taken under local anaesthetic, before the meal and at 30 minutes and 2 hours after the meal
·         A total of 9 small blood samples (3 ml) that will be taken before the meal and at regular intervals after the meal
·         A total of 5, 5 minute periods of sitting underneath a metabolic hood before the meal and at regular intervals after the meal

You will receive $60 for your time and would benefit by receiving feedback on your: metabolic rate and glucose, insulin and other physiological responses to a meal.

Results from this study will provide crucial information about the normal physiological responses to a meal that may assist in the treatment of pre-diabetes and type 2 diabetes

This study has been approved by the Deakin University Human Research Ethics Committee (Ref No: 2015-078)

If you are interested in participating or would like more information please contact:
Dr. Clinton Bruce or Mr. Steven Hamley
School of Exercise and Nutrition Sciences, Deakin University
Email: clinton.bruce@deakin.edu.au or shamley@deakin.edu.au

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