Sunday, June 19, 2016

My Honours Project: Pre-Diabetes and Insulin Resistance Completely Normalised after Just 1 Week

My honours project recently got published in a peer-reviewed journal [1].  Academic publishing can take a while, but it gets done eventually.  It’s a good project and may be of interest, but at times it’s not quite written for the general public, so I’ll summarise it here:


We put 20 mice on the standard laboratory low fat chow diet (CHOW) and put 40 mice on a high fat diet (HFD)*.  After 8 weeks we switched 20 mice on the high fat diet to the chow diet (HFD→CHOW).  We did oral glucose tolerance tests (OGTT) before and after the diet switch, and took tissues at the end of the study

We used:

·         OGTTs to measure glucose tolerance and how insulin and free fatty acids changed during the OGTT
·         Stable isotopes of glucose to measure the source of plasma glucose during the OGTT – whether it came from the body (normal glucose) or from the OGTT (heavy glucose).  This can give an indication of whether the defect is from excess glucose production or impaired glucose uptake by tissues
·         Heavy water (2H2O) to measure lipid synthesis and the sources (but not the absolute amount) of glucose production (glycogenolysis vs. gluconeogenesis)
·         Tissue samples to measure glycogen, triglycerides, other tissue lipids, lipid synthesis and various metabolites (metabolomics)

* I’ve written before about how the mice strain we used (C57Bl/6) was bred to be susceptible to develop obesity and the metabolic syndrome on a high fat diet [2].  So think of this as a model of diet induced obesity and pre-diabetes rather than being prescriptive for human diets


When the mice were on the high fat diet:

·         They ate more calories (but slightly less volume, data not shown) (figure 1)
·         They gained more weight as fat mass (and had no change in the weight of their quads) (figure 1)
·         They developed impaired fasting glucose and impaired glucose tolerance (pre-diabetes) and insulin resistance (figure 2)
·         They developed fat accumulation in their liver (figure 5) and muscle (data not shown)

When the mice were switched back to standard laboratory chow they

·         Substantially reduced their energy intake voluntarily and even ate less than the chow group, and as a result lost about half their excess weight in 9 days (figure 1)
·         After 1 week they completely normalised their pre-diabetes and insulin resistance (figure 2)
·         Lost some, but not all the fat in their adipose tissue (figure 1), liver (figure 5) and muscle (data not shown)
·         Got more accumulation of some ceramide species in the liver (ceramide accumulation is a proposed mechanism of insulin resistance, so this surprised me) (figure 5)

The Energy State as a Mechanism of Pre-Diabetes and Insulin Resistance

A potential mechanism of pre-diabetes and insulin resistance could be that they are caused by an elevated energy state in the liver (low AMP or a low AMP:ATP ratio) as a consequence of excess energy intake:

Excess energy intake >>> elevated hepatic energy state >>> inhibited glucose metabolism and insulin resistance >>> hyperglycemia

This is based on:

·         Changes in fasting glucose, glucose tolerance and/or insulin resistance occur within ≤ 1 week in humans and mice in response to overfeeding, calorie restriction and bariatric surgery.  So you need a mechanism that can be manipulated very quickly and the hepatic energy state can be
·         The energy state of the cell negatively regulates glucose metabolism [3]
·         Previous research that found the hepatic energy state was elevated in HFD mice

We used targeted metabolomics to measure AMP and the intermediates of glycolysis (G6P, F6P, 3PGA, PEP) in the liver.  The HFD group had lower AMP and intermediates of glycolysis, but higher glucose in the liver* (figure 4).  The higher glucose and lower concentrations of glycolytic intermediates could suggest impaired glucose metabolism, but this isn't a imprecise measure as differences in static concentrations don’t necessarily parallel differences in metabolic fluxes

However, the HFD→CHOW group only had a partial normalisation of AMP and intermediates of glycolysis (figure 4).  Considering they completely normalised their pre-diabetes and insulin resistance, this casts doubt onto the mechanism

* The primary glucose transporter is in the liver GLUT2, which doesn’t require insulin and has a high capacity and low affinity for glucose.  This means the glucose concentration in the liver will be similar to the blood, so this result is to be expected, and this enables the liver to act as a glucose sensor (like the beta cells of the pancreas)


Metabolic stuff generally happens very quickly.  This is the case whether you’re talking about cholesterol [4], blood pressure [5], or in this case, pre-diabetes and insulin resistance (which I’ll discuss more of later).  The rapid timeframe and results of this study suggest that mechanisms relying on overweight/obesity and fat accumulation in either the liver or muscle are probably not major mechanisms in the development and normalisation of pre-diabetes and insulin resistance

Whatever the mechanisms are, they have to be capable of acting very quickly.  Based on some plausibility and the timeframe, some possible mechanisms could be based in the brain, liver and gut, but probably not muscle.  More on this later

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