Wednesday, July 27, 2016

Why Liver is Likely More Important than Muscle for Pre-Diabetes: Part 2

Muscle is argued to be the primary defect in insulin resistance.  The main evidence for this comes from the presence of impaired insulin signalling and glucose metabolism in people with insulin resistance and type 2 diabetes, and their first degree relatives [1].  Whether you think the contribution of muscle to postprandial glucose uptake is closer to 75% or 30% (see earlier post), it’s a decent contribution.  However, this line of research is limited as similar studies haven’t been done in the liver, since liver biopsies are far more invasive than muscle or fat biopsies (and unfortunately my PhD won’t resolve this)

One of the difficulties with mechanistic research in disease models is that you take a snapshot in time and find several pathologies are present.  The trick then is to figure out what are the possible causes of disease as opposed to the consequences of disease or just unrelated effects.  A method of doing this is to investigate what’s happening at the earliest time points in the progression of disease, rather than drawing conclusions from biomarkers in poorly controlled diabetes

My honours project followed on from a time course study in mice that found the following [1]:

·         Glucose intolerance was present when first measured after 3 days on a high fat diet (HFD)
o   Whole body insulin resistance was present when first measured at 1 week with the euglycemic-hyperinsulinemic clamp (higher glucose infusion rate)
§  This was due to liver insulin resistance  (this wasn’t associated with a defect in Akt activation or inflammation, but with a relatively mild increase in lipid accumulation)
§  Adipose tissue was also insulin resistant, but muscle wasn’t yet
·         Muscle insulin resistance was present when next measured at 3 weeks
o   This was associated with no further deterioration in glucose tolerance, but worsened whole body insulin resistance and increased fasting insulin
·         Glucose tolerance or tissue insulin resistance didn’t worsen even several weeks after the initial defect

This is consistent with other studies in rodents and humans:

·         Liver, but not muscle insulin resistance was present after mice [2] and rats [3] were put on a HFD at 3 days when first measured with the clamp (and associated with liver ER stress [2]).  Muscle insulin resistance was present when next measured at 3 weeks (which was associated with muscle lipid accumulation) [3]
·         Short-term overfeeding (3-7 days) in healthy humans causes hepatic insulin resistance [4] [5] [6] [7] [8], whereas muscle insulin resistance was not present at this point [4] [6]
·         Short term energy restriction almost normalises fasting glucose after 2-7 days, before significant weight loss, and this is associated with improvements in liver insulin sensitivity, but not muscle insulin sensitivity, which improves several weeks later [9] [10]

Since pre-diabetes and insulin resistance develop very quickly in mice on a HFD, one of the hypotheses of my honours project was that the pre-diabetes and insulin resistance would be normalised in a similar timeframe.  This was one of the novel aspects of my honours project, as earlier diet reversal studies in rodents haven’t looked at early points, but instead after 3, 4 and 16 weeks respectively [11] [12] [13].  We found pre-diabetes and insulin resistance was normalised in 7 days after switching mice from a high fat diet to the standard laboratory low fat chow diet.  We didn’t directly measure changes in insulin sensitivity in the liver and muscle, but some evidence suggests it was likely due to changes in liver glucose metabolism rather than muscle:

·         The HFD group had a higher change from baseline in endogenous glucose at 15 minutes, suggesting elevated glucose production between 0-15 minutes.  This was completely normalised in the HFD→CHOW group (figure 3B)
·         The HFD group had higher exogenous glucose at 30 and 60 minutes but not at 120 minutes, indicating a defect in early glucose disposal (15-60 minutes).  As the liver gets first access to glucose, this could suggest a defect primarily in liver glucose uptake that was completely normalised in the HFD→CHOW group (figure 3C and 3E)
·         The HFD group showed evidence of elevated futile glucose cycling in the liver, indicating impaired glucose metabolism, and this was completely normalised in the HFD→CHOW group (figure 3G)
·         While tissue lipid accumulation isn’t always associated with insulin resistance, the HFD→CHOW group normalised 60% of their liver triglycerides (figure 5A) and 32% of their muscle triglycerides (data not shown)

The takeaway from this is not so much that muscle isn’t important, just that it seems that changes in muscle insulin resistance generally occur more slowly and can be sufficiently compensated for by the beta cells or other organs**.  Metabolic changes happen quickly.  Whatever the mechanisms behind the rapid changes in glucose control are they have to be capable of changing quickly.  Lipid accumulation might be too slow and obesity is definitely too slow.  And these mechanisms are more likely to originate in the liver rather than muscle.

If this stuff interests you I strongly recommending reading this review

* Muscle insulin receptor knockout (MIRKO) mice have normal glucose levels and insulin levels, but have elevated plasma triglycerides and free fatty acids [14].  This is in contrast to liver insulin receptor knockout (LIRKO) mice that prior to liver failure have severe insulin resistance and hyperglycemia, but reduced or normal circulating fatty acids and triglycerides [15] [16]

** Also, in figure 1B bellow, note the absence of impaired glucose tolerance and insulin resistance people with duchenne muscular dystrophy, and the profound insulin resistance but normal glucose tolerance of those who were wheelchair bound (WC) 

“With such a severe reduction in muscle mass, glucose intolerance rather than insulin resistance would be the expected consequence. Therefore, the loss of muscle mass in this group is probably unrelated to their insulin resistance. Instead, the inactivity itself, which accompanies the loss in muscle tissue, is probably a major factor in the development of the insulin resistance.” [17]

Sunday, July 24, 2016

Why Liver is Likely More Important than Muscle for Pre-Diabetes: Part 1

My honours project recently got published.  I recommend reading the paper [1], but if that is too technical you can read my summary of it here.  A reasonable question you might have is why we focussed on the liver and not muscle, because after all, isn’t muscle supposed to be the main organ involved in glucose control?

The contribution of muscle to whole body glucose uptake is often over-exaggerated.  People bring up the fact that muscle is responsible for ~70-80% of insulin stimulated glucose uptake and use this to claim that muscle is responsible for ~70-80% of glucose uptake in the postprandial state (following a meal or an oral glucose tolerance test (OGTT))

The ~70-80% figure is correct, but the problem is that is that it comes from studies using a technique called the ‘euglycemic-hyperinsulinemic clamp’.  The euglycemic-hyperinsulinemic clamp is the gold standard to measure insulin resistance** and involves infusing glucose and insulin into the blood to maintain euglycemia (normal glucose levels ¬5 mmol/l) and hyperinsulinemia (high insulin levels), often higher than what occur following an oral glucose tolerance test (see below [2] [3])


This method clearly doesn’t accurately represent what happens following a meal or OGTT, where muscle is responsible for ~30% of glucose uptake* [4].  The conditions of the clamp favour muscle glucose uptake over other key organs such as the liver for a few of reasons:

·         The main glucose transporter in muscle (GLUT4) has a higher affinity for glucose than the main glucose transport in the liver (GLUT2) [5] and the muscle version of hexokinase (the enzyme catalysing the first step of glycolysis) has a higher affinity for glucose than liver glucokinase [6].  These differences mean muscle takes up more glucose during euglycemic conditions than the liver [6]
·         When you eat a meal or do an OGTT the liver has the benefit of glucose and insulin levels being ¬2-3x higher in the portal vein compared to systemic circulation.  This enhances liver glucose uptake and proportionally decreases glucose uptake by other tissues [4, 7, 8], but doesn’t occur when glucose and insulin are infused directly into the bloodstream

So it’s somewhat correct to say that muscle is responsible for ~70-80% of insulin stimulated glucose uptake, but this shouldn’t be extrapolated to what happens following a meal or OGTT where both insulin and glucose transiently increase 

* In the fasted state the brain takes up most of the glucose and muscle is responsible for ~18% of glucose uptake (see below) [9]

Thursday, June 30, 2016

A Letter to the Editor Criticising the Paleo Mouse Study

A paper was published in February 2016 claiming that they tested the effect of a Paleo diet in mice and found that it causes excess weight gain, impaired glucose tolerance and insulin resistance [1].  I dubbed the paper ‘the Paleo mouse study’ and have written about it earlier in the year.  I discussed that mice respond differently to higher fat diets than humans*.  And also, that the LCHF diet is the Paleo mouse study sucks as it was largely comprised of added fats, casein and sucrose whereas the standard low fat diet was a lot more whole foods based

A letter to the letter by Nathan Cofnas** was recently published that brought up some issues with the Paleo mouse study [2] (it’s short, open access and I recommend reading it):

  • The representation of the study in the media*** and on Melbourne University’s Youtube channel was that this study was a test of a LCHF Paleo diet (never mind the details on what the mice actually ate), but the paper didn’t even include the word ‘Paleo’ or ‘Paleolithic’
  • The Paleo concept is based on evolution and genetic adaptation and would make the hypothesis that animals do best on the diets that they are most genetically adapted to, which can be largely inferred from what they eat in the wild.  For mice, this is a low fat, high carbohydrate, largely plant based diet, so it would be expected that the chow diet would be better for them 

But one thing the letter didn’t mention much of are results from human RCTs, which trump and contradict and mouse studies.  Meta-analyses of low carb vs. low fat RCTs [3] or Paleo vs. conventional dietary advice RCTs [4] both find that low carb and Paleo diets result in greater weight loss than the alternative, and more recent studies continue to support these findings [5, 6]

* For example, the senior author of the Paleo mouse study recommends the Mediterranean diet, but the amount (~40% of total calories) and type (olive oil/MUFA) of fat promoted in the Mediterranean diet causes obesity, glucose intolerance and insulin resistance in mice [7, 8]

** He is part of the Department of History and Philosophy of Science at the University of Cambridge and has published some earlier work on evolutionary mismatch [9].  Leave it academics outside the field and bloggers to apply some common sense in translating studies to the real world 

*** For example: “To put that in perspective, a 100 kilogram person on a Paleo diet could pile on 15 kilograms in two months” [10] (good luck achieving half that effect with a deliberate overfeeding study in humans)

Wednesday, June 29, 2016

Moderation is Subjective and Susceptible to Bias

I discussed the concept of moderation in an earlier post.  My problems with moderation as dietary advice include: that moderation is poorly defined and very subjective; it can be used by the food industry to legitimise their unhealthy products; and some people do better with abstinence than with moderation

The problems with the first point – the subjectiveness of moderation – was well illustrated in a recent paper [1].  In the introduction, this paper cites earlier research that found: (1) people are poor judges of food intake; (2) people look to their peers and themselves to determine what ‘moderate’ consumption should be; (3) people’s beliefs are biased to favour themselves

Study 1

The first study involved asking female students about how many cookies: (1) one should eat; (2) would be moderate consumption; and (3) would be considered indulgent

On average, the participants reported that they should eat 2.25 cookies and that a ‘moderate consumption’ was 3.17 cookies

Study 2

The second study involved asking people taking a survey how many candies would be: (1) a reasonable amount to eat in one sitting; (2) eating in moderation in one sitting; or (3) what they should eat in one sitting.  Then they reported how much they like the candies

On average, the participants reported they should eat 8.87 candies, moderation consumption was 10.93 candies, and a reasonable amount was 14.17 candies.  There was also a correlation between the number of candies reported as moderate consumption with the liking of the candies (r = 0.38, p < 0.0001) and the reported consumption of those candies (r = 0.27, p = 0.007), whereas correlations were weaker or not significant for ‘should eat’ and ‘reasonable amount’

Study 3

The third study involved asking people taking a survey to report their average consumption of various foods and how moderate they thought their consumption of those foods

On average, the participants rated their consumption as moderate (4.50 and 4.47 on a 1-7 point scale, where 1 = ‘not at all’, and 7 = ‘very much’).  Across the 12 food and beverage categories, participants on average defined moderate consumption as greater than their personal consumption

There was also a correlation between reported personal consumption and what was reported as ‘moderate consumption’ (r = 0.52, p < 0.001 and r = 0.50, p < 0.001).  This correlation wasn’t disproportionate seen in people who were overweight.  However, there was no correlation between (1) the participants’ personal consumption with the participants’ rating of their consumption as moderate; and (2) the participants’ rating of their consumption as moderate with how much they believed was moderate consumption


Moderation could work as a concept, but not in situations where people’s reference point is one where they should be eating unhealthy food and that moderation involves eating a greater consumption of unhealthy food; or where most people believe they are already doing so well (when most people clearly aren’t) that moderation would once again involve a greater consumption of unhealthy food

For moderation to work, I think you would need to define ‘should’ as 0 and moderation as only slightly above 0, and for most people for moderation to mean something closer to self-discipline than a self-rationalised or socially acceptable indulgence 

If you want to adopt the recommendation of moderation, then I suggest you acknowledge then try to account for your biases and operationalise what moderation means.  Then most importantly, make adjustments based on the feedback your body is giving you

Tuesday, June 28, 2016

Other Publications in Peer-Reviewed Journals

After the publication of my honours project, I think this is a good time to summarise some other papers I’ve contributed to (as a general rule the first author made the most contribution and the last is the senior author).  These papers aren’t open access, but you can find them all at ResearchGate (links included).  You can also find them on PubMed by searching ‘hamley s’ (the perks of having an uncommon last name)

Overexpression of sphingosine kinase 1 in liver reduces triglyceride content in mice fed a low but not high-fat diet [1]


  • Half the mice were put on a high fat diet (HFD) and half on a standard low fat chow diet (LFD)
  • Some of mice were injected with a non-infectious virus to increase the expression of sphingosine kinase 1 


  • The HFD group developed obesity, impaired glucose tolerance, insulin resistance and fat accumulation in the liver
  • The HFD group had lower expression of sphingosine kinase 1 but not sphingosine kinase 2
  • Overexpression of sphingosine kinase 1 reduced liver triglycerides, de novo lipogenesis and lipogenic gene expression in the LFD group but not in the HFD group
  • Overexpression of sphingosine kinase 1 did not alter glucose tolerance or insulin sensitivity in mice on the LFD or HFD groups 

Implications: The idea of this study was that overexpression of sphingosine kinase 1 may reduce lipid accumulation in the liver, which would protect against the development the development of glucose intolerance and insulin resistance in the HFD group.  The first step on this process didn’t happen in the HFD group, which was unexpected.  So the fact that glucose intolerance and insulin resistance wasn’t altered in the overexpressing HFD group shouldn’t be surprising

Application of dynamic metabolomics to examine in vivo skeletal muscle glucose metabolism in the chronically high-fat fed mouse [2]


  • Half the mice were put on a HFD and half on a LFD
  • An OGTT was used with the glucose coming from a glucose isotope with all carbon atoms having an extra neutron (U-13C).  The use of U-13C glucose enables the metabolic fate of the ingested glucose to be tracked 


  • The HFD group developed obesity, impaired glucose tolerance and insulin resistance
  • The HFD group had less heavy carbon labelling in 3PGA (an intermediate of glycolysis), lactate, alanine and the TCA cycle, except for citrate 

Implications: The labelling data suggests that glucose metabolism in muscle is impaired with insulin resistance.  The labelling data also indicates that almost all the pyruvate enters the TCA via pyruvate dehydrogenase (PDH, cataplerosis) rather than pyruvate carboxylase (CK, anaplerosis)

In vivo cardiac glucose metabolism in the high-fat fed mouse: Comparison of euglycemic-hyperinsulinemic clamp derived measures of glucose uptake with a dynamic metabolomic flux profiling approach [3]


  • Half the mice were put on a HFD and half on a LFD
  • An OGTT was used with the glucose coming from a glucose isotope with all carbon atoms having an extra neutron (U-13C)
  • The mice were infused with insulin and glucose to maintain elevated insulin levels and fasting glucose levels (hyperinsulinemic-euglycemic clamp) 


  • The HFD group developed obesity, impaired glucose tolerance and insulin resistance
  • Insulin resistance in the heart was present at 3 weeks after being on the HFD and does not get worse over time.  This is similar to muscle, but the liver develops insulin resistance earlier (present at 1 week)
  • There were no significant differences between the diets in heavy carbon labelling in the intermediates and products of glycolysis and TCA cycle, except for lower alanine in the HFD group 

Implications: The labelling data suggests that glucose metabolism in the heart isn’t reduced.  This is likely to be due to the higher glucose levels, which compensates for the insulin resistance.  Why this happens in heart but not muscle is unknown.  The labelling data also indicates that most of the pyruvate enters the TCA via PDH rather than PC anaplerosis.  The heart is a relatively small contributor to energy expenditure and whole body insulin sensitivity/glucose uptake, but this might be important for some heart diseases