The group which demonstrated that exogenous insulin induces insulin resistance in T1DM NOD mice went on to demonstrate that exogenous insulin induces T2DM in normal* mice on a normal chow diet.
*If you can describe Bl/6 mice as normal, with their failure to assemble mitochondrial super complexes and all of the potential implications that has on all sorts of metabolic effects. But I digress, as always.
Exposure to excess insulin (glargine) induces type 2 diabetes mellitus in mice fed on a chow diet
So you can imagine that I quite like this group. But they are naughty and the naughtiness is very annoying.
If you read about insulin administration in the current paper you will be presented with this bollocks:
"The dose of glargine was determined according to our previous experiments (Liu et al. 2009a )..."
What they did in Liu et al 2009a was to titrate the dose of insulin determir upwards to just achieve normoglycaemia. Different doses were needed in individual mice because that's what diabetes is like in NOD mice. That is NOT what they did in this current paper to the Bl/6 mice. Here they used glargine and they went straight in at a massive supraphysiological dose:
"C57BL/6 (B6) mice (male, 6–9-week old) from Charles River were treated with either saline or a long- and slow-acting insulin reagent, glargine (50 Unit/kg body weight, s.c. injection, once a day), for 8 weeks".
Even allowing for metabolic scaling from humans down to mice this is a massive dose of glargine. It's not remotely what they did with the NOD mice.
EDIT: Going back through the first paper the NOD T1 mice did actually end up with the average group dose of detemir being 25iu/kg twice daily. The difference in protocol is that the group is assuming that glargine 50iu/kg once daily is equivalent to detemir 25iu/kg twice daily. A big assumption. And that this would be fine for all mice. And that going in at the full dose rate on day one rather than titrating up over two weeks would also be equivalent. But the dose rate is more reasonable than I expected. END EDIT.
So there are fibs in the methods. Can you really trust these folks? Not much choice really...
None of the mice died on the glargine 50iu/kg dose, so I think we can assume that they developed a marked and rapid onset insulin-induced insulin resistance.
When you wade through the IRS1 serine phosphorylation, IRS1 tyrosine phosphorylation, Akt phosphorylation, total Akt etc etc etc in the results section the end conclusion is that the liver became insulin resistant but the gastrocnemius muscle (representing what I called "systemic" tissues) did not. Bummer for my nice, plausible and apparently incorrect ideas.
So to ease my cognitative dissonance I went back to our initial paper and waded through the IRS1 serine phosphorylation, IRS1 tyrosine phosphorylation, Akt phosphorylation, total Akt etc etc etc and discoved that, lo and behold, that under a clinical protocol the gastrocnemius did become insulin resistant. So did the liver but I can live with that, after all the liver does have a (bloody enormous) arterial blood supply in addition to receiving flow from the portal vein.
Phew, biases in-tact.
It's interesting to see in the paper that exogenous glargine actually destroys the pancreas. Beta cell mass falls, their mitochondria undergo marked oxidative stress and this allows the failure to deal with the hepatic insulin resistance induced by glargine 50iu/kg, hence the induction of diabetes.
I think the take home message is that taking glargine 50iu/kg won't help your own clinical diabetes, should you be so affected. It's also too high a dose if you are attempting to do harm!
Anyhow. Growth hormone and insulin resistance next, probably.
Peter
Saturday, January 20, 2018
Friday, January 19, 2018
Metformin (06) Insulin-induced insulin resistance is real
When I started reading about insulin-induced insulin resistance I began with this paper:
Insulin Is a Stronger Inducer of Insulin Resistance than Hyperglycemia in Mice with Type 1 Diabetes Mellitus (T1DM)
It's a nice paper. They took NOD mice which had developed their NOD mouse version of T1DM and either treated them with insulin detemir, or didn't. They had a third group which never developed T1DM so were never treated and these served as a control group.
The treated diabetic NOD mice were gradually stabilised over a two week period then kept normoglycaemic for a further two days. They were assessed for insulin sensitivity using an insulin tolerance test, where a dose of neutral insulin is injected then you track what happens to the blood glucose concentration. The more insulin sensitive the animal, the more the glucose level drops:
It's pretty obvious that the detemir treated mice (top line) have absolutely no response to neutral insulin and that both non-treated diabetic mice and never-diabetic mice drop their blood glucose levels by about 50% on this particular dose of neutral insulin.
I could stop this post here. Exogenous insulin induces insulin resistance in T1DM mice, as it does in people. This is fact.
But of course you should not just accept this. The question is: Why?
Why does "enough" insulin as secreted by the pancreas to produce normoglycaemia (in the never-diabetic control group of mice) cause no insulin resistance whereas insulin detemir given to produce the same level of normoglycaemia induces striking insulin resistance in those treated NOD mice?
Recall that the hyperglycaemia in T1DM has little to do with the lack of insulin per se. The hyperglycaemia is caused by an excess of glucagon from the alpha cells of the pancreas. Insulin starts its control of hyperglycaemia by the suppression of pancreatic glucagon secretion, it's a local action within the islets. How high this concentration of insulin is under normal physiological conditions is quite hard to determine but it is likely to be a lot higher than the diluted insulin concentration in the portal vein, heading towards the liver.
The diluted insulin within the portal vein arrives at the liver where its next job is to suppress hepatic glucose output, again in antagonism to glucagon.
Finally, if glucose from the liver continues to enter the systemic circulation, the function of insulin here is to push that glucose in to any cells that will take it. Muscle and adipose tissue being two major targets.
So under normal physiology there is a gradient of insulin concentrations from very high within the Islets of Langerhans, to significantly lower at the hepatocytes, down to much lower in the systemic circulation.
Exogenous insulin produces no such gradient. It drains from its injection site into the systemic veins and is then redistributed, at a single concentration, throughout the body.
This will never effectively suppress alpha cell glucagon secretion and will only do a modestly effective job of suppressing hepatic glucose output. So glucose will be continuously secreted in to the systemic circulation. The dose of detemir used has to be enough to mop up this excess glucose supply, and it can only put it in to cells sensitive to insulin throughout the body. Muscle cells. Adipocytes.
Now look at it from recipient cell's point of view. Glucagon is high, hepatic glucose output is high and this continuous supply of glucose is being allowed in to systemic cells by the exogenous insulin. To these cells the glucose supply looks like a meal being digested (high glucose, high insulin). The cells rapidly realise that they have enough calories (High NADH levels, high mitochondrial delta psi, reduced electron transport chain). They don't want any more. Their solution: insulin-induced insulin resistance (ie reverse electron transport to generate H2O2 at complex I and so inactivate insulin signalling).
That's what happens.
So the very effective control of blood glucose in these NOD mice is at the cost of continuous exposure to supraphysiological insulin levels coupled with a supraphysiological glucose supply, because the systemic cells are "covering for" the failure to replicate the normal gradient from islet to liver to systemic circulation.
Exogenous insulin can never be physiological.
Aside: Except, of course, under deeply ketogenic eating where only minimal insulin is ever secreted, very little is metabolised, the gradients between alpha cells, hepatocytes and adipocytes flattens out and the correct physiology is for glucagon to be elevated with minimal insulin. I've posted this before. T1DM patients have no choice, ketosis is the only physiological state which can be fairly well mimicked using very low doses of exogenous insulin. End aside.
I would never suggest that exogenous insulin has no effect on pancreatic glucagon secretion or elicits no suppression of hepatic glucose output. It will always have some effect, but there will always be an abnormal emphasis of its effect on systemic tissues.
This is the situation in NOD mice and T1DM people. They become insulin resistant by simply using exogenous insulin to ensure normoglycaemia.
I guess the next question, which was asked by the same group whose paper we've just been looking at, is whether simply injecting exogenous insulin in to normal mice induces insulin resistance. That's the next paper, in the next post.
Spoiler: Of course it does.
Peter
Insulin Is a Stronger Inducer of Insulin Resistance than Hyperglycemia in Mice with Type 1 Diabetes Mellitus (T1DM)
It's a nice paper. They took NOD mice which had developed their NOD mouse version of T1DM and either treated them with insulin detemir, or didn't. They had a third group which never developed T1DM so were never treated and these served as a control group.
The treated diabetic NOD mice were gradually stabilised over a two week period then kept normoglycaemic for a further two days. They were assessed for insulin sensitivity using an insulin tolerance test, where a dose of neutral insulin is injected then you track what happens to the blood glucose concentration. The more insulin sensitive the animal, the more the glucose level drops:
It's pretty obvious that the detemir treated mice (top line) have absolutely no response to neutral insulin and that both non-treated diabetic mice and never-diabetic mice drop their blood glucose levels by about 50% on this particular dose of neutral insulin.
I could stop this post here. Exogenous insulin induces insulin resistance in T1DM mice, as it does in people. This is fact.
But of course you should not just accept this. The question is: Why?
Why does "enough" insulin as secreted by the pancreas to produce normoglycaemia (in the never-diabetic control group of mice) cause no insulin resistance whereas insulin detemir given to produce the same level of normoglycaemia induces striking insulin resistance in those treated NOD mice?
Recall that the hyperglycaemia in T1DM has little to do with the lack of insulin per se. The hyperglycaemia is caused by an excess of glucagon from the alpha cells of the pancreas. Insulin starts its control of hyperglycaemia by the suppression of pancreatic glucagon secretion, it's a local action within the islets. How high this concentration of insulin is under normal physiological conditions is quite hard to determine but it is likely to be a lot higher than the diluted insulin concentration in the portal vein, heading towards the liver.
The diluted insulin within the portal vein arrives at the liver where its next job is to suppress hepatic glucose output, again in antagonism to glucagon.
Finally, if glucose from the liver continues to enter the systemic circulation, the function of insulin here is to push that glucose in to any cells that will take it. Muscle and adipose tissue being two major targets.
So under normal physiology there is a gradient of insulin concentrations from very high within the Islets of Langerhans, to significantly lower at the hepatocytes, down to much lower in the systemic circulation.
Exogenous insulin produces no such gradient. It drains from its injection site into the systemic veins and is then redistributed, at a single concentration, throughout the body.
This will never effectively suppress alpha cell glucagon secretion and will only do a modestly effective job of suppressing hepatic glucose output. So glucose will be continuously secreted in to the systemic circulation. The dose of detemir used has to be enough to mop up this excess glucose supply, and it can only put it in to cells sensitive to insulin throughout the body. Muscle cells. Adipocytes.
Now look at it from recipient cell's point of view. Glucagon is high, hepatic glucose output is high and this continuous supply of glucose is being allowed in to systemic cells by the exogenous insulin. To these cells the glucose supply looks like a meal being digested (high glucose, high insulin). The cells rapidly realise that they have enough calories (High NADH levels, high mitochondrial delta psi, reduced electron transport chain). They don't want any more. Their solution: insulin-induced insulin resistance (ie reverse electron transport to generate H2O2 at complex I and so inactivate insulin signalling).
That's what happens.
So the very effective control of blood glucose in these NOD mice is at the cost of continuous exposure to supraphysiological insulin levels coupled with a supraphysiological glucose supply, because the systemic cells are "covering for" the failure to replicate the normal gradient from islet to liver to systemic circulation.
Exogenous insulin can never be physiological.
Aside: Except, of course, under deeply ketogenic eating where only minimal insulin is ever secreted, very little is metabolised, the gradients between alpha cells, hepatocytes and adipocytes flattens out and the correct physiology is for glucagon to be elevated with minimal insulin. I've posted this before. T1DM patients have no choice, ketosis is the only physiological state which can be fairly well mimicked using very low doses of exogenous insulin. End aside.
I would never suggest that exogenous insulin has no effect on pancreatic glucagon secretion or elicits no suppression of hepatic glucose output. It will always have some effect, but there will always be an abnormal emphasis of its effect on systemic tissues.
This is the situation in NOD mice and T1DM people. They become insulin resistant by simply using exogenous insulin to ensure normoglycaemia.
I guess the next question, which was asked by the same group whose paper we've just been looking at, is whether simply injecting exogenous insulin in to normal mice induces insulin resistance. That's the next paper, in the next post.
Spoiler: Of course it does.
Peter
Sunday, January 14, 2018
A wander off in to dietary protein calories
There is prize for developing the longest-lived mouse in the world. It was set up in 2003 and the first award went to Dr Bartke.
"On June 8th, 2003, the inaugural Methuselah Prize was awarded to Dr. Andrzej Bartke for the "Methuselah Mouse" that lived the equivalent of 180 human years".
You can read a bit more about growth hormone receptor knockout mice and other forms of dwarf mice in Dr Bartke's review, written soon after winning the prize:
Life extension in the dwarf mouse.
It's now 2018 and no one appears to have improved on the Laron mouse model which won that initial prize. Over the last 15 years there has been a lot of interesting research but no numerical progress. I think it is worth noting that Laron mice are not GH deficient, they have tons of the stuff. They simply do not have the receptor to do anything with it. Which, in particular, means they cannot generate IGF-1.
How do Laron humans fare? The best studied group live in Colombia. They're of very short stature. They have no recorded cases of diabetes and only one recorded cancer, which was non lethal*. Their every biochemical parameter is exemplary, especially insulin level and HOMA score. Do they all live to be centenarians? Apparently not. Being a dwarf in Columbia requires alcohol in large amounts to render life tolerable, plus accidental trauma is another huge problem. Quite what would happen if these people lived under similar conditions to the Laron mice in Dr Bartke's laboratory is a question which is unlikely ever to be answered! Longevity in the real world vs what works under ideal conditions...
*Dr Laron has reported two cases of Laron Syndrome people developing diabetes (and there are others), including the complications such as atherosclerosis, renal disease and diabetic retinopathy. This is an interesting observation and might be worth a post on its own some time.
There are tantalising suggestions from other GH modifying mutations in humans. One of the better studied of these is carried by the "Little people" of Krk in Croatia. More from Dr Laron:
Do deficiencies in growth hormone and insulin-like growth factor-1 (IGF-1) shorten or prolong longevity?
Longevity of the hypopituitary patients from the island Krk: a follow-up study
They have a mutation which causes multiple pituitary hormone deficits, ACTH secretion excepted. There are too few documented people with this genetic problem to say a great deal about longevity but ages of 68, 77, 83 and 91 years have been recorded in the four individuals to have died since detailed observations began. The equivalent syndrome in mice under lab conditions promotes longevity.
One of the nicer studies looking at human height (viewing this as a GH/IGF-1 signalling surrogate) and longevity is this one:
Shorter Men Live Longer: Association of Height with Longevity and FOXO3 Genotype in American Men of Japanese Ancestry
It found, as you might expect, an inverse relationship between height at enrolment and longevity. They also tied the relationship, observationally, to a down-regulating SNP of the FOXO3 gene, FOXO genes being major controllers of the insulin/IGF-1 signalling system.
Which genes you have is not under your control. What you do with then might well be...
Let's finish this post with the LoBAG diet. It's modest (20% of calories) in carbohydrate, has 30% of calories from protein and the rest as fat. It's being compared to a diet with similar carbohydrate content, 15% of calories from protein, with the rest as fat. Lots of details in here:
The metabolic response to a high-protein, low-carbohydrate diet in men with type 2 diabetes mellitus
As they say in the discussion:
"The present data indicate that the increase in IGF-1 is the result of the increase in protein content. The further decrease in carbohydrate did not result in a further increase in IGF-1. In fact, the increase was approximately the same (138% and 136%, respectively)".
What interested me initially (and had made me chase the paper) was the effect on GH itself. The LoBAG diet actually drops GH levels, admittedly by a ns amount. What turns out to be a much more interesting incidental finding is that, despite the downward trend in GH, IGF-1 rises by a statistically significantly and possibly by a biologically significant amount. Especially when you consider a whole slew of cancers sprout IGF-1 receptors on their surface.
Brief aside. You have to be very careful with GH and IGF-1 levels in papers like this one as both hormones come with a whole load of plasma binding proteins which very few people, including the LoBAG folks, ever measure. These may well alter the effective concentration of the hormone either upwards or downwards. Caution is needed with simple measurements like those in this paper. End aside.
So. Folks should eat whatever they feel comfortable with, protein-wise. I probably eat a little more protein than I would prefer, but then I'm no perfectionist. What I wouldn't do is to add protein gratuitously to any meal...
But that's just me I guess.
Peter
"On June 8th, 2003, the inaugural Methuselah Prize was awarded to Dr. Andrzej Bartke for the "Methuselah Mouse" that lived the equivalent of 180 human years".
You can read a bit more about growth hormone receptor knockout mice and other forms of dwarf mice in Dr Bartke's review, written soon after winning the prize:
Life extension in the dwarf mouse.
It's now 2018 and no one appears to have improved on the Laron mouse model which won that initial prize. Over the last 15 years there has been a lot of interesting research but no numerical progress. I think it is worth noting that Laron mice are not GH deficient, they have tons of the stuff. They simply do not have the receptor to do anything with it. Which, in particular, means they cannot generate IGF-1.
How do Laron humans fare? The best studied group live in Colombia. They're of very short stature. They have no recorded cases of diabetes and only one recorded cancer, which was non lethal*. Their every biochemical parameter is exemplary, especially insulin level and HOMA score. Do they all live to be centenarians? Apparently not. Being a dwarf in Columbia requires alcohol in large amounts to render life tolerable, plus accidental trauma is another huge problem. Quite what would happen if these people lived under similar conditions to the Laron mice in Dr Bartke's laboratory is a question which is unlikely ever to be answered! Longevity in the real world vs what works under ideal conditions...
*Dr Laron has reported two cases of Laron Syndrome people developing diabetes (and there are others), including the complications such as atherosclerosis, renal disease and diabetic retinopathy. This is an interesting observation and might be worth a post on its own some time.
There are tantalising suggestions from other GH modifying mutations in humans. One of the better studied of these is carried by the "Little people" of Krk in Croatia. More from Dr Laron:
Do deficiencies in growth hormone and insulin-like growth factor-1 (IGF-1) shorten or prolong longevity?
Longevity of the hypopituitary patients from the island Krk: a follow-up study
They have a mutation which causes multiple pituitary hormone deficits, ACTH secretion excepted. There are too few documented people with this genetic problem to say a great deal about longevity but ages of 68, 77, 83 and 91 years have been recorded in the four individuals to have died since detailed observations began. The equivalent syndrome in mice under lab conditions promotes longevity.
One of the nicer studies looking at human height (viewing this as a GH/IGF-1 signalling surrogate) and longevity is this one:
Shorter Men Live Longer: Association of Height with Longevity and FOXO3 Genotype in American Men of Japanese Ancestry
It found, as you might expect, an inverse relationship between height at enrolment and longevity. They also tied the relationship, observationally, to a down-regulating SNP of the FOXO3 gene, FOXO genes being major controllers of the insulin/IGF-1 signalling system.
Which genes you have is not under your control. What you do with then might well be...
Let's finish this post with the LoBAG diet. It's modest (20% of calories) in carbohydrate, has 30% of calories from protein and the rest as fat. It's being compared to a diet with similar carbohydrate content, 15% of calories from protein, with the rest as fat. Lots of details in here:
The metabolic response to a high-protein, low-carbohydrate diet in men with type 2 diabetes mellitus
As they say in the discussion:
"The present data indicate that the increase in IGF-1 is the result of the increase in protein content. The further decrease in carbohydrate did not result in a further increase in IGF-1. In fact, the increase was approximately the same (138% and 136%, respectively)".
What interested me initially (and had made me chase the paper) was the effect on GH itself. The LoBAG diet actually drops GH levels, admittedly by a ns amount. What turns out to be a much more interesting incidental finding is that, despite the downward trend in GH, IGF-1 rises by a statistically significantly and possibly by a biologically significant amount. Especially when you consider a whole slew of cancers sprout IGF-1 receptors on their surface.
Brief aside. You have to be very careful with GH and IGF-1 levels in papers like this one as both hormones come with a whole load of plasma binding proteins which very few people, including the LoBAG folks, ever measure. These may well alter the effective concentration of the hormone either upwards or downwards. Caution is needed with simple measurements like those in this paper. End aside.
So. Folks should eat whatever they feel comfortable with, protein-wise. I probably eat a little more protein than I would prefer, but then I'm no perfectionist. What I wouldn't do is to add protein gratuitously to any meal...
But that's just me I guess.
Peter
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