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Sunday, 11 January 2015

The $64,000 Question


I've been obsessed with this question. It all started after reading the literature of pre-insulin treatment of diabetes and insulin-free animal models of the disease. Feeding fats and restricted protein, with no or minimal carbohydrate, gives the best prognosis without insulin; also fasting, which tends towards a similar mix of substrates. Then I read the literature showing inferior prognosis of type 2 diabetes with higher-carbohydrate, post-1977 diets; the longer carbohydrate is fed, the more fasting glucose climbs. Most of the glucose in the blood of diabetics comes from hepatic GNG, not dietary carbohydrate (I'm grateful to Carbsane for pointing this out to me originally - it's an important point).
I read the Richard K. Bernstein paper which describes tight control of glucose using low doses of insulin and a low carb diet, and then I watched the Robert Unger lecture I linked to in the previous post, and saw a slide of blood glucose levels in a mouse with no beta cells, given insulin normally (wide fluctuations ranging into hypo- and hyperglycaemia) or given insulin plus a glucagon antagonist.



Also compare the recent case study "Type 1 diabetes mellitus successfully managed with the paleolithic ketogenic diet" by  Tóth and Clemens.

"He was put on insulin replacement therapy (38 IU of insulin) and standard conventional diabetes diet with six meals containing 240 grams carbohydrate daily. He followed this regime for 20 days. While on this regime his glucose levels fluctuated between 68–267 mg/dL.
Average blood glucose level while on the standard diabetes diet with insulin was 119 mg/dL while 85 mg/dL on the paleolithic-ketogenic diet without insulin. Fluctuations in glucose levels decreased 
as indicated by a reduction of standard deviation values from 47 mg/dL on the standard diabetes diet to 9 mg/dL on the paleolithic-ketogenic diet. Average postprandial glucose elevation on the standard diabetes diet was 23 mg/dL while only 5.4 mg/dL on the paleolithic-ketogenic diet." 

Again the question - why does LCHF (or fasting) act like the glucagon receptor antagonist? Why does feeding glucose worsen hyperglycaemia and ketoacidosis, and fat improve them, when fat, and protein, not glucose, are the gluconeogenic and ketogenic substrates?
Could it be that in diabetes - when there is no insulin present, or when the cells of the liver are highly insulin-resistant, or when subcutaneous insulin fails to give attain an adequate concentration in the portal vein feeding the liver - glucose itself in some way promotes gluconeogenesis and ketogenesis?
Consider first that in uncontrolled diabetes blood glucose is very high and becomes even higher after carbohydrate feeding. This is especially so in the portal vein feeding the liver. Hepatocytes without insulin are not resistant to glucose, especially at high concentrations. The Glut2 receptor is not wholly controlled by insulin, though the metabolism of glucose within the cell is.


Concentrations of glucose approaching 10 mM are pre-diabetic levels. Concentrations of glucose above 10 mM are analogous to a diabetic condition within the cell culture system. This is important because the same processes that can affect cells and molecules 
in vivo can occur in vitro. The consequence to growing cells under conditions that are essentially diabetic is that cells and cell products are modified by the processes of glycation and glyoxidation. These processes cause post-translational secondary modifications of therapeutic proteins produced in cell cultures. [Sigma cell culture guide]
This excess glucose is getting into the cell, and is modifying its metabolism in ways that promote and increase the hormonal action of glucagon.

For example, in this mouse study.

Glucotoxicity Induces Glucose-6-Phosphatase Catalytic Unit Expression by Acting on the Interaction of HIF-1α With CREB-Binding ProteinA. Gautier-Stein et al. 2012.

We deciphered a new regulatory mechanism induced by glucotoxicity. This mechanism leading to the induction of HIF-1 transcriptional activity may contribute to the increase of hepatic glucose production during type 2 diabetes.

If that's true in humans (and I have to say it's very unlikely that glucotoxicity will do anything good for you) then minimising post-prandial glucose spikes is going to help keep a lid on fasting glucose levels as well.

There's also the concept of reductive stress; the metabolism of excess glucose will result in a buildup of NADH and a relative deficiency of NAD+. The cell copes with this by a number of mechanisms. Ketogenesis itself helps, because the conversion of acetoacetate to Beta- hydroxybutyrate generates NAD+. 
Under conditions of high glucose, glyceraldehyde-3-phosphate will build up in the cell unless cytoplasmic NADH is continuously re-oxidized. Cells oxidize cytoplasmic NADH by a combination of three pathways, the aspartate:malate shuttle, the glycerol:phosphate shuttle and during the conversion of pyruvate to lactate.Pyruvate may not enter the mitochondria. It may be reduced to lactic acid by lactic acid dehydrogenase. This reaction is driven when the cell’s need to oxidize NADH to NAD for use as a substrate to keep glycolysis working. Pyruvate reacts with hydrogen peroxide and forms water, carbon dioxide and acetic acid. This non-enzymatic reaction helps the cell defend itself from oxidative intermediates.

Now, in our model, pyruvate will not enter the mitochondria, because that step is controlled by insulin. This means that lactate will either be recycled to glucose or exported. So what is the link between lactate and diabetes?
Plasma lactate predicts type 2 diabetes here.
And lactic acidosis is a common finding in cases of diabetic ketoacidosis, here.
In starvation (very good account here, thanks to Ash Simmonds for the link),
 pyruvate, lactate, and alanine are exported to the liver for conversion into glucose. So, glucose is a gluconeogenic substrate. Meanwhile the poor hepatocyte is trying to oxidise fatty acids, making some ketone bodies in the process, but also struggling with the need to fend off, by metabolizing, devastatingly high glucose concentrations.I speculate that the liver's ATP needs are not being met under these conditions (of futile cycling), and that this is a trigger that increases sensitivity to the lipolytic effect of glucagon in adipocytes (as it is supposed to increase appetite in the liver homeostasis model of appetite regulation, how no-one knows).
And that ketogenesis is also increased by glucotoxicity. But the mechanism of all this is beyond me at present, I'm just sayin' that these are possibilities.

I don't feel that I've answered the $64,000 question yet. But I do think that idea of a glucose -> gluconeogenesis vicious cycle has merit in the type of imbalanced systems we've been looking at, where adding glucose has been a bad idea, and removing it a good one, since history began.

Now it may be that the answer is very obvious and doesn't need any of these baroque explanations.
In which case, please feel free to tell me. All I want is a formula that's consistent with every fact. Is that too much to ask?

17 comments:

Steve Parker, M.D. said...

I've been struggling with some of these concepts, too. I found a potentially interesting tidbit in a 2012 Unger article, under the heading "Glucagon and the glycemic volatility of T1DM":

"For example, it is not widely appreciated that, when hyperglycemia is unaccompanied by an increase in insulin, it stimulates rather than suppresses glucagon secretion. This paradoxical increase in glucagon could be an important factor in the exaggerated postprandial hyperglycemia of T1DM. If β cells are not juxtaposed to α cells to provide a glucose-stimulated paracrine “squirt” of insulin, postprandial hyperglycemia will stimulate a paradoxical rise of glucagon secretion, rather than trigger suppression of its release. This adds an endogenous source of glucose to the exogenous glucose from the meal."

As always when I read Unger, I'm not always sure when he's referring to humans or a particular rodent.

Here's the article: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3248306/

-Steve

Steve Parker, M.D. said...

Just one more thing...
That case report of T1DM treated with a ketogenic/paleo diet is odd in that the authors didn't even discuss the possibility that their patient was simply experiencing the well-known "honeymoon" or remission phase of T1 DM, which can last for weeks, months, or a few years.

George Henderson said...

They do mention it, but say that honeymoon differs in that it results from good response to insulin and doesn't usually dispense with it.

George Henderson said...

The Unger quote is perfect.
In humans, carb feeding does the same (see last post). Might be the gut glucagon too from the timing of the curve.

George Henderson said...

Competing hypotheses so far;

Leptin as a partial glucagon antagonist (like insulin it signals the fed state, but at a later, post-absorptive stage). See
http://www.sciencedaily.com/releases/2013/09/130903123358.htm

This might explain an added benefit of fat
http://www.jstor.org/discover/20347115?sid=21105051366771&uid=2&uid=3738776&uid=4

Second, the glucotoxicity hypothesis. In this regard,
Wiki has this curious line about Glut2
"GLUT2 appears to be particularly important to osmoregulation, and preventing edema-induced stroke, transient ischemic attack or coma, especially when blood glucose concentration is above average.[11] GLUT2 could reasonably be referred to as the "diabetic glucose transporter" or a "stress hyperglycemia glucose transporter"

- glucose stimulates glucagon release, without insulin this isn't reined in


Other hypothetical mechanisms -

- at lower blood glucose more ketone bodies are being oxidised by neurons and other cells, lowering level in blood

- ketoacidosis is manly due to changes in fluid volume and pH for which glycosuria is more proximal cause than ketosis (this seems fairly sound but doesn't explain decreased ketonuria and glycosuria)

- amino acids are a significant source of ketone bodies in T1D, also protein elevates glucagon (effect of protein here is easy to explain - pre-insulin diets were protein-restricted)
see that great 1944 paper linked above.

- adipose lipolysis is perhaps inhibited by dietary fat independent of insulin
(see this paper on FFAs)
http://www.ncbi.nlm.nih.gov/pubmed/22338010


George Henderson said...

Also, in this famous mouse polyol pathway paper there is a rise in serum BOHB when glucose is fed to the knockout mice.
http://www.ncbi.nlm.nih.gov/pubmed/24022321

PhilT said...

I got stuck at "Most of the glucose in the blood of diabetics comes from hepatic GNG". Maybe this is lacking in context, for a T2D on a standard diet of around 50% carbohydrate it cannot be correct, surely.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC443176/ reports 10 micromols/kg of body weight per minute of GNG in a fasted state. So in 100kg person that's 1 mmol/min or 1.44 mol/day = 260 grams/day which is about the same as the glucose intake of such a person in the diet.

Given the GNG would be depressed by eating, at least postprandially, and also by release from glycogen reserves in fed subjects, I don't see how it can be even half of the blood glucose supply.

Larcana said...

Interesting series. Thanks for the effort. I had thought some of the lactic acid build up was from muscle secondary to vasoconstriction during the DKA. I know that when we administer glucose/insulin drips to bring down the high blood glucose that the pH stay elevated form a time due to a delay in reperfusion. Does this add to the elevation and could it be a confounder?

George Henderson said...

@PhilT, yes there is plainly going to be a difference between poorly controlled T1D and T2D where there is some insulin secretion.
There will be no post-prandial decrease in GNG when glucose is fed w/o insulin and an increase instead.
@)larcana, it occurred to me that the lactate could be an artefact from muscle, but it showing up early in disease progression of T2D might indicate glucotoxicity.
The scenario has a metabolic effect of excessive glucose concentrations superimposed upon a hormonal effect of excess glucagon.
These two conditions do not coexist in normal metabolism.

George Henderson said...

If we come back to aetiology, which is of prime concern in a reversible disease like T2D, we find that glucotoxicity leads lipotoxicity in beta-cell failure. This would also apply to Unger's ceramide model of alpha-cell insulin resisstance.
http://www.ncbi.nlm.nih.gov/pubmed/11796484
"We propose that chronic hyperglycemia, independent of hyperlipidemia, is toxic for beta-cell function, whereas chronic hyperlipidemia is deleterious only in the context of concomitant hyperglycemia."

We are taught that metabolism is controlled by hormones. But what happens to a cell's metabolism when the force majeure of substrate availability is an egregious mismatch with hormonal instructions, i.e. hyperglycaemia plus glucagon?
"Uniquely, glucose entry into liver cells may occur by simple passive diffusion" - Principles of Biochemistry 5th Edition

George Henderson said...

Here's a good paper about glucotoxicity causing insulin resistance in hepatoma cells (the cell culture version of hepatocytes). It discusses the GLUT2 uptake of glucose being insulin-independent.
http://www.jbc.org/content/275/27/20880.full.pdf

George Henderson said...

A nice Unger quote
"As insulin is a powerful inhibitor of glucagon, we propose that within-islet inhibition of α-cells by β-cells creates an insulin-to-glucagon ratio that maintains glycemic stability even in extremes of glucose influx or efflux. By contrast, in type 1 diabetes mellitus, α-cells lack constant action of high insulin levels from juxtaposed β-cells. Replacement with exogenous insulin does not approach paracrine levels of secreted insulin except with high doses that “overinsulinize” the peripheral insulin targets, thereby promoting glycemic volatility. Based on the stable normoglycemia of mice with type 1 diabetes during suppression of glucagon with leptin, we conclude that, in the absence of paracrine regulation of α-cells, tonic inhibition of α-cells improves the dysregulated glucose homeostasis."

"It is not widely appreciated that, when glucose increases without a parallel insulin increase, hyperglycemia stimulates glucagon. This is crucially important in T1DM, in which insulin levels do not increase when glucose increases. The hyperglycemia will therefore paradoxically stimulate glucagon secretion (21, 22), as illustrated in Fig. 4"

From http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2941311/

That is really most of the answer I wanted. Hepatic glucotoxicity (vicious cycle) adds another mechanism, and then there's the fact that pretty much any intake of exogenous carbs (including fructose) will delay the clearance and oxidation of endogenous (GNG) glucose and ketone bodies.

The link between diet and leptin in diabetes is another route to follow.

George Henderson said...

From 1973 (Unger started publishing in 1970 but this is earliest free abstract)

The prevalence of hyperglucagonemia was studied in twenty-six consecutive patients admitted to Dallas hospitals in diabetic ketoacidosis. Plasma glucagon averaged 390 pg/ml, ranging from 120 to 1,290 pg/ml, significantly greater than the fasting level of 118 pg/ml in diabetic subjects without ketoacidosis. Absolute hyperglucagonemia, i.e., levels over 240 pg/ml, was present in sixteen; all had “relative hyperglucagonemia.” Glucagon levels declined after four hours of therapy and were normal at discharge. The plasma glucagon level was significantly correlated with the blood glucose level, respiratory rate and hours of insulin therapy required to correct the ketonemia. Patients with absolute hyperglucagonemia required significantly more insulin than patients without absolute hyperglucagonemia.

The results indicate that glucagon excess is present in most patients hospitalized with diabetic ketoacidosis and are compatible with the view that glucagon, an insulin-opposing hormone, may increase the severity of the disease and its insulin requirements.

http://www.sciencedirect.com/science/article/pii/0002934373900831

George Henderson said...

Correction - I have traced Roger Unger's publishing history back to 1958, where he is measuring portal insulin vs injected. He is credited with proving that glucagon is a true hormone, for which he received the Banting Medal of the ADA in 1988.
An intimate 2001 interview with Roger Unger which discusses the history of his researches.
https://www.endocrine.org/~/media/endosociety/Files/About%20Us/Sawin/roger-unger-031309.pdf

PhilT said...

I'm still not buying ""Most of the glucose in the blood of diabetics comes from hepatic GNG"

On a 50% carbohydrate diet there's 200 -300 grams a day of glucose from the diet.

As 50% of the energy need is coming from dietary carbohydrate, the quantity of GNG can't even be as much as that so certainly not "most".

What am I missing.

PhilT said...

Found one data point - "Total and endogenous rates of glucose appearance (3,091±523 and
1,814±474 mg/kg per 8h) in the diabetic subjects were significantly (P<0.02) greater than those in non-diabetic subjects (1,718±34 and 620±98 mg/kg per 8h, respectively)"

60 / 40 and 64 / 36 resp (endogenous production from GNG and glycogen vs from food).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC425257/pdf/jcinvest00135-0325.pdf

George Henderson said...

OK I'm not sure I remembered the line clearly, but the gist of it was that endogenous glucose was something that we low carbers (at the time) were overlooking. Which as far as it goes was a very useful insight.
It would depend on how much carbohydrate and protein one ate and how much beta-cell function one had.
Even without diabetes, if you are eating <40g carb your GNG production will be greater than 40g, unless perhaps you are deeply ketoadapted.

What complicates things is a high cycling flux of dietary glucose through GNG. I've seen both 65% and 85% cited. That is, that this % of glucose that is taken up by hepatocytes will be cycled through the TCA back to glucose.
So, is this to be counted as dietary or endogenous gluucose?