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Monday, 23 February 2015

Why the High-Fat Hep C Diet? Rationale and n=1 results.



I originally started this blog to publicise the hypothesis that a diet low in carbohydrate and linoleic acid, but high in saturated fat and long-chain PUFA, will inhibit HCV replication.

The blog header with the pig above is the abstract for this hypothesis.

I first worked this out in 2010 after reading Dr Atkins New Diet Revolution while studying HCV replication. The lipid patterns in low-carb dieters - low TG and VLDL, high HDL, normal or high LDL - are those associated with lower viral load and improved response to treatment in HCV cases.
The mechanics of HCV replication and infection support this link.


HCV inhibits PPAR-a, a ketogenic diet reverses this inhibition

I wrote a fairly comprehensive version of the hypothesis in 2012:
http://hopefulgeranium.blogspot.co.nz/2012/02/do-high-carbohydrate-diets-and-pufa.html

Recently I was sent a link to an article that cited this paper:
http://www.journal-of-hepatology.eu/article/S0168-8278(11)00492-2/pdfHCV and the hepatic lipid pathway as a potential treatment target. Bassendine MF, Sheridan DA , Felmlee DJ, et al. Journal of Hepatology 2011 vol. 55 j 1428–1440

This review compiles a great deal of supporting evidence regarding the interaction between HCV and lipids, and between lipids and HCV. The only thing missing is the role of carbohydrate. It mentions multiple lipid synthetic pathways as targets for indirect-acting antiviral drugs (IDAA), pathways which are also well documented as targets of low carbohydrate ketogenic diets, or of saturated fat in the diet (in the case of the LDL-receptor complex).

From 2012:
A little n=1 experimental data; 4 years ago (2008) my viral load was 400,000 units, now after 2 years of low carb dieting and intermittent mild ketosis (2012) it is 26,000.

Later in 2012:
Total Cholesterol:  6.7  H     
Triglyceride:          0.8         
HDL:                     1.63              (63.57)
LDL (calc.)            4.7   H    
Chol/HDL ratio:     4.1          

HCV viral load on this day (21st May 2012): 60,690 IU/mL (4.78 log)



Lipid panel from 07 Feb 2012, during ketogenic diet phase (non-fasting)

Total Cholesterol: 8.9   HH  (347.1)
Triglyceride:         1.3          (115.7)
HDL:                    1.65         (64.35)
LDL (calc):           6.7    H    (261.3)
Chol/HDL ratio:     5.4   H

HCV viral load on this day: 25,704 IU/mL (4.41 log)

From 2014:
On a personal note, I have started an 8-week trial of Sofosbuvir and GS-5816 (Vulcan). It is day 11 and it seems tolerable so far.
A pre-trial blood test on 22nd October was normal except for these counts:
AST 74
ALT 174

and viral load was 600,419 (log 5.78), counts consistent with the tests I've had done this last year.

But the day the trial started, 18th November, before my first dose, things were different:
AST 21

ALT 32
Viral load 27,167 (log 4.43)

The low viral load is easy to explain; I get a consistent 1 log drop (to 14,000-60,000*) when I try to eat very low carb (50g/day or lower) and an elevation to 400-600,000 when my carbohydrate intake is over 50g/day. When I ate very high carb (but took antioxidant supps) it was as high as it was on 22nd October. So for me the tipping point seems to be where ketosis begins, and other variations don't have much effect; it's an on/off switch, not a dial (and the name of that switch is PPAR-alpha).
[edit: though the very low scores are at ketogenic, or nearly so, carb intakes, the exact increase in carbohydrate needed to cause a significant increase in viral load seemed to vary]
(I do however, according to CAPSCAN elastography, have zero excess fat in my liver, which is an effect of low carb in general, as well as avoiding vegetable seed oils).

My belief is that my viral load was much higher than any of these counts previous to 2003. This was the year I started taking antioxidant supplements, eating a bit better (in a normal, confused "healthy eating" pattern), and using herbal antivirals like silybin. Prior to that I was seriously ill, and I believe that my viral load would have reflected my extra autoimmune symptoms, signs of liver failure, and elevated enzymes. Unfortunately in those days one didn't get a PCR unless one was considering donating one's body to interferon, which I was not.

* I don't seem to have a record of the date of the 14,000 VL reading, but will include it when I find it.

Summary:
A very low carbohydrate ketogenic diet, without enough PUFA to lower LDL artificially, had a significant inhibitory effect on HCV viraemia in my case.
Effective DAA drugs for HCV infection are now available. There is a ~98% SVR rate at present. These drugs are expensive, they sometimes have side effects (though much less so than interferon + ribavirin), and interferon + ribavirin is still being used.
If my results are more generally applicable, VLCKD diet offers an adjunct therapy for patients with a high viral load, steatosis that relates to diet and lifestyle as well as HCV infection, or a need to postpone treatment. In people who oppose or cannot complete or afford treatment, it offers a way to manage the disease, and in particular to reverse the autoimmune syndromes caused by immune complexes when viraemia is excessive.


Friday, 20 February 2015

Gluconeogenesis Drives Ketogenesis - role of the Nutritional Prometheus.

In trying to explain the findings of Newburgh and Marsh*, and of Karl Petren, from 1923 that switching to a high fat, restricted protein, and very low carbohydrate diet - a ketogenic diet - suppresses diabetic ketoacidosis (DKA) in diabetics without access to insulin, I can't help noticing that gluconeogenesis is a driver of ketogenesis. DKA is a dehydrating syndrome characterized by hyperglycaemia, due in large part to runaway gluconeogenesis, plus levels of ketone bodies, much higher than those seen in starvation or nutritional ketosis, which result in a lethal acidosis. And excess glucose and excess ketones are linked metabolically.

Remember the old saw, that fat burns in a carbohydrate flame? Laugh all you like, but this is true. And it is even more true when the flame is taken away - when carbohydrate (glucose) is being stolen from mitochondrial metabolism. Gluconeogenesis involves a direct loss of oxaloacetate from the citric acid (Krebs, TCA) cycle. Without this oxaloacetate, the fat-burning flame sputters; the smoke that escapes from incomplete combustion is the ketone bodies. I'm not a chemist, but this seems to me a pretty certain way of interrupting the TCA cycle. And a very convenient one in evolutionary terms; at times when you need endogenous glucose, you can use a few extra ketones as well.




Pyruvate from glucose can supply acetyl-CoA or oxaloacetate, fatty acids can only supply acetyl-CoA, if there's no oxaloacetate acetyl-CoA can't be converted to citrate and is converted to ketone bodies instead (not shown).


[In Starvation] degradation of fatty acids in the liver proceeds more rapidly than usual, with augmented production of of acetoacetyl-CoA and acetyl-CoA and their products.
In addition there is a deficit of oxaloacetate and thus a decrease in formation of citrate.
The low level of oxaloacetate is further accentuated because it is being utilized for gluconeogenesis.
This further impairs operation of the citric acid cycle.

Ketosis incident to starvation is most frequently encountered clinically in gastrointestinal disturbances in infancy or pregnancy. Other circumstances in normal individuals in which excessive lipid and diminished carbohydrate are being metabolised may also lead to ketosis, e.g. renal glycosuria and abrupt replacement of a normal diet by one low in carbohydrate and very rich in lipid.

Clinically, the most important cause of ketosis is diabetes mellitus. In the diabetic individual, in contrast to the above situations, glucose is present in excessive amounts in the fluids of the body; however, the metabolic defect, viz., insulin deficiency, prevents glucose utilization from operating at a normal rate. From the point of view of the effect upon lipid metabolism, diabetes and starvation resemble one another.

In diabetic individuals with severe ketosis, urinary excretion of ketone bodies may be as high as 5,000mg/24 h and the blood concentration may reach 90mg/100ml, in contrast to normal values of less than 125mg and less than 3mg respectively.


Ketogenesis, from Principles of Biochemistry, 5th Edn, White A, Handler P, Smith EL. McGraw Hill, 1973, p577-578.


[NB: acetyl-CoA is also a precursor for cholesterol;
"The data suggest that, although acetyl-CoA is channeled towards ketone body formation in both diabetes and fasting, augmented cholesterol synthesis is evident only in diabetes." This suggests that the closer the diabetic diet gets to a ketogenic diet, the less cholesterol synthesis will be augmented - as does seem to be the case in practice.]




So what happens when a diabetic without insulin eats carbohydrate or excess protein?
As we saw in earlier posts, glucagon is released from pancreatic alpha cells in response to carbohydrate and protein. This elevates gluconeogenesis in the liver. Blood glucose is elevated by the meal and by GNG, and hyperglycaemia itself increases hepatic GNG further. Lipolysis is increased by the glucagon, so the liver has additional fatty acids to metabolize. Perfect conditions for ketogenesis to be enhanced above normal levels, because oxaloacetate is being extracted from the TCA in record amounts as this fat is being burned.

What happened when diabetics, in acidosis and without insulin, were switched to the Newburgh and Marsh ketogenic diet in 1923?
With no glucose and minimal protein to trigger glucagon, hepatic GNG is lower. With no glucose to add its load to hyperglycaemia, there is less portal hyperglycaemia to additionally drive GNG.
Less GNG = less ketogenesis.
And, as a bonus, it is likely that dietary fat has an inhibitory effect on lipolysis that is independent of hormonal controls. As long as it's saturated, or not polyunsaturated - in 1923, endocrinologists favoured butter as a source of fat.


Beef tallow diet decreases beta-adrenergic receptor binding and lipolytic activities in different adipose tissues of rat.
Matsuo T, Sumida H, Suzuki M. Metabolism. 1995 Oct;44(10):1271-7.

Abstract
The effects of dietary fats consisting of different fatty acids on lipolytic activity and body fat accumulation were studied in rats. Sprague-Dawley male rats were meal-fed an isoenergetic diet based on either beef tallow or safflower oil for 8 weeks. Lipolytic activities in epididymal and subcutaneous adipose tissues were lower in the beef tallow diet group than in the safflower oil diet group. Body fat accumulation was greater in rats fed the beef tallow diet versus the safflower oil diet. Norepinephrine (NE) turnover rates used as an index of sympathetic activities in adipose tissues were lower in the beef tallow diet group. beta-Adrenergic receptor binding was determined with [3H]dihydroalprenolol. Binding affinities of beta-receptors in adipose tissues were significantly lower in the beef tallow diet group. Membrane fluidities of adipose tissues were also lower in the beef tallow diet group. Membrane fluidities were correlated with the affinities of the beta-receptor. We believe from these correlations that the decreases in beta-receptor binding affinities are due to the changes in membrane fluidities. The results of the present study suggest that intake of the beef tallow diet promotes body fat accumulation by reducing lipolytic activities resulting from lower beta-receptor binding and sympathetic activity in adipose tissues.

Dr Bernstein describes the mechanism of DKA differently; he doesn't consider that the liver is the main source of ketones, or that gluconeogenesis drives ketogenesis. His description addresses the pathology of DKA well, but not I think the early links in the chain of causality. Perhaps the difference is that he is describing the failure of insulin to work, and I am describing the long-term absence of insulin. But we are both agreed; dietary carbohydrate is the cause of DKA in diabetics.

"Furthermore, the higher your blood sugars go, the more insulin resistance you will experience. The more insulin-resistant you are, the higher your blood sugars are going to be.

A vicious circle. To make the circle even more vicious, when you have high blood sugars, you urinate—and of course what happens then is that you get even more dehydrated and more insulin-resistant and your blood sugar goes even higher. Now your peripheral cells have a choice—either die from lack of glucose and insulin or metabolize fat. They’ll choose the latter. But ketones are created by fat metabolism, causing you to urinate even more to rid yourself of the ketones, taking you to a whole new level of dehydration."
See also http://www.diabetes-book.com/ketoacidosis-hyperosmolar-coma/
and http://www.diabetes-book.com/diabetes-dehydration/

Edit: here's a bit more on starvation, from this book
After about 3 days of starvation, the liver forms large amounts of acetoacetate and d-3-hydroxybutyrate (ketone bodies; Figure 30.17). Their synthesis from acetyl CoA increases markedly because the citric acid cycle is unable to oxidize all the acetyl units generated by the degradation of fatty acids. Gluconeogenesis depletes the supply of oxaloacetate, which is essential for the entry of acetyl CoA into the citric acid cycle. Consequently, the liver produces large quantities of ketone bodies, which are released into the blood. At this time, the brain begins to consume appreciable amounts of acetoacetate in place of glucose. After 3 days of starvation, about a third of the energy needs of the brain are met by ketone bodies (Table 30.2). The heart also uses ketone bodies as fuel.

And diabetes:
Because carbohydrate utilization is impaired, a lack of insulin leads to the uncontrolled breakdown of lipids and proteins. Large amounts of acetyl CoA are then produced by β-oxidation. However, much of the acetyl CoA cannot enter the citric acid cycle, because there is insufficient oxaloacetate for the condensation step. Recall that mammals can synthesize oxaloacetate from pyruvate, a product of glycolysis, but not from acetyl CoA; instead, they generate ketone bodies. A striking feature of diabetes is the shift in fuel usage from carbohydrates to fats; glucose, more abundant than ever, is spurned. In high concentrations, ketone bodies overwhelm the kidney's capacity to maintain acid-base balance. The untreated diabetic can go into a coma because of a lowered blood pH level and dehydration.

Note for future research: Mammals can synthesise oxaloacetate from pyruvate, but what if this step depends on insulin (which suppresses ketogenesis) and the conversion of pyruvate to acetyl-CoA doesn't?
The diabetic hepatocyte is swamped with glucose, it can't resist metabolising it, and 65-85% of the carbon from this glucose is recycled as GNG glucose.
What if this glucose, without the guiding hand of insulin, is, like fatty acids, a poor source of oxaloacetate and a good source of acetyl-CoA? After all, its metabolism is not suppressing ketogenesis - the opposite seems to be true.
Ketone bodies for use by heart muscle in normal hepatic metabolism are produced from glycogen, according to the first text I quoted.
So - is glucose itself a ketogenic substrate under certain conditions?
The quest continues...

Sunday, 1 February 2015

The Guinea Pig Model of Atherosclerosis


This is the kind of research that doesn't get as much attention as it used to.
Animal models are vital ways of testing theories that it would be unethical to test on humans, and when theories or novel chemicals are tested on unsuspecting humans, we refer to those humans as "guinea pigs". Not mice, rabbits, rats or any other rodent, but cavia porcellus. The guinea pig was used by Lavosier to demonstrate, by melting snow around his calorimeter, that respiratory gas exchange is a combustion, and by Pasteur, Roux, and Koch in their germ experiments, but fell into neglect at the end of the 20th century: only 2% of laboratory animals in the USA are currently guinea pigs.

In the early history of the lipid hypothesis, the rabbit model of atherosclerosis was developed by Anitschkow in 1911:

"When fed fat and cholesterol, rabbits develop high TC levels and subsequent fatty deposits in their blood vessels. When cholesterol is taken out of their diet, TC levels generally reduce and the fatty deposits may regress. If not used as conclusive evidence as to the process in humans, such experiments are said to be supportive of the theory that under conditions of high TC, cholesterol is more likely to be deposited in human arteries."[1]

The amount of cholesterol fed in these experiments - 0.2% or 0.25% of dry matter - probably exceeds what a human could consume, and of course rodents in nature have a minimal exposure to dietary cholesterol. The lesions seen do not correspond exactly to human atherosclerosis, and the role of saturated and unsaturated fats, or of foods like butter, with regard to progression in rabbits does not always match the lipid hypothesis predictions.

In 2006 Maria Luz Fernandez and Jeff Volek published a paper which should have stirred things up:
"Carbohydrate restricted diets have been shown to reduce plasma triglycerides, increase HDL cholesterol and promote the formation of larger, less atherogenic LDL. However, the mechanisms behind these responses and the relation to atherosclerotic events in the aorta have not been explored in detail due to the lack of an appropriate animal model. Guinea pigs carry the majority of the cholesterol in LDL and possess cholesterol ester transfer protein and lipoprotein lipase activities, which results in reverse cholesterol transport and delipidation cascades equivalent to the human situation. Further, carbohydrate restriction has been shown to alter the distribution of LDL subfractions, to decrease cholesterol accumulation in aortas and to decrease aortic cytokine expression. It is the purpose of this review to discuss the use of guinea pigs as useful models to evaluate diet effects on lipoprotein metabolism, atherosclerosis and inflammation with an emphasis on carbohydrate restricted diets."[2]

Rats and rabbits, on the other hand, don't resemble humans in the way they disburse lipids. The LDL fraction is tiny, they lack CETP and lipoprotein lipase, and generally diverge from human measurements in ways guinea pigs, it seems, don't. Even though rabbits and guinea pigs have pretty much the same natural diet - grasses, and their own poop. Guinea pigs and humans, unlike rats and rabbits, also can't synthesise vitamin C. Is there an orthomolecular connection here?
OMG put that pig on statins stat!
Long story short, guinea pig lipids react to lipid lowering drugs, PUFA and fibre in the same way and via the same mechanisms that produce effects in humans. If you feed a guinea pig cholesterol, fat, and carbohydrate it gets atherosclerosis. If you feed the guinea pig a very low carbohydrate diet (20%E SFA in these experiments - 10%CHO,65%FAT,25%PRO ) it's protected from cholesterol-induced atherosclerosis and inflammation. If you feed it a low-fat diet (55:20:25) the atherosclerotic syndrome progresses [3].

Most recently, the low carb diet reverses the metabolic alterations induced in guinea pigs by high-cholesterol feeding: the high-carb diet doesn't.

"Higher concentrations of total (P < 0.005) and free (P < 0.05) cholesterol were observed in both adipose tissue and aortas of guinea pigs fed the HC compared to those in the LC group. In addition, higher concentrations of pro-inflammatory cytokines in the adipose tissue (P < 0.005) and lower concentrations of anti-inflammatory interleukin (IL)-10 were observed in the HC group (P < 0.05) compared to the LC group. Of particular interest, adipocytes in the HC group were smaller in size (P < 0.05) and showed increased macrophage infiltration compared to the LC group. When compared to the H-CHO group, lower concentrations of cholesterol in both adipose and aortas as well as lower concentrations of inflammatory cytokines in adipose tissue were observed in the L-CHO group (P < 0.05). In addition, guinea pigs fed the L-CHO exhibited larger adipose cells and lower macrophage infiltration compared to the H-CHO group."[4]
Why the guinea pig is such a good model is explained by Maria Luz Fernandez in this 2001 paper, which predates her collaboration with Jeff Volek.[5]
Researchers at our laboratory and other investigators have found that guinea pig cholesterol metabolism does indeed have some analogies to human cholesterol metabolism that merit discussion.
Some of these similarities include the following:
  1. Guinea pigs have high LDL-to-HDL ratios (Fernandez et al. 1990a).
  2. They have higher concentrations of free than of esterified cholesterol in the liver (Angelin et al. 1992).
  3. They have plasma cholesteryl ester transfer protein (CETP)2 (Ha et al. 1982), lecithin-cholesterol acyltransferase (LCAT) (Douglas and Pownell 1991) and lipoprotein lipase (LPL) (Olivecrona and Bengsston-Olivecrona 1993) activities for intravascular processing of plasma lipoproteins.
  4. They exhibit comparable moderate rates of hepatic cholesterol synthesis (Reihner et al. 1990) and catabolism (Reihner et al. 1991).
  5. Similar to humans, the binding domain for the LDL receptor differentiates between normal and familial binding defective apolipoprotein (apo)B-100 (Corsini et al. 1992).
  6. Apo B mRNA editing in the liver is negligible (Greeve et al. 1993)
        7. They require dietary vitamin C (Sauberlich 1978).       
         8. Females have higher HDL concentrations than males (
Roy et al. 2000).
         9. Ovariectomized guinea pigs have a plasma lipid profile similar to that of postmenopausal women (
Roy et al. 2000).         
         10. During exercise in guinea pigs, plasma triacylglycerol (TAG) decreases and plasma HDL cholesterol (HDL-C) increases (
McNamara et al. 1993).
           11. Guinea pigs respond to dietary interventions (
Fernandez and McNamara 1992b1992a and 1995aHe and Fernandez 1998a) and drug treatment (Berglund et al.1989Hikada et al. 1992) by lowering plasma LDL cholesterol (LDL-C)

What we have here is a story of good science that should be better known. Maria Luz Fernandez develops the modern guinea pig lipoprotein model, Jeff Volek recognises its value for testing the carbohydrate hypothesis of atherosclerosis and the safety of LCHF diets, and Fernandez sees the value of such testing in adding to our knowledge of cardiovascular disease and lipid and carbohydrate contributions to inflammation.






[1] Flaws, Fallacies and Facts: Reviewing the Early History of the Lipid and Diet/Heart Hypotheses, Elliot J 2014. Food and Nutrition Sciences. Vol.5 No.19, October 2014 link

[2] Guinea Pigs: a suitable animal model to study lipoprotein metabolism, atherosclerosis and inflammation. Fernandez ML and Volek J. 2006  Nutrition and Metabolism 3:17 doi:10:1186/1743-7075-3-17 link
[3] Low-carbohydrate diets reduce lipid accumulation and arterial inflammation in guinea pigs fed a high-cholesterol diet. Leite JO et al. 2009 Atherosclerosis. 2010 Apr;209(2):442-8. doi: 10.1016/j.atherosclerosis.2009.10.005 link

[4] 
Cholesterol-induced inflammation and macrophage accumulation in adipose tissue is reduced by a low carbohydrate diet in guinea pigs. Aguilar D. et al. 2014 Nutr Res Pract. 2014 Dec;8(6):625-631   http://dx.doi.org/10.4162/nrp.2014.8.6.625  link
[5]  
Guinea Pigs as Models for Cholesterol and Lipoprotein Metabolism. Fernandez, ML 2001 J Nutr. Jan 1 2001 Vol 131 no.1 10-20 link


Friday, 23 January 2015

Testing the effect of a high fat diet in severe type 2 diabetes - Garg and Unger metabolic ward study from 1988



It's all very well to test the metabolic effects of high-fat diets in RCTs. There are usually beneficial results in type 2 diabetes, but compliance is limited. The trial isn't showing what the diet does, but what effect the advice has on people who may be more or less indifferent to it. In fact, it's amazing these trials produce the positive results they do.
A metabolic ward study involves subjects who follow the diet because they have nothing else to eat; all variables such as exercise are kept constant. Because you don''t get huge numbers volunteering for these studies, and the cost is high because of the round-the-clock supervision and testing, the crossover method is normally used. Half the subjects eat the test diet, the other half the control, then they switch over. Results from the end of each period in both groups are averaged.
This is a 1988 study authored by Abhimanyu Garg, Roger Unger and 3 colleagues.


N Engl J Med. 1988 Sep 29;319(13):829-34.

Comparison of a high-carbohydrate diet with a high-monounsaturated-fat diet in patients with non-insulin-dependent diabetes mellitus.

Garg A1, Bonanome A, Grundy SM, Zhang ZJ, Unger RH

Abstract






We compared a high-carbohydrate diet with a high-fat diet (specifically, a diet high in monounsaturated fatty acids) for effects on glycemic control and plasma lipoproteins in 10 patients with non-insulin-dependent diabetes mellitus (NIDDM) receiving insulin therapy. The patients were randomly assigned to receive first one diet and then the other, each for 28 days, in a metabolic ward. In the high-carbohydrate diet, 25 percent of the energy was in the form of fat and 60 percent in the form of carbohydrates (47 percent of the total energy was in the form of complex carbohydrates); the high-monounsaturated-fat diet was 50 percent fat (33 percent of the total energy in the form of monounsaturated fatty acids) and 35 percent carbohydrates. The two diets had the same amounts of simple carbohydrates and fiber. As compared with the high-carbohydrate diet, the high-monounsaturated-fat diet resulted in lower mean plasma glucose levels and reduced insulin requirements, lower levels of plasma triglycerides and very-low-density lipoprotein cholesterol (lower by 25 and 35 percent, respectively; P less than 0.01), and higher levels of high-density lipoprotein (HDL) cholesterol (higher by 13 percent; P less than 0.005). Levels of total cholesterol and low-density lipoprotein (LDL) cholesterol did not differ significantly in patients on the two diets. These preliminary results suggest that partial replacement of complex carbohydrates with monounsaturated fatty acids in the diets of patients with NIDDM does not increase the level of LDL cholesterol and may improve glycemic control and the levels of plasma triglycerides and HDL cholesterol.

Thanks to Ivor Cummings, I have the full-text pdf, and it's very interesting.
The other dietary variables are well controlled for.


The types of fatty acids, if that makes any difference, are also well-matched between diets (low fat diet used corn and palm oils, high fat diet used olive oil, so neither was high omega-3).
The results are fascinating (this is the average from the last week of each period, days 21-28).


Who knew that a urinary glucose output of 142 mg/day was normal on a high-carbohydrate diet in subjects with "non-insulin dependent diabetes mellitus treated with insulin" - to disappear completely on a diet with 50% of calories from olive oil?
Oh, and the base line? That was after a week on the diet recommended by the ADA in 1988, which was the lead-in diet.
What about lipids? They improved too:




What's especially interesting aboout these lipid results is the comparison between this study (second phase T2D) and Garg and Unger's 1992 study of the same diets in mild (first phase) T2D. In mild T2D, a high MUFA diet improved lipids but did not influence insulin sensitivity. This seems consistent with high-carb/high-calorie diets and hyperinsulinaemia in those prone to diabetes driving lipotoxicity, when then produces the phase 2 phenomenon of hyperglycaemia plus hyperlipidaemia by altering the ratio of alpha- to beta- cell sensitivity and activity. Dietary carbohydrate drives fat which drives endogenous glucose.

The authors of the 1988 paper sum up thus:


Abhimanyu Garg has authored this convenient review of all the studies using a high-MUFA diet for Type 2 NIDDM.
It includes this classic line:
 "The improvement in the glycemic profile with high-monounsaturated-fat diets may not be related to changes in insulin sensitivity but to a reduction in the carbohydrate load, which patients with type 2 diabetes may not be able to handle readily because of severe insulin resistance and b cell defects."
Amen.


Of course what we lack is a comparative series of studies with high SFA diets, or indeed diets in the normal range of mixed SFA, MUFA and PUFA. Does the type of fat matter if carbohydrate is low enough? Quite possibly not, at least for the majority. Is 35% carbohydrate low enough to see the full benefit of a high-fat diet? Maybe not, but the results, after only 28 days, were impressive enough.





Friday, 16 January 2015

Some Answers to that Question





Let's back the truck right up to 1923. Insulin was introduced the year before and is now being mass produced by Eli Lilly & Co. Diet research into diabetes has crystallized into a proven diabetic diet (the diet that will best extend life in the absence of insulin). This is defined by Ladd and Palmer in the American Journal of Medical Science of August 1923 with this formula.



Thus we have the classic 1:4 ratio of carbohydrate to fat that defines the limits of ketosis, with the refinement that protein straddles the separation, with 58% of protein counting as carbohydrate (gluconeogenic) and the remainder as fat (ketogenic).
Note the epilogue; "we feel that adequate dietary control will remain the basis of treatment for many cases, especially those of the milder type".
As in this example from David Unwin's 2014 case series. This is the sort of paper I like best today; what it lacks in randomised, controlled rigour (ffs people, the point has already been proven) it makes up for in realism - these types of papers show what it takes to treat real people in clinical practice, it's hard work sometimes but the results are clearly worthwhile.



And here's a picture of Dr Unwin, getting the point across. 

How does this fit with the glucagonocentric restructuring of diabetes by Roger Unger (the "father of glucagon" and 1975 Banting Medal recipient)?

When someone with deficient insulin response (like the woman in Dr Unwin's example - polydipsia is a sign of beta-cell failure) eats carbohydrate, these things happen:

1) Glucose enters the blood and glucagon is elevated as a result.



High glucagon concentrations stimulate the liver to break down glycogen, releasing glucose, and to make more glucose from protein (58%) and triglycerides (10%), as well as ketone bodies.

2) If the blood sugar concentration rises far enough - more likely after a carbohydrate meal - glucose enters the liver cells at an excessive rate. This glucotoxicity increases gluconeogenesis further. In fact I suspect this is the tipping point, and that this high blood glucose and its effect on the liver (and kidneys) is what separates the ketoacidosis of starvation from the lethal ketoacidosis of diabetes.

3) because extra carbohydrate has been consumed in the meal, the clearance by cellular oxidation of hepatic glucose and ketone body output is proportionately delayed, maintaining a higher level of glycaemia than would otherwise be the case.

In 1923 ketogenic diets (then known for the treatment of epilepsy and tested alongside diabetic diets) were not considered suitable for diabetics.
[Edit: this is incorrect as the diabetic diet of Newburgh and Marsh, 1923 is a true ketogenic diet at 35g carbohydrate per day, 0.67g protein per Kg per day, and the remainder of 30-40 calories per Kg per day from fat. Protein is restricted to 35g day in the induction period with 85-95 g fat per day. Newburgh and Marsh report the complete absence of acidosis in 180 patients, including juvenile cases, maintained on their diet, which would be considered a ketogenic diet in non-diabetics. "Not only does acidosis not develop in patients who are living on 
this diet, but it is a fact that all our patients showing at admission an acidosis short of coma rather promptly lost their acidosis while taking the high fat diet."]
At some point in the 70's or 80's they became acceptable (that the brain can run on ketones was discovered in 1967). The added benefit of a ketogenic diet is a further restriction of the glucagon curve, less competition from dietary glucose, less glucotoxicity, and, in animal experiments, a diminishing of pancreatic alpha cell to beta cell ratio.
In normal starvation metabolism a high level of ketone bodies stimulates the release of insulin from beta cells, just as a high level of glucose does, so that the action of glucagon is kept optimal, resources are not wasted, and toxicity is minimised.

A quick search shows that insulin is not universally available today, and not always affordable by everyone. In communities where this is the case the research of 1923 is still relevant for type 1 diabetics, in terms of survival until medicine is available and affordability (making a limited supply of insulin last longer). Safety is also an issue - the lower the dose of insulin required, the lower the risk of hypoglycaemia. It is certainly still relevant for type 2 diabetics. Low carbohydrate, high fat diets will probably become the norm again sooner than we think.
Even the Daily Mail has dispensed with the usual "experts warning about all that fat" add on when discussing LCHF therapeutic diets.






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?

Tuesday, 6 January 2015

Further Notes on Glucagon Dominant Hepatic Metabolism


The idea of the various forms of diabetes and pre-diabetes as a too ready susceptibility to glucagon dominance of hepatic metabolism, as described in the previous post, is not intended to diagnose or treat any disease (which I am not qualified to do, but I could not afford to buy the iamnotadoctor.com web address). It's a heuristic, a rule of thumb which puts what I've been reading into context, and which hopefully encapsulates a useful way of thinking about these diseases.
It produces many other thoughts, questions, and findings that seem consistent with the facts.

Firstly, a question; if the liver is producing higher than normal blood glucose levels, even between meals, why is there any need for glucose in the diet at all?
Secondly, a fact and a question; not all glucose usage depends on insulin. The brain uptake doesn't, and the liver uptake doesn't either, but in the case of the liver important pathways of glucose usage (glycogenisis and lipogenesis) do require insulin. So what happens to glucose taken up by the liver when these pathways are blocked? I am guessing that glycolysis generates excess NADH - the reductive stress that high glucose concentrations generate in cell cultures - which slows down fatty acid oxidation, which requires NAD+. This may be why feeding a little glucose decreased ketonemia even in pre-insulin diabetes. However, then we have to explain Petren's 1924 finding that a high fat, restricted protein diet suppressed ketonaemia. I wish I had a translation of the original paper, which was in German - all I have is this tantalizing hint. Was he talking about the patients in his practice (Karl Petren was a famous diabetes clinician), or some one-off short-term experiment?
Edit: Ketogenesis consumes NADH and relieves reductive stress. This is more in line with the Petren finding.

If you had no, or low, insulin production from beta cells, or severe insulin resistance, it seems likely to me that carbohydrate itself would contribute to elevated glucagon. This seems wrong, but in fact a rise in glucagon is the first step in the insulin production cascade. Glucagon stimulates its counter-hormone insulin, which then suppresses glucagon.





 So, imagine what happens in column one without insulin to bring down the glucagon, or if the phase 1 insulin response is delayed (as in pre-diabetes) or if the alpha cells of the pancreas have become insulin resistant. Glucose in this context is contributing to an elevation of glucagon, yet it's not a substrate that glucagon-dominated metabolism can act on. Instead, the rise in glucagon is going to result in even more glucose being produced from hepatic metabolism.

What is the requirement for carbohydrate? I see it as a requirement for those useful and essential nutrients that are highly associated with carbohydrate. Magnesium, ascorbate, folate, potassium (which meat also supplies), carotenoids, fibre, and a different variety of trace elements to supplement those found in meat.
From this perspective it makes sense to include non-starchy vegetables and fruit in the diet if possible. Fruits and sweet root veges, in a low carb diabetic diet, may have advantages over starches (and are certainly not inferior to them, unless sweetness is an appetite trigger). Pre-insulin diets for diabetics were woefully lacking in micronutrients and this may well have produced inferior results to those seen today.
There are other reasons why we see better results today than was generally the case historically. The general diet is higher in carbohydrate and sugar and lower in fat, so there is more room for improvement. Diabetes is usually diagnosed sooner, pre-diabetes is diagnosed more often, there are multiple noninvasive biofeedback devices to check sugar and ketones with, and there is the safety net of insulin. It is hard to avoid the conclusion that insulin, if needed, is the ideal drug treatment for diabetes. Drugs which stimulate beta cell insulin secretion give inferior results and seem liable to exacerbate the loss of beta cell function, especially if they simultaneously upregulate amylin secretion; whereas insulin, like a ketogenic diet, is giving the beta cells a rest.
And what effect does this have? Hat tip to the astute Melchior Meijer for pointing out the relevance of this study on the Hyperlipid blog.

Long-term ketogenic diet causes glucose intolerance and reduced β and α-cell mass but no weight loss in mice.
(Sounds bad! What happened????)
 Long-term KD resulted in glucose intolerance that was associated with insufficient insulin secretion from β-cells. After 22 wk, insulin-stimulated glucose uptake was reduced. A reduction in β-cell mass was observed in KD-fed mice together with an increased number of smaller islets. Also α-cell mass was markedly decreased, resulting in a lower α- to β-cell ratio.
That sounds like an effect that might rein in glucagon a little, if it translates to humans.

And here we have Calorie's Proper's 2012 take on this; Gluca-Gone Wild!

And Peter D.'s post on insulin that got me started on this road.

Also, a video lecture by Robert Unger - "A New Biology for Diabetes".



And an AHSC2012 presentation by Maelán Fontes about antinutrients which, among other things, dysregulate glucagon.


These antinutrients are opioids, and, curiously, opiates and other psychoactive drugs were once used in attempts to control diabetes.




(excerpt from Fatal Thirst - Diabetes in Britain until Insulin, 2010 by Elizabeth Lane Furdell)