Those NEFA are Pesky Things!

NEFA = Non-Esterified Fatty Acids aka Free Fatty Acids (FFA)

(By the way, I've just always preferred the NEFA acronym because in my head it sounds out more nicely than "ef ef ay" -- and for whatever reason, I sound it out "knee fah", though a reader once wondered about "neh fay".  I don't know there's a correct pronunciation for acronyms like this!)

On a hypothetical Metabolic SAT test NEFA are to lipids what glucose is to carbohydrates and amino acids are to proteins.  These are the forms of the three macronutrient classes that are absorbed/transported into and out of cells and circulation and the forms that enter into the energy-producing pathways.  By contrast, lipids are stored as triglycerides (aka triacyl glycerols, TAG), while carbs are stored in rather more limited quantities as glycogen, and there exists essentially no true storage depot for protein in excess of "tissue maintenance" needs.

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Caffeine and Insulin Sensitivity

Caffeine and Insulin Sensitivity  (full text PDF free till end of the month)

A number of reports have observed that acute caffeine ingestion decreases glucose tolerance and insulin sensitivity, and have raised the question whether its increased consumption throughout the world in the form of coffee and cola beverages might be of public health concern in the development of type 2 diabetes. Although some epidemiologic studies have found strong associations between coffee intake and detrimental lifestyle factors that favor obesity and diabetes, it is interesting that in spite of this, they have demonstrated that increased coffee consumption is associated with a decreased risk of developing type 2 diabetes.  When lifestyle confounders are taken into account, individuals consuming 6 cups coffee per day have at least 50% less risk of developing type 2 diabetes than those consuming 2 cups per day. Although it is perhaps premature to recommend increased coffee or caffeine intake to prevent the development of type 2 diabetes, there is little or no evidence to warrant the recommendation that it should not be a part of a normal healthy diet.

This is an interesting summary article.  In the section entitled Caffeine and Carbohydrate Metabolism, a case for acute caffeine consumption impairing glucose tolerance by inducing insulin resistance (decreases glucose uptake) is laid out which goes counter to the results of the coffee study highlighted above.   Perhaps it's something else in the coffee?  

More modest inverse associations were also observed for decaffeinated coffee consumption, caffeine intake from noncoffee sources, and total caffeine intake, as well as the incidence of type 2 diabetes, suggesting that caffeine and other components of coffee contribute to this inverse relationship.

Seems in part the case.  The authors discuss the conflicting short term "laboratory measured" effects and those seen in epidemiologic studies.

... it is very obvious that the results and conclusions of the acute and epidemiologic studies do not agree, and this illustrates the problem of extrapolating shortterm observations to a chronic disease (with an etiology that is influenced by a variety of interacting genetic and lifestyle factors.)

This seems even more surprising given the rather strong correlation between coffee intake and other lifestyle factors that are deleterious such as drinking, poor diet, etc.  Below I've summarized in bullet point fashion how the authors believe increased coffee intake decreases the risk of developing T2:

• Caffeine stimulates resting metabolic rate.  It could be as simple as fewer coffee drinkers getting overweight?
• Caffeine increases epinepherine
• Caffeine + epinepherine act together to promote lypolysis leading to an increase in plasma free fatty acid levels. (<- at first glance this might seem to not be a good thing)
• C+E have a thermogenic effect.  (thus the FFA's are likely readily oxidized with the boosted metabolism)
• The combined effects increase lipid turnover (less ROS hanging around??) "which may in the long-term have beneficial effects on body weight, body composition, and substrate use that could help to prevent the development of glucose intolerance, insulin resistance, and diabetes."

They go on:

However, such arguments are very speculative, and it may well be that the observed acute effects of coffee or caffeine on glucose tolerance and insulin sensitivity are suppressed by habituation to its repeated consumption.  Indeed, repeated caffeine consumption over 5 days induces complete tolerance to its effects on blood pressure, heart rate, and, in particular, blood glucose concentrations.52 

They also consider the other components of coffee:

• Coffee contains many bioactive compounds, most of which have as yet unknown metabolic effects.
• Coffee contains a quinide that improves insulin-mediated glucose uptake in rats
• Phenolic compounds in coffee influence GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide I) levels.  (These two peptides are called incretins and are associated with beta cell proliferation and decreased apoptosis (death) 

"Consequently, the combined physiologic effects of coffee’s many components may well be very different from that of one of the components studied alone."

Posted by CarbSane after her third very large mug of coffee 8*)

Fat Futile Cycling ~ From Carb Excess??!!

This post may well make heads spin.  It sure did mine.

A common claim in LC circles is that we "waste" excess fats through futile cycling.  Although this has not been demonstrated in any significant amount in humans except in massive fat overfeeding, it is still incorporated into books and blog posts by the likes of Dr. Mike Eades (futile cycling to "blow off" low carb excesses is stated as if fact in The 6 Week Cure).

Inherent in these statements is the implication that this only happens for excess dietary fat when carbs are low ... insulin would be trapping the fats in the cells as the theories go.  Well ...

Substrate cycling between de novo lipogenesis and lipid oxidation: a thermogenic mechanism against skeletal muscle lipotoxicity and glucolipotoxicity  (2004 Review Article,  PDF)

Life is a combustion, but how the major fuel substrates that sustain human life compete and interact with each other for combustion has been at the epicenter of research into the pathogenesis of insulin resistance ever since Randle proposed a ‘glucose–fatty acid cycle’ in 1963. Since then, several features of a mutual interaction that is characterized by both reciprocality and dependency between glucose and lipid metabolism have been unravelled, namely:

(i) the inhibitory effects of elevated concentrations of fatty acids on glucose oxidation (via inactivation of mitochondrial pyruvate dehydrogenase or via desensitization of insulin-mediated glucose transport),

(ii) the inhibitory effects of elevated concentrations of glucose on fatty acid oxidation (via malonyl-CoA regulation of fatty acid entry into the mitochondria), and more recently

(iii) the stimulatory effects of elevated concentrations of glucose on de novo lipogenesis, that is, synthesis of lipids from glucose (via SREBP1c regulation of glycolytic and lipogenic enzymes).  

This paper first revisits the physiological significance of these mutual interactions between glucose and lipids in skeletal muscle pertaining to both blood glucose and intramyocellular lipid homeostasis. It then concentrates upon emerging evidence, from calorimetric studies investigating the direct effect of leptin on thermogenesis in intact skeletal muscle, of yet another feature of the mutual interaction between glucose and lipid oxidation: that of substrate cycling between de novo lipogenesis and lipid oxidation. It is proposed that this energy-dissipating substrate cycling that links glucose and lipid metabolism to thermogenesis could function as a ‘fine-tuning’ mechanism that regulates intramyocellular lipid homeostasis, and hence contributes to the protection of skeletal muscle against lipotoxicity.

The figure below describes the mechanisms by -- as the figure caption states -- nutrient OVERSUPPLY can lead to insulin resistance.

I like schematics like this because even though I'm scientifically-minded, sometimes reading about various reactions gets really confusing.  It helps to just see it.  The possible ways all three macronutrients can lead to IR in excess are described:

• The fatty acids are shown in the upper left, with the fats that are "burned" proceeding down the left arrow through ß-oxidation (this metabolic process is often referred to as the fatty-acid spiral).  An excess of FA's leads to accumulation of "lipid pools" in the cell:  that little cycle in the upper left including DG = diacylglycerol (2 FA's on a glycerol) and FA-CoA. In my posts on lipotoxicity and intramyocellular lipids (IMTG or IMCL), I've discussed the implications of this previously.  The metabolites DG and FA-CoA activate various enzymes that inhibit glucose uptake.  They also lead to cell dysfunction and/or death when they undergo conversion to ceramides and/or peroxidation (formation of ROS).
• Amino acids, surprisingly, can inhibit insulin mediated glucose uptake through the mTOR pathway.
• Glucose can inhibit its own uptake by glucosamine synthesis and its action.

There are two central molecules in the metabolic engines of the cells:  Pyruvate and Acetyl CoA.  Pyruvate is an intermediary coming from glycolysis (glucose breakdown) and some amino acids, whereas Acetyl CoA is the ultimate "end product" of the preliminary breakdown of many amino acids, glucose and lipids.  From Ac-CoA on, the rest of the "metabolic engine" is the same regardless of the source of this molecule.  

• The authors describe a state of gluco-lipotoxicity and/or proteo-lipotoxicity that could develop when excesses in either glucose or AA's or both stimulate the conversion of Acetyl CoA to Malonyl CoA that can suppress fatty acid oxidation leading to a vicious cycle of lipid accumulation in the cellular pools.

Malonyl CoA is usually considered a "commitment step" in de novo lipogenesis - fatty acid synthesis - but has previously been ignored in skeletal muscle as DNL wasn't thought to occur at any significant rate in these cells.  This is the groundbreaking revelation of this paper ... it DOES occur!   The discussion describes how there are two different enzymes, Acetyl CoA Carboxylases (ACC's) that convert Acetyl to Malonyl CoA.  ACC-1 expressed primarily in liver and adipose tissue, and ACC-2 in skeletal muscle and other "non-lipogenic" tissues.  The two ACC's are compartmentalized in cells so that:

• ACC-1:  Generates the cytoplasmic (cellular) pool of Malonyl CoA used for DNL
• ACC-2:  Associated with mitochondria and governs this pool of Malonyl CoA to control ß-oxidation

Excerpt:  

In skeletal muscle, the suppressive effect of an elevated concentration of glucose on fatty acid oxidation is now recognized to occur ... but the question of whether a cytoplasmic pool of malonyl-CoA might be utilized for fatty acid synthesis has rarely been invoked most probably because of the long-held assumption that skeletal muscle is not an organ where de novo lipogenesis occurs. ... Consequently, the importance of skeletal muscle substrate metabolism in the homeostatic control of blood glucose has been viewed entirely from the reciprocal nature of interactions between glucose and lipid metabolism.  Recent evidence, however, suggests otherwise. As discussed below, de novo lipogenesis can indeed be shown to occur in muscle cells and to be modulated by factors influencing nutritional status, thereby revealing another facet of interactions between glucose and lipid metabolism that shows dependency between these two fuel substrates in skeletal muscle.

Here's where this gets really interesting.  The discussion continues:

The recent recognition that de novo lipogenesis might have relevance for lipid homeostasis in skeletal muscle stems from the realization that Sterol regulatory element binding protein-1c (SREBP-1c), a member of the family of transcription factors that regulate the expression of genes involved in lipid storage in liver and adipose tissue, is also present in skeletal muscle at a level close to that observed in the liver,41,42 and that its dysregulation might lead to increased lipid storage, and hence contribute to the pathogenesis of insulin resistance. There is now evidence both in humans and in rodents that SREBP-1c mediates insulin upregulation of genes encoding glycolytic and lipogenic enzymes in skeletal muscle,42–46 but most fascinating are the very recent demonstrations that glucose alone (in the absence of insulin) can stimulate de novo lipogenesis in skeletal muscle cells. 

Translation:  Lipid storage pathways thought previously to only be active in significant levels in liver/adipose tissues have now been shown to be active in muscle cells.  What they are seeing is that the DNL stimulated by glucose in this manner is NOT accompanied by the expected suppression of ß-oxidation.  They speculate on possible explanations for this, but the bottom line is summarized:

Whatever the explanation, it is clear that de novo lipogenesis, although low in skeletal muscle, can be markedly stimulated in muscle cells, particularly under conditions of high glucose (and/or high insulin) concentrations.

Hmmmmmm.....  The discussion goes on to speculate on the physiological significance for DNL in muscles, and that it may contribute yet another "sink" for temporary glucose excesses (e.g. after a carby meal), particularly when glycogen depots are full.  However there's a "glitch" because lipid accumulation is associated with insulin resistance.  IOW, synthesizing more would seem counter-productive to glucose clearance/disposal  unless the synthesized lipids were also "disposed of".  Well here's where the observation that ß-oxidation is not suppressed comes into play.

... recent work from our laboratory investigating the mechanisms by which leptin ... may interact with insulin to stimulate thermogenesis in skeletal muscle, suggests the possible existence of a thermogenic pathway of substrate cycling in which lipids derived from glucose... are subsequently oxidized.

So the carbs are thermogenic, not the fats?  Looks like it!  The discussion gets a bit complicated because it names enzymes and pathways, etc.  I'll let the more scientifically inclined amongst you read that part for yourselves.  

My summary of "Substrate cycling between de novo lipogenesis and lipid oxidation"

One role of leptin has been demontrated to be it's regulation of thermogenesis.  This hormone is recognized as being involved, in conjunction with insulin, in glycemic control and in preventing excessive IMCL accumulation.  Leptin has been shown to stimulate both glucose utilization and lipid oxidation.   This research group demonstrated that leptin can stimulate thermogenesis by a pathway requiring an enzyme known as P13K, and P13K is stimulated by insulin (insulin is described as a "potent activator" of P13K).   Leptin apparently also stimulates AMPK, an enzyme involved in lipid oxidation.  Therefore, these researchers have demonstrated a concurrent stimulation of glucose and fatty acid oxidation resulting in thermogenesis, and that evidence is consistent with DNL involved in the thermogenesis - experiments with DNL suppressing agents and leptin demonstrate this as w/o DNL, thermogenesis was not observed.  As the authors summarize:

Taken together, these studies suggest that the direct effect of leptin in stimulating thermogenesis in skeletal muscle could be mediated by substrate cycling between de novo lipogenesis and lipid oxidation, and that the orchestration of this substrate cycling requires both PI3K and AMPK signaling.

The discussion continues on the possible control mechanisms for this proposed futile cycle of DNL/ß-oxidation that are likely initiated with Acetyl CoA produced by both glycolysis and fatty acid oxidation "backing up" as it overwhelms the Krebs Cycle.    This futile cycle is established in brown adipose tissue.  I was surprised to learn that this thermogenesis has pretty much been established to involve DNL from glucose:

... analogous to the relation between substrate metabolism and thermogenesis in brown adipose tissue. In this tissue, whose primary function is to produce heat for thermoregulation ... it has long been known that much of the fuel for thermogenesis also derives from glucose being first converted to lipids before being oxidized.70–73  {They go on to describe mouse studies demonstrating that suppressing DNL in BAT leads to hypothermia.} 

Furthermore, this ‘dependency’ interaction between these two substrates and thermogenesis is well recognized at the whole-body level, and is attributed to activation of a neuroendocrine network (comprising insulin, leptin and the sympathoadrenal system), which plays a pivotal role in several overlapping regulatory systems: that of blood glucose, body temperature, body weight and more recently intramyocellular lipids.56–58

... The energy-dissipating substrate cycle that links glucose and lipid metabolism to thermogenesis in skeletal muscle (depicted in Figure 3) provides a novel molecular mechanism of thermogenesis through which this abovementioned neuroendocrine network operating through insulin, leptin and catecholamines overlaps in the regulation of body weight, blood glucose and intramyocellular lipids, and hence in the protection against obesity, hyperglycemia and lipotoxicity. 

Take away message here?  Well, the futile cycle is lipids.  But what stimulates and/or is required for this futile cycle to "waste" energy?  Who'da thunk it.  CARBS!!!

Mitochondrial H2O2 Emission, Cellular Redox State and Insulin Resistance - Part I

Reader Ryan emailed me this link a while back and I've been remiss in getting around to it.  Better late than never!  Thanks for the link Ryan!

Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans

Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the H₂O₂-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial H₂O₂ emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial H₂O₂ emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity.

The introduction is chock-full of background information (references) regarding the etiology of IR in skeletal muscle.

The accumulation of lipid in skeletal muscle has long been associated with the development of insulin resistance (1), a maladaptive response that is currently attributed to the generation and intracellular accumulation of proinflammatory lipid metabolites (e.g., fatty acyl-CoAs, diacylglycerols, and/or ceramides) and associated activation of stress-sensitive serine/threonine kinases that antagonize insulin signaling (2–4). Skeletal muscle of obese individuals is also characterized by profound reductions in mitochondrial function, as evidenced by decreased expression of metabolic genes (5, 6), reduced respiratory capacity (7–9), and mitochondria that are smaller and less abundant (9), leading to speculation that a decrease in the capacity to oxidize fat due to acquired or inherited mitochondrial insufficiency may be an underlying cause of the lipid accumulation and insulin resistance that develops in various metabolic states (10, 11).

H2O2 is hydrogen peroxide.  Dip your finger into even the dilute OTC prep, and we see what it can do.  Obviously we're not talking those concentrations in the cells or we'd all be dead.  But H2O2 is chemically unstable (which is why it is sold in brown opaque bottles with directions to store in a cool place).  H202 is a reactive species (RS) that can cause damage.

Here is a link to but one summary article on H2O2, and another on RS and antioxidants in general.

Hydrogen Peroxide– and Peroxynitrite-Induced Mitochondrial DNA Damage and Dysfunction in Vascular Endothelial and Smooth Muscle Cells

Free radical tissue damage: protective role of antioxidant nutrients 

Back to the article and some more background:

In addition to providing energy for the cell, mitochondria are now recognized as an important site for the generation, dispensation, and removal of a number of intracellular signaling effectors, including hydrogen peroxide (H₂O₂), calcium, and nitric oxide. In fact, the emission rate of H₂O₂ from mitochondria, which reflects the balance between the rate of electron leak/superoxide formation from the respiratory system and scavenging of H₂O₂ in the matrix, varies over a remarkably consistent range across diverse forms of aerobic life (20). Once in the cytosol, H₂O₂ can alter the redox state of the cell by either reacting directly with thiol residues within redox-sensitive proteins or shifting the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG), the main redox buffer of the cell. Thus, the rate at which H₂O₂ is emitted from mitochondria is considered an important barometer of mitochondrial function and modulator of the overall cellular redox environment (21).

For the non-chemists in the audience, oxidation involves the loss of electrons from a molecule, reduction involves a gain.  The term "redox" is a contraction of reduction and oxidation and, since electrons do not exist as separate particles (well, in the solution chemistry sense which is what our bodies are) these reactions occur in "redox" pairs -- where one molecule loses electrons picked up by the other -- a species is oxidized when another is reduced.  Which direction these reactions go depends on the relative oxidation potentials (a measure of reactivity) of the chemicals involved.  Rusting iron is a classic example everyone is familiar with.  Put an iron nail into water and come back in a week or so and the water will be brown and there will be rust plumes on the nail.  Add salt and it will go faster.  This is because dissolved oxygen in the water has a higher oxidation potential than the iron, therefore in the reaction, oxygen is reduced while iron is oxidized.  That orange stuff is the product of this reaction.  We can think of this as environmental oxidative damage b/c the result is to convert a bright shiny strong nail into a rusty nail that can eventually degrade to where the head falls off, etc.  In our bodies, O2 is an essential oxidizing agent, and metabolically it serves a key role so we don't consider it damaging.   Reactive Oxidative Species (ROS or sometimes just RS) are molecules with high oxidation potentials.  In our bodies, they can have the effect of "rusting" critical components of cell membranes, signalling proteins, DNA, etc.  Back to my Bill Nye the Science Guy experimentation.  Take two nails and put salt water in two glasses.  In one just put the plain steel nail.  In the other, wrap a similar nail with aluminum foil.  Report back in a week.  What you'll see is that the nail wrapped in foil is virtually free from rust but you might see a bit of white cloudiness around the foil.  Aluminum in this context acts as the antioxidant.  It essentially reacts preferentially with the oxygen (the white stuff is the less aesthetically offensive product) protecting the iron.  While not a perfect analogy (or, for that matter a perfect description of galvanic corrosion which is what the iron/aluminum scenario is), this is the role antioxidants play in our bodies.   Antioxidants "scavenge" ROS and take the hit (are oxidized) so that the ROS won't damage critical molecules.  The oxidized form of the antioxidant is usually harmless and either eventually excreted or "recycled" back to it's reduced form by some biochemical mechanism.

End of chemistry lesson ...

What this article is saying is that H2O2 is one such RS/ROS produced in the mitochondria (under perfectly normal conditions), but that in balance we produce natural antioxidants (glutathione, etc.) that serve to "remove"  these ROS before they can damage critical molecules.

Here's where obesity and high fat diet figure in.  (Summarized from:  Obesity/diet alter mitochondrial H2O2 emission in humans.)  The comparison was between lean insulin sensitive (I'll call these LIS) males and obese insulin resistant males (OIR) in skeletal muscle.

• H2O2 emission was 4X greater in OIR vs. LIS at basal ("fat burning") rates
• H2O2 emission was 2X greater in OIR vs. LIS in response to stimulation
• The difference in H2O2 emission did not correlate with O2 utilization which was not different between the two groups.
• Maximum stimulated O2 consumption was ~35% less in OIR vs. LIS indicating reduced respiratory capacity in the obese.

Figure 5 from the article:  Caption:  H2O2 emission elevated in obese men and lean men following a high fat meal.

Part II to follow as a separate post.

  

High-fat diet, muscular lipotoxicity and insulin resistance

High-fat diet, muscular lipotoxicity and insulin resistance

A high dietary fat intake and low physical activity characterize the current Western lifestyle.  Dietary fatty acids do not stimulate their own oxidation and a surplus of fat is stored in white adipose tissue, liver, heart and muscle. In these organs intracellular lipids serve as a rapidly available energy source during, for example, physical activity. However, under conditions of elevated plasma fatty acid levels and high dietary fat intake, conditions implicated in the development of modern diseases such as obesity and type 2 diabetes mellitus, fat accumulation in liver and muscle (intramyocellular lipids; IMCL) is associated with the development of insulin resistance. Recent data suggest that IMCL are specifically harmful when combined with reduced mitochondrial function, both conditions that characterize type 2 diabetes. In the (pre)diabetic state reduced expression of the transcription factor PPARg co-activator-1a (PGC-1a), which is involved in mitochondrial biogenesis, has been suggested to underlie the reduced mitochondrial function. Importantly, the reduction in PGC-1a may be a result of low physical activity, consumption of high-fat diets and high plasma fatty acid levels. Mitochondrial function can also be impaired as a result of enhanced mitochondrial damage by reactive oxygen species. Fatty acids in the vicinity of mitochondria are particularly prone to lipid peroxidation. In turn, lipid peroxides can induce oxidative damage to mitochondrial RNA, DNA and proteins. The mitochondrial protein uncoupling protein 3, which is induced under high-fat conditions, may serve to protect mitochondria against lipid-induced oxidative damage, but is reduced in the prediabetic state. Thus, muscular lipotoxicity may impair mitochondrial function and may be central to insulin resistance and type 2 diabetes mellitus.

Caveat:  This is a summary paper on how fats in the Western Diet "behave".  However even on a zero carb diet, our metabolic pathways, receptors and such do not change.   VLC diets switch us to an "alternate metabolism" based more on lipid oxidation by skeletal muscles.  In that context, UP3 should be upregulated and there shouldn't be "idle" fatty acids lying around to be prone to peroxidation.  There is, however, a rationale for a low fat diet and exercise approach to reversing the condition.

Obesity, energy balance and fat balance

By definition, the development of obesity and overweight is characterized by a positive energy balance. Numerous investigations (Schutz et al. 1989; Bennett et al. 1992) have shown that in the long term an imbalance between energy intake and energy expenditure is reflected in a positive fat balance. ... In addition, in human subjects there is evidence for a clear substrate hierarchy for the utilization of macronutrients, in which fat balance is least regulated. For example, the human body responds only very slowly by increasing fat oxidation when fat intake is increased (Thomas et al. 1992; Schrauwen et al. 1997a), leading to a deposition of dietary fat in the fat stores. On the other hand, the storage capacity for carbohydrate and protein in the human body is limited and therefore carbohydrate and protein oxidation are very well and rapidly adjusted to their respective intake (Abbott et al. 1988). As a consequence, a positive energy balance will be reflected in a positive fat balance.

This is a nice summary of the nutrient heirarchy and, although we can clearly consume excess calories on a low fat diet, it would require excessive carbohydrate consumption in positive caloric balance.

Fat oxidation on a high-fat diet

Although there is ample evidence that the adaptation of fat oxidation to increased fat intake is slow in man, the reason for this slow adaptation is relatively unknown. According to the two-compartment model of Flatt (1987), whole-body fat oxidation can be increased via an expansion of fat mass, leading to increased plasma NEFA levels available for oxidation. In this model the body is divided into two compartments, fat mass and glycogen stores, and the oxidation mixture of fatty acids and glucose depends on the size of these two compartments. As the glycogen stores are very limited in size, small changes in the size of the glycogen stores will affect glucose oxidation. In contrast, as the fat mass can be relatively unlimited in size, a large expansion of fat mass is needed before changes in fat oxidation will occur. This model can explain why the addition of a surplus of fat to a single meal, which will not result in a change in fat mass, does not affect fatty acid oxidation rates, and why obese subjects have relatively high fat oxidation. In fact, expansion of fat mass (obesity) could be considered as an adaptation of the body to increase fat oxidation to a level that matches a high dietary fat intake. 

This is interesting to me and makes sense.  It would not surprise me to find leptin controlling this.  I highlighted, however one statement that is often presumed opposite in nutritional circles of all stripe, LC in particular.  It turns out that the obese tend to "burn fat" just fine and fatty acids are available for the burning regardless of diet.

However, it has been shown (Schrauwen et al.1997a) that healthy human volunteers who consume a high-fat diet for 7 d, while being in energy balance, are able to slowly increase their fat oxidation to a level that equals the high-fat intake. As no substantial expansion of fat mass can be expected after 7 d of a high-fat diet, these results seem to contradict Flatt’s (1987) model. These findings have, however, been explained in terms of the changes in glycogen stores that may have occurred (Schrauwen et al. 1997b, 1998). During the first days on a high-fat diet, when fat oxidation does not equal fat intake, subjects are in negative carbohydrate balance (carbohydrate oxidation>carbohydrate intake), leading to a decrease in the body’s glycogen stores. According to Flatt’s (1987) two-compartment model, a decrease in glycogen stores would result in a decrease in glucose oxidation and would therefore be another way to increase fat oxidation.  Indeed, it has been shown (Schrauwen et al. 1997b, 1998) that lowering glycogen stores by exhaustive exercise markedly improves the rate at which subjects are able to adapt their fat oxidation to an increased fat intake.

In order to further investigate the mechanisms by which fat oxidation increases on a high-fat diet, a more detailed determination of fatty acid oxidation has been conducted in subjects consuming high-fat diets (Schrauwen et al. 2000). Interestingly, it was observed that the increase in fat oxidation after 7 d of a high-fat diet is completely accounted for by an increase in TAG-derived fatty acid oxidation {...}  mainly intramyocellular lipids (IMCL)) {and not plasma fatty acids). IMCL are small lipid droplets that are located in the sarcoplasm and predominantly found in the vicinity of mitochondria, suggesting that they may serve as a rapidly available energy source for the muscle. {...}it has recently been shown (Schrauwen-Hinderling et al. 2005) that the amount of IMCL is already markedly increased after 7 d of a high-fat diet in healthy lean subjects. {...} these combined observations of an increased IMCL mass and increased IMCL oxidation indicate that increases in IMCL content are also needed to drive increased IMCL oxidation. Thus, the slow rate at which fat oxidation adapts to increased fat intake when a high-fat diet is consumed may also be attributed to the time needed to increase IMCL content. {...}  Interestingly, it has been found (Schrauwen et al. 2002c) that when sedentary middle-aged subjects follow an endurance training programme for 3 months whole-body fat oxidation increases, and again this increase is completely accounted for by an increase in TAG-derived fatty acid oxidation. In accordance with the earlier mentioned hypothesis, endurance training is also known to increase IMCL content (Goodpaster et al. 2001; Schrauwen-Hinderling et al. 2006a), suggesting that similar mechanisms may be involved in the training- and diet-induced increase in fat oxidation.

So even in the presence of carbohydrates, our bodies adapt our substrate oxidation rates to our macronutrient intake.  Those consuming more fat will burn more fat provided it is an energy balanced diet.  It is net caloric excesses that throw things out of whack, and it would seem that most Western diets contain an excess of both fat and carbs.

OK, so what of IMCL and lipotoxicity?  Lipotoxicity is a term used to describe various detrimental effects of accumulated lipid in tissues not intended for storage.  The most toxic effect of which may be apoptosis -- cell death.

IMCL and IR

Evidence gathered in recent decades has pointed towards an important causal role of disturbed fatty acid metabolism in the development of type 2 diabetes mellitus. Not only are plasma glucose levels increased in uncontrolled type 2 diabetes, but also plasma NEFA, and the storage of fatty acids in non-adipose tissues such as pancreas, liver and muscle is elevated in patients with type 2 diabetes (Schalch & Kipnis, 1965). Moreover, a strong negative correlation has been found between the level of IMCL and insulin sensitivity in non-trained subjects (Perseghin et al. 1999), and levels of IMCL are increased in first-degree relatives of patients with type 2 diabetes who are insulin resistant, but not diabetic (Jacob et al. 1999). These data suggest that IMCL accumulation may be a primary factor in the development of type 2 diabetes.

The highlighted part is my cause for concern as this also occurs in the "pre diabetic" insulin resistant state.  In self-treating T2 -- especially if one is in early stages and hyperinsulinemic (IOW they can still make plenty of insulin -- is a low carb/high fat diet harmful but the the effects masked by the improved BG control?  There are two issues here:  1. Elevated NEFA and direct effects on circulatory system, etc. and 2. IMCL -- I think the question here is will IMCL "accumulate" in a VLC fat burner.  I've posted a lot of info (and more to come) on the plasma NEFA.  

The article goes on to present data on the impact and fates of IMCL.  IMCL is a source of energy -- readily available fatty acids.  But if there are too many and they are not burnt for energy, they are stored as triglycerides (TAG) in the muscle (or organ) cells instead of in the adipose tissue.  It is under these conditions that IMCL's cause deleterious effects.

I'm pretty much convinced that a high fat diet induces insulin resistance.  The question remains if this IR is relevant to low carbers who don't rely on a postprandial insulin response to clear glucose from the blood.  I have two thoughts on this as well:  1.  What of protein transport?   and 2.  What of those who do not adhere strictly to low carb?  Is something like the LoBAG diet (50% fat / 30% protein / 20% carbs) better if it can be adhered to more consistently?  It is still high fat, but there should be enough carbs in this diet to get NEFA under control once a modest weight loss has been achieved.

It seems to me that if someone becomes insulin resistant, the goal should be to reverse that IR, not merely mask the effect.  Here is where I have questions about VLC (that necessarily becomes high fat) diets for the long term.  More importantly, I think there's a lesson to be learned about EXERCISE -- something many in the LC community seem to have an aversion too.  In the article it is stated that T2's have impaired lipid oxidation capability -- dysfunctional mitochondria.  This seems to be due to expression of PGC-1alpha.  But this can be upregulated by both acute exercise and endurance training.  As the discussion states, the reduced expression seen in healthy relatives of T2's might tempt one to believe this is totally genetic, but this would not explain the rise in T2 and the earlier onset we are now seeing.  They postulate that reduced activity could play a role.  Of course that is speculation, but, especially in children, I see a lot less acute activity going on.  It would also seem that on a LC diet, exercise might even be MORE important than on a low fat calorie restricted diet!

A role for oxidative stress in the development of insulin resistance

Since the finding that mitochondrial function may be impaired in the (pre)diabetic state, most studies have focused on PGC-1a and mitochondrial biogenesis. However, mitochondrial function is not only determined by mitochondrial biogenesis, but also by mitochondrial quality. In the latter context, it has been shown (Kelley et al. 2002) that mitochondria from patients with type 2 diabetes are smaller and show morphological abberations when compared with controls, and mitochondrial area correlates positively with insulin sensitivity. The smaller and damaged mitochondria in skeletal muscle of patients with diabetes also result in an impaired functional capacity (Kelley et al. 2002). Thus, mitochondria of patients with type 2 diabetes have a reduced electron transport chain capacity{...}

To explain the observed mitochondrial damage, elevated production of reactive oxygen species (ROS) and its by-products (e.g. lipid peroxides) has been suggested. In addition to the production of ATP, mitochondria are also the major contributor to the production of ROS. Mitochondrial ROS can react rapidly with DNA, protein and lipids, thereby leading to so-called oxidative damage.  Recent evidence points towards a causal role for ROS in the development of insulin resistance. {...}

Fatty acids are especially very prone to ROS-induced oxidative damage, resulting in the formation of lipid peroxides, which in turn can induce damage to proteins and DNA. Thus, accumulation of fatty acids in the vicinity of the mitochondrial matrix, where ROS are formed, increases the likelihood of lipid peroxidation. As discussed earlier, patients with type 2 diabetes are characterized by the accumulation of IMCL and these lipid droplets are located close to the mitochondria. To prevent simple diffusion of fatty acids into the mitochondria their entry is regulated{...} however, this system cannot completely prevent the diffusion of fatty acid into the mitochondria. {...}  It can easily be imagined that this ‘passive diffusion’ is more likely to occur under conditions of a high IMCL concentration{...}  Consistent with this notion, skeletal muscle of subjects who are obese and insulin resistant not only contains a higher amount of IMCL, but these lipids also show a higher extent of lipid peroxidation (Russell et al. 2003b). Potentially, these lipid peroxides could lead to oxidative damage to mitochondrial structures and explain the increased mitochondrial damage observed in patients with type 2 diabetes (Kelley et al. 2002).

This is sobering food for thought.

The article goes on to discuss UCP3 - Uncoupling Protein 3 in the mitochondria.  This protein seems to be involved in transporting LCFA's that are not oxidized out of the mitochondria so that they don't undergo lipid peroxidation and do damage.  This is a theory but one that seems to agree with the evidence presented.  UCP3 is suppressed in pre(diabetics) but lifestyle intervention and/or an endurance training program restore this to normal levels.

Deleterious Effects of Elevated NEFA - I: Monocytes and Vascular Adhesion

Elevated Concentrations of Nonesterified Fatty Acids Increase Monocyte Expression of CD11b and Adhesion to Endothelial Cells

First, a layperson friendly description of Monocytes:

Monocytes are a type of leukocyte or white blood cell which play a role in immune system function. Depending on a patient's level of health, monocytes make up between one and three percent of the total white blood cells in the body. They can be counted as part of a blood test, and changes in their levels can indicate changes in a patient's health. As a general rule, a low monocyte count is a good sign, and a high count indicates that a problem is present...

... Levels of monocytes in the blood tend to rise when someone has an infection, because more of these cells are needed to fight it. Monocytes can also increase in response to stress and other factors. A high monocyte count may be referred to as monocytosis, and it is typically addressed by determining why the count is so high, and addressing the problem. For example, if monocytes are elevated because of an inflammation caused by a viral infection, the patient would be given medication to kill the virus and bring down the inflammation.

My Study Summary:  The investigators incubated human monocytes with NEFA (physiological FA composition) and measured adhesion and the expression of a protein associated with adhesion.  NEFA increased adhesion in a dose and time related manner.  In other words, the more NEFA in the incubation medium and the longer the incubation, the greater the increase in adhesion (although it did peak at 48 hrs then fall off at 72 hrs).  NEFA also increased CD11b, a protein termed an integrin involved in the adhesion of monocytes to endothelial cells (vessel walls).

From the Discussion:

Monocytes from subjects with diabetes have been demonstrated to bind to endothelial cells in greater numbers than monocytes isolated from subjects without diabetes.¹⁵ ....However, there is increasing evidence that elevated levels of NEFA may have numerous proinflammatory effects on vascular cells in subjects with insulin resistance and diabetes. The goals of this study were to test whether elevated levels of NEFA could contribute to the enhanced adhesion of monocytes to endothelial cells and to ascertain the mechanism by which NEFA may achieve this effect.

We first demonstrated that exposure of monocytes to a physiological mixture of NEFA for 48 hours led to maximum monocyte adhesion; adhesion increased in a concentration-related fashion. This is the first report to our knowledge of increased monocyte adhesion resulting from prolonged exposure to a physiological mixture of fatty acids. Although NEFA-treated monocytes showed increased adhesion to unstimulated endothelial cells, pretreatment of endothelial cells with LPS greatly enhanced monocyte binding as has been previously reported.¹⁷This indicates that one consequence of prolonged exposure of monocytes to NEFA may be to prime these cells to bind to activated endothelial cells. This may be particularly relevant for the development of atherosclerosis where monocyte accumulation is enhanced at sites of vascular inflammation, where upregulation of a variety of adhesion molecules occurs. Monocyte firm adhesion usually requires interaction of integrins ^... Our studies indicate that NEFA stimulates the expression of both message and protein for CD11b [one such integrin]...

Our studies also demonstrate that NEFA-induced generation of ROS may mediate monocyte adhesion to endothelial cells. This was demonstrated by several lines of evidence. First, maximum stimulation of ROS by NEFA occurred after the same duration of exposure and at the same concentration as that of monocyte adhesion (Figure 1). Second, addition of glutathione or BHT, 2 structurally different antioxidants, prevented both production of monocyte ROS and monocyte adhesion (Table). Moreover, depletion of GSH with diethyl maleate before addition of NEFA further increased ROS generation and monocyte adhesion. Third, inhibitors of NADPH oxidase (a major producer of ROS in monocytes), but not those of nitric oxide synthase, xanthine oxidase or the mitochondrial electron transport pathway were shown to be effective inhibitors of monocyte adhesion. These latter experiments also demonstrate that NADPH oxidase appears to be an important and specific source of NEFA induced ROS in monocytes. Our results are consistent with those of previous studies that have indicated that inhibitors of NADPH oxidase, but not various mitochondrial complex inhibitors, inhibit ROS release from THP-1 cells induced by high glucose conditions, and that inhibitors of PKC, a recognized stimulator of NADPH oxidase, also reduced ROS generation and monocyte adherence.¹⁸ ...

Although levels of NEFA are increased in individuals with diabetes, similar degrees of elevation are frequently present in individuals with insulin resistance and might be expected to increase adhesiveness of monocytes in individuals with insulin resistance as well as in those with diabetes. Consistent with this notion, insulin resistance, as measured by a direct measure of insulin mediated glucose uptake, was a significant predictor of monocyte adhesion to endothelial cells.¹⁹ Although adipose tissue is a major source of serum NEFA, triglyceride-rich lipoproteins may also provide free fatty acids directly to the artery wall. Insulin resistance and type 2 diabetes are associated with increased levels of just such lipoproteins, resulting primarily from increased hepatic secretion of triglyceride-rich very-low-density lipoprotein and exaggerated postprandial hyperlipidemia.²⁰ ...

In summary, these studies demonstrate that elevated levels of NEFA, as frequently occurs in conditions of obesity, insulin resistance and type 2 diabetes, may contribute to increasedmonocyte expression of CD11b and enhance their adhesion to activated endothelial cells. These data provide another example of elevated levels of free fatty acids inducing inflammation and support the concept that modalities that will diminish levels of NEFA or inhibit their intracellular signaling may contribute to reduced atherogenesis in these individuals.

There's a LOT of info in this discussion -- I excised some of it to focus on the direct NEFA response.  If a low carb diet results in chronically elevated NEFA this does not seem to be a healthy outcome.  If, however, the impact is more acute, perhaps this is not sufficient to "prime" the monocytes.  However since the carb-restricted state is metabolically analogous to the fasted state, I fear this is more chronic than acute.

Therefore in deciding the proper macronutrient composition of a "healthy diet", NEFA levels, IMHO, should not be overlooked.

Fructose -- Protective?

Fructose and Tagatose Protect Against Oxidative Cell Injury by Iron Chelation

Abstract

To further investigate the mechanism by which fructose affords protection against oxidative cell injury, cultured rat hepatocytes were exposed to cocaine (300 ) or nitrofurantoin (400 ). Both drugs elicited massively increased lactate dehydrogenase release. The addition of the ketohexoses -fructose (metabolized via glycolysis) or -tagatose (poor glycolytic substrate) significantly attenuated cocaine- and nitrofurantoin-induced cell injury, although both fructose and tagatose caused a rapid depletion of ATP and compromised the cellular energy charge. Furthermore, fructose, tagatose, and sorbose all inhibited in a concentration-dependent manner (0–16 mM) luminol-enhanced chemiluminescence (CL) in cell homogenates, indicating that these compounds inhibit the iron-dependent reactive oxygen species (ROS)-mediated peroxidation of luminol. Indeed, both Fe²⁺ and Fe³⁺ further increased cocaine-stimulated CL, which was markedly quenched following addition of the ketohexoses. The iron-independent formation of superoxide anion radicals (acetylated cytochrome c reduction) induced by the prooxidant drugs remained unaffected by fructose or tagatose. The iron-chelator deferoxamine similarly protected against prooxidant-induced cell injury. In contrast, the nonchelating aldohexoses -glucose and -galactose did not inhibit luminol CL nor did they protect against oxidative cell injury. These data indicate that ketohexoses can effectively protect against prooxidant-induced cell injury, independent of their glycolytic metabolism, by suppressing the iron-catalyzed formation of ROS. 

Cell protection by fructose is independent of adenosine triphosphate (ATP) levels in paracetamol injury to rat liver slices

Abstract

Fructose protects cells against several types of injury but the mechanism of protection is uncertain. We have used paracetamol injury in rat liver slices as a model system to investigate the role of ATP levels in protection by fructose. Fructose depletes ATP levels in a concentration-dependent fashion in liver slices obtained from non-induced rats. Liver slices recover their ATP levels in the presence of fructose concentrations up to 10 mM. However, in the presence of 20 mM fructose, ATP levels are depleted for the duration of 240 min incubation. Adenine at 100 μM reverses the ATP depletion induced by 20 mM fructose in slices over 240 min incubation. Liver slices obtained from phenobarbitoneinduced rats were exposed to 10 mM paracetamol for 120 min and, then, incubated without paracetamol, with or without fructose for another 240 min. Introduction of 10 mM or 20 mM fructose in the second stage of incubation prevents paracetamol-induced injury. Fructose at 20 mM induces a rapid and marked depletion in slice ATP levels and these remain low throughout the second 240 min incubation period. Fructose at 10 mM maintains high ATP levels, even in paracetamol-treated slices. There is a profound protective effect against paracetamol-induced injury by either concentration. This suggests that protection is not dependent on high or on low ATP levels. Incubation of paracetamoltreated slices in the presence of 20 mM fructose plus 100 μM adenine in the second 240 min incubation period still results in the same level of protection as with 20 or 10 mM fructose alone while reversing the ATP depletion observed with 20 mM fructose.

Protection of cellular and mitochondrial functions against anoxic damage by fructose in perfused liver

Abstract

In anoxic perfused liver, conversion of fructose to lactate was greatly increased to about 3 μmol/min per g liver. This increase in lactate implied that the same amount of ATP was also produced. The rate of metabolism of glucose was less than 10% of that of fructose, as judged by rate of production of lactate. In anoxic liver perfused with fructose, the ATP levels of both the tissue and mitochondria remained high, despite lack of oxygen, thus preventing enzyme leakage and preserving processes requiring ATP, such as bile excretion and urea formation. The mitochondrial oxidative phosphorylation capacity of anoxic liver perfused with fructose was also unimpaired. Spectral analysis of light transmitted through the liver revealed that the mitochondrial electron transfer system was in the completely reduced state during anoxia, indicating that the mitochondria were incapable of synthesizing ATP. These results suggest that fructose metabolism during anoxia resulted in sufficient production of ATP for maintaining the physiological functions of the cells and the oxidative phosphorylation capacity of their mitochondria.