Where do triglycerides come from? Part I (Updated)

This post contains sufficient updates from the original (dated 5/11/11) that I consider it more of a fully updated version vs. a bump.   This began with my intent to link to this post in an upcoming discussion of triglycerides, when I noticed that Dr. Ronald Krauss was amongst the authors.   This study originally caught my eye because of   Marc Hellerstein's name, he of DNL not a major pathway in humans fame, that I've blogged on previously.

Now I have excerpted copiously from the discussion because the authors make several points relevant to the discussion of what comprises a healthy lipid profile.  As part of updating, I am breaking those excerpts up a bit more and adding some/more emphasis and additional commentary.

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Robb Wolf: On Palmitic Acid, Carbs, LDL and CVD

Palmitic acid is 16 carbons long, fully saturated, and commonly found in palm oil and animal products, including beef, eggs, milk, poultry, and seafood.  Palmitic acid has long been implicated in CVD, as it tends to raise LDL cholesterol.  Among the saturated fats, it would appear palmitic acid does pose the greatest likelihood of increasing LDL cholesterol.  However, palmitic acid has also recently been shown to be vital both to forming new memories and accessing long-held memories.  As we shall see when we investigate how our diet has changed, a Paleo diet supplies an adequate amount of palmitic acid for optimum cognitive function while limiting the intake to levels that are not harmful to the cardiovascular system.  It is also important to note that excessive carbohydrate intake leads to palmitic acid production.  If you recall from the insulin chapter, when liver glycogen is full, additional carbohydrate is converted to palmitic acid.  This process appears to blunt our sensitivity to leptin, which then inhibits our satiety to a normal meal.  This is the beginning of insulin resistance and is at the heart of the mechanism of how we cease to respond to food by feeling "full."

From The Paleo Solution by Robb Wolf 2010, p. 108.

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Comparative Fatty Acid Toxicity on Macrophages

Comparative toxicity of fatty acids on a macrophage cell line (J774)

In the present study, the cytotoxicity of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on a macrophage cell line (J774) was investigated. The induction of toxicity was investigated by changes in cell size, granularity, membrane integrity, DNA fragmentation and phosphatidylserine externalization by using flow cytometry. Fluorescence microscopy was used to determine the type of cell death (Acridine Orange/ethidium bromide assay). The possible mechanisms involved were examined by measuring mitochondrial depolarization, lipid accumulation and PPARγ (peroxisome-proliferator-activated receptor γ ) activation. The results demonstrate that fatty acids induce apoptosis and necrosis of J774 cells. At high concentrations, fatty acids cause macrophage death mainly by necrosis. The cytotoxicity of the fatty acids was not strictly related to the number of double bonds in the molecules: palmitic acid>docosahexaenoic acid>stearic acid=eicosapentaenoic acid=arachidonic acid>oleic acid>linoleic acid. The induction of cell death did not involve PPARγ activation. The mechanisms of fatty acids to induce cell death involved changes in mitochondrial transmembrane potential and intracellular neutral lipid accumulation. Fatty acids poorly incorporated into triacylglycerol had the highest toxicity.

 I could C&P the entire introduction to this paper but don't want to do that, so please go read it.  Summary:

• The FFA/NEFA are generally implicated in having toxic effects in non-adipose tissues, aka lipotoxicity
• Saturated fatty acids tend to be more lipotoxic
• Ectopic (non-adipose) triglyceride storage is somewhat protective but this remains under consideration as metabolites/intermediates of triglycerides (ceramides) are implicated in toxicity.
• Cell death contributes to the inflammatory properties of FA's
• PPAR-ɣ increases reduce inflammatory cytokines like IL's and TNF-α

Macrophage infiltration into adipose tissue in obesity is implicated in the inflammatory state associated with obesity.   It should be noted that this study was in vitro (e.g. culture dish) on a non-human (murine to be exact) derived cell line.   But the elevated NEFA associated with insulin resistance and T2 Diabetes would produce a "toxic" state within adipose tissue leading to macrophage death (and adipocyte death?). 

Cell death types:  

• Apoptosis, aka programmed cell death:  When functioning properly, this is the "natural" death of cells for cellular turnover in tissues, etc.  In cancer, apoptosis is short circuited leading to so-called immortal cells.  Some toxic conditions lead to disruption of the normal signals and pre-mature apoptosis.  
• Necrosis:  Premature cell death due to some - always detrimental - external source.  Necrosis initiates a greater immune response as dead cells must be engulfed and removed, and such cells rupture and "spill" more "stuff" into the surroundings than cells undergoing PCD.

Some exerpts:

In the present study, we evaluated whether the induction of cell death could be a mechanism by which FAs modulate macrophage function. Indeed, treatment with different concentrations of FAs was toxic to the macrophage cell line J774, as assessed by loss of membrane integrity and DNA fragmentation.  In most cases, the lowest concentration that caused loss of membrane integrity was the same that induced DNA fragmentation (Table 3), and the percentages were similar. These findings are indicative that necrosis and apoptosis occurred concomitantly.

... high FA concentrations cause macrophage death mainly by necrosis. This effect was also observed by others after treatment of different cell types, such as melanoma, leukaemia cell lines, lung carcinoma and fibroblasts, with high concentrations of FA [49–51].  The results of both loss of membrane integrity and/or DNA fragmentation shown in the present study suggest the following rank of toxicity on J774 cells: PA>DHA>SA=AA=EPA>OA>LA 

... The relationship between lipid accumulation and apoptosis has been demonstrated through a series of experiments with different cell lines [63–65]. Accumulation of excess FAs into the TAGpool has been postulated to divert these molecules from pathways that lead to toxic effects and, thus, lipid bodies may serve as buffers against lipotoxicity [19]. Treatment with all FAs led to an increase of lipid bodies inside J774 cells (Figure 2B).  Cells treated with non-toxic concentrations of the FAs exhibited higher granularity, indicating accumulation of lipid droplets that was observed by fluorescence microscopy. 

A look at Figure 1 indicates a threshold behavior for the toxic effects.

I have not addressed everything in this article and this post is sort-of half book marking, half just putting this out there as I had not seen a discussion of this nature before.

Long term exposure to fatty acids and ketones inhibits B-cell functions in human pancreatic islets of Langerhans

Long term exposure to fatty acids and ketones inhibits B-cell functions in human pancreatic islets of Langerhans

We previously demonstrated in the rat that long term exposure to fatty acids inhibits B-cell function in vivo and in vitro. To further assess the clinical significance of these findings, we tested in human islets the effects of fatty acids on glucose-induced insulin release and biosynthesis and on pyruvate dehydrogenase (PDH) activity.

PDH is the enzyme thought to control the entry of acetyl CoA from glycolysis into the Kreb's / TCA /Citric Acid Cycle.

These authors did an in vitro study with human cells to see if the results compared with those seen both in vitro and in vivo in rats.  While in vitro observations don't always correlate with what we see in whole organisms, this did correlate for the rat.  Therefore it is reasonable to believe that the results of this experiment are applicable to human metabolism.

Human islets were obtained from the β-Cell Transplant Unit (Brussels, Belgium). Exposure to 0.125 mmol/l palmitate or oleate for 48 h during tissue culture (RPMI-1640 and 5.5 mmol/l glucose) inhibited the postculture insulin response to 27 mmol/l glucose by 40% and 42% (P<0.01 for difference). Inhibition was partly prevented by coculture with 1 μmol/l etomoxir, a carnitine-palmitoyl-transferase-I inhibitor (P<0.05 for effect of etomoxir).

Etomoxir inhibits fatty acid oxidation.  Recent developments employ this to treat chronic heart failure associated with Type II Diabetes.   

Inhibitory effects on glucose-induced insulin secretion by previous palmitate were additive to the inhibitory effects exerted by previous high glucose (11 and 27 mmol/l). Palmitate-induced inhibition of insulin secretion was evident after exposure to 25 μmol/l added fatty acid. The insulin content of islets exposed to fatty acids was significantly reduced, and glucose-induced proinsulin biosynthesis was inhibited by 59% after palmitate addition and by 51% after oleate exposure (P<0.01). These effects were partly prevented by etomoxir (P<0.05). The activity of PDH in mitochondrial extracts of islets preexposed for 48 h to palmitate was decreased by 35% (P<0.05) υs. that in control islets, whereas the activity of PDH kinase (which inactivates PDH) was significantly increased in the same preparations (P<0.05).

This underlines that high levels of both -- glucose/carbs and fatty acids -- wreak havoc on the body.

The effects of ketones were tested by 48-h exposure to β-hydroxybutyrate (β-D-OHB). Ten millimoles of D-β-OHB per L inbibited the subsequently tested insulin response to 27 mmol/L glucose by 56% (P<0.001). Half-maximal inhibitory effects of D-β-OHB on insulin secretion and insulin content were seen at concentrations between 0.5-2.5 mmol/l. Inhibition by D-β-OHB was partially reversed by etomoxir, whereas exposure to D-β-OHB failed to affect PDH and PDH kinase activities. 

We conclude that fatty acids as well as ketone bodies diminish B-cell responsiveness to glucose in human islets by way of a glucose-fatty acid cycle. Increased plasma concentrations of fatty acids and ketones are likely to be important factors behind the negative influences on B-cell function exerted by a diabetic state in botb type 1 and type 2 diabetes.

I don't have access to the full-text.  Seems "long term exposure" consisted of 48 hours.  I am curious what long term exposure of a ketogenic VHF diet  does to someone in the long haul.  At least early on, Type II's make insulin just fine -- just that they make too much of it b/c their cells are insulin resistant.  In researching to determine for myself if my low carb diet is healthy for the long haul, I've come across a lot of disturbing information on free fatty acids.  If by lowering BG we send FFA levels skyrocketing through the roof, do we mask a problem that still exists?  

I see no reason why IR cannot be reversed.  Indeed I believe the "lose 10% of weight cure" for T2 basically demonstrates this for persons whose pancreas has not yet been compromised (e.g. still able to produce insulin).  Indeed even the T2's with insufficient insulin responses may well have that inhibited by high NEFA/FFA -- restore fuel balance and there's no reason to believe this too can be reversed.

The relationship between high NEFA and sudden cardiac death continues to haunt me, and VLC/VHF diets have been shown to dramatically increase these!  So I remain skeptical of LC for weight maintenance and/or especially if one is gaining weight on such a diet.