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.

Adiposopathy v. Obesity ~ I

I just came across the following article, and haven't quite digested the whole thing just yet.  Still, it is interesting so I thought I would share it here.  This post will be about the most curious topic in this paper, but I hope to revisit this in a series of future blog posts (hence the "I" in the title).  

Adiposopathy Is a More Rational Treatment Target for Metabolic Disease than Obesity Alone

Near as I can tell, the lead author, Harold Bays, is the doctor who coined the term "adiposopathy" or "sick fat".  

Adiposopathy is pathologic adipose tissue dysfunction that may be initiated and/or exacerbated by fat accumulation (adiposity) in genetically susceptible patients [1••].  Adipocytes are metabolically active and adipose tissue is an important endocrine organ (Table 1) [2••]. Abnormalities of adipocyte factors contribute to dysmetabolism (Fig. 1), and adiposopathy [1••,3•] promotes some of the most common metabolic diseases encountered in clinical practice, including type 2 diabetes mellitus (T2DM), hypertension, and dyslipidemia.

This is a summary paper focusing on the fact that it is dysfunction of the adipose tissue rather than the amount of adipose tissue that is responsible for Metabolic Syndrome.  Thus explaining your "metabolically obese thin people" and "metabolically normal obese people", etc.   The focus, as the title implies, is MetS treatments that target the fat cell dysfunction rather than simply the person's fat mass.  

Sick fat appears to be related to adipocyte hypertrophy -- an enlarged fat cell:

It has been known since the 1970s that adipocyte hypertrophy increases the lipid/protein ratio of the adipocyte (through a relative consistency in protein content coupled with increased fat content) [13], decreases the responsiveness of adipose tissue to insulin [14], and increases the risk of metabolic diseases such as T2DM [15] and dyslipidemia, even if adipocyte hypertrophy is found in only slightly overweight individuals [16]. In fact, adipocyte hypertrophy is more closely linked to metabolic abnormalities, such as insulin resistance, than is an increase in total body fat [17].

An increase in fat cell size represents a failure of adipose tissue to adequately proliferate and differentiate [18] (as found with obesity and T2DM [19]) and, therefore, a failure to inadequately accommodate a further increase in energy influx [20]. Adipocyte hypertrophy may indicate a resistance or inability to store triglycerides beyond some maximal amount [21]. 

(This is consistent with the Critical Visceral Fat Theory I blogged on previously.)

For this post, I want to focus on the discussion of one of the pharmaceutical treatments:  PPAR-gamma agonists.  (Here's a LINK to general information on these drugs --  thiazolidinediones or “glitazones.” -- Actos and Avandia).  I'm not promoting pharmaceutical therapy, and Avandia has seen a lot of negative press of late, but nonetheless I find the mechanism of action of these drugs to be interesting.

These compounds appear to "cure" sick fat by stimulating the proliferation of new, small, young adipocytes and/or promote the death (apoptosis) of dysfunctional hypertrophied large fat cells.  Since SCAT has a greater ability to differentiate, and VAT cells are more metabolically active, this seems to have differential effects on the two types of adipose tissue.  These drugs tend to cause fat mass gain in SCAT, and loss in VAT, but appear to be most effective in the patients who are fatter to begin with and who gain more fat.  Yes, you read that right.  

From an adipose tissue metabolism standpoint, PPARγ agents have been shown to reduce free fatty acids [23••,91], increase adiponectin [92–94], and reduce leptin [93] (although not consistently so [95]), with unconfirmed effects upon resistin [95,96], IL-6 [97], and tumor necrosis factor-α [95,97]. Thus, it appears that many of the favorable effects of PPARγ agents upon glucose metabolism and adipocyte function may be most related to improvements in free fatty acid metabolism

I've seen diabetics list these meds in their treatment regimes and yet be on weight loss regimes.  In some ways this seems counter productive.  One way to try to cure sick fat is to try to reverse the dysfunction by emptying out the cells.  Another, it appears, would be to replace sick adipocytes with healthy ones.  While weight gain may be the last thing a person wants, I know I probably would resist it were I diabetic, it is interesting to consider.