Fat Tissue Regulation ~ Part VI: Journey & Fate of Dietary Fat

Regulation of fatty acid transport and storage: influence of acylation-stimulating protein

Katherine Cianflone and Sabina Paglialunga, 2006

Commonly, the dietary sources of fat exceed the actual needs and the tissues are faced with dealing with the excess. Under these circumstances, the removal process of dietary triglycerides and fatty acids becomes overloaded, resulting in excessive postprandial lipemia and accumulation of chylomicrons, remnant particles and non-esterified fatty acids. These particles are associated with disruptions in lipoprotein metabolism and changes in inflammatory factors, thus their association with cardiovascular disease, metabolic syndrome and diabetes is not surprising. Dietary factors, not just fat, influence postprandial fluxes. This leads to the question: do we need a standardized fat tolerance test?

I've been reading a lot of studies lately dealing with postprandial clearance of fats from the blood and it certainly seems to me that these are coalescing to a hypothesis that the fat tissue fails first.  Oh but CarbSane, you've been saying this for over a year now  This is news?  Well, yes, in a way.  Gratuitous third person self referencing aside, it does still appear that a breakdown in fat tissue regulation is the precipitating factor in the metabolic dysfunction cascade in the majority of cases.  However this initial breakdown appears to occur on the uptake side of the adipocyte.  Impaired fatty acid uptake by adipose tissue leads to elevated postprandial circulating NEFA that are:

1. Excessive in the postprandial phase when they should be low, and/or 
2. Different in composition, reflecting dietary intake, from the types of fatty acids released from stored body fat.

This paper "puts it all together" with a focus on the role ASP, amongst a myriad of other factors, plays in this whole scenario.  Between this and some of the reading I've been doing on blood glucose and such, it's enough to shake one into at least reconsidering a low fat diet.  

The paper contains a wonderful graphic that I've included below:

Fig. 1. Determinants of fatty acid fluxes: transporter, enzymes and hormones directly influencing postprandial metabolism, including absorption, lipolysis and hepatic remnant clearance are shown, emphasizing those that play a ‘‘gatekeeper’’ role.  Each protein appears to have a specific and individual role in the overall process.

ASP: acylation-stimulating protein; Chol: cholesterol; CHYLO: chylomicron; CR: chylomicron remnant; FATP: fatty acid binding protein; GLP: glucagon-like peptide; HL: hepatic lipase; INS: insulin; INSR: insulin receptor; LDL: lowdensity lipoprotein; LDLR: low-density lipoprotein receptor; LPL: lipoprotein lipase; LRP: LDL receptor-like protein; MTP: microsomal triglyceride transport protein; NPC1L1: Niemann-Pick C1-like 1; NEFA: non-esterified fatty acid; TG: triglyceride; TNFa: tumor necrosis factor-a; TNFR: tumor necrosis factor receptor; VLDL: very low-density lipoprotein; VLDLR: very low-density lipoprotein receptor.

Overview of Fatty Acid Fluxes:  A Bullet-Point Summary:

• Fatty acids are absorbed by small intestine cells through diffusion and assisted by fatty acid transporters (FATP).
• FA's are esterified to form triglycerides, TAG's.
• TAG's are packaged in the core of a lipoprotein particle called chylomicrons
• Chylomicrons are formed with the action of fatty acid binding protein-2 (FABP-2) and microsomal
• triglyceride transfer protein (MTP)
• Chylo contain apolipoprotein B48 (apoB48), these are released into circulation.
• Circulating chylo also contain other apoproteins including: apoCI, apoCII, apoCIII, apoE and the recently
• identified apoAV
• Hormones such as glucagon-like peptide (GLP)-1 and -2 may be involved in absorption.
• Lipoprotein lipase (LPL) associated with the endothelial cell surface in the capillaries binds to the chylomicrons.  LPL hydrolizes the triglycerides to glycerol and free fatty acids.
• LPL-liberated fatty acids have two fates:  uptake to adipose tissue for storage or uptake and use for energy by muscle and other cells. 
• As TAG are lost, chylo get smaller and shed excess phospholipids and apoproteins which are transfered to HDL particles.
• What is left after 70-90% of the triglyceride has been hydrolyzed, the particle is called a chylomicron remnant, CR.
• "LPL is a key gatekeeper of this clearance process, and in the absence of LPL there is no chylomicron hydrolysis.  However, in addition to LPL, there are many collateral players that influence the activity of LPL, including very low-density lipoprotein receptor (VLDLR), apoE, apoCII, apoCIII and apoAV."
• Fatty acid uptake by peripheral tissues, primarily muscle, is facilitated by various transporters:  CD36, FABPpm and FATP-type proteins.  Note:  This is one of several references I've seen to active transport of fatty acids -- implying a regulatory opportunity (e.g. hormonal, signalling molecule stimulation of FA uptake) -- although the literature is pretty clear that a passive "flip flop" mechanism is also involved.  Thus FA uptake by cells is more complicated than is usually discussed, a topic I hope to get around to publishing some blog posts on.  Selective uptake of certain fatty acids may well be explained by the involvement of transporters and this may well explain why body fat and NEFA from body fat tends to have a relatively constant fatty acid compositional profile.   
• Normally, a little more than one-third of LPL-liberated FA's remain in circulation and are not immediately taken up by adipose and peripheral tissues.  Note:  This is a very interesting phenomenon that could go a long way in explaining impaired glucose tolerance with high fat diets.  If the FA uptake mechanisms are saturable -- and I'm seeing evidence that at least some are (future blog posts) -- this would mean there is some maximum uptake possible over the short time-course, and the percentage of "escapees" would likely exceed the cited 36%.
• LPL is subject to what is called product inhibition.  The product of LPL, the NEFA, inhibit further lipolysis.  If uptake is impaired, NEFA levels rise.  Rising NEFA then inhibit further lipolysis of chylo triglycerides causing an accumulation of chylo in circulation as well (and likely more triglyceride-rich CR delivered to the liver).
• Both circulating NEFA and CR deliver fatty acids to the liver and are the primary sources of fatty acids for VLDL.
• CR uptake by the liver is mediated by apoE, enzymes and is receptor mediated by low-density lipoprotein receptor-related protein (LRP) and low-density lipoprotein (LDL) receptor.  The VLDL receptor, which binds apoE, and scavenger  receptor (SR)-B1, which binds multiple particles, may also be involved.  
• The liver also takes up NEFA via numerous fatty acid transporters.
• Increased postprandial NEFA and CR lead to increased hepatic VLDL output.  I note that VLDL are essentially equivalent to circulating triglycerides in the fasted state.  In the fed state, the lipoproteins carrying the majority of the triglyceride load are chylo, CR and VLDL.
• VLDL particles contain triglycerides, cholesterol ester, phospholipid and apoB100. The more fatty acids available the larger the VLDL particle. 
• LPL acts on VLDL particles to release fatty acids to cells for uptake.  The liver recycles the CR and NEFA if you will.  In the case of the latter, to transport them in a less biologically active form.
• As with chylo, as triglycerides are released, VLDL becomes smaller.  Apoprotein excesses are transferred to HDL and VLDL remnants are converted to LDL particles in the liver.
• LDL contains primarily apoB and is taken up by the liver and other cells by a receptor mediated process, the receptor aptly named LDL receptor, LDLR.

The authors discuss the need for some sort of standardization for an oral lipid tolerance test by which to assess the efficacy of an individual's postprandial lipid metabolism.  They cite wide variation in test meals --  size, composition, liquid v. solid, and fatty acid type -- used in the plethora of studies available.  They also discuss a period of more than just an overnight fast to reduce the variability of diet on the results.  

... changes in dietary patterns and exercise before the fatload test may influence both fasting lipoproteins and postprandial fat responses.  The chylomicron response to a fat challenge can vary by as much as 50% according to the fat, carbohydrate and protein content of daily food intake. Chronic consumption of reduced calorie, high-carbohydrate diets can exaggerate plasma TG levels.

The authors identify LPL as a major "gatekeeper" but describe an intricate web of proteins and hormones that regulate LPL.  Therefore one might envision LPL as the gate by which various gate operators regulate lipid metabolism.  The discussion continues with the role of the various receptors and proteins and how they impact LPL action.  In the interests of brevity (or limiting length - grin) I'll not include this here.

I suppose this paper is not really so much about fat tissue regulation, and therefore should probably not be a part of this series.  But I put it here anyway due to the authors' focus on Acylation Stimulating Protein, ASP.  Thus we get to the section entitled  Hormones and acylation-stimulating protein.  I'm going to excerpt this part in its entirety and intersperse some comments of my own.

Various hormones contribute to modulation of overall LPL activity. These include insulin, tumor necrosis factor-α  (TNF-α), ASP and possibly adiponectin.   Insulin is well documented as a major LPL modulator.  In the postprandial state, adipose tissue LPL is increased relative to fasting levels, whereas muscle LPL tends to be reduced.  In adipocytes insulin acutely increases LPL activity and secretion, mostly related to changes at the post-translational level.  This raises the question: are the postprandial increases in LPL solely mediated by the postprandial increases in insulin?  Based on studies by Deshaies and coauthors in rodents, the simple answer would appear to be: ‘‘yes’’.  Changes in adipose tissue LPL activity were proportional to the changes in insulin, even in the absence of nutrient absorption. Conversely, the usual postprandial changes in adipose and muscle LPL did not occur in the absence of an increase in insulinemia.  Although insulin clearly plays a dominant role in influencing LPL activity, it is not the only component that influences postprandial fat clearance.  

Clearly the work of Cianflone's group in collaboration with Frayn's group demonstrates this.  See:  Fat Accumulation: Taubes v. Frayn ~ ASP in vivo in humans.  Bottom line, the period of greatest postprandial triglyceride clearance does not coincide with the insulin "peak".  Still, there can be no denying the major role insulin plays in regulating circulating lipids.

TNF-α is a cytokine with a wide range of activities.  It is produced primarily by monocytes/macrophages, although significant amounts are secreted by other cell  types, including adipocytes, and increased production has been implicated in the pathogenesis of insulin resistance and type 2 diabetes.   Several variants have been identified that modify gene transcription. TNF-α can potently suppress lipid genes, including LPL and glycerol phosphate dehydrogenase, thereby slowing down not only extracellular lipolysis, but also intracellular processing.

This is the first I've read of TNF-α having a role in fatty acid metabolism related to LPL.  Most of what I've read deals with apoptosis.  This might be an additional avenue of inquiry.

It is important to stress that TG clearance occurs as a two-step process: first, the lipoproteins are hydrolyzed by LPL releasing NEFAs; secondly,cNEFAs are taken up into the cell and re-esterified to a storage TG molecule. Numerous studies in vitro and in vivo have demonstrated that excess generation of NEFAs by LPL, without prompt and rapid clearance into cells, will result in product inhibition of LPL. Thus, processes that amplify the ability of the adipocyte rapidly to take up and store NEFA as TG will indirectly increase the hydrolytic efficiency of LPL. ASP is one such hormone. Also known as C3adesArg, ASP is an adipokine produced through the cleavage of complement C3 by adipsin. ASP interacts with C5L2, a G-protein coupled receptor, activating an intracellular pathway that leads to increased glucose transport and TG storage. Studies in ASP-deficient, C3 knockout mice demonstrate delayed postprandial TG and NEFA clearance (in spite of normal insulin levels), which are normalized with injection of ASP prior to fat load. This effect is maintained in mice treated with a high-fat diet, and in genetically obese ob/ob mice deficient in ASP (double knockout).  This delay in TG clearance leads to a leaner mouse with reduced adipose tissue, which is resistant to diet-induced obesity. In vitro studies demonstrated that while ASP has no direct effect on LPL activity (in contrast to insulin), the clearance of TG-rich lipoproteins is enhanced by ASP through increasing intracellular TG synthesis, which relieves NEFA inhibition of LPL. The effectiveness of adipose tissue trapping of LPL-derived NEFAs is determined by overall LPL activity, which in turn determines the efficiency of postprandial TG clearance. In contrast to the effects of ASP on adipose tissue, ASP decreased in situ muscle LPL activity, similar to the effects of insulin.

The above paragraph is a synopsis of much of the work of Cianflone and colleagues, some blogged on in the past, and some of which is still in the draft bin.   It seems almost too obvious that if the uptake/esterification by adipocytes is impaired, this will cause an upstream "backup" of circulating lipids.  Is it possible that, although larger adipocytes have been shown to remain ASP sensitive, that at some point they become resistant?  Or that the enzyme in the rate limiting step of esterification (conversion of the di to the triglyceride by diacylglycerol transferase) is also product inhibited by too much triglyceride in the fat cell?  

In human studies, fasting ASP is influenced by diet, body size (obesity), exercise and metabolic status (presence of cardiovascular disease and/or diabetes).  In normal healthy men and women, stratification of fasting ASP by tertiles demonstrated a delayed postprandial TG and NEFA clearance in the subjects with the highest fasting plasma ASP, a correlation that remained even after correction for differences in fasting TG. The precursor to ASP, complement C3, also demonstrates these similar associations with TG clearance, as well as correlations with metabolic syndrome indices.   The association between increased fasting ASP and delayed TG clearance probably reflects ASP resistance, as proposed and described elsewhere, similar to the insulin resistance paradigm. Fasting plasma ASP and C3 may be useful markers to identify subjects with postprandial delayed TG and NEFA fluxes, such as those associated with metabolic syndrome.

Cianflone's group is rather prolific in its generation of peer review research articles.  I've got a few related to this discussion yet to discuss.  This notion of ASP resistance is intriguing.  In his more recent works, Keith Frayn has focused on the role of adipose tissue as a "buffer" for dietary fluxes of fat, but oddly he's seemingly dropped even the mention of ASP from his lexicon.  I can't help but wonder if there wasn't some sort of falling out between rival research groups?  The evidence for ASP's involvement in fatty acid flux into fat cells is overwhelming, and various knockout mice targeting ASP/pathways produced adipose tissue anomalies seen with the various insulin receptor knockouts (those IRKO mice).

Dietary glucose is disposed of and utilized in rather short order.  Although fatty acids are constantly being oxidized for energy, it seems more that dietary fats are not immediately used for energy, even when taken up by the peripheral tissues.  My reading of the lipid droplet literature that caught my eye (as well as all of this mitochondrial buzz) a while back seems to favor a cellular TAG/FA cycle that sequesters fatty acids in the less reactive form (triglycerides) until release (lipolysis to fatty acids) is warranted to meet immediate needs and immediate transport to mitochondria for oxidation.

Because of this recycling and transfer of apoproteins (chylo → CR + NEFA → VLDL/trigs → LDL) with HDL being higher, apparently, when more triglycerides are used in a timely manner (thus apoproteins "disposed of" post haste), fasting lipids seem more telling -- in conjunction with hormone levels -- than fasting blood glucose likely tells us much about our metabolic status.  

It seems reasonable that a VLC diet in a hypocaloric (weight loss) context will initially improve lipids.  More fat = more chylo = more apoproteins to be transferred to HDL.  That many LC'ers see LDL increase in the face of lower fasting triglycerides (for all intents and purposes VLDL) may be further signal of improvements. A greasing of the works so to speak.  In the face of glucose restriction and energy deficit, dietary fat may well be cleared more efficiently, and the LDL a byproduct of eating a lot of fat.  This would be in contrast to elevated LDL and VLDLseen in the context of an ordinary weight maintaining (or energy surplus) diet of a person with impaired adipose tissue fat uptake (clearance).  Hellerstein has demonstrated that higher VLDL/triglycerides on high carb diets are attributable mostly to decreased clearance of VLDL.  This is not impaired uptake of dietary influx, but rather leaving some stuff out in circulation to be recycled.  

One interesting thing about the action of ASP as discussed in this article was that it increases adipocyte-associated LPL but decreases muscle-cell-associated LPL activity.  ASP increases fat storage and decreases fat uptake for burning.  Umm... Well let's do a little TWICHOO Mad Libs, shall we?  Nothing stimulates ASP like chylomicrons -- aka dietary fat -- nothing.  Not even protein.   Dietary carbs stimulate carbohydrate oxidation.  Dietary fats?  Umm ... they stimulate fatty acid uptake into adipose tissue.  We can think of ASP acting like a sump pump pulling fatty acids into your cells by repeatedly emptying the well by stimulating esterification.  If the sump can't keep up with the water flow into the well, it doesn't transfer any more water out and the pump may well malfunction.  On the other hand if the pump is working properly and can handle the water influx, it transfers a lot of water in spurts.  This seems an apt analogy to ASP.

It bears repeating that the dietary NEFA fluxes that would occur with inefficient post-prandial plasma lipid clearance would not be reflected in fasting levels.  In the context of a "usual Western diet" (e.g. one that averages in the mid-to-high 30's % fat, mid-to-high 40's % carb and around 15% protein) this is reflected in elevated fasting LDL and VLDL -- from recycled NEFA & degradation -- rather than persisting elevated fasting NEFA per se (that should actually be suppressed somewhat with nutrient excess).  Further, fasting levels are elevated in normal individuals because we shift fuel to fat burning in the unfed state naturally.

However post-prandial NEFA spikes can contribute considerably to the fatty acid supply to lean/ectopic tissues, where they will accumulate if energy needs are such that they are not needed for energy.  If these are going to be used in short order for energy (e.g. endurance athletes have high IMTG/IMCT/IMCL) they are not problematic (the athlete's paradox).  In the context of chronic energy excess where these (a) hang around for long periods subject to peroxidation, and/or (b) deliver fatty acid excess to the mitochondria where they -- for lack of a better term -- gum up the works.   Furthermore, as stated previously, the ppNEFA could easily exceed normal physiological fasting levels.  Unlike glycation, the transport into non-adipose cells (liver the exception) is not reversible when NEFA levels subside.  No, once in the cells they are esterified and stored in lipid droplets if not used.   Perhaps a defect in this, leaving intracellular (reactive) NEFA levels high as a result, contributes to lipotoxicity as well.  Although uptake is receptor/transporter mediated, there is ample evidence for passive diffusion whenever NEFA levels are elevated.

In other words, this paper aligns with others in implicating a defect in postprandial lipid disposal as the precipitating factor in the cascade of metabolic mahem.   Fats stimulate disposal into fat tissue.  When this is impaired, all heck breaks loose.  Obviously I'm thinking out loud here with a couple of other papers, Frayn's most recent probably foremost, on my mind.  I hope to get the time and focus to produce a summary post on these thoughts.  So ... to be continued ...

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For Part VII, click here.