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Welcome all seeking refuge from low carb dogma!

“To kill an error is as good a service as, and sometimes even better than, the establishing of a new truth or fact”
~ Charles Darwin (it's evolutionary baybeee!)

Sunday, October 2, 2011

Fat Tissue Regulation: Part IV ~ How Acylation Stimulating Protein Works

Lipases are a tricky bunch of enzymes when one is looking to the action of an enzyme to extrapolate to overall regulation of fat mass.    What do lipases do?  They are enzymes that facilitate lipolysis, which is the breaking apart of triglycerides into glycerol and three fatty acids.  We have digestive lipases that break dietary triglycerides down so that they can be absorbed, but once absorbed they are packaged again back into triglycerides for transport to cells.  These triglycerides are packaged in chylomicrons.  There are lipases both in circulation and attached to all of our different cells, called lipoprotein lipases (LPL's) that break down triglycerides to free up fatty acids.  Those associated with the cells are doing so to facilitate uptake of the fatty acids.  Here's where it gets tricky, because lipases associated with, say, muscle cells, are acting to bring fatty acids into the cell to be oxidized for energy.  But the lipases associated with fat cells?  These are acting to bring fatty acids into the cells for the purposes of being re-esterified into triglyceride stores.  Then there are lipases within cells, like hormone sensitive lipase, HSL, in fat cells.  The function of this lipase is "mobilization" of fat stores -- breaking down triglycerides to release fatty acids.

If you're locked into a "fat burning" paradigm then, you want high HSL and LPL activity in fat cells and non-fat tissue cells respectively, and low adipocyte LPL activity.  Fats can't get into cells without lipolysis.  But this isn't how our human metabolism works.


ASP enhances in situ lipoprotein lipase activity by increasing fatty acid trapping in adipocytes

This study from Katherine Cianflone's research group used adipocyte tissue cultures (in vitro) to look at the activity of secreted and cell-associated LPL. 
The conventional view is that LPL activity in WAT is the rate-limiting step in the hydrolysis of TG-rich lipoproteins and the uptake of derived NEFAs.  In fact, this has not always been supported by in vivo findings.  In a study examining the effect of epinephrine on LPL activity in humans, the extraction of plasma TG across a subcutaneous WAT bed was increased with epinephrine infusion, indicating an increase in WAT LPL activity.  However, LPL-derived NEFAs were not taken up into WAT but released into the circulation.  The authors concluded that the coordinated reciprocal regulation of NEFA influx and NEFA efflux (resulting from the activation of hormone sensitive lipase (HSL) by epinephrine) is an essential determinant of the efficient uptake and esterification of LPL-derived NEFAs.  This suggests that factors other than the activity of LPL can determine overall TG clearance and the uptake of released NEFAs.
Epinepherine clearly is another hormone regulating the TAG/FA, but that is a topic for another day.  What this demonstrates, however, is that when epinepherine acts on LPL it's purpose is to increase the availability of NEFA in circulation.  The NEFA liberated in the above scenario was not trapped in the fat cells.  
LPL activity is known to be inhibited by NEFAs (the main product of lipolysis). Therefore, an alternative hypothesis is that the rate-limiting step in the hydrolysis and clearance of TG-rich lipoproteins by endothelial LPL is the capacity of underlying adipocytes to take up, esterify, and trap generated NEFAs. That is, effective or in situ LPL activity in WAT will not only be determined by LPL activity in the adipocytes but also by the effectiveness of fatty acid trapping within them. Factors that enhance NEFA uptake and esterification into the adipocytes would then relieve LPL inhibition by NEFAs. The aim of the present study is to determine whether acylation-stimulating protein (ASP) acts in this way.
The inhibition of LPL by NEFA is classic product inhibition of enzyme activity.  When NEFA are in high supply, one would not want LPL creating more, rather we want the reverse, or esterification to be upregulated if anything.  This is a sort of Catch 22 for the LPL, because in order to get fatty acids into the fat cell to be stored, they must be in the free form -- in other words the same form that can get out of the fat cells is required in order for them to get in.    At right is a crude graphic of this process.  Triglycerides (TAG) are acted upon by LPL and the resulting NEFA can be released into circulation or taken up by the fat cell.  DAGAT is the acronym for diacylglycerol acyltransferase.  It is the enzyme that catalyzes the last step in the esterification process, attaching the last fatty acid to convert a di- to a tri-glyceride.  I've got some information on NEFA transport into fat cells in the pike, but although this is a facilitated process it does rely on a favorable concentration gradient.  In other words, as NEFA move into the cells it would reduce the difference in NEFA concentration from outside-to-inside the cell thus slowing transport into the cell.  However, when the NEFA are esterified to TAG, this prevents this from happening.  In other words, esterification essentially removes NEFA from the inside of the cell, maintaining the concentration gradient.  Therefore, esterification lowers NEFA levels inside the cell, this facilitates more NEFA to be transported into the cell, and this removes NEFA from the proximity of LPL thereby relieving the product inhibition of the enzyme.  One can think of DAGAT as a pump out of an underground tank into which water can flow by gravity.  Water flows into the tank until it's full, then no more flows in, but pump the water out and more can flow in.   From the article:
ASP increases TG synthesis in a concentration- and time-dependent manner in two ways. First, ASP stimulates the activity of diacylglycerol acyltransferase, the last and possibly the rate-limiting enzyme involved in TG synthesis. Hence, ASP indirectly increases NEFA uptake and esterification without increasing the delivery of NEFAs or acyl-CoA to microsomal enzymes .  Second, ASP directly increases glucose uptake in cultured human skin fibroblasts and human adipocytes. Moreover, ASP also decreases NEFA release from human adipocytes by increasing the fractional reesterification of NEFAs (to the same extent as insulin) and decreasing lipolysis (to a lesser extent than insulin). Thus, the ASP pathway in the adipocytes interacts in a positive feedback mechanism that increases ASP secretion, increases lipogenesis, and decreases lipolysis, the net effect being increased NEFA trapping (i.e., TG storage) within the adipocytes.
So in this study they cultured fat cells and measured LPL activity in the medium (akin to circulation) and associated with the cells.  The results are shown at right {click to enlarge}.  The ASP appears to have no impact on cell-associated LPL, and a minor (described as 5%) contribution to LPL activity in the medium.  Therefore it is concluded that ASP does not exert its triglyceride clearing behavior through stimulating LPL activity.  

Next they incubated the cells with triglycerides, the results are shown in the graphic at right, and 
Both ASP and insulin significantly increased in situ LPL activity (Vmax) by 60% and 41%, respectively
The authors also point out that only about 10% of the radiolabel was released as NEFA and taken up into adipocytes at even the lowest concentration so that the fatty acid substrate concentration was not rate limiting even at those low concentrations.  Therefore the observations are attributable to action by ASP and insulin.  They also performed two experiments:   1. An LPL inhibitor and 2. Incubation with free fatty acids (unbound oleate).  Inhibiting LPL diminished both ASP and insulin stimulated in situ (cell-associated) LPL activity.  Increasing NEFA inhibited LPL stimulation by both ASP and insulin,  but the ASP stimulatory effect on uptake was not eliminated.

From the Discussion:
The conventional view is that LPL is the gatekeeper of NEFA distribution, as LPL activity is essential for hydrolysis; it is also the rate-limiting step in the clearance of TG-rich lipoproteins and for the storage of released NEFAs in adipocytes.
Hmmmm ... is TWICHOO really "challenging conventional wisdom" after all?  
In the present study, we have shown that insulin, but not ASP, increases cell-associated LPL activity. On the other hand, ASP, through a stimulatory effect on NEFA trapping ... enhances the hydrolysis [lipolysis] of ... TG-rich lipoproteins to the same level as insulin. [LPL inhibitor]  markedly reduces [3H]NEFA release and incorporation into the adipocytes and abolishes ASP and insulin stimulatory effects. This result suggests that the stimulatory effects of ASP and insulin are not based on enhanced substrate bridging and uptake of whole nonhydrolyzed particles but on [radio labeled triglyceride] hydrolysis, which represents a prerequisite for the detection of the measured label. Unquestionably, LPL activity is an indispensable first step in the clearance of TG-rich lipoproteins. However, our data demonstrate that the clearance of TG from the medium can be enhanced by increasing the trapping of LPL-derived NEFAs within the adipocytes without necessarily increasing LPL in the adipocytes. Efficient postprandial TG clearance requires the coordination of both steps, and both insulin and ASP enhance the process through different mechanisms.
Insulin increases cell-associated LPL activity by ∼3-fold, a finding that is consistent with previous data demonstrating that insulin is a primary regulator of LPL activity  
Well, there you have it folks.  Even the ASP Queen Katherine  acknowledges the supremacy of insulin in the regulation of fat tissue.  Not so fast ;-)  ASP is no shrinking violet in the fat cell, and, quite frankly, I've no idea why anyone would think that it would be.  After all, ASP is actually made by the adipocytes themselves!  There's no reason to think it might play an integral role in fat tissue regulation ... right?  The discussion continues:
By contrast, ASP does not enhance cell-associated LPL activity.  Nevertheless, both ASP and insulin increase in situ LPL activity through increased NEFA trapping as stored TG.  It should be pointed out, however, that the increase in in situ LPL activity produced by insulin was smaller than anticipated by the increase in in vitro LPL activity alone (41% versus 100% increase, respectively, at an insulin concentration of 1 nM).   Thus, LPL activity alone is unlikely to be the rate-limiting step in the clearance of TG-rich lipoproteins; otherwise, the hydrolysis of TG-rich lipoproteins in situ should parallel the amount of available LPL activity in the adipocytes.   Furthermore, overloading the adipocytes with unbound NEFAs diminished in situ LPL activity, but to a lesser extent in the presence of ASP.   Thus, the efficiency of NEFA trapping by adipocytes appears to contribute to the rate of LPL activity and, consequently, the clearance of TG-rich lipoproteins.   Further supporting this finding is the result that the amount of LPL-released NEFAs remaining in the medium is small (15–30%) compared with the amount of NEFAs incorporated into adipocyte lipids (70–85%) even after short incubation times. Thus, these findings emphasize the importance of cellular uptake and storage in the overall regulation of TG clearance
ASP plays a clear role in the clearance of dietary fat from circulation.  I thought I'd harken back to my discussion of another study on ASP action in vivo in humans (a collaboration between Frayn's and Cianflone's research groups) in conjunction with my discussion of Frayn's more recent offering on fat tissue as a buffer for lipid influxes.   From the first study, we have the graphics below-left of ASP and triglycerides after a mixed meal, and from the second study we have the NEFA levels/net flux below right.

Just some thoughts:  It almost seems that insulin serves to "purge the tank" -- the NEFA pool surrounding/associated with the relevant enzymes -- shortly after a mixed meal, in preparation for the dietary fat chylomicron "assault".  When that arrives?  ASP takes over.   To say that either is necessarily the primary agent is misleading, IMO, as they seem to be two hormones serving the same purpose by differing mechanisms.   And to ignore the role of ASP in triglyceride clearance (e.g. uptake) following a meal is, in the words of Taubes, inexcusable for any scientist.  Good thing scientists aren't the one's making this inexcusable display of suboptimal intelligence.

To sum up, ASP clearly plays an important role in uptake and storage of fat, and therefore cannot be ignored in any comprehensive discussion of the regulation of fat tissue mass.


Click for Part V

3 comments:

Todd said...

Evelyn,
Is it fair to conclude from this post that both insulin and ASP are required for uptake and storage of fat? If so, that does not contradict what I call the "weak" carbohydrate-insulin hypothesis", namely that insulin is a necessary, but not sufficient condition for fat storage. This should be distinguished from the "strong" carbohydrate-insulin hypothesis, which holds that insulin is a sufficient condition for fat storage. But that hypothesis is largely a caricature, based on a misunderstanding of what we usually mean by "cause".

For example, to say that an ignition source causes a fire does not mean that it is sufficient condition. A fire results from three necessary but individually insufficient conditions: ignition source, fuel source and oxygen. When a match is struck we say it causes the fire, even though it does act alone and requires the oxygen and fuel. Likewise, insulin is a necessary but insufficient condition. Looks like ASP is required, as well as countless other metabolic factors. To highlight insulin's “driving” role is by no means to diminish that of ASP, which as you say is another hormone serving the same purpose by a different mechanism But precisely because insulin is necessary and controllable through diet, we can typically lose weight by dialing insulin levels much lower, e.g. by eating fewer insulinogens like carbs.

Conversely, the supposed “counterexamples” of the high carb eating Kitavans or Okinawans does not disprove the weak C-I hypothesis. Nor does the requirement for ASP. Striking a match does not always cause a fire, but that does not mean that ignition sources don't cause fires.

Evelyn aka CarbSane said...

Hi Todd, In citing this blog post, I realized I missed this comment. If you read this, my apologies! Based on the previous two parts of this series, together with the FIRKO mouse (lacks insulin receptors), it would appear that you are correct. Because one way to look at this is that the presence of either does not allow for proper triglyceride uptake necessary for fat to accumulate. Insulin receptors intact w/o ASP or ASP receptors intact w/o insulin both lead to the same adipose phenotype and obesity resistance. You only need to knock out one to disrupt the triglyceride uptake/storage.

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