Fat Burning 101 -- The Biochemistry

Laugh if you get it!

In comments on my last Thermodenyics post, I made the following statement:

The β-oxidation + Krebs part is the "metabolism" of fatty acids resulting in production of some heat, some ATP, and a large number of so-called "reducing equivalents" that will produce much more ATP (cellular energy currency) in the Electron Transport Chain. You don't see mass escaping your body, but the carbons that were originally contained in the larger fatty acid molecules are exhaled as carbon dioxide. Atkins' original claim was that enough molecules escaped the Krebs cycle and were excreted before being fully metabolized for their caloric content. The vast majority of β-oxidation to burn fat for energy occurs in organs like the heart and skeletal muscle. Once a fatty acid is committed to the β-oxidation pathway they are oxidized completely down to carbon dioxide.

In comments, Kindke quoted that last bolded line and inquired:

I'm interested in this comment, can it be explained in more detail, ......where does it come from? ..... and from what references does it gain support?

I began responding in regards to just that statement, but it requires context and thus I thought it better to make a blog post out of the expanded topic instead.

Liver vs. Everything Else

As a first point of clarification, I want to amend my sentence with context:

• I'm talking about the fate of a fatty acid that is oxidized by the metabolic pathway called β-oxidation (largely the long chain fatty acids) or otherwise known as the Fatty Acid Spiral, FAS ... and,
• I'm talking about β-oxidation in all cells capable of metabolizing fatty acids OTHER THAN those capable of ketogenesis.  Thus not the liver (and to a lesser extent perhaps the kidney).

Under most conditions, the liver will also completely oxidize fatty acids down to carbon dioxide and use them to fuel its synthetic duties.  But in some circumstances, the liver is capable of stopping this at the door to the Krebs cycle and exporting the ketones for use by other cells of the body.   It does this because the enzyme responsible for the first step in "ketone" production -- HMG CoA Synthase -- is only present in significant quantities in mitochondria of the liver (and perhaps kidney).

There are only a few cell types in the body capable of two-way metabolic substrate traffic that can be both givers and takers.  All of the others, the whole greedy bunch of them, are simply takers.   Where glucose is concerned, the cells of the liver (and to lesser extent the kidney) are the only ones capable of two-way trafficking.  Where fatty acids are concerned, it is the liver and fat cells that are the only ones capable of two-way trafficking.    

All other cells, what goes in that is not used to synthesize cell structures, hormones, etc., MUST be oxidized -- or "burned" -- to get out (as carbon dioxide).  

Metabolic Cycles and Sequential Chemical Reactions:

When I discussed the Krebs/TCA Cycle in this post, I wrote:

I like to liken the Krebs Cycle to a giant revolving door with many entrances and exits about the rotation at which people can get on and off. ... When the "door" is filled with people in each compartment, it revolves around and the coordinated "pushing" of all the people imparts a momentum to the door itself, while any single person can pretty much be swept along and round and round without even trying.

It's not a perfect analogy, but it illustrates what goes on in *bio* chemistry where virtually every reaction that occurs is mediated by relatively giant protein structures called enzymes.  These enzymes have different shapes and sites that attract and bind molecules and at times quite literally push them together so the reaction happens.  Often these are arranged in complexes where the products of one are passed down to the next.  Whether cyclic (revolving door) or linear (any waiting line where things are forced single file will do), the reactions that occur seem to develop somewhat of a chemical momentum.  When you think of a single file line, say to get into a crowded hot spot, when people get on at the end they push forward wanting to move those in front along.  Similarly when the bouncer opens the door and lets a few in, those behind them in line are practically "sucked along" until the door slams in their faces.   

In many of the "cattle coral" lines at banks, amusement parks, and the DMV, once you commit to getting in line, you're pretty much stuck there until you get to the destination and "out".   An even better analogy would be going over a bridge or into a tunnel.  Once you pass that last exit, there's no going back, and once you get on/in, you have committed to the journey to the other side.  In biochemistry, we can often identify a "commitment step" in a metabolic pathway akin to deciding to go into the tunnel.  

Another important step in most metabolic pathways is the rate-limiting step.  Any sequence or cycle of events can only go so fast as the slowest step.   Rate-limiting steps are often identified as those which are most heavily regulated by a variety of means (enzyme activity, substrate concentration, hormonal stimulus, etc.).  When you "turn on" this step, it's like turning on the whole pathway, and vice versa if you inhibit it to a crawl.  

The commitment step and the rate-limiting step are sometimes, but not always, the same step.  When they are not, it is not unusual to see some regulatory feedback from the rate-limiting step to the commitment step thus refusing entry of new substrate into the pathway and/or rerouting to another pathway to avoid a "metabolic traffic jam".    Much like during rush hour congestion or construction you see "Use Alternate Route" signs on the highway when a particular bridge is backed up by the limits of processing at the toll gates.

The Matrix -- Where Fat Burning Happens!

For all the confusing and gimmickified rhetoric surrounding this notion of fat burning, much of it is nothing more than a semantics game and has little to do with fat burning itself.  For the purposes of this post, realize that it is as close to scientific fact as possible that no cell is ever deficient in fatty acids to burn.  Period!  Especially the obese  (see this post and others linked to therein).  So I'm going to pick up the storyline at the point of a free fatty acid in the cytosol (fluid inside the cell) of a non-hepatic cell -- let's use a muscle cell.    So we begin at Step 1 in the figure below.

Figure 23.1 Marks Basic Medical Biochemistry (my edition p. 419) 

Free fatty acids, or non-esterified fatty acids (NEFA) are indeed "toxic" to cells.   If one considers all of the evidence and biophysical realities, one can understand why.  Free fatty acids are like soldiers in guerilla/paramilitary armies.  They can operate independently, without obeying treaties established between recognized states, and answer to no central government that can be held accountable for their actions.  This is because fatty acids can cross lipid membranes without assistance and without resistance, and in biochemistry we absolutely cannot have an open borders policy!  And yet, they cannot participate in civilized society or formal military organizations as they often lack the necessary "documentation" to participate in certain activities.  This leads to metabolic havoc.

This nature of NEFA is exactly why we have such relatively infinitesimal amounts of fat in this form in circulation, and what we do have is to ensure adequate supply to and uptake by cells.  Once taken up, as Keith Frayn wrote in Metabolic Regulation (I need to get you the page number), the most likely fate of the fatty acid is immediate esterification, as NEFA inside cells is even lower still than in circulation.

The first step involves the FA being "activated" by the addition of a coenzyme-A moiety to form a Fatty acyl-coA.  (Marks' p. 421)

The acyl CoA synthetase that activates long-chain fatty acids, 12 to 20 carbons in length, is present in three locations in the cell: the endoplasmic reticulum, outer mitochondrial membranes, and peroxisomal membranes.  This enzyme has no activity toward C22 or longer fatty acids, and little activity below C12.

There are three fates of FA-coA as shown in the figure at right, also from Marks'.  For the most part the split is between oxidation for energy or storage as triglycerides (triacylglycerols).  In muscles, the "Energy" pathway is just β-oxidation followed by Krebs/TCA.  Yes, there are storage sites in the cell for excess fatty acids -- called non-descriptly "lipid droplets" or LD.   So even getting into a cell doesn't guarantee "burning".  There are multiple mitochondrion (furnaces/generators) and lipid droplets (storage tanks) throughout the cytosol (or cytoplasm) of all but a very few cell types in the body.

So here's why I went into the commitment and rate-limiting steps and such.  Below is Figure 23.5 from Marks' that I've "colorized" and enhanced a bit to make it easier to follow the fatty acid through the steps and to show the location of the reactions with respect to the mitochondrial membranes.  For the whole picture, see the cutaway of the mitochondria at right.  

What you have going on above is often referred to as the "carnitine shuttle"  you have the FA-CoA handing off its fatty acid to carnitine, between membranes, and then the FA-carnitine handing the fatty acid back to a CoA in the matrix.  One might ask themselves "why?".  Why not have a system where the Fatty acyl-CoA just passes through a single membrane or both straight into the matrix without all of this fuss?    Well, in biochemistry, anything that doesn't make sense from a superficial practical point of view almost always makes sense when you think about regulating reactions.   Selective transport across membranes is one way to make sure that you only send the fatty acids into the mitochondria that are needed to be oxidized for energy.  

Can you see already how absurd it is to focus on the release of fatty acids from fat cells as being anything approaching some "fine tuned" regulator of fat burning (β-oxidation)?  Never mind that well-in-excess-of-need levels of fatty acids are continually released and excesses taken back up as a matter of routine.

Fatty Acid Oxidation:  Commitment and Rate Limiting Steps

The exchange of a CoA for the carnitine moiety on the fatty acid catalyzed by the carnitine palmitoyl transferase I (CPTI, also known as CATI - carnitine acyl transferase I) enzyme is believed to be the rate-limiting step in the above process.  I boxed that enzyme in green to highlight this.  The Acyl CoA Synthetase is in the outer mitochondrial membrane with its active region facing the cytosol (therefore the fatty acyl-CoA is formed outside the mitochondria)that one might consider the activation of the fatty acid to fatty acyl-CoA as being the "commitment" step in the oxidation of the fatty acid.  The FA-CoA is manufactured outside the mitochondria but the outer membrane is permeable to it.  If CPTI is actively "removing" FA-CoA from the other side of the membrane -- by converting it to fatty acyl-carnitine --  then the FA-CoA is "sucked" into the mitochondria by virtue of a favorable concentration gradient.    Ultiimately: 

"Control of fatty acid oxidation is exerted mainly at the step of fatty acid entry into mitochondria"

Marks' Fig. 33.16
with CPTI modified 

The product of the first step in fatty acid synthesis (de novo lipogenesis), malonyl-CoA, inhibits the activity of CPTI thereby "governing" the direction of fat metabolism (from synthesis to breakdown).  When malonyl-CoA is high, a "signal of plenty", CPTI is inhibited.  Incidentally, high malonyl-CoA is associated with elevated cytosolic fatty acyl-CoA -- close some of the toll gates and traffic backs up!   Perhaps we should rename the carnitine transferase enzyme in the Asylum Dictionary for Calorie Denialists as CR1 - Calorie Receptor 1!  Through malonyl-CoA the mitochondria "sense" the energy state of the cell and decide whether or not they need more ATP from fatty acids.  If they do, then the gates open and fatty acids are imported to be burned.  If not, the gates stay shut.

There's a ton on this as regards diabetes and energy regulation, including other regulatory factors for CPTI ... hopefully a subject to be tapped for some 2015 rants!

A (Fatty) Acid Trip Into the Matrix

I was dabbling in the idea of making analogies to the Matrix movies, but I just don't have it in me for now, and I'm not sure they would work well anyway.   The major purpose of fatty acids delivered to cells is as a source of energy to produce ATP.   Although on a whole body level, ATP can be thought of as a rechargeable battery, ATP is more like quarters required to operate all the quarters-only coin-op machines that perform all the necessary functions of the cell.  ATP is the "end of the line" for energy,  and there's not a lot of change jars lying around the cells so it's generated on a somewhat "as needed" basis to replenish a cellular pool.

Once pyruvate (from glucose metabolism) or a fatty acid is transported into the mitochondrial matrix, the intent is to convert them to ATP.  In this respect, the mitochondria are like change machines.  No matter if you put a $1, $5 or $10 bill in, you only get quarters out for the full amount.   If you don't need the change, you don't put the bills in the machine.    If you don't need $10 in quarters you'll probably scrounge around for some in your pockets or see if you can get find a smaller bill somewhere.  Dealing with just the fatty acids then, I've finally gotten around to Kindke's question.  To repeat from above:  "Control of fatty acid oxidation is exerted mainly at the step of fatty acid entry into mitochondria."   If you don't need the ATP, the fatty acid is not transported into the matrix.  The rest is "go to completion".

Once inside the mitochondria of most cells,  there's no way out but to go completely through the β-oxidation + Krebs/TCA pathways emerging as CO2 and producing ATP "change".  To repeat, this is because most mitochondria lack the HMG CoA Synthase to divert acetyl-CoA away from Krebs/TCA to ketone synthesis.  Ketogenesis is a whole body adaptive pathway to spare protein during starvation.  There would be no evolutionary or energetic advantage or purpose to diverting the energy stream within mitochondria.

Without an off ramp at Exit CoA, the fatty acid is as fully committed to being metabolized to completion as you are to going through a tunnel.   At this point, with proper energy (fatty acid delivery rate) to to mitochondria, this is what is supposed to happen.  The β-oxidation pathway is cyclical in nature in that the output/product of one "pass" through is the input/reactant for the next until you've chewed up the whole long chain.  Biochemical momentum, if you will, once again.*  Thus once inside the mitochondrial matrix, the activated fatty acyl-CoA is destined for extinction.  The acetyl-CoA products of β-oxidation have nowhere to go but into the Krebs cycle.

* EDIT:  The paragraph above has been edited slightly from the original.  Thanks to Jane Karlsson for sending me this study:  Mitochondrial Overload and Incomplete Fatty Acid Oxidation Contribute to Skeletal Muscle Insulin Resistance.  It directly counters this statement I made:

But I can back this assertion up by pointing to what you DON'T see in mitochondria: a build up of shorter chain fatty acyl-CoAs resulting from incomplete β-oxidation. Particularly in cases of mitochondrial disruption/dysfunction -- such as in obese or diabetic people -- you would expect to see this if incomplete β-oxidation occurred. You don't.

In this study, they created mice with metabolic syndrome by feeding them a high fat diet.  This resulted in excessive adiposity, hyperinsulinemia, etc., and:

As expected, total serum nonesterified free fatty acids (NEFA) were elevated in response to an overnight fast and prolonged HF feeding; however, in the present study, the diet-induced increase was apparent only in the postprandial state

They then proceeded ...

... to analyze 36 independent acylcarnitine species ranging in size from 2 to 22 carbons, representing byproducts of substrate catabolism. These acylcarnitine esters are formed from their respective acyl-CoA intermediates by a family of carnitine acyltransferases that reside in subcellular organelles (primarily mitochondria), where they catalyze the exchange of CoA for carnitine (Ramsay, 2000). Whereas acyl-CoAs cannot cross the mitochondrial membrane, the acylcarnitines do so efficiently. Subsequently, cytosolic acylcarnitines can be exported into the blood. The serum acylcarnitine profile therefore provides an integrated systemic snapshot of in vivo substrate flux through specific steps of β-oxidation ...

So ... we DO see partially oxidized fatty acids in the dysfunctional state when Krebs is overwhelmed and/or throughput in the fatty acid spiral leads to fragments escaping the next "turn".  The carnitine acyltransferases in the matrix, then, appear to act like scavengers to remove these from the matrix (and subsequently they can escape the cell entirely into circulation.   What is interesting is that the fragments are somehow "escaping" from the β-oxidation "spiral" ... one would think it more practical to have these scavengers guide the fragments back into the spiral rather than escorting them out of the mitochondria.  This supports the idea of β-oxidation as an intended "one shot deal" to completion ... but apparently the limits of this pathway are tested when too many fatty acids are fed in at once.  This feeds into the next section which picks up with what I wrote originally ... /EDIT

When Things Go Wrong ...

In the realm of scientists studying mitochondria and particularly in diabetes, there is much that is unknown, but there is also a lot that is extremely well characterized.  One thing that the scientists agree on is that mitochondrial dysfunction brought on by diet is induced by the excess.  EXCESS.  Too many calories.  To a greater or lesser extent, different people are able to shield the cells from this excess by sequestering the energy in adipose tissue.  When this "safety net" gets overwhelmed, that's where the problems begin due to excess fatty acid (NEFA) delivery to the cells.  Long time readers of this blog know that I've been beating this drum for a very long time.  I took the time to write this post as it will hopefully provide the basis upon which to connect some dots and re-visit some old posts.  In light of changes in the above section, I also plan to revisit that paper and hopefully get to a few more regarding mitochondrial fatty acid overload.  

Funny, I was getting to the point about uncoupling and futile cycles in the course of the thermodynamics discussions.  This will not be such a tangent after all ... :-)