Never forget, never forgive

For recreational purposes only, from here:



How toxic is palmitate at any concentration from zero to 0.4mmol/l if glucose is held at 5mmol/l? It's not.

How toxic is glucose at 25mmol/l in the presence of increasing palmitate? Glucose at this concentration becomes progressively more toxic within increasing but still physiological levels of palmitate.

Glucose at 25mmol/l is UTTERLY non physiological.

There is potentially a huge amount to discuss from this paper, one day, maybe. Even though they never delve in to the ETC, which they should do. But anyway, someone commented that papers using 5mmol/l glucose in the culture medium were rare. This one is a hen's tooth.

Back to thread next.

Peter

Protons: The pancreas

We've seen the concept of superoxide being used to produce insulin resistance as a means of limiting (glucose derived) energy input in to cells which really don't want it. Superoxide appears to be the primary marker of energy excess at the cellular level.

We know from isolated mitochondrial preparations that superoxide is physiologically produced by reverse electron transport through complex I and is driven, gently, by succinic acid alone working through complex II. Far more is produced when the NADH level is high as well as having a reduced CoQ couple through FADH2 input, be that from complex II or from fatty acid oxidation products. Macroscopically fat and glucose together should produce enough superoxide to show as cellular insulin resistance, rejecting glucose from the cell, while allowing continued fatty acid oxidation. That's simple and logical.

But if you are building an energy sensor, it would be a bit dumb to restrict access to the very energy molecules which you are trying to look at to judge overall energy status, especially when energy status is high: You need to decide when to store calories...

The beta cells appear to use both fatty acids and glucose to generate superoxide, but instead of signaling beta cell insulin resistance, they signal insulin secretion. Several lines of evidence fit in with this.

You can get succinic acid itself directly in to beta cells by providing it as a methyl or ethyl ester. As a metabolic fuel source this acts as a near pure complex II substrate, pushing electrons in to the ETC through the FADH2 of succinate dehydrogenase to reduce the CoQ couple and set the scene for reverse electron transport and superoxide production, especially when NADH from glucose metabolism rises. In a commonly used model of functional beta cells, succinic acid methyl ester is a marked insulin secretion potentiator, especially at higher glucose concentrations. Glucose supplies NADH, succinate supplies FADH2, they clash at the CoQ couple and the generation of superoxide signals that there is a ton of energy available. Better store it. Better secrete insulin.

Succinic acid methyl ester drives complex II. This drives insulin secretion in response to glucose. But it's a drug. There is nothing physiological about this drug. So shall we go a little more physiological?

To recap from previous posts: Superoxide generation is directly proportional to the ratio of FADH2 generated to the amount of NADH generated for a given substrate, the F:N ratio.

Here's a nice graph of insulin secretion stimulated in response to 12.5mmol glucose on a background of assorted free fatty acids from an isolated pancreas preparation:



If you can't be bothered to work out the F:N ratios (shame on you), here they are added to the graph:



Please excuse the C8 value; as we all know, MCTs are shunted directly to the liver via the portal vein. They do not seem to feature too prominently in pancreatic superoxide generation and insulin secretion. It would take a ton of reading to see why and how they are handled differently to longer chain fatty acids. For the time being let's stay looking at C16 and longer as these make a much tidier story...

So, for the four longer fatty acids tested, the amount of insulin secreted is remarkably closely associated with the F:N ratio of the fatty acid available.

Does this work in people?

Of course it does. Remember the Spanish study? I lo0ked at it in some detail here.

In particular look at this graph:



From the top downwards we have butter, high palmitic acid seed oil, refined olive oil and a mix of fish and vegetable oils as the white triangles. It is very clear that the insulin secretion here is in direct proportion to the saturation and length of the fatty acids in the meal, in an intact group of volunteers..

Aside: Obviously, there is a glaring error in the graph. All of the curves except the control use 800kcal of total food, of which 40g is carbohydrate/protein and the bulk is fat. The graph is missing a group where 800kcal was supplied as pure carbohydrate. We can all imagine where this much bulk glucose would have put the insulin curve, needless to say there is absolutely no way it would fit on to the presented graph. We would need a much taller vertical axis, which would show the mixed meals in their true context!

But the principle, that insulin secretion at a given level of glucose is elevated in direct proportion to the F:N ratio of the background fat, holds perfectly well in this carefully contrived human study.

BTW, lucky for me they didn't include a coconut oil group!

The obvious conclusion from this finding is that to lower insulin maximally we should, taking as given that replacing carbohydrate with fat is the biggest step by far, all go for vegetable oil with some fish oil. Not butter. But in the original post on the Spanish paper I went on to discuss what appeared to be happening to the lipid from the meal. It could stay in the bloodstream and be used for metabolism, as the butter did, or it could be cleared rapidly in to adipocytes allowing metabolism to return to being glucose based. At the cost of expanding the adipocyte stores of fat.

The high PUFA meal really was rapidly stored as fat in adipocytes. The F:N based explanation is because we are supplying a low F:N ratio fat and so not generating insulin resistance phyiologically; we are allowing lipid easily in to adipocytes because the lipid does not generate adipocyte insulin resistance. We are going back to glucose metabolism as rapidly as possible. PUFA facillitates fat storage and glucose based metabolism. All is fine until you can't get any fatter. Butter limits fat storage and runs metabolism of palmitic and stearic acids. Those high PUFA-fed mice generate obesity when fed their high PUFA diets from pre-conception onwards:



In the butter group there is some excess insulin. Does this matter if no one (cellularly) is listening to it?

I next want to look at the flip side, the reduction of supply of free fatty acids to the pancreas. This was done in the same paper. You can certainly do this in intact rats (and humans if you so wish). Then we might get back to the fat mice.

I think that had better be another post as this one is getting overly long and it's light enough to let the chickens out.

Peter

Protons: Linoleic acid in the hypothalamus

Hi all, just getting my head above water now that we have two or three locums at work to cover some of the (rather difficult) gaps in the rota!



Before we look at the fat mouse study which wins the prize for most miserly hoarding of data, I just wanted to put up a brief post, based on that paper, about breaking your hypothalamus with a high fat diet. Just to re emphasis: This is NOT what happens to a human after 7 days on a high fat diet.

Remember Schwartz's rats? Put them on a high fat diet and this happens to food intake:



Note the very sudden and dramatic spike in the intake of food, shown by the red line which I've added to emphasise the abrupt change from baseline chow consumption. We can ignore the red oval for this post. What happens in the VMH neurons of these rats?

This is what happens:



The dark brown staining cells on the right are dying, the rats have been eating "cookie dough", which they "can't get enough of", for seven days. The nice healthy cells in the left hand photomicrograph are from rats on crapinabag. The basic idea appears to be that feeding rats a bit of fat and sugar makes them eat so much, starting in just one day, that by seven days their VMH is killed by over indulgence. You eat too much, you kill your brain. Simple. This is, of course, absolute bollocks.

At the risk of repetition, we can produce exactly the same lesions in the VMH with MSG or gold thioglucose (or an ice pick if you must be crude and don't want nice pictures). This injury results in fat gain which must be compensated for by overeating. Rats will gain weight more slowly if they are on low fat diets than on high fat diets because of the effects of increased de novo lipogenesis which I've discussed in previous posts.

Want pretty pictures from GTG injured rats? Here's some random immuno from a random paper, there's a lot of it around, only black and white though:



Gold thioglucose on the right, arrow marks the injury area. And I just noticed the same pics, also in black and white, from fat injured rats from elsewhere in the Schwartz paper (mirror imaged compared to the GTG pics, random choice of which side of brain got sectioned!) after just a week on their high fat diet:



So we can produce the pretty black stains of dying cells with gold thioglucose (or MSG if we looked at neonatal immuno) but this injury preceeds the loss of calories in to adipocytes and subsequent "hyperphagia". THE INJURY COMES FIRST.

Let's really look at the bizarre idea that non-forced "overeating" causes subsequent damages your VMH. This is how it works for over eating by a gold thioglucose injected rat, no yummie high fat diet needed: It simply decides to over eat crapinabag because this has suddenly and randomly become delicious and so it becomes obese. We all know overeating CAUSES the VMH injury in fat fed rodents. So how do GTG injured rats get the injury first and over eat secondarily? Gold thioglucose obese rodents might SEEM to have a chemical lesion causing obesity but clearly they get fat first, travel back in time (squeezing in to a time machine as obese chrononaughts) and retrospectively force the researchers to give them the injection of GTG to obtain the lesion in their VMH which they are going to produce in the future by eating too much crapinabag. Got that? You've all watched Back to the Future. I watched parts I and II but never managed part III. It's simple time travel. Ditto MSG and ice-pick (ouch!) obese rodents. Self inflicted injuries using time travel.

Or we could abandon such stupidity and say that high fat diets injure the VMH first and this injury increases fat storage by decreasing sympathetic tone to adipocytes, as it does.

And I suspect it's superoxide, generated by a high F:N ratio (classically derived from palmitic acid at an F:N ratio of 0.47) in POMC neurons, which probably does the damage. You all know POMC neurons, the ones in the VMH with both gluokinase to sense (via metablism) glucose and CD36 to monitor FFA status (via metabolism again). No lactate for the energy status sensing neurons of the VMH...

So the question is, as always, what happens to the VMH of a C57BL/6 mouse (bred to get fat on a high fat diet) when put on a high fat diet which does NOT generate superoxide in POMC neurons? You can do this.

No one has done the necessary immuno staining under these conditions to get the pretty pictures of dying (or non dying) cells, as far as I know. But it's easy to look at the weight gains, which are a reasonable surrogate for POMC injury. Schwartz again using rats:



Not the most lucid graph, but it gives the basic idea. The control weight gains on the left are comparable to the weight gains shown for day 14.

Now, here is what happens if you take a C57BL/6 mouse and put it on to 35% of calories from fat if you keep the F:N ratio of that fat well below 0.47, using omega 6 PUFA with an F:N ratio of 0.42, as the primary source of fat:



Ignore the top two lines (for now) and look at the weight gain of the mice in the bottom two lines. One group weaned on to crapinabag, the other weaned on to 35% of calories from fat, but a fat with a low F:N ratio. There is zero, zilch, nil difference in weight gain over three weeks. There is no excess weight because there is no VMH injury. No one generates significant superoxide from a low F:N ratio fat like linoleic acid. That appears to include the POMC neurons of C57BL/6 mice.

C57BL/6 mice (and Long Evans rats) are specifically bred to get fat on palmitic acid (sometimes plus fructose) based diets. They fail to deal with the absolutely normal levels of superoxide produced in POMC neurons in the VMH which are crucial to energy status sensing. They do not have the luxury of developing insulin resistance as their job is to monitor both glucose and fatty acid levels. They are not allowed to run on lactate with an F:N ratio of 0.2 the way much of the brain does. They take whatever plasma gives them and do their best to cope with it. Or, in the case of rodents bred to become fat on high fat diets, not cope with it.

Before we go looking at the linoleic acid paper a bit more carefully I think it's worth trying to look at energy sensing rather more peripherally than the POMC neurons of the VMH. Then we can come back to the fat mice and try to think about what's going on using the meagre data available. Because it's quite interesting.

Peter