OBESITY AND FREE FATTY ACIDS IN SEVERE ACUTE PANCREATITIS,
VIJAY P. SINGH, MD
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I’ll be focusing on the mechanisms by which obesity may be worsening pancreatitis and more so on
the local effects. As we heard from George, obesity is a risk factor for severe acute pancreatitis, it’s
an epidemic globally. And we’ve seen the numbers. For example, acute pancreatitis was quoted as
being between 200,000 to 300,000 cases per year or admissions. And mortality from severe
pancreatitis can range anywhere from 15% to 45% quoted in various studies and what we have so far
in conservative management is just fluid replacement, antibiotics, giving the patient NPO. Now
what exactly exacerbates pancreatitis in an obese patient is not known. Visceral fat has been
implicated suggested by CD findings showing increased visceral fat in obese patients. And if you
look at the locations where visceral fat could reside, it could be in the liver, the retroperitoneal
places, intrapancreatic or peripancreatic locations. Now if you want to put this in a perspective of
what’s happening clinically, I would break this down into the two types of mortality which we
typically see in acute pancreatitis. There’s an early mortality typically within the first two weeks or
some places at one week, and a later mortality. The early mortality is typically implicated on
multisystem organ failure. Late mortality usually has necrosis or its complications like infected
necrosis and eventually there is organ failure that develops because most deaths are a
cardiopulmonary event. And if you look at the mortality it’s kind of equally split between the two so
my focus is going to be what is highlighted in the pink area which is the late mortality which is
typically a complication of necrosis or its complications.
Now there are two recent studies I’ll be quoting over here which suggests the relationship between
obesity and how the outcomes may be getting worse. The first one is – which came out this year on
OBESITY AND FREE FATTY ACIDS IN SEVERE ACUTE PANCREATITIS,
VIJAY P. SINGH, MD
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the role or how risk visfatin is the marker of local complications and all three scoring systems
whether we use the shorter score, the Balthazar or the necrosis score, all three were higher in
patients who had higher levels of visfatin in our admission. And similarly the same group published
late year on Resistin and showed that the scores are higher in patients who have elevated Resistin
levels and if you just kind of focus on the Resistin levels themselves – sorry. We will see that they
are typically about two-fold elevated above their baseline compared to the milder group or the ones
with the lower scores. And I’ll come back to this later.
If we take what is common in Resistin, Visfatin and adipokine in general, they come from
adipocytes, some studies say it is the macrophages in that adipose tissue. Recent studies have shown
that even Resistin which was so far characterized when it came from, in humans, has now been
characterized to be coming from pre-adipocytes differentiating into adipocytes.
So, yes, adipokines whether they be Resistin, Visfatin, all come from adipose tissue. And when we
started on this project and tried to understand how this may be happening, my background was
typically working in animal models of acute pancreatitis but then we started saying okay, let’s see
what’s happening in the human pancreas. And first thing when I looked down the microscope I had
to take my eyes off the scope and then come back again, it took me a few days to reconcile that there
as a fundamental difference between what we see in the rodent pancreas and the human pancreas and
that is intrapancreatic fat. These are just some examples of it. So on the left side in the upper panel
lobar you can see, you know, there’s practically no intrapancreatic fat, the lower panel is the hyper
OBESITY AND FREE FATTY ACIDS IN SEVERE ACUTE PANCREATITIS,
VIJAY P. SINGH, MD
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image. The middle one shows about 10% and the third one shows about 70% intrapancreatic fat and
as you can see the lower panels, these are all adipocytes.
So there is intrapancreatic fat that is present in the human pancreas if we look at it. See whether this
was associated with obesity we kind of quantified this as just as a percentage of total area in the
histological section. And if you break it down into two categories, that is the obese versus the nonobese. The ones who have BMIs greater than 30 versus those who are less than 30, whether we took
controls who had died from unrelated caused or patients who had had an autopsy diagnosis of
pancreatitis which is in the middle panel or those who did fulfill 2 out of 3 criteria of pancreatitis at
the time of death thereby had clinical acute pancreatitis. Among those who had a BMI above 30, all
of them universally had increased intrapancreatic fat compared to the ones who had a BMI less than
30. And this was about a two-fold elevation, the dashed lines show this being about 20% of the
pancreatic area was fatty.
And when you compare this and study the relationship and the linear correlation between BMI and
intrapancreatic fat whether we looked at control patients or those with acute pancreatitis we found a
moderately strong correlation between BMI and amount of intrapancreatic fat. The R value was
about .45 and all of the associations were significant.
So amount of intrapancreatic fat goes up with BMI. Since we are having – examining all of this on
histological samples there’s always a possibility of sampling error. Thirteen of our cases had had CT
OBESITY AND FREE FATTY ACIDS IN SEVERE ACUTE PANCREATITIS,
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scans which are noncontrast within the three months before they died. And these were patients who
had never had an episode of pancreatitis at the time the CT scan was done or the CTs were done
proceeding the episode of pancreatitis. And when we looked at the CT and asked the radiology
colleagues to correlate what their measure of intrapancreatic could be, they used two measures. One
was the Hounsfield units shown on the right side and the other one was by a thresholding method
shown on the left side. We found a very strong correlation between what we had quantified on
histology and the amount of fat measured on CT scans. As you can see by the – just measuring
Hounsfield units versus the score on histology, there on the right side there was an R value of 0.677
and on the left side you can see it was about 0.76 by a thresholding method, that is seeing the amount
of pancreas which had a Hounsfield unit value of less than zero and taking that as a total area and
seeing that – what proportion of the total area had Hounsfield units less than zero. And so the
histological results were verified on CTs, so yes there is by two independent methods a decrease – or
so you increase an IPF or intrapancreatic fat with BMIs.
Again, when we looked at the cases who had had severe pancreatitis in autopsies, so at the time of
death and then look at their BMIs. The ones with severe had a higher amount of intrapancreatic fat
which is shown on the right side and they also had the higher BMIs and that correlates very well with
what John had shown earlier that patients who have BMIs above 30 have a much higher mortality.
Well, to dissect this a little more mechanistically we said what’s going on? Fat can reside in the
pancreas in two manners. Here in the left panel you can see in the upper portion fat which seems
OBESITY AND FREE FATTY ACIDS IN SEVERE ACUTE PANCREATITIS,
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very peacefully residing with the pancreatic parenchyme or these empty spaces you see are
adipocytes and the parenchyme looks normal but then you’ll look at the lower portion of the same
image, there’s this bluish cheesy appearance in the fat, that’s necrose fat. Pathologist were calling
that fat necrosis and it took me a while to understand what they’re actually talking about because we
never saw that in animal models which we had studied earlier.
And when we looked at the amount of fat necrosis again in our patients we found that the ones who
had pancreatitis and were obese, which is shown in the middle graph had a higher amount of fat
necrosis and the ones who were nonobese and had acute pancreatitis had less amount of fat necrosis
and in the extreme right you can see the graph showing that the ones who had severe pancreatitis had
much more fat necrosis than the ones who were mild controls, did not have any significant amount of
fat necrosis.
Kind of zooming into this a little bit more and saying okay if we are having fat necrosis and there is
damage going on what is the relationship between two things. If we again focus on the left panel
first and look at the empty spaces on the top, those are normal adipocytes and they have normal
parenchymal surrounding them. But then you look at the dash oval which has this bluish, cheesy
material inside them and then if we zoom into it and go and focus on the image on the right, you’ll
find that area of the necrosed adipocytes has necrosed parenchyme around it and the damage is the
most, closet to the necrosed adipocytes. As you go away from the necrosed adipocytes we run into
normal parenchyma. So there seems to be this lake effect, kind of look to the whole damage
OBESITY AND FREE FATTY ACIDS IN SEVERE ACUTE PANCREATITIS,
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phenomenon taking place that yes, you can have normal adipocytes sitting in a normal person but
once the adipocytes get damaged and necrosed they start damaging the parenchyme around them and
the damage it the worst proximal to the fat and goes away as we go away from the fat. So there was
this lake effect kind of look to it.
And if we were to quantify this fat necrosis which surrounded the fat which we call peri-fat acinar
necrosis again this was more in the patients who were obese because they had more intrapancreatic
fat, it’s very intuitive there. And if we just look at the amount of acinar necrosis itself, as you can
see in the dashed line on the extreme right, the obese patients with acute pancreatitis had about as
much acinar necrosis, 50% of which was peri-fat acinar necrosis. So that, so fat necrosis seems to
contribute to about half the damage we saw in these patients. And if we were to look at it from the
standpoint of severity again the severe patients had an average of about 25% necrosis on histology as
we saw it and half of it was around, it was peri-fat acinar necrosis. The mild ones had much less and
the controls did not have it.
Now these findings are not really novel or super new, people have alluded to this in other – for
example Kloppel, this is a paper from the 80’s alluded to it in the pancreas paper saying that there
was perilobular fat necrosis and damage to the parenchyma. The question is mechanistically how is
this taking place. What is the basis of this lake effect? So to understand this a little bit more we said
fine, it boils down to an adipocyte acinar interaction that’s parenchyma and fat cells there, can we
simulate this in vitro. So if you take acinar cells alone here on the left side and we leave them for a
OBESITY AND FREE FATTY ACIDS IN SEVERE ACUTE PANCREATITIS,
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period of time on their own, they look very fine. And we put a blue dye around them, they do not
take up that blue dye, they glow normally, they look yellow as we see it over here. Blue dye means
holes in the cell membrane and dead cells. But if we to co-culture them together with the adipocytes
there’s a lot of cell death taking place. It’s all blue. So there did seem to be some form of hostile
interaction between the adipocytes and acinar cells, taking place if we did allow to co-culture and left
the two compartments communicate.
And when we tried to see what kind of cell that this was, this was a complicated image but if we kind
of break it down, this is basically looking at mode of cell death. The image on the top shows the
cells with red nuclei, that is propidium iodide uptake, necrotic cells take up propidium iodide and
when you coculture them there was a massive increase in propidium iodide uptake suggesting
necrotic cell death which was what we were seeing histologically. And the lower panel over here is
basically – the blots allude to the mode of cell death. The first one looks at cytochrome C at the
upper panel and the lower panel looks at the two fractions, the mitochondrial and cytosolic fractions.
And what you can see is that the cytochrome C leaks out into the cytosolic fraction when we have the
acinar cells and adipocytes together, that’s on the left lower side.
And the other two graphs, I’m sorry the other two plots simply say that’s its not apoptotic or
autophagic cell death, it’s necrotic cell death. So eventually based on these findings when we
cocultured them we found that it was a necrotic mode of cell death similar to what we’re seeing in
the histologic sections of the patients who had died from pancreatitis.
OBESITY AND FREE FATTY ACIDS IN SEVERE ACUTE PANCREATITIS,
VIJAY P. SINGH, MD
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When we quantified the free fatty acids in the medium, grossly also when you cocultured them you
could see this medium turning turbid white, chalky, so there was something, some saponification,
something taking place at this time and intuitively it was this fat, so there’s going to be fatty acid
formation and some sort of formation there. And then we quantified fatty acids, yes there was a
massive increase in fatty acids in the medium. And an increase in Resistin in the medium, very
similar to the two-fold increase which is noted on admission in the patients who have severe local
complications.
So we said okay, if this is fatty acids in the in vitro environment what correlative proof do we have
on the histological sections of these patients who have severe pancreatitis. So on the left side here
you see a hematoxylin, an eosin image with bluish cheesy material in these empty spaces in the
center which is fat necrosis and right around that you can see this area where the cell outlines are lost
and that is necrotic parenchyma. When we did Von-Kossa stains, Von-Kossa is a stain for calcium.
Calcium in the pancreas is not normally going to be deposited unless there is some form of
saponification taking place. And yes indeed right around the areas of fat necrosis there was this
massive deposition of calcium taking place and it spread into the dead parenchyma. So there was
this toxic cell death taking place, leakage of whatever components was coming out from the dead fat
damaging the surrounding parenchyma. And if this was fatty acids, we said let’s characterize what
fatty acids it could be so we took five different fatty acids, all of them are long chain fatty acids. On
the top LLA is linolenic acid, linoleic acid, oleic stearate and palmitic. And this is a calcium flux
OBESITY AND FREE FATTY ACIDS IN SEVERE ACUTE PANCREATITIS,
VIJAY P. SINGH, MD
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measurement. As you can see it is the unsaturated fatty acids which are linolenic, linoleic and oleic
which caused the calcium flux whereas the others do not. And if we were to measure LDH leakage
which is a marker of just how many – what is the proportion of cells with holes in their membranes
which can leak out the cytosolic enzyme called lactic dehydrogenase into the medium, we find again
it is the unsaturated fatty acids which cause cell death. And irrespective of whether it is an early
event at 3 hours or at 5 hours, irrespective of the concentration of the fatty acids we chose it was the
unsaturated fatty acids which were causing cell death.
Again this image over here merely shows that linoleic acid shown in LA causes PI positivity which is
propidium iodide uptake which is a marker of necrosis, ATP levels drop again that happens in
necrosis with linoleic acid. Cytochrome C leaks again into the cytosol and it is not autophagic or
apoptotic cell death, so unsaturated fatty acids were causing straight necrosis of acinar cells.
Well we said okay, if such a thing is happening in the in vitro environment, we’ve seen in autopsy,
what’s happening in the real patient? We got debridement fluid samples from patients with severe
pancreatitis, so they were undergoing surgical debridement and we said okay, if such is the scenario,
let’s measure the fatty acids in the necrotic debridement fluid. And there are two messages in this.
One is again in the pink highlighted area, it is the C18-1 and C18-2 which are the dominant
population and again there’s a C16-1 and these are all unsaturated fatty acids so if you add the
numbers together it comes to about 75% unsaturated fatty acid contained in the debridement fluid
whereas 25% is saturated. So most of it is unsaturated. The average concentration of this in the
OBESITY AND FREE FATTY ACIDS IN SEVERE ACUTE PANCREATITIS,
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debridement fluid was about 7.4 millimolars so it’s such a high concentration you find that it’s really
relevant to what we used in vitro in what is going on there. So yes, these relevant concentrations
were causing necrotic cell death. If that was the case than this seemed to be - okay, adipocytes have
a big lump of fat inside them, triglycerides and they are generating fatty acids, where is it coming
from. So there has to be some interplay between the two compartments. Acinar cells have a lot of
lipases sitting there and Dr. Lowe is the expert in lipases over here, I discussed the findings with him
and then when we added orlistat which is an inhibitor of lipases to the medium we could block fatty
acid generation very effectively.
And we could restore viability of the cells very effectively. So, again, here in the big image over
here, the three panel image you see here. The first two columns show just cells which have died
when we have co-cultured with adipocytes and if you put orlistat again which is an inhibitor of
lipases we could restore viability. The graph shown over here merely quantify the phenomenon of
lipolysis taking place or the mode of cell death. The top graph which shows, on the right side show
propidium iodide uptake, the third bar which is massive is basically a lot of propidium iodide uptake
in the co-culture system which is blocked when we block lipolysis.
The second graph, the middle one shows that ATP levels drop again signifying acinar cell death as
necrotic and then the bottom graphs verify that this is actually necrotic cell death.
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Not only were the cells morphologically better, the graph on the right bottom shows that they were
functionally normal too. The first, this is a typical amylase secretory pattern which we see with
acinar cells, those three bars you see in the bottom right graph is a normal secretory pattern which is
completely abolished with the co-culture system and restored with orlistatin environment.
So blocking lipolysis restored viability, prevented necrosis of these cells and brought down the
Resistin levels interestingly. So the cell death that was taking place was being mediated by the fatty
acids most likely unsaturated fatty acids that were being generated over there in the co-culture
system. Yes, Resistin was going up but at least in our system it seems more to be a marker than a
mediator. Well, we can explore this further.
And what we – we have gone further now and I’ll just show you one image and this is representative
of what happens in animal models. Again, animal models which have increased intrapancreatic fat.
If we were to look at them histologically on the left side, just plain hematoxylin and eosin staining or
with a calcium stain which is the Von-Kossa staining on the right side, again you’ll see there is
saponification in the right side of the image, you see that black cheesy material around fat and fat
necrosis signifying saponified fat in the animal models and that significantly is in the areas where
there is surrounding pancreatic necrosis.
So the paradigm based on this that we have come up with, there can be two kind of outcomes in
pancreatitis mechanistically speaking. One will be the typical lean patient in the upper schematic.
OBESITY AND FREE FATTY ACIDS IN SEVERE ACUTE PANCREATITIS,
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They have an insult like gallstones or whatever x, y, z insult and in the lean pancreas where little or
few adipocytes, there will be some damage which the primary insult causes but in the absence of
adipocytes very low levels of unsaturated fatty acids are going to be generated, there’s going to be
very little lipolysis and less injury and less inflammation.
In the obese patient the scenario changes, once the adipocytes come into the picture the primary
insult still does cause the same damage to the pancreas but the adipocytes sitting there are like fuel.
The spark which had set off the fire in the wood now with the fuel around causes a much bigger fire
causing much more damage, increased injury inflammation and severe local injury. So this is a
developing model which are studying now and there’s substantial proof we have gone further to see
that this is actually taking place and is preventable by blocking the deleterious effects of unsaturated
fatty acids.
So just to summarize what we have seen in this is that as a person becomes obese the amount of
intrapancreatic fat increases and those who have clinically severe pancreatitis have more
intrapancreatic fat, they have more fat necrosis and this fat necrosis has acinar areas bordering these
which have necrosis called peri-fat acinar necrosis. It seems that it is the lipolyses which causes the
generation of fatty acids and it is the unsaturated fatty acids which cause most of the deleterious of at
least the necrosis part of acinar cells. They do so partly maybe via the increasing calcium which we
saw in that color graph and to conclude based on what we have seen in the autopsy samples as well
as in our in vitro studies and animal studies, we think that the obesity associated increase in
OBESITY AND FREE FATTY ACIDS IN SEVERE ACUTE PANCREATITIS,
VIJAY P. SINGH, MD
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intrapancreatic fat is associated with worse fat necrosis, peri-fat acinar necrosis and damage by
unsaturated fatty acids.
Thank you.