Obesity/adipose distr in relation to nutrition James Minchin

JAMES MINCHIN: Thank you. So, thank you to the organizers for allowing
me to contribute to this workshop. It’s been really interesting and really
helpful so far. So, we’re looking at adipose tissue biology
and primarily, you know, how this is regulated genetically, and we’re using zebrafish as
a model system to really look … you know, answer these questions, and we are using zebrafish
for two primary reasons. Number one is that they’re transparent and,
as Steve Farber and John said, that you can incubate the fish in these nice kind of fluorescent
mid-microaerophilic dyes, and you can very nicely see the lipid that’s accumulating
in live animals. So, this is a Nile red stain here, which shows
neutral lipid in yellow, and the second reason is that they’re, you know, amenable to genetic
chemical screens, so it’s a, it’s a good tractable model for kind of probing the genetic
basis for kind of various adiposity traits. And so, I’m a developmental biologist by
training, and what I’m going to talk to you today is kind of about the development
of adipose tissue, so during these kind of fast growing rates that we have heard about,
so in juveniles or postembryonic larvae, generally around between 1 and 2 months of age. And really the take-home is that zebrafish
adipose develops in different stages, and diet affects adipose development in a kind
of stage-specific manner; so, potentially very important on adipose development. So, I think … there we go. So, just to give a brief intro on adipose
tissue, I’m sure you’re all aware, but adipose tissue is this kind of distinct tissue—this
distinct organ distributed throughout the body and accumulates lipid. So, you can see here in this DXA scan of a
human, lipid is in this kind of yellow color here, and it’s in all of these anatomically
diverse places. And so, structurally, adipose tissue is formed
from … by primarily by fat cells, also called adipocytes, which are these large spherical
cells shown here in green that kind of inflates with lipid and can grow to be very large cell
diameters. You can see that this cell here is possibly
a 100, 150 microns in diameter, and so these are incredibly morphologically unique cells,
and these cluster together … to form an adipose tissue. You can see here these are both in zebrafish
and see that there’s a big adipose tissue actually associated with pancreas in the zebrafish. Alright, so medically we’re, you know, we’re
interested in adipose tissue because of obesity and body fat distribution, and these are two
things that were really looking at in zebrafish. Just to give you a little bit of background
on what’s known about zebrafish adipose tissue. It’s a very small field, and actually, there’s,
you know, very few papers on the subject, but one of the first papers was done by John,
who showed that zebrafish adipose tissue’s morphologically, molecularly, and actually
functionally very similar to mammalian white adipose tissue. That is, it accumulates lipids in times of
energy excess, and it mobilizes lipid during times of energy or caloric depletion—energy
depletion. So, morphologically zebrafish adipose … adipocytes,
fat cells, are labeled here with the asterisks, and you can see that they are huge, and they
seem to express similar markers to mammalian adipocytes, so things like PPARγ and fapb11,
which are kind of, you know, very, you know, established markers of mammalian white adipocytes,
and so, these are the same animal here in the kind of vertical columns when they’re
alive, they’ve been stained using Nile red, so you can see the lipids stored in lipid
droplets, and then they’re fixed and processed to look at PPARγ, mRNA, and you can kind
of track where the cells are with a lipid expressing, say, PPARγ or fapb11a. So, it looks as though these zebrafish did
{indiscernible} because they’re morphologically and molecularly similar to mammalian white
adipose, and this is really … you know, one of the reasons why diet is really important,
you know, for our research, and that is that the zebrafish adipose tissue doesn’t really
appear until, you know, postembryonic stages; so, 10 days postfertilization or 8 days postfertilization. So, this just means that, you know, experimentally,
when we look at days … traditionally, when we look at days postfertilization in zebrafish,
but that’s not really sufficient to explain a lot of the variability that we see with
adipose tissues. So, again, these are nice Nile red images
from John’s lab showing that, initially, at 5 days postfertilization, you see lipid
in the intestine and no real kind of lipid stored in the adipocytes, but then this postembryonic
stage, so these guys are getting fed from 5 days postfertilization, but then shortly
after that or a good few days after that, they start to store lipid in adipocytes and
this kind of expands. And so a different study—another study from
Kirsten Sanders’ lab—basically showed that, again, if you use DPF as a metric to
study adipose tissue in zebrafish, there’s huge variability. You can see the 12 DPF fish of this length
has a huge variation in size of fish, for starters, but only a few of them have adipocytes,
so there’s huge variability. And this is primarily because the fish are
independently feeding and they’re growing at vastly different rates. So, Dave Parichy did a study where he looked
at the relationship between fish size and fish development during these postembryonic
stages, and he showed that a … fish that were 20 days old, shown here in black dots,
covered a huge range—size range, so there are massively different sizes ranging from,
you know, 5 millimeters in length to 10 millimeters in length, and they also, you know, kind of
encompass this huge developmental range, as well. You can see here these are kind of postembryonic
developmental stages, these kind of DR/AR stages. You can see here, even within that system,
there’s huge variability, as well, so a fish that’s 6.3 millimeters could be any
one of these developmental stages here. So, it’s really important, and I think that
this … this needs to be incorporated into any kind of, you know, evaluation of dietary
success is using this, you know, established kind of staging criteria that was established,
you know, a few years ago by the Parichy lab. Then so diet and other kind of environmental
factors can affect this interaction between size of the fish and developmental stage of
the fish. For two different traits, both melanophore
development and also the appearance of adipose tissue, fish that are fed … that are grown
in different densities, so low density and high density, and that’s the same for this
experiment here and … as a proxy for kind of food per capita, so food availability. So obviously, low density … fish grown at
low density have more food available to them and grow at a faster rate. What can be seen for both of these traits
is the fish with … which had grown at a high density that have a low food availability
actually develop … or developmental traits appear at smaller sizes. So, there’s a disconnect between size of
the fish and developmental progress of the fish, and so this makes for, you know, extremely
complicated comparisons to be made at these stages and should definitely be included in
any kind of future diet study. Just to kind of concentrate a little bit more
on adipose tissue. We kind of carefully characterize … where
adipose tissue is stored in zebrafish, and there’s lots of different anatomical sites
of adipose accumulation in zebrafish, but they can be broadly classified into internal
adipose tissues or subcutaneous adipose tissues, so just like in humans or a mouse, essentially. And the internal adipose tissues are primarily
composed of visceral adipose tissue associated with the internal visceral organs. And we see very interesting dynamics in how
these adipose issues grow. So, these are … on the x-axis are the size
of the fish, and then on y-axis, this upper graph is the, kind of, growth in body area
of the fish, and this is about, you know, 300, 400 fish here. This is this kind of a line plotted to show
the growth. See, there’s very consistent growth in body
area across these, kind of, fish sizes, but if we look at the growth of the different
adipose tissues, you can see that visceral adipose tissue undergoes this … appears
first but undergoes this very quick expansion that slows and plateaus out to essentially
not growing at all, and then kind of opposite to that or complementary to that, subcutaneous
adipose tissue appears slightly later but then kind of grow … as visceral adipose
issue is slow … the growth of visceral adipose is slowing, subcutaneous adipose tissue is
kind of expanding at a very fast rate, quickly catching up to that. If you look at—so these are the same graphs
that I’ve just shown—but if you look at body fat percentage across the different sizes
of fish, you can see that there’s, during this kind of early phase when visceral adipose
tissue is expanding really quickly, there’s a very fast rate of body fat accumulation. Then this seems to slow when subcutaneous
… when lipid storage changes to subcutaneous adipose tissue, this kind of rate of body
fat accumulation slows. So, there seem to be two different, kind of,
growth phases of adipose tissue in zebrafish. The first one, which is, you know, thinking
before around about 8 millimeters, is kind of characterized by very fast visceral adipose
tissue growth and a very high rate of fat deposition. But then the later stage of adipose growth
in zebrafish is characterized by this kind of plateauing of visceral adipose tissue growth
and lipids starting to being stored in subcutaneous adipose tissue but at a lower rate. What I’ll talk about now is how a high-fat
diet affects adipose growth in these two different stages. So, as mentioned by Steve Farber and by John,
we use this chicken egg yolk that’s kind of like a crude high-fat diet, obviously a
very lipid-rich diet, and experimentally we … the fish are fed our normal regimen, but
they’re … everyday, they’re immersed in this high-percent chicken egg yolk, almost
as a supplement, and just kind of give them a lipid load, essentially. You can see that we’ll raise our fish normally
until a certain developmental stage, and then we’ll split the siblings into either a group
that’s immersed in system water, so a standard fed group, or a group that’s immersed in
system water plus the high-fat diet. And then we do that typically for, you know,
1 to 3 days or 7 days or 2 weeks or 3 weeks. Those are typically the kind of durations
that we … that we can immerse them in the high-fat diet. You can see that … while the effect of this
high fat diet doesn’t really have any effect on somatic growth. so the fish don’t grow larger, but there’s
a small bump in adiposity. So, it basically increases … it produces
a response in the adipose tissue and leads to increase adipose storage. In no way does this resemble obesity, but
it’s a very early kind of marker of adipose remodeling in response to a high-fat diet. Really, the take-home is that if we do that
during this early kind of visceral centric growth, we get, you know, all of the growth
… all of the high-fat induced growth occurs in internal and visceral adipose tissues. And there’s almost no response in subcutaneous
adipose tissues. Whereas if we do it in later stages when subcutaneous
adipose tissues are the primary type of adipose tissue that’s growing, we see a response
in subcutaneous fat and not visceral fat. So it seems that the different stages … if
you give the fish a high-fat diet at these different stages, different adipose tissues
respond in a different way. And so, that’s a high-fat diet, but we’ve
also done a similar thing with starvation and then looking at how adipose tissues re-deposit
lipid in response to refeeding. And we see a similar thing in that during
… and this is an example of starvation or refeeding during the later stage, the kind
of the subcutaneous adipose growth stage. And we see that … when you starve fish—so
you can see here they’re losing lipid—and then when you start to refeed the fish. they’re regaining lipid, and if we split
this out into internal and subcutaneous adipose tissues, the colored bars represent the size-
and stage-batched normal kind of expected levels of lipid. You can see that the internal adipose tissues
have lost the ability to recover to their normal fed size, whereas the subcutaneous
adipose tissues respond really nicely, and this leads to changes in body fat distribution. So basically, you know, I guess what I’d
like to kind of emphasize is that zebrafish adipose develops in two different phases,
and if you if you manipulate the diet during these distinct phases, you get a different
response. So, I’ll just leave it at that. A lot of this work was done in John’s lab. Thank you. {applause}
UNIDENTIFIED MALE: Is there a thought or evidence that visceral fat … It’s still thought
that in like people, their belly fat is more contributing to like … to like, you know
Syndrome X or, like, the … JAMES MINCHIN: Yeah, it’s the bad fat. UNIDENTIFIED MALE: Yeah, that bad fat. {laughter} Even if it’s all white fat, but
it’s still the location? JAMES MINCHIN: I mean, so I think it’s interesting. We don’t have a lot of data or real, kind
of, causative, like, strong data that the visceral fat is causing a lot of metabolic
dysregulation. But I think it’s interesting that zebrafish
initially stores … fat around the visceral organs. We’re probably all aware that when you start
feeding the fish at 5 days, it takes them maybe a week to … for a week they’re very
vulnerable I think, and then, basically, as soon as they start getting more stronger and
start growing really well, they’re storing fat around visceral organs, so I think that
might … signify a beneficial effect for the fat. UNIDENTIFIED MALE: {off microphone} Is there
any evidence or relationship between adipose tissue and egg-associated inflammation? JAMES MINCHIN: Yeah, so I haven’t shown
it, but if you put these fish on a high-fat diet, then there’s increased inflammation
in the adipose tissue. And you see that very early on from, like,
maybe 3 days after they’ve been on a high-fat diet, which is something that is, you know,
occurs in mammalian … in mouse and human obesity as you get this kind of {indiscernible}. So that’s definitely something that we’re
interested in. UNIDENTIFIED MALE: {off microphone} I have
one more question. Does the demarcation that you offered, down
to 8 millimeters plus/minus … do you have ideas on what, anatomically or physiologically,
is separating that? And then, I guess I would … are there other
events that are happening around that stage that may explain why there’d be a shift
there? JAMES MINCHIN: Yes, so I really don’t know
why there’s a shift from visceral to subcutaneous and why it does seem to be probably quite
a wide window that it, you know, that it takes to fully … for subcutaneous fat to fully
catch up … to this kind of exponential expansion. UNIDENTIFIED MALE: That’s interesting, because
total yolk sac absorption at 28 is around 7 days. JAMES MINCHIN: Yeah. UNIDENTIFIED MALE: This is much later, right? UNIDENTIFIED MALE: So, you said what day was
it? JAMES MINCHIN: We … typically do, this is
probably around a month of age. {multiple conversations}
UNIDENTIFIED MALE: 8 milliliters. JAMES MINCHIN: Yeah. I don’t know why. My feelings are that the visceral fat has
a beneficial role, you know, for organ function early on. Then once the fish is through this kind of
new dicey phase and they’re growing nicely and they’re, you know, getting overfed on
these zebrafish diets that we feed them, they have all this excess energy that then gets
kind of redeposited into subcutaneous fat, but I don’t know; that’s all handwaving.

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