Zebrafish Mod for Obesity/Type 2 Diabetes Mellitus Res Liqing Zang
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Zebrafish Mod for Obesity/Type 2 Diabetes Mellitus Res Liqing Zang

December 3, 2019

LIQING ZANG: Good afternoon, everyone. My name is Liqing Zang from Mie University
in Japan. Today I would like to talk about zebrafish
models for obesity and diabetes research. A simple introducing of our university. It established in 1949, and not a big university. There are two facilities in Mie University:
One is the Medical Zebrafish Research Center, and the second is the Zebrafish Drug Screen
Center. We started to use zebrafish for medical research
from 2004. I think it’s a little bit late than American
or Europe. As I am sure you know, the BMI mass … okay
… the BMI is a value derived from the weight and height of an individual. A person can be categorized as underweight,
healthy weight, overweight, or obese based on these values. According to recent data in America, the adult
obesity rate exceeds about 25 percent in 43 states in 2016. We can see in Maryland the obesity rate is
around 30 percent and is highly increased from 1990. So, it is well-known that zebrafish is a good
animal model for human disease. How should we do to prevent the prevalence
of obesity? So first, we need obesity models of zebrafish. There are three approaches to create an obesity
model in zebrafish, including induced models, transgenic lines, and mutant lines. This part is our risk models, which published
in 2010. We developed this model by overfeeding Artemia. As we all know, Artemia is an easily accessible
food and is usually used in the zebrafish facility worldwide. We first investigated the nutrition facts,
so Artemia … this is our original data, and the conclusion is first, the energy of
100 gram of dry Artemia is 390 kilocalorie, and one cyst of Artemia is 0.0095 calorie. And I’m not sure if this information is
useful. So finally, the fat contents of Artemia is
just 15 percent, so we conclude that Artemia cannot be defined as a high-fat diet. We then divided adult zebrafish—this adult
is over 3 months of age—and we divided it into a normal feeding group and overfeeding
group. The normal feeding zebrafish were fed 5 milligrams
of Artemia once daily, and the overfeeding zebrafish were fed a total of 60 milligrams
of Artemia and were fed three times daily. So … what’s that … After 8 weeks of
overfeeding, we can see that both the male and female have significantly increased BMI. And the plasma lipids are also significantly
increased after 8 weeks of overfeeding feeding, both the male and the female. We performed 3D micro-CT to evaluate the adipose
tissue volumes of our DIO zebrafish. These are the 3D micro-CT images for normal
feeding and overfeeding zebrafish. Green color indicates the bones, and the yellow
color indicates the adipose tissues. So, these are a 2D image after the analysis. We can see the yellow color indicates the
visceral adipose tissue, and the orange color indicates the subcutaneous adipose tissue. So, after quantification of adipose tissue
volume, here is the result. The total body fat visceral adipose tissue
and the subcutaneous adipose tissue are great higher than that of control group, and the
{indiscernible} of the …for dissection also reveal the lipid accumulation in liver. This is transcriptome analysis of the visceral
adipose tissue. We can see the obesity … in obese zebrafish
animal model shares a common pathology pathways in lipid metabolism. So, therefore we used these obesity models
to evaluate the anti-obesity functions of numeral … of natural products. For example, this is a tomato … green tea,
or the compounds extracted from seaweed. So, these are all our achievements. So, as we all know, obese people have a high
risk of developing type 2 diabetes, and the United States has a double epidemic of diabetes
and obesity. So, this is 20 percent of adults are obese,
and 7 percent have diabetes in 2006, and this data depicts the relationship between weight
gain in adulthood and the relative risk of type 2 diabetes. So, the concurrences of risk is increased
exponentially with larger gains in weight. Also, we successfully created our best model
of zebrafish. It has one weak point is that it is normal
glycemic. So, how can we get hyperglycemic zebrafish? We think it may be related to food. So, we gathered what we can do … the current
diets in zebrafish and, we investigated the nutrition facts. To be noted that the HFD32 is a high-fat diet
used rodents. We have fed it to the zebrafish, but it seemed
that they didn’t like the taste, so we focused on these seven diets from TetraMin to Artemia,
and there’s no big difference in energy. So, we next focused on the palatability and
the food intake volume. This data … six diets we’ve tested. Here BTs— beef cattle—we mixed beef cattle
into the powder diets to make a high-fat diet, and we can see the best three diets in food
into volume is Otohime B2, BT-Otohime B2, and Zeigler. The calorie intake is also higher … I’m
sorry, there’s no data on BT-Otohime B2 because Zeigler … older Zeigler in Japan
is troublesome, and the BT-Otohime’s sticky, and it’s hard to feed the zebrafish in the
quantitative amount, so we finally decided Otohime B2 as our test diet. It’s made by a Japan company and designed
for freshwater fish and not specially for zebrafish. Next we use Otohime B2 to measure the food
intake volume in the short term—this is short term for 3 hours. We can say the maximum food intake in 5 minutes
is 10 milligrams and the maximum food intake in 1 hour is 15 milligrams. There is no big difference between male and
female in 3 hours. And next, we performed the continuous feeding
at 1-hour interval. The total time … the total feeding times
is 11. So, according to this data, because the maximum
food intake in 1 hour is 15 milligrams, so the simply calculated result of 1-day maximum
food intake should be 825 milligrams for five fish. But the result of the male is consistent with
this conclusion. However, the female exceeds this standard. For this reason, the gender difference appears
in our 1-day food intake volume. To modify the old feeding method, we also
tried automated feeding system. This is a food timer. It can apply the same amount of food every
time, and it can feed fish five or more times daily, and just set it on the little tank,
and it’s very convenient. So, combined the Otohime B2 and the automated
feeding system, we fed the two groups with this protocol, and to be surprised we got
the hyperglycemic zebrafish. They also have a high … increased body weight,
and the fasting blood glucose after 1 week of overfeeding is increased. This trend is continued until the end of the
study. Because caloric restriction is the most frequently
treatment for diabetes, we also performed caloric restriction for 2 and 4 weeks. After this treatment, the fasting blood glucose
is … I’m sorry … is reduced but still higher than that have no DIO group. By the way, I was usually asked, how can I
collect blood from zebrafish without killed him? Play us this video, after the feed {indiscernible},
using a glass capillary connected with an {indiscernible} tube, then insert into the
middle side … look for the dorsal aorta, and then take suction … I think it may be
5 microliters. So, after blood collection, just press the
puncture to stop the bleeding, and then recover the fish into system water … alive. This is very simple to master … I can’t
wait. Okay. So, next we measured the glucose … we performed
the intraperitoneal and overall glucose tolerance test. This … we can see … in DIO zebrafish the
blood glucose level is higher … increased … beyond the point, and cannot back to the
basal level, so we decided our fish is glucose intolerant. Because glucose intolerance is associated
with insulin resistance, we quantified the incidence of production in DIO zebrafish. There’s no commercially available antibody
for zebrafish insulin suitable for Western blot analysis, so we used insulin EGFP transgenic
line to quantify insulin levels by EGFP expression. The average EGFP intensity is higher in … is
significantly higher than non-DIO group. And the Western blot analysis revealed the
abundance of DFP protein is also higher than that of control group. To evaluate if our model can respond to human
drugs, we treated fish with anti-diabetic drugs, both metformin and glimepiride … both
reduced blood glucose levels. Next, we performed the transgenic … no,
we performed the RND sequencing and process to pathway analysis. The GSEA analysis revealed that the gene sets
related to immune response, nervous system, and vascularization are all listed as common
as human and zebrafish, either in diabetic pancreas or diabetic liver. This insulin secretion pathways are also revealed
at similar gene expression alterations between zebrafish and human. These are our ongoing projects. We got candidate genes related to diabetes,
and firstly screening them with vivo-morpholino by evaluate that their effects on the blood
glucose levels, and then the positive genes are picked out to make CRISPRs. Finally, we performed mouse experiments using
siRNA-liposome complex, and as a result, we have found one therapeutic target gene that
we are knock down its expression level, it can ameliorate the hyperglycemic. I am preparing the manuscript now for publish. Okay. In summary, we created two zebrafish models
and demonstrated they are useful tools for human obesity and diabetes research. I would like to acknowledge my professor and
my colleagues. Thank you for attention. {applause}
UNIDENTIFIED MALE: We have time for a couple questions. UNIDENTIFIED MALE: How did you go about measuring
the intake? LIQING ZANG: {off microphone} Yeah. We had two methods. You can see that … in {indiscernible} within
3 hours we just fed them … for example, we first divided both into very, very small
amount, and then put in the tank. After the fish completely ate them and … this
is a manual method. And second, for our 1-day, we just put … a
big amount of food, and then we have the {indiscernible} food, we come to {indiscernible} and then
dry, dry them, and then measure the weight. UNIDENTIFIED MALE: Do you know … you mentioned
the initial diet that you’re using for diet-induced obesity did not induce hyperglycemia, right? Do you know what it is in these new diets
that are inducing the hyperglycemia? Is that something you’re interested in? LIQING ZANG: Yeah, I’m sorry, we just … we
just did the Otohime B2, I didn’t … maybe Zebra is also that? I don’t know. UNIDENTIFIED MALE: Okay.

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