Cannon & Nedergaard 2012 in Nature

Hi! My personal, original plan was to have a new post every week, but I am already behind.
*face palm*
But you know what they say "sometimes plan change", I thinkkkk.
*scratches head*
Anyways, I'm going to squeeze out this post, because I was behind last week.

I'm pretty excited about this short, simple review that presented much to think about from Drs. Barbara Cannon and Jan Nedergaard, both from the Wenner-Gren Institute of Stockholm University.

Dr. Barbara Cannon

Dr. Jan Nedergaard

1. Citation:

Cannon B & Nedergaard J (2012). Neither brown nor white. Nature 286(488):286-287. 

2. Purpose:  

The purpose of this review is to provide a brief introduction about the beige/brite fat cells based on different works from other researchers. In the review, Drs. Cannon and Nedergaard specifically referred to the work of Wu et al [Wu J et al (2012).Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell 150(2): 366-376.]
as providing convincing evidence for the new type of fat cells. 

3. Data presentation: 

They included a concept figure demonstrating the differences between classical brown, classical white, and the new beige fat cells. 

 4. Conclusions:
"The identification of a third type of fat cell in mice and humans might open up new avenues for combating obesity."

Drs. Cannon and Nedergaard raised many good points to consider:

"These results raise several questions. What is the origin of the two types of cell? Are there
two distinct types of pericytes (cells that are wrapped along the blood capillaries and supposedly give rise to fat cells)? Or does one cell type derive, for example, from the bone marrow? How can certain white-like adipocytes, which in general possess very few mitochondria, suddenly enhance their mitochondrial complement during the ‘beiging/britening’ process?" 

5. Novelty:
I felt that the novelty of this review lies in the thought provoking questions they provided in the conclusion of what this beige cell can mean for human treatment of obesity. 

6. Future directions: and 7. Money line: 

Hahaha, from my perspective, I still think it's questions that I previously mentioned in the conclusions and novelty parts. 

Cinti 2011 in the Annals of Medicine

So, the last paper and the first paper, hehe, we left off on was from the lab of Dr. Spiegelman. They found that there are three kinds of distinct types of fat cells in the body: classical brown, white, and the new and oh so hot, beige/brite cells. In the previous paper, they specifically focused on the beige cells and how those cells can be induced by Prdm16.

Today's paper is from Dr. Saverio Cinti. Some quick words about him, is that he is a professor at the University of Ancona in Italy. I had the pleasure to meet him one summer. ^_^ Hahaha, I don't think he remembered though. -___- But it was a great experience nonetheless! Dr. Cinti is a fantastic histologist, his papers always have wonderful histology pictures of adipose tissue. Here is a link to his university webpage: http://www.med.univpm.it/?q=node/305

Here is a quick video with Dr. Cinti explaining his research in Italian! :D

I chose his paper, because it slightly contrasts on Dr. Spiegelman's work. The hot news in the field is that there are 3 distinct types of fat cells as I have previously mentioned. Not all, however, agree with this view. I believe Dr. Cinti has a different perspective on it that we will see in his very nice review that was simple and easy to read, as well as the great descriptions of histology from electron microscopy.

You may think I have clearly chosen a side here, but I haven't! These two views are valid and are quite interesting leaving much to think about.

Since Dr. Cinti's paper is a review, I will follow a different format to tackle review papers, somewhat different than from primary research papers. Let's go!!!

1. Citation:

Cinti S (2011). Between brown and white: Novel aspects of adipocyte differentiation. Ann Med 43:104-115. 

2.  Purpose:

It is previously known that there are two types of fat cells: brown and white. The purpose of this review is to examine the evolutionary functions of these two types of fat cells, as well as a discussion on the distinct differences between them in regards to how they can be used to treat the epidemic of obesity and type 2 diabetes, and also give a survey of different fat depots in the body and where they are located. Dr. Cinti, interestingly, also touched on his view of transdifferentiation, which he has a slight different from Dr. Spielgeman.  

3. Data presentation: (Do the authors give enough specifics for you to understand their conclusions?)

Yes, he did. This review is fill with histology pictures of adipocytes from either electron microscopy or immunohistochemical staining, which lies in line with the intent of the review, that is to examine the differences between brown and white fat cells using the method of microscopy.

I do a little bit of microscopy and tissue staining, so I'm kind of stoked about some nice figures Dr. Cinti has.

Figure 1: Light microscopy of a section where the
transition between brown (BAT) and white adipose tissue (WAT)
is visible. Note the different cytoplasmic lipid accumulation:
mostly unilocular in WAT and multilocular in BAT. Only BAT is
intensely stained by UCP1 antibodies. Bar 25 μm.

Figure 2: This is my most favorite figure in this review, because of how big the lipid droplet looks compared to the organelles in the fat cell! Electron microscopy is so beautiful, yielding such crisp images that always look like they're hand drawn to me. O_O Electron microscopy of the peripheral part of a white adipocyte. Elongated mitochondria with randomly oriented cristae are visible (arrows). N nucleus; RER rough endoplasmic reticulum; large L main lipid droplet; small L peripheral lipid droplet; BM basal membrane. 
Bar 0.7 μm.

Figure 3: Electron microscopy of cytoplasm of a brown
adipocyte. Several big mitochondria packed with cristae are
visible. L lipid droplet (some indicated). Bar 1 μm. For goodness sake, look at those cristae!!!

4. Conclusions: (What are the main conclusions from this review?)

• There are two types of fat cells: brown (energy burning) and white (energy storage).
• These fat cells are plastic/modifiable.
• According to Dr. Cinti, they can convert from white to brown or brown to white depending upon the stimulus, either cold exposure or diet induced obesity, respectively. He called this conversion "transdifferentiation".
• Macrophage infiltration is triggered by the death of fat cells and results in inflammation, which can contribute to the complications associated with obesity like type 2 diabetes. 

5. Novelty: (Do any of these conclusions represent new ideas not expressed comprehensively in the reviewed literature?)

Yes, the transdifferentiation view is very different from the work of some researchers. From my interpretation of this review, I believe that according to Dr. Cinti, there are ONLY 2 types of fat cells, brown and white. The new "beige" cells are not considered a distinct group of cells, but instead as a "transitory" cells during the transdifferentiation process. 

"In the opinion of some authors the multilocular cells expressing UCP1 — the BAT marker gene and protein — arising in the depots traditionally viewed as ‘ white ’ cannot be considered as ‘classic ’ brown adipocytes; accordingly, terms such as brite (brown to white) and beige (the intermediate colour between white and brown) have been coined for them, essentially to stress their different molecular and embryological lineage features compared with the cells found in the interscapular depot, which is traditionally considered as typical
BAT. 

We believe that such differences could reflect the presence in the tissue of cells in intermediate transdifferentiation stages and that these very elements can be responsible for the molecular differences described."

Something else novel to me from this review, is the presence of what he termed "crown like structure", when he stained for macrophages in dead fat tissue. He also used a distinct term for adipose tissue, referring to it as an organ, "the adipose organ".

6. Future directions:

He suggests that by learning more about the transdifferentiation process, inflammation (common in obese individuals) can be addressed  along with incorporating the molecules regulating differentiation (Prdm16, BMP7). 

7. Money line: 

I am beginning to think that murine adipose tissue are not the best model for studying, adipose tissue, but in the conclusion of the review he did not think that.

Spiegelman et al., 2011 in The Journal of Clinical Investigation

So, the first article that I chose to share with you about is from the laboratory of Dr. Bruce Spiegelman at Harvard Medical School, who is very well recognized regarding his work on regulation of fat cell differentiation and transcriptional basis of energy metabolism. Here is a link to his lab webpage for anyone interested: http://research4.dfci.harvard.edu/spiegelmanlab/research.htm 

:) :) :)

I will break down his paper in this format that my neuoimmunology professor provided that I think is very helpful and simple for digesting primary research articles.

1. Citation:

Seale P, Conroe HM, Estall J, Kajimura S, Frontini A, Ishibashi J, Cohen P, Cinti S, Spiegelman BM (2011). Prdm16 determines the thermogenic program of subcutaneous white adipose tissue in mice. J Clin Invest 121:96-105.

2. Purpose:

Weight is due to an energy imbalance with an increase in energy intake and decrease in energy expenditure. White adipose tissue (WAT) predominantly stores energy, while brown adipose tissue (BAT) burns it as heat in the form of non-shivering thermogenesis. Subcutaneous WAT has been shown to be more beneficial than visceral WAT and even being involved in insulin sensitivity. Transplanting subcutaneous WAT into the abdominal cavity yielded insulin sensitivity as compared to transplantation of visceral WAT. This demonstrates that there are some differences perhaps at the gene level between subcutaneous WAT and visceral WAT.

Unlike the traditional just WAT and BAT, within in these past few years, beige or brite fat cells or brown-fat like adipocytes have been discovered. These cells do not arise from the same lineage as the classical BAT, which is from Myf-expressing embryonic progenitors. These brown-fat like adipocytes have multilocular brown like morphology and express uncoupling protein 1 (Ucp1). They are found in both rodents AND humans. The brown like transition was most predominantly seen in inguinal WAT not periogonadal WAT around the testes/ovaries.

Prdm16 is a transcriptional coregulator that controls the development of brown adipocytes in classic BAT depots.

The purpose of this paper is to investigate the involvement of Prdm16 in inducing brown adipose tissue found within WAT, specifically comparing between inguinal WAT and perigonadal WAT since this was not looked into detail before. 

Hypothesis: Prdm16 is necessary for the induction of brown-fat like adipocytes in WAT.

3. Novelty: I think that it is novel that inguinal WAT have more brown fat genes or gene involved in the thermogenic program when compared with the perigonadal WAT. Prdm 16 was required/necessary for induction of thermogenic gene program in subcutaneous WAT, and when the Prdm16 is induced to be on, those mice have an increase in energy expenditure and resistance to diet induced obesity compared to the wildtype mice.

4. Design/steps: 

The authors observed the expression of brown fat like gene program in subcutaneous adipocytes of wildtype mice at 24C in different fat tissues in the body. They also did a gain of function study, where they induced the promoter of Prdm16 to be on in transgenic mice, and they measured the expression of brown fat like gene program as well as typical fat cell genes. They also provided fat histology of Ucp1 staining in the inguinal WAT and perigonadal WAT of the transgenic and wildtype mice. The authors quantified the number of tyrosine hydroxylase fibers in the inguinal WAT tissue of the transgenic mice and wild type mice. After the gain of function approach with the promoter of the Prdm16, the authors provided functional in vivo data on those mice to show how they regulate their blood glucose levels, insulin sensitivity as well as food intake and energy expenditure. Finally to complement the gain of function approach, they did a loss of function approach as well, using short hair pin RNA to knock down Prdm16. In addition to in vivo data, the author provided in vitro data from primary stromal vascular cells.

5. Data/Analysis: 

For this portion, please download the paper and follow with me as I go through the results from each figure.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3007155/pdf/JCI44271.pdf

Figure 1 (a) - Inguinal WAT has a greater amount of Prdm16 mRNA when compared with the intra-abdominal WAT depots, but much lesser than BAT.
Figure 1 (b) - Shows the western blot results to complement the graph in part a.
Figure 1 (c) - Inguinal WAT has greater levels of brown fat like genes (Ucp1, Cidea, Cox8b) than the intra-abdominal WAT depots, but much lesser than BAT.
Figure 1 (d) - The inguinal WAT has greater levels of Prdm16 mRNA in the SV fraction and the adipocyte fraction it equal the Prdm16 mRNA level of BAT. This difference was lost for the expression of Ucp1 in the SV fraction of inguinal WAT compared to BAT. When they looked at a mature adipocyte marker like GLUT4, they saw no differences among the different fat depots.
Figure 1 (e) - Expression levels of Prdm 16 and Ucp1 mRNA levels increased at day 8 of differentiation for the fat cells.

Figure 2 (a) - The gain of function of Prdm16 significantly increased the mRNA levels of Prdm16 in the transgenic mice for epididymal and inguinal WAT and BAT as opposed to the wildtype mice. This was also seen in the protein expression levels of these fat depots (figure 2 (b)).
Figure 2 (c) - But when they compared the other genes involved in thermogenic gene program like Ucp1, Cidea, and Ppargc1a, only inguinal WAT had a greater increase in those genes than epididymal WAT. As a control, I supposed they looked at other general fat markers to demonstrate that only the brown like genes are increased with this gain of function model.

Figure 3 (a-g) - They presented some fat histology to complement their mRNA data. The transgenic mice with the increase in Prdm16 production had a greater level of brown like morphology compared to the wildtype mice, but only inguinal WAT had that result more than epididymal WAT.
Figure 3 (h-j) - They quantified the sympathetic fibers in inguinal WAT, by indirectly staining for tyrosine hydroxylase (TH), and transgenic mice has 4 fold increase of TH fibers over the wildtype mice.  

The authors tested for the effect of diet induced obesity by giving the mice a high fat diet and measuring their metabolic responses. 
Figure 4 (a-b) - The trangenic mice had significantly lower body fat than the wildtype mice and they had greater lean mass.
Figure 4 (c) - The energy expenditure was much greater also in transgenic mice, but the food intake did not change.
Figure 4 (d-e) - The transgenic mice had better regulation of their blood glucose level than the wildtype mice with the glucose injections and insulin sensitivity test.
Figure 4 (f) - Even with a high fat diet, all of the fat depots had significantly greater amount of Prdm16 compared with the wildtype mice.

Figure 5 (a-c) - The short hair pin RNA was efficient as removing the presence of Prdm16, and the sympathetic nerves were unresponsive to isoproterenol treatment. It was not able to increase the level of Prdm16.
Figure 5 (d-g) - Without Prdm16, the thermogenic gene program cannot be activated. As you can see, I am getting kind of tired now.

6. Conclusions: 
"Transgenic expression of Prdm16 in all adipose tissues caused a selective transformation of subcutaneous WAT to a brown-like phenotype. The development of brown-like cells in the subcutaneous adipose of aP2-Prdm16 animals was associated with a rise in whole-body energy expenditure and a suppression of weight gain in response to a high-fat diet. Importantly, Prdm16 was required for the induction of a thermogenic gene program in isolated, WT subcutaneous adipocytes and in vivo. Thus, our results identify Prdm16 as a critical mediator of adaptive thermogenesis in subcutaneous WAT."


7. Money line:

The involvement of the sympathetic nerves possibly drawing the brown-fat like cells is interesting. 

An update about this blog (that may be confusing to some readers)

Hi there!

I started this blog about 3 years ago, it has been so long! In that time this blog has undergone many different directions with some earlier posts about tips on pipetting and ammonium sulphate precipitation for my undergraduate molecular cell lab, followed by posts regarding different techniques that scientists use such as, RNA interference and parabiosis.

I have now (permanently :)) decided that this blog will be dedicated to my interest on adipose tissue, which will be helpful for me to kill 2 birds with one stone forcing me to read relevant journal articles more regularly (bleh!) and analyzing the information, so that I will be well informed enough to share with you! And you! And you too!

Congratulations to Drs.Lefkowitz and Kobilka!!! :)

It's been a while since I have written anything, because graduate school is keeping me very very busy.
I am currently taking a molecular cell biology class, which explains my interest in the news about two U.S. chemists,  Robert Lefkowitz and Brian Kobilka, being awarded the Nobel Prize for their study of G protein coupled receptors (by using radioactivity and imaging of the adrenergic receptor).

Dr. Lefkowitz is an investigator at the Howard Hughes Medical Institute and professor at Duke University Medical Center. Dr. Kobilka is a professor at Stanford University School of Medicine.

Link to original news article: http://www.msnbc.msn.com/id/49354751/ns/technology_and_science-science/t/us-chemists-win-nobel-study-protein-receptors/#.UHdox1GrGSp

Here are citations/links to some of their papers:

Bouvier, M., Hnatowich, M., Collins, S., Kobilka, B.K., Deblasi, A., Lefkowitz, R.J., & Caron, M.G.
Expression of a human cDNA encoding the beta 2-adrenergic receptor in Chinese hamster fibroblasts (CHW): functionality and regulation of the expressed receptors. Mol Pharmacol, ;33(2):133-9, 1998.

Lefkowitz, R.J. Historical review: A brief history and personal retrospective of seven-transmembrane receptors. TRENDS in Pharmacological Sciences Vol.25 No.8 August 2004
http://www.cse.nd.edu/courses/cse598k/www/dropbox/jtan1/FinalProject/GPCRs.pdf

http://www.gmca.anl.gov/publication/Rasmussen-Kobilka_et.al._Nature_2007.pdf

Information about the Nobel Prize:
The Nobel Prize is awarded for achievements in physics, chemistry, physiology or medicine, literature and for peace. It's an international award administered by the Nobel Foundation in Stockholm, Sweden. It started in 1901, between 1901 and 2011, there have been 549 Nobel Prizes awarded. The Nobel laureates receive a diploma, a medal, and a document confirming the prize amount. They attend an award ceremony in Stockholm. The prize money comes from the fortune of Alfred Nobel whom the award is named after.

Information was obtained from: http://www.nobelprize.org/nobel_prizes/about/

just for fun: Nikon's small world photomicrography competition

I was perusing on the good ol' internet when I stumbled upon this really neat competition that Nikon has every year.

Quick introduction (taken from the website http://www.nikonsmallworld.com/):

"Small World is regarded as the leading forum for showcasing the beauty and complexity of life as seen through the light microscope. For over 30 years, Nikon has rewarded the world's best photomicrographers who make critically important scientific contributions to life sciences, bio-research and materials science."

Rules: http://www.nikonsmallworld.com/rules 

  

There are some really cool pictures. 

The winning picture (shown below) for 2011 was from Anna Franz. 

Ink injection into yolk sac artery of 72 hour-old chick embryo to visualize the beating heart and the vasculature (reflected light technique)

 

Technique: What is RNA interference?

The central dogma consists of
DNA --> mRNA (via transcription) --> protein (via translation)

RNA interference/RNAi is the silencing of RNA for controlling gene expression. This occurs naturally. Scientists have been able to use RNAi as a tool for studying gene expression, drug target screening, and for development of therapeutic drugs. 

1. Double stranded RNA is cleaved into small interfering RNA/siRNA by the RNAseIII enzyme called Dicer.
2. The siRNA are incorporated into silencing complexes that will target messenger RNA/mRNA for cleavage.
3. The mRNA that is coding for a specific protein is degraded, and there won't be any protein expression.

Information was obtained from: http://www.vet.uga.edu/id/tripp/RNAi.php

http://www.vet.uga.edu/id/tripp/RNAi.php

This video was obtained from youtube. The original link for this video is: http://www.nature.com/nrg/multimedia/rnai/animation/index.html

http://www.nature.com/news/2009/090624/images/_tmp_articling-import-20090624083219872961_4591047a-i1.0.jpg

Richard Jorgensen is a molecular geneticist, who is an early pioneer in the study of RNAi. In the 1980s, he was asked to create purple petunias by a biotechnology company. He introduced the gene that coded for the color purple into the plant, and unexpectedly the petunias did not turn purple. A defense mechanism called RNAi was set off, and it deactivated all pigment genes in the plant.

This information was obtained from: http://www.oprah.com/health/RNAi-Based-Treatments-and-Possible-Cures-for-HIV-and-Cancer

Here is a link to the video from Nova: http://www.pbs.org/wgbh/nova/body/rnai.html

A few years after, another group of scientists also noticed this gene silencing effect in C. elegans. Andrew Fire and Craig Mello, were awarded the 2006 Nobel Prize in physiology or medicine for the discovery that RNAi is triggered by double stranded RNA (paraphrased from http://www.nigms.nih.gov/News/Extras/RNAi/factsheet.htm).