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.