Protein produced in adipose tissue triggers inflammation that causes diabetes

A Brazilian is the principal author on a Harvard University study published in journal Cell Metabolism.

By Karina Toledo

Agência FAPESP – The relationships among obesity (mainly visceral fat), chronic systemic inflammation and the development of metabolic disorders such as diabetes are well established in the scientific literature.

In an article published in the journal Cell Metabolism in March, researchers at Harvard University describe the role of a protein secreted by adipose tissue and the liver – RBP4 – in the activation of defense cells that produce inflammatory substances and, as a result, induce insulin resistance.

“We showed that RBP4, a protein found at concentrations two or three times higher in the obese and diabetics, functions as a trigger for inflammation in the adipose tissue. This molecule is therefore a target for new medicines,” said Brazilian Pedro Moraes-Vieira, the principal author of the article.

Moraes-Vieira earned his master’s and doctoral degrees at the University of São Paulo (USP), with funding from FAPESP. He is currently pursuing a post-doctoral degree at Harvard under the supervision of Barbara Kahn, a professor in the Division of Endocrinology.

According to Moraes-Vieira, until the 1990s, it was thought that the only function of the RBP4 protein in the body was to transport vitamin A. However, recent epidemiological studies in humans have indicated that there is a correlation between insulin resistance, systemic inflammation and elevated concentrations of RBP4.

In 2005, research led by Kahn showed that RBP4 protein expression in mice increased as healthy animals became resistant to insulin.

“Up until that time, the mechanism through which elevated RBP4 induced the metabolic disorder was not clear. Our study was designed to understand how this protein could modulate inflammation, mainly in the adipose tissue,” Moraes-Vieira explained.

To conduct the study, the group used a transgenic mouse model capable of expressing the RBP4 protein in muscle cells. “Our animals presented the same degree of elevation in the blood concentration of RBP4 as that observed in obese or diabetic humans: in other words, nearly three times the normal [concentration]. The transgenic mice became diabetic around the sixth week of life, although they remained thin,” said Moraes-Vieira.

Whereas the RBP4 elevation in the mice was due to genetic modification, in the case of humans, RBP4 elevation is thought to be caused by an increase in visceral adipose tissue or by metabolic stress caused by the accumulation of fat in the liver, explained Moraes-Vieira.

The analyses

When the transgenic animals had completed their eighth to tenth week of life, the researchers measured their body mass index (BMI), the percentage of lean and fat mass, and the concentrations of fatty acids and cholesterol in the blood and conducted glucose tolerance and insulin tests.

On comparing the results with those of the control group, composed of mice that had normal concentrations of RBP4, the transgenic group presented a difference in only the glucose tolerance and insulin tests, which confirmed diabetes.

After sacrificing the rodents, the researchers assessed the presence of inflammatory substances in the visceral and subcutaneous adipose tissue, the spleen, the lymph nodes and the liver.

“We noted significant inflammation in the visceral adipose tissue, with activation of cells of both the innate immune system and the adaptive immune system. We also saw moderate inflammation in the liver, with activation of the innate immune system only. This is because RBP4 tends to accumulate more in the adipose tissue,” Moraes-Vieira commented.

Using a technique known as flow cytometry to count, phenotype, examine and classify the cells, the researchers analyzed the leukocytes present in the inflamed tissues.

“The scientific literature reports the existence of two types of macrophages in visceral adipose tissue: pro-inflammatory and anti-inflammatory. We observed that the RBP4 protein causes the anti-inflammatory macrophages to begin to produce pro-inflammatory cytokines. This type of converted macrophage activates the adaptive immune system and induces the production of Th1-type CD4 T lymphocytes,” Moraes-Vieira explained.

According to Moraes-Vieira, the Th1-type cells specialize in secreting an inflammatory substance known as interferon-gamma (IFN-γ). When in excess, this cytokine interferes with adipocyte signaling, further activates the macrophages and impedes the effect of insulin.

The group conducted another experiment to test the hypothesis that it was the macrophages – considered a type of antigen-presenting cell (APC) – that were responsible for activating the adaptive immune system and consequently inducing the inflammation.

“We isolated dendritic cells, which are a type of APC, from mice; activated the cells with RBP4; and implanted them into healthy mice. After six weeks of weekly infusions, the animals became diabetic, developed inflammation in the visceral adipose tissue and had large numbers of Th1-type lymphocytes,” explained Moraes-Vieira.

A third experiment, also conducted using transgenic animals, revealed that a cell signaling pathway mediated by the JNK protein is essential for the inflammatory effect unleashed by RBP4.

“Through a partnership with researchers from the University of Massachusetts, we developed a knockout-transgenic mouse model for JNK [in which the gene responsible for JNK protein expression was silenced] only in macrophages. We then isolated pro- and anti-inflammatory macrophages from the visceral adipose tissue of these animals and treated these cells with RBP4, but the adaptive immune system was not activated. In other words, without the JNK pathway, the inflammation is not unleashed,” explained Moraes-Vieira.

According to Moraes-Vieira, the key role of RBP4 in the development of type 2 diabetes in the obese has already aroused the interest of the pharmaceutical industry.

“There’s a company investigating immunobiological drugs that are potentially capable of reducing the concentration of RBP4 in the blood. This would help to decrease inflammation in the adipose tissue and, theoretically, to reduce resistance to insulin,” he stated.

The article RBP4 Activates Antigen-Presenting Cells, Leading to Adipose Tissue Inflammation and Systemic Insulin Resistance (doi: 10.1016/j.cmet.2014.01.018) can be read at www.cell.com/cell-metabolism/abstract/S1550-4131(14)00024-2.