Anti-TGF-beta antibody suppressed body weight gain, size of fat depots, and fat mass in mouse models.
Scientists claim blocking the TGF-β/Smad3 pathway could provide a therapeutic approach against type 2 diabetes and obesity. Research in mice led by a team at the National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK) has shown that Smad2-deficient mice are not only protected from diet-induced obesity and diabetes but that in these animals white adipose tissue (WAT) starts to take on the characteristics of brown adipose tissue (BAT).
Sushil G. Rane, M.D., Ph.D., and colleagues in addition showed that systemically blocking TGF-β signaling in mice protected them from obesity, diabetes, and hepatic steatosis. Their findings are reported in Cell Metabolism in a paper titled “Protection from Obesity and Diabetes by Blockade of TGF-β/Smad3 Signaling.”
The primary purpose of WAT is as a store of surplus energy, while the primary function of BAT is to generate heat. Brown adipocytes are densely packed with mitochondria and contain iron, which leads to their brown coloration, Dr. Rane and colleagues report. Brown adipocytes have also been found interspersed with WAT in response to cold exposure or on stimulating the β-adrenergic pathways. The origin of these cells and their regulation at the genetic level is unclear, however.
TGF-β and its related factors control the development, growth, and function of diverse cell types. TGF-β transmits its signals via dual serine/threonine kinase receptors and transcription factors called Smads, with Smad3 serving as the principal facilitator of TGF-β signals, the researchers explain. Previous research has also found that TGF-β levels correlate with obesity in mice and humans, while studies by Dr. Rane’s team has identified an important role for the TGF-β/Smad3 pathway in the regulation of insulin gene transcription and β cell function.
The NIDDK researchers and their collaborators’ newest research was designed to evaluate the importance of TGF-β signaling in energy homeostasis and the pathogenesis of diabetes and obesity.
They found that mice in which the Smad3 gene was globally knocked out exhibited enhanced insulin sensitivity and increased whole-body glucose uptake, which was particularly marked in WAT compared with other metabolically active tissues such as skeletal muscle.
Smad3-deficient mice also gained less weight when fed a high fat diet (HFD), were protected from hepatic steatosis, and demonstrated better glucose tolerance and insulin sensitivity, leading to lower fasting blood glucose levels and insulin levels. Although there was no difference in calorific intake between the wild-type mice and those lacking the Smad3 gene, the engineered animals displayed significantly reduced fat mass and smaller white adipocytes.
Mouse embryonic fibroblasts (MEFs) from these mice were impaired in their ability to differentiate into white adipocytes, and both MEFs and WAT showed reduced expression of white adipocyte-specific genes. Interestingly, white adipocytes from the Smad3 knockout animals were also dark red rather than the pale color of white adipocytes from wild type animals. They also demonstrated a morphology more akin to that of brown adipocytes. The cells in addition displayed increased mRNA expression of markers of brown adipogenesis.
The researchers confirmed these findings in cultured 3T3-L1 cells, an MEF adipose like cell line that can be triggered to differentiate into adipocyte-like cells. When 3T3-L1 cells in which smad3 had been knocked down using an shRNA were treated using a white adipocyte differentiation medium, they expressed elevated mRNA and protein expression of BAT-specific markers.
The results hinted that loss of Smad3 promotes acquisition of brown fat features in white fat, a suggestion supported at the physiological level by the finding that Smad3 knockout mice exhibited higher basal body temperatures than wild-type mice during day and night conditions and were able to maintain significantly higher body temperatures when exposed to cold for an extended period of time.
Genetic data indicated that that WAT in mice without Smad3 expressed increased levels of PGC-1α, which represents the master regulator of mitochondrial biogenesis, as well as increased mitochondrial DNA copy number and mitochondrial-specific transcripts under basal and cold-exposure conditions. The mitochondria in this tissue also demonstrated morphological features such as densely packed cristae, which are characteristic of brown adipocyte mitochondria. Increased expression of mitochondrial-specific transcripts and essential mitochondrial gene products was also observed in the Smad3-knockout 3T3-L1 cells, suggesting the phenomenon was cell-autonomous, according to the investigators.
“While the findings thus far supported the concept that reduced TGF-β/Smad3 signals are beneficial to glucose and energy homeostasis, they also suggested that elevated TGF-β levels might promote glucose intolerance and obesity,” the authors note. To investigate this notion further the team examined circulating TGF-β1 levels in 184 nondiabetic subjects of diverse ethnic origin.
After adjusting for factors such as age and fasting insulin levels, they found a significant correlation between TGF-β1 levels and body mass index, with TGF-β1 levels increasing proportionately with adiposity in overweight and obese subjects. Positive correlations were in addition observed with fat mass, fasting insulin levels, and HOMA insulin resistance index, but not with blood pressure and levels of fasting glucose, triglyceride, free fatty acids, and insulin sensitivity.
The association of TGF-β1 levels with adiposity were confirmed in additional mouse models. In a leptin-deficient obese mouse model TGF-β1 levels increased seven-fold over the course of the animals doubling their bodyweight. In wild-type mice fed a HFD, TGF-β1 levels also increased five- to seven-fold compared with levels in animals fed a regular diet.
Studies up until this point had demonstrated a beneficial effect of suppressing TGF-β/Smad3 signals on glucose tolerance, body weight gain, and energy homeostasis. To examine the therapeutic relevance of the findings, they treated two mouse models of obesity and type 2 diabetes using an anti- TGF-β1 antibody (designated 1D11) that is closely related to fresolimumab, an antibody currently being tested in humans against pulmonary fibrosis, renal disease, and cancer.
As expected antibody treatment resulted in reduced levels of phosphorylated Smad3 in the WAT of leptin-deficient and in diet-induced obese mice, and suppressed body weight gain, size of fat depots, and fat mass. The antibody also led to reductions in triglyceride levels and resistin.
Also, consistent with the data from the Smad3 knockout mice, WAT from mice treated with 1D11 showed significant increases in mitochondrial DNA copy number as well as increased levels of transcripts that regulate BAT, mitochondrial function, and skeletal muscle biology. Importantly, treatment with 1D11 improved glucose and insulin tolerance, suppressed hyperglycemia and hyperinsulinemia , ameliorated hepatic steatosis , and increased protein levels of BAT/mitochondrial markers in the WAT.
Global microarray analyses of the WAT from both the Smad3 knockout mice and those treated with 1D11 antibody, showed evidence of a unique signature of 103 genes, 60% of which are involved in BAT, mitochondrial biology, and skeletal muscle development and function, “a finding that is consistent with the nexus between brown fat and skeletal muscle,” the team writes.
“It is possible that a small pool of brown adipocyte precursors or a shared WAT/BAT/skeletal muscle progenitor may reside in the white fat environment. Thus, it is plausible that the TGF-β1 effect could be at the level of the common progenitor for white, brown, and muscle cells, and we are examining this possibility,” the authors note.
“The occurrence of elevated TGF-β1 levels in obese individuals combined with the beneficial effect of the anti-TGF-β neutralization antibody in mouse models of obesity and diabetes offer treatment alternatives for these diseases.”
