M. Rodrigues1, A. Whittam1, M. Sorkin1, M. Januszyk1, R. Kosaraju1, R. C. Rennert1, A. McArdle1, Z. Maan1, D. Duscher1, G. C. Gurtner1 1Stanford University,Department Of Surgery, Division Of Plastic Surgery,Palo Alto, CA, USA
Introduction: Chronic low-grade inflammation that develops during obesity is postulated to contribute to insulin resistance. Although there is significant interest in understanding how inflammation contributes to diabetes, the cellular and molecular mechanisms that underlie these processes remain unclear. In this study we test the contribution of macrophages to insulin resistance.
Methods: Using a vav-mtmg murine model with hematopoietic cells labeled with GFP, and non-hematopoietic cells labeled with RFP, combined with single cell transcriptional analyses, we first developed a marker profile for macrophages in murine adipose tissue. These markers were used to fluorescently sort and analyze the distribution of macrophages in murine and human subcutaneous (SAT) or visceral adipose tissue (VAT). Next, to determine the role of macrophages during the onset of obesity, mice were either fed with normal chow, or high-fat diet, to induce obesity, and diabetes. A second well-established diabetic transgenic murine model Leprdb was used for the isolation of diabetic macrophages. Single-cell transcriptional analyses determined changes in distribution of macrophage-subtypes upon obesity, and during diabetes. Finally, macrophages isolated from adipose tissue of diabetic or non-diabetic mice were injected into the visceral adipose tissue of non-diabetic wild type mice. These mice were either fed with a high fat diet, or normal chow. Blood glucose, insulin, cholestrol and triglyceride levels were measured as an indication of diabetes and diabetic complications.
Results: Single cell analysis of vav-mtmg adipose tissue revealed that macrophages comprise 64% of hematopoietic cells in the VAT and 36% of hematopoietic cells in the SAT. These cells positively expressed cd45 cd14 cd11b emr1 cd64 csf1r cd33 fcgr3 msr1 cd36. Onset of diabetes increased the number of macrophages (CD45+ CD11b+ F4/80+) in murine VAT by 4-fold with no significant increase in the macrophages in the SAT. A similar trend was seen in human adipose samples from non-diabetic and diabetic patients. Single-cell analysis revealed that diabetes induced an increase in the number of pro-inflammatory M1 macrophages in the VAT, highly expressing il1b and il6. Transplantation of these macrophages into the perigonadal adipose tissue of non-diabetic mice in the presence of a high fat diet led to significant increases in body weight, adipose tissue and liver mass, as well as increased blood triglyceride and cholestrol levels. These mice showcased hyperglycemia within 4 weeks, significantly faster than mice injected with macrophages from non-diabetic mice, which took 8 weeks to become hyperglycemic.
Conclusion: Pro-inflammatory M1 macrophages in the visceral adipose tissue increase with obesity, and quicken the onset of diabetes in the presence of high-fat food. Specific targeting of these cells will lead to ways of containing diabetic onset.