Bronson A. Haynes

STAT4 Deficiency Reduces Obesity-Induced Insulin Resistance and Adipose Tissue Inflammation

Signal transducer and activator of transcription (STAT) 4 is one of the seven members of the STAT family. STAT4 has a prominent role in mediating interleukin-12–induced T-helper cell type 1 lineage differentiation. T cells are key players in the maintenance of adipose tissue (AT) inflammation. The role of STAT4 in obesity and AT inflammation is unknown. We sought to determine the role of STAT4 in AT inflammation in obesity-induced insulin resistance. We studied STAT4-null mice on the C57Bl6/J background. We have found that STAT4−/−C57Bl6/J mice develop high-fat diet–induced obesity (DIO) similar to wild-type controls, but that they have significantly improved insulin sensitivity and better glucose tolerance. Using flow cytometry and real-time PCR, we show that STAT4−/− mice with DIO produce significantly reduced numbers of inflammatory cytokines and chemokines in adipocytes, have reduced numbers of CD8+ cells, and display increased alternative (M2) macrophage polarization. CD8+ cells, but not CD4+ cells, from STAT4−/− mice displayed reduced in vitro migration. Also, we found that adipocyte inflammation is reduced and insulin signaling is improved in STAT4−/− mice with DIO. We have identified STAT4 as a key contributor to insulin resistance and AT inflammation in DIO. Targeting STAT4 activation could be a novel approach to reducing AT inflammation and insulin resistance in obesity.

Adipose Tissue 12/15 Lipoxygenase Pathway in Human Obesity and Diabetes

CONTEXT Visceral adipose tissue (VAT) is a key contributor to chronic inflammation in obesity. The 12/15-lipoxygenase pathway (ALOX) is present in adipose tissue (AT) and leads to inflammatory cascades that are causal for the onset of insulin resistance in rodent models of obesity. OBJECTIVE The pathophysiology of the ALOX 12/15 pathway in human AT is unknown. We characterized the ALOX pathway in different AT depots in obese humans with or without type 2 diabetes (T2D). DESIGN This study includes a cross-sectional cohort of 46 morbidly obese (body mass index >39 kg/m(2)) nondiabetic (n = 25) and T2D (n = 21) subjects. SETTING This study was conducted at Eastern Virginia Medical School (Norfolk, Virginia) in collaboration with Sentara Metabolic and Weight Loss Surgery Center (Sentara Medical Group, Norfolk, Virginia). PATIENTS Twenty-five obese (body mass index 44.8 ± 4.4 kg/m(2)) nondiabetic (hemoglobin A1c 5.83% ± 0.27%) and 21 obese (43.4 ± 4.1 kg/m(2)) and T2D (hemoglobin A1c 7.66% ± 1.22%) subjects were included in the study. The subjects were age matched and both groups had a bias toward female gender. MAIN OUTCOMES AND MEASURES Expression of ALOX isoforms along with fatty acid substrates and downstream lipid metabolites were measured. Correlations with depot-specific inflammatory markers were also established. RESULTS ALOX 12 expression and its metabolite 12(S)-hydroxyeicosatetraenoic acid were significantly increased in the VAT of T2D subjects. ALOX 15A was exclusively expressed in VAT in both groups. ALOX 12 expression positively correlated with expression of inflammatory genes IL-6, IL-12a, CXCL10, and lipocalin-2. CONCLUSIONS ALOX 12 may have a critical role in regulation of inflammation in VAT in obesity and T2D. Selective ALOX 12 inhibitors may constitute a new approach to limit AT inflammation in human obesity.

Activation of the 12/15 lipoxygenase pathway accompanies metabolic decline in db/db pre-diabetic mice

The 12-lipoxygenase (12LO) pathway is a promising target to reduce islet dysfunction, adipose tissue (AT) inflammation and insulin resistance. Optimal pre-clinical models for the investigation of selective12LO inhibitors in this context have not yet been identified. The objective of this study was to characterize the time course of 12LO isoform expression and metabolite production in pancreatic islets and AT of C57BLKS/J-db/db obese diabetic mouse in a pre-diabetic state in order to establish a suitable therapeutic window for intervention with selective lipoxygenase inhibitors. Mice have 2 major 12LO isoforms -the leukocyte type (12/15LO) and the platelet type (p12LO) and both are expressed in islets and AT. We found a sharp increase in protein expression of 12/15LO in the pancreatic islets of 10-week old db-/- mice compared to 8- week old counterparts. Immunohistochemistry showed that the increase in islet 12/15LO parallels a decline in islet number. Analysis of 12- and 15-hydroperoxytetraeicosanoid acids (HETE)s showed a 2-3 fold increase especially in 12(S)-HETE that mirrored the increase in 12/15LO expression in islets. Analysis of AT and stromal vascular fraction (SVF) showed a significant increase of platelet 12LO gene expression along with 12- and 15- HETEs. The data demonstrate that the db/db mouse is a suitable model for investigation of 12/15LO inhibitors in the development of inflammatory mediated type 2 diabetes, with a narrow window of therapeutic intervention prior to 8 weeks of age.

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease. This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) >35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2-3 g of fat. These cells can be expanded in culture over 10-14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5-6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPARγ agonist-rosiglitazone. This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.