Objective The relationship between adipose tissue fibrosis, adipocyte hypertrophy, and preadipocyte hyperplasia in the context of obesity, and the correlation of these tissue-based phenomena with systemic metabolic disease are poorly defined. The goal of this study was to define clarify the relationship between adipose tissue fibrosis, adipocyte hypertrophy, and preadipocyte hyperplasia in human obesity and determine the correlation of these adipose-tissue based phenomena with diabetes. Methods Visceral and subcutaneous adipose tissues from humans with obesity collected during bariatric surgery were studied with QRTPCR, immunohistochemistry, and flow cytometry for expression of collagens and fibrosis-related proteins, adipocyte size, and preadipocyte frequency. Results were correlated with clinical characteristics including diabetes status. Results Fibrosis was decreased, hypertrophy was increased, and preadipocyte frequency and fibrotic gene expression were decreased in adipose tissues from diabetic subjects compared to non-diabetic subjects. These differences were greater in visceral compared to subcutaneous adipose tissue. Conclusions These data are consistent with the hypothesis that adipose tissue fibrosis in the context of human obesity limits adipocyte hypertrophy and is associated with a reciprocal increase in adipocyte hyperplasia, with beneficial effects on systemic metabolism. These findings suggest adipose tissue fibrosis as a potential target for manipulation of adipocyte metabolism.

Mitochondrial calcium uptake stimulates bioenergetics and drives energy production in metabolic tissue. It is unknown how a calcium-mediated acceleration in matrix bioenergetics would influence cellular metabolism in glycolytic cells that do not require mitochondria for ATP production. Using primary human endothelial cells (ECs), we discovered that repetitive cytosolic calcium signals (oscillations) chronically loaded into the mitochondrial matrix. Mitochondrial calcium loading in turn stimulated bioenergetics and a persistent elevation in NADH. Rather than serving as an impetus for mitochondrial ATP generation, matrix NADH rapidly transmitted to the cytosol to influence the activity and expression of cytosolic sirtuins, resulting in global changes in protein acetylation. In endothelial cells, the mitochondrion-driven reduction in both the cytosolic and mitochondrial NAD ؉ / NADH ratio stimulated a compensatory increase in SIRT1 protein levels that had an anti-inflammatory effect. Our studies reveal the physiologic importance of mitochondrial bioenergetics in the metabolic regulation of sirtuins and cytosolic signaling cascades.

No abstract

ObjectiveNK cells are understudied in the context of metabolic disease and obesity. The goal of this study was to define the effect of NK cell ablation on systemic inflammation and glucose homeostasis in murine obesity.MethodsA transgenic murine model was used to study the effect of NK cell ablation on systemic inflammation and glucose homeostasis in the context of diet-induced obesity using flow cytometry, QRTPCR, and glucose tolerance and insulin sensitivity testing.ResultsNK cell ablation achieved a 3-4 fold decrease in NK cells but had no effect on T-cell levels in adipose tissues and spleen. NK cell ablation was associated with decreased total macrophage infiltration in intra-abdominal adipose tissue, but macrophage infiltration in subcutaneous adipose tissue and spleen was unaffected. NK cell ablation was associated with modest improvement in insulin sensitivity but had no effect on tissue transcript levels of inflammatory cytokines.ConclusionNK cells play a role in promoting intra-abdominal adipose tissue macrophage infiltration and systemic insulin resistance in obesity.

Breast cancer is a heterogeneous disorder in which most tumors display some degree of aneuploidy, especially those at later stages of the disease. Aneuploidy and associated chromosome instability may be important in the progression of mammary tumorigenesis. Aneuploidy is prevented during normal cell division in part through regulation of a mitotic spindle checkpoint where mitotic arrest prevents segregation of misaligned chromosomes into daughter cells at anaphase. Mitotic arrest genes, including the MAD family, which was originally characterized in yeast, help regulate normal function of the mitotic spindle checkpoint. Decreased expression of the human gene MAD2L1 was previously reported in a breast cancer cell line exhibiting chromosome instability and aneuploidy. To explore further the potential role of MAD2L1 in breast cancer, we analyzed MAD2L1 gene expression in 13 minimally to grossly aneuploid human breast cancer cell lines and found significant differences of expression in three lines. Sequence analysis of MAD2L1 cDNA in these as well as nine additional aneuploid breast cancer and five immortalized normal human mammary epithelial cell lines revealed one heterozygous frameshift (572 del A) mutation in a cancer cell line that demonstrated a high level of transcript expression. In addition, two 3′UTR sequence variants were noted in breast cancer cell lines. The 572 del A mutation creates a truncated MAD2 protein product. Further functional studies in primary breast tumors are therefore warranted to determine the potential role MAD2L1 may play in breast cancer. © 2000 Wiley‐Liss, Inc.

Adipocytes promote progression of multiple cancers, but their role in pancreatic intraepithelial neoplasia (PanIN) and ductal adenocarcinoma (PDAC) is poorly defined. Nutrient transfer is a mechanism underlying stromal cell-cancer crosstalk. We studied the role of adipocytes in regulating in vitro PanIN and PDAC cell proliferation with a focus on glutamine metabolism. Murine 3T3L1 adipocytes were used to model adipocytes. Cell lines derived from PKCY mice were used to model PanIN and PDAC. Co-culture was used to study the effect of adipocytes on PanIN and PDAC cell proliferation in response to manipulation of glutamine metabolism. Glutamine secretion was measured with a bioanalyzer. Western blotting was used to study the effect of PanIN and PDAC cells on expression of glutamine-related enzymes in adipocytes. Adipocytes promote proliferation of PanIN and PDAC cells, an effect that was amplified in nutrient-poor conditions. Adipocytes secrete glutamine and rescue PanIN and PDAC cell proliferation in the absence of glutamine, an effect that was glutamine synthetase-dependent and involved PDAC cell-induced down-regulation of glutaminase expression in adipocytes. These findings suggest glutamine transfer as a potential mechanism underlying adipocyte-induced PanIN and PDAC cell proliferation.

Context:The role of the extracellular matrix (ECM) in regulating adipocyte metabolism in the context of metabolic disease is poorly defined.Objective:The objective of this study was to define the metabolic phenotype of adipocytes associated with human diabetes (DM) and the role of the ECM in regulating adipocyte metabolism.Design:Adipose tissues from obese patients were studied in standard 2-dimensional (2D) cell culture and an in vitro model of decellularized adipose tissue ECM repopulated with human adipocytes, and results were correlated with DM status.Setting:This study was conducted at the Academic University Medical Center and Veteran’s Administration Hospital.Patients:Seventy patients with morbid obesity undergoing bariatric surgery were included in the study.Interventions:Visceral and subcutaneous adipose tissues were collected at the time of bariatric surgery.Outcome measures:This study used metabolic assays for glucose uptake, lipolysis, and lipogenesis in adipocytes in 2D cell culture and 3-dimensional ECM culture.Results:Adipocytes from subjects with DM manifest decreased glucose uptake and decreased lipolysis in 2D culture. ECM supports differentiation of mature adipocytes and recapitulates DM-specific differences in adipocyte metabolism observed in 2D culture. ECM from subjects without DM partially rescues glucose uptake and lipolytic defects in adipocytes from subjects with DM, whereas ECM from subjects with DM impairs glucose uptake in adipocytes from subjects without DM.Conclusions:DM is associated with adipocyte metabolic dysfunction. The ECM regulates adipocyte metabolism. Nondiabetic ECM rescues metabolic dysfunction in DM adipocytes, whereas DM ECM imparts features of metabolic dysfunction to nondiabetic adipocytes. These findings suggest the ECM as a target for manipulating adipose tissue metabolism.

The adipose tissue extracellular matrix (ECM) regulates adipocyte cellular metabolism and is altered in obesity and type 2 diabetes, but mechanisms underlying ECM-adipocyte metabolic crosstalk are poorly defined. Advanced glycation end-product (AGE) formation is increased in diabetes. AGE alter tissue function via direct effects on ECM and by binding scavenger receptors on multiple cell types and signaling through Rho GTPases. Our goal was to determine the role and underlying mechanisms of AGE in regulating human ECM-adipocyte metabolic crosstalk. Visceral adipocytes from diabetic and non-diabetic humans with obesity were studied in 2D and 3D-ECM culture systems. AGE is increased in adipose tissue from diabetic compared to non-diabetic subjects. Glycated collagen 1 and AGE-modified ECM regulate adipocyte glucose uptake and expression of AGE scavenger receptors and Rho signaling mediators, including the DIAPH1 gene, which encodes the human Diaphanous 1 protein (hDia1). Notably, inhibition of hDia1, but not scavenger receptors RAGE or CD36, attenuated AGE-ECM inhibition of adipocyte glucose uptake. These data demonstrate that AGE-modification of ECM contributes to adipocyte insulin resistance in human diabetes, and implicate hDia1 as a potential mediator of AGE-ECM-adipocyte metabolic crosstalk.