Tae Hwa Chun

A Pericellular Collagenase Directs the 3-Dimensional Development of White Adipose Tissue

White adipose tissue (WAT) serves as the primary energy depot in the body by storing fat. During development, fat cell precursors (i.e., preadipocytes) undergo a hypertrophic response as they mature into lipid-laden adipocytes. However, the mechanisms that regulate adipocyte size and mass remain undefined. Herein, we demonstrate that the membrane-anchored metalloproteinase, MT1-MMP, coordinates adipocyte differentiation in vivo. In the absence of the protease, WAT development is aborted, leaving tissues populated by mini-adipocytes which render null mice lipodystrophic. While MT1-MMP preadipocytes display a cell autonomous defect in vivo, null progenitors retain the ability to differentiate into functional adipocytes during 2-dimensional (2-D) culture. By contrast, within the context of the 3-dimensional (3-D) ECM, normal adipocyte maturation requires a burst in MT1-MMP-mediated proteolysis that modulates pericellular collagen rigidity in a fashion that controls adipogenesis. Hence, MT1-MMP acts as a 3-D-specific adipogenic factor that directs the dynamic adipocyte-ECM interactions critical to WAT development.

MT1-MMP-dependent neovessel formation within the confines of the three-dimensional extracellular matrix.

During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix–degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP–dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., β3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.

Genetic Link Between Obesity and MMP14-Dependent Adipogenic Collagen Turnover

OBJECTIVE In white adipose tissue, adipocytes and adipocyte precursor cells are enmeshed in a dense network of type I collagen fibrils. The fate of this pericellular collagenous web in diet-induced obesity, however, is unknown. This study seeks to identify the genetic underpinnings of proteolytic collagen turnover and their association with obesity progression in mice and humans. RESEARCH DESIGN AND METHODS The hydrolysis and degradation of type I collagen at early stages of high-fat diet feeding was assessed in wild-type or MMP14 (MT1-MMP)-haploinsufficient mice using immunofluorescent staining and scanning electron microscopy. The impact of MMP14-dependent collagenolysis on adipose tissue function was interrogated by transcriptome profiling with cDNA microarrays. Genetic associations between MMP14 gene common variants and obesity or diabetes traits were examined in a Japanese cohort (n = 3,653). RESULTS In adult mice, type I collagen fibers were cleaved rapidly in situ during a high-fat diet challenge. By contrast, in MMP14 haploinsufficient mice, animals placed on a high-fat diet were unable to remodel fat pad collagen architecture and display blunted weight gain. Moreover, transcriptional programs linking type I collagen turnover with adipogenesis or lipogenesis were disrupted by the associated decrease in collagen turnover. Consistent with a key role played by MMP14 in regulating high-fat diet–induced metabolic programs, human MMP14 gene polymorphisms located in proximity to the enzymes catalytic domain were closely associated with human obesity and diabetes traits. CONCLUSIONS Together, these findings demonstrate that the MMP14 gene, encoding the dominant pericellular collagenase operative in vivo, directs obesogenic collagen turnover and is linked to human obesity traits.

Peri-adipocyte ECM remodeling in obesity and adipose tissue fibrosis.

Adipocytes differentiate and function in environments rich in extracellular matrix (ECM) proteins. The phenotypes of genetically modified mice have aided in recognizing the importance of ECM proteins and their modifiers, e.g., proteinases, in the regulation of obesity and metabolism. Most of the molecular mechanisms through which ECM proteins and modifiers regulate adipogenesis or adipocyte function have not been fully defined. Adipose tissue fibrosis may be a factor that links obesity to diabetes or cardiovascular disease risk in conjunction with tissue inflammation. Defining the molecular mechanisms through which the ECM environment regulates adipogenesis and adipocyte function should provide us with a better understanding of the disease link between obesity and diabetes or cardiovascular diseases.

Thrombospondin 1 Mediates High-Fat Diet-Induced Muscle Fibrosis and Insulin Resistance in Male Mice

Thrombospondin 1 (THBS1 or TSP-1) is a circulating glycoprotein highly expressed in hypertrophic visceral adipose tissues of humans and mice. High-fat diet (HFD) feeding induces the robust increase of circulating THBS1 in the early stages of HFD challenge. The loss of Thbs1 protects male mice from diet-induced weight gain and adipocyte hypertrophy. Hyperinsulinemic euglycemic clamp study has demonstrated that Thbs1-null mice are protected from HFD-induced insulin resistance. Tissue-specific glucose uptake study has revealed that the insulin-sensitive phenotype of Thbs1-null mice is mostly mediated by skeletal muscles. Further assessments of the muscle phenotype using RNA sequencing, quantitative PCR, and histological studies have demonstrated that Thbs1-null skeletal muscles are protected from the HFD-dependent induction of Col3a1 and Col6a1, coupled with a new collagen deposition. At the same time, the Thbs1-null mice display a better circadian rhythm and higher amplitude of energy expenditure with a browning phenotype in sc adipose tissues. These results suggest that THBS1, which circulates in response to a HFD, may induce insulin resistance and fibrotic tissue damage in skeletal muscles as well as the de-browning of sc adipose tissues in the early stages of a HFD challenge. Our study may shed new light on the pathogenic role played by a circulating extracellular matrix protein in the cross talk between adipose tissues and skeletal muscles during obesity progression.

Thrombospondin 1 as a novel biological marker of obesity and metabolic syndrome

CONTEXT Thrombospondin 1 (THBS1 or TSP-1) is an adipose-derived matricellular protein, which has recently been highlighted as a potential mediator of insulin resistance and adipose inflammation in obesity. OBJECTIVE In this study, we aimed to determine the clinical significance of THBS1 as a novel biological marker of visceral obesity, metabolic syndrome, and diabetes. METHODS The THBS1 mRNA level was quantified with real-time PCR in human adipose tissues obtained from 16 non-obese subjects. The relationships between serum THBS1 level and obesity/diabetes traits as well as the diagnostic components of metabolic syndrome were assessed in 164 normal-weight or overweight/obese subjects (78 males and 86 females; mean age, 50.4; mean BMI, 29.8) with analysis of covariance (ANCOVA) and regression analyses. RESULTS THBS1 was predominantly expressed in visceral adipose tissues relative to subcutaneous adipose tissues (P<0.001). The visceral THBS1 expression was positively associated with the body mass index (BMI; γs=0.54, P=0.033). ANCOVA demonstrated that the THBS1 level is associated with abdominal obesity (P<0.001), hyperglycemia (P=0.02), and hypertension (P=0.04). Multivariable regression analysis suggested an association between serum THBS1 and fasting plasma glucose levels. The associations between serum THBS1 levels and obesity/diabetes traits were found preferentially in women (BMI, γs=0.30, P=0.05; FPG, γs=0.26, P=0.016). Subanalyses demonstrated that the association with obesity traits was predominantly found in premenopausal women (BMI, γs=0.41, P=0.007), whereas the association with diabetes traits was predominant in postmenopausal women (HbA1c, γs=0.38, P=0.01). During medical weight reduction treatment, the change in the serum THBS1 level was associated with the change in BMI and HbA1c in pre- and postmenopausal women, respectively. CONCLUSIONS Serum THBS1 is a useful biological marker of obesity and metabolic syndrome in Japanese subjects, particularly in women. THBS1 may act as a critical circulating factor that couples obesity with metabolic syndrome and diabetes in humans.

Adipogenic Histone Mark Regulation by Matrix Metalloproteinase 14 in Collagen-Rich Microenvironments

Adipogenesis is directed by both transcriptional network and posttranslational modification of chromatin structure. Although adipogenesis in vivo proceeds in collagen-rich extracellular matrix (ECM) environments, the impact of ECM proteins and their modifying enzymes on the epigenetic regulation of adipogenesis has been largely unknown. We aimed to define the role of fibrillar type I collagen and its modifying enzymes in regulating adipogenic chromatin signatures and gene regulation in the in vivo-like settings. Adipogenic cocktail induces a robust increase in the level of protranscriptional acetylated histone H3 at lysine 9 (H3K9ac) within 24 h. When cultured atop fibrillar type I collagen gel, however, H3K9ac levels in differentiating 3T3-L1 cells are substantially reduced. The suppression of adipogenic histone mark in differentiating 3T3-L1 cells is type I collagen density dependent and released by heat denaturing of the subjacent collagen substratum, pointing to the critical role played by the triple-helical structure of type I collagen. By probing adipogenic collagenolysis with a series of proteinase inhibitors, matrix metalloproteinase (MMP) family members are found to be responsible for adipogenic collagenolysis. At the same time, MMP inhibitor specifically blocked the adipogenic induction of H3K9ac. By targeting individual MMP using small interfering RNA oligos, MMP14 was identified as the major adipogenic MMP critical for H3K9 acetylation. Consistently, MMP14-null adipose tissues display diminished protranscriptional histone mark H3K9ac while maintaining repressive histone mark tri-methylated histone H3 at lysine 9 (H3K9me3). Taken together, MMP14-dependent collagenolysis plays the major role in regulating adipogenic histone marks by releasing the epigenetic constraints imposed by fibrillar type I collagen.

Fat depot-specific gene signature and ECM remodeling of Sca1high adipose-derived stem cells

Stem cell antigen-1 (Sca1 or Ly6A/E) is a cell surface marker that is widely expressed in mesenchymal stem cells, including adipose-derived stem cells (ASCs). We hypothesized that the fat depot-specific gene signature of Sca1(high) ASCs may play the major role in defining adipose tissue function and extracellular matrix (ECM) remodeling in a depot-specific manner. Herein we aimed to characterize the unique gene signature and ECM remodeling of Sca1(high) ASCs isolated from subcutaneous (inguinal) and visceral (epididymal) adipose tissues. Sca1(high) ASCs are found in the adventitia and perivascular areas of adipose tissues. Sca1(high) ASCs purified with magnetic-activated cell sorting (MACS) demonstrate dendrite or round shape with the higher expression of cytokines and chemokines (e.g., Il6, Cxcl1) and the lower expression of a glucose transporter (Glut1). Subcutaneous and visceral fat-derived Sca1(high) ASCs particularly differ in the gene expressions of adhesion and ECM molecules. While the expression of the major membrane-type collagenase (MMP14) is comparable between the groups, the expressions of secreted collagenases (MMP8 and MMP13) are higher in visceral Sca1(high) ASCs than in subcutaneous ASCs. Consistently, slow but focal MMP-dependent collagenolysis was observed with subcutaneous adipose tissue-derived vascular stromal cells, whereas rapid and bulk collagenolysis was observed with visceral adipose tissue-derived cells in MMP-dependent and -independent manners. These results suggest that the fat depot-specific gene signatures of ASCs may contribute to the distinct patterns of ECM remodeling and adipose function in different fat depots.

3-D adipocyte differentiation and peri-adipocyte collagen turnover.

Peri-adipocyte extracellular matrix (ECM) remodeling is a key biological process observed during adipose tissue development and expansion. The genetic loss of a pericellular collagenase, MMP14 (also known as MT1-MMP), renders mice lipodystrophic with the accumulation of undigested collagen fibers in adipose tissues. MMP14 is not necessary for adipocyte differentiation (adipogenesis) per se under a conventional two-dimensional (2-D) culture condition; however, MMP14 plays a critical role in adipogenesis in vivo. The role of MMP14 in adipogenesis and adipocyte gene expression was uncovered in vitro only when tested within a three-dimensional (3-D) collagen gel, which recapitulated the in vivo ECM-rich environment. Studying adipogenesis in 3-D may serve as an effective experimental approach to bridge gaps in our understanding of in vivo adipocyte biology. Moreover, by assessing the content of collagen family members and their rate of degradation in adipose tissues, we should be able to better define the role of dynamic ECM remodeling in the pathogenesis of obesity and diabetes.

Cellular and Molecular Aspects of C-Type Natriuretic Peptide (CNP)

C-type natriuretic peptide (CNP) was originally isolated from porcine brain extracts (1). CNP has a primary structure similar to α-ANP and BNP consisting of 22 amino acids, and the ring portion consisting of 17 amino acids is highly homologous to α-ANP and BNP (Fig. 1). Different from ANP and BNP, CNP lacks the C-terminal tail, and has a Cys residue at the C-terminus. Another species of CNP is CNP-53, which has the N-terminal extension of 31 amino acids compared with CNP-22 (2). Molecular cloning of the CNP precursor in the pig, rat, human, and mouse has revealed that the primary structure of CNP-22 is identical in these species (1,3–5). However, two amino acid substitutions are noted in CNP-53 between human and porcine/rat/mouse precursors. ProCNP consists of 103 amino acids and a single arginine residue precedes CNP-53 and CNP-22. Using reverse-phase high-performance liquid chromatography (HPLC), we demonstrated that the major molecular forms in the human brain are CNP-22 and CNP-53 (4).