Kevin Y. Lee

Sirtuin-3 (Sirt3) regulates skeletal muscle metabolism and insulin signaling via altered mitochondrial oxidation and reactive oxygen species production

Sirt3 is a member of the sirtuin family of protein deacetylases that is localized in mitochondria and regulates mitochondrial function. Sirt3 expression in skeletal muscle is decreased in models of type 1 and type 2 diabetes and regulated by feeding, fasting, and caloric restriction. Sirt3 knockout mice exhibit decreased oxygen consumption and develop oxidative stress in skeletal muscle, leading to JNK activation and impaired insulin signaling. This effect is mimicked by knockdown of Sirt3 in cultured myoblasts, which exhibit reduced mitochondrial oxidation, increased reactive oxygen species, activation of JNK, increased serine and decreased tyrosine phosphorylation of IRS-1, and decreased insulin signaling. Thus, Sirt3 plays an important role in diabetes through regulation of mitochondrial oxidation, reactive oxygen species production, and insulin resistance in skeletal muscle.

Indian hedgehog is a major mediator of progesterone signaling in the mouse uterus

The hedgehog family of morphogens are regulators of cell proliferation, differentiation and cell-cell communication. These morphogens have been shown to have important roles in organogenesis, spermatogenesis, stem cell maintenance and oncogenesis. Indian hedgehog (encoded by Ihh) has been shown to be expressed in the uterine epithelium under the control of the steroid hormone, progesterone. Although in vivo and in vitro studies have shown that progesterone achieves its effects on uterine function through epithelial-stromal cross-talk, molecular mediator(s) for this cellular communication pathway have not been elucidated. Using new experimental approaches that ablate Ihh specifically in Pgr-positive uterine cells of the mouse, we demonstrate that Ihh is an essential mediator of Pgr action in the uterus, and expression of this factor is critical in mediating the communication between the uterine epithelium and stroma required for embryo implantation.

Bmp2 Is Critical for the Murine Uterine Decidual Response

ABSTRACT The process of implantation, necessary for all viviparous birth, consists of tightly regulated events, including apposition of the blastocyst, attachment to the uterine lumen, and differentiation of the uterine stroma. In rodents and primates the uterine stroma undergoes a process called decidualization. Decidualization, the process by which the uterine endometrial stroma proliferates and differentiates into large epithelioid decidual cells, is critical to the establishment of fetal-maternal communication and the progression of implantation. The role of bone morphogenetic protein 2 (Bmp2) in regulating the transformation of the uterine stroma during embryo implantation in the mouse was investigated by the conditional ablation of Bmp2 in the uterus using the (PR-cre) mouse. Bmp2 gene ablation was confirmed by real-time PCR analysis in the PR-cre; Bmp2fl/fl (termed Bmp2d/d) uterus. While littermate controls average 0.9 litter of 6.2 ± 0.7 pups per month, Bmp2d/d females are completely infertile. Analysis of the infertility indicates that whereas embryo attachment is normal in the Bmp2d/d as in control mice, the uterine stroma is incapable of undergoing the decidual reaction to support further embryonic development. Recombinant human BMP2 can partially rescue the decidual response, suggesting that the observed phenotypes are not due to a developmental consequence of Bmp2 ablation. Microarray analysis demonstrates that ablation of Bmp2 leads to specific gene changes, including disruption of the Wnt signaling pathway, Progesterone receptor (PR) signaling, and the induction of prostaglandin synthase 2 (Ptgs2). Taken together, these data demonstrate that Bmp2 is a critical regulator of gene expression and function in the murine uterus.

Lessons on Conditional Gene Targeting in Mouse Adipose Tissue

Conditional gene targeting has been extensively used for in vivo analysis of gene function in adipocyte cell biology but often with debate over the tissue specificity and the efficacy of inactivation. To directly compare the specificity and efficacy of different Cre lines in mediating adipocyte specific recombination, transgenic Cre lines driven by the adipocyte protein 2 (aP2) and adiponectin (Adipoq) gene promoters, as well as a tamoxifen-inducible Cre driven by the aP2 gene promoter (iaP2), were bred to the Rosa26R (R26R) reporter. All three Cre lines demonstrated recombination in the brown and white fat pads. Using different floxed loci, the individual Cre lines displayed a range of efficacy to Cre-mediated recombination that ranged from no observable recombination to complete recombination within the fat. The Adipoq-Cre exhibited no observable recombination in any other tissues examined, whereas both aP2-Cre lines resulted in recombination in endothelial cells of the heart and nonendothelial, nonmyocyte cells in the skeletal muscle. In addition, the aP2-Cre line can lead to germline recombination of floxed alleles in ∼2% of spermatozoa. Thus, different “adipocyte-specific” Cre lines display different degrees of efficiency and specificity, illustrating important differences that must be taken into account in their use for studying adipose biology.

WNT4 is a key regulator of normal postnatal uterine development and progesterone signaling during embryo implantation and decidualization in the mouse

WNT4, a member of the Wnt family of ligands, is critical for the development of the female reproductive tract. Analysis of Wnt4 expression in the adult uterus during pregnancy indicates that it may play a role in the regulation of endometrial stromal cell proliferation, survival, and differentiation, which is required to support the developing embryo. To investigate the role of Wnt4 in adult uterine physiology, conditional ablation of Wnt4 using the PRC” mouse model was accomplished. Ablation of Wnt4 rendered female mice subfertile due to a defect in embryo implantation and subsequent defects in endometrial stromal cell survival, differentiation, and responsiveness to progesterone signaling. In addition to altered stromal cell function, the uteri of PITe/+ Wnt4f/f (Wnt4d/d) mice displayed altered epithelial differentiation characterized by a reduction in the number of uterine glands and the emergence of a p63‐positive basal cell layer beneath the columnar luminal epithelial cells. The altered epithelial cell phenotype was further escalated by chronic estrogen treatment, which caused squamous cell metaplasia of the uterine epithelium in the Wnt4d/d mice. Thus, WNT4 is a critical regulator not only of proper postnatal uterine development, but also embryo implantation and decidualization.—Franco, H. L., Dai, D., Lee, K. Y., Rubel, C. S., Roop, D., Boerboom, D., Jeong, J.‐W., Lydon, J.‐P., Bagchi, I. C., Bagchi, M. K., DeMayo, F. J. WNT4 is a key regulator of normal postnatal uterine development and progesterone signaling during embryo implantation and decidualization in the mouse. FASEB J. 25, 1176–1187 (2011). www.fasebj.org

COUP-TFII Mediates Progesterone Regulation of Uterine Implantation by Controlling ER Activity

Progesterone and estrogen are critical regulators of uterine receptivity. To facilitate uterine remodeling for embryo attachment, estrogen activity in the uterine epithelia is attenuated by progesterone; however, the molecular mechanism by which this occurs is poorly defined. COUP-TFII (chicken ovalbumin upstream promoter transcription factor II; also known as NR2F2), a member of the nuclear receptor superfamily, is highly expressed in the uterine stroma and its expression is regulated by the progesterone–Indian hedgehog–Patched signaling axis that emanates from the epithelium. To further assess COUP-TFII uterine function, a conditional COUP-TFII knockout mouse was generated. This mutant mouse is infertile due to implantation failure, in which both embryo attachment and uterine decidualization are impaired. Using this animal model, we have identified a novel genetic pathway in which BMP2 lies downstream of COUP-TFII. Epithelial progesterone-induced Indian hedgehog regulates stromal COUP-TFII, which in turn controls BMP2 to allow decidualization to manifest in vivo. Interestingly, enhanced epithelial estrogen activity, which impedes maturation of the receptive uterus, was clearly observed in the absence of stromal-derived COUP-TFII. This finding is consistent with the notion that progesterone exerts its control of implantation through uterine epithelial-stromal cross-talk and reveals that stromal-derived COUP-TFII is an essential mediator of this complex cross-communication pathway. This finding also provides a new signaling paradigm for steroid hormone regulation in female reproductive biology, with attendant implications for furthering our understanding of the molecular mechanisms that underlie dysregulation of hormonal signaling in such human reproductive disorders as endometriosis and endometrial cancer.

Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle through Reversible Enzymatic Deacetylation

Sirt3 is an NAD+-dependent deacetylase that regulates mitochondrial function by targeting metabolic enzymes and proteins. In fasting mice, Sirt3 expression is decreased in skeletal muscle resulting in increased mitochondrial protein acetylation. Deletion of Sirt3 led to impaired glucose oxidation in muscle, which was associated with decreased pyruvate dehydrogenase (PDH) activity, accumulation of pyruvate and lactate metabolites, and an inability of insulin to suppress fatty acid oxidation. Antibody-based acetyl-peptide enrichment and mass spectrometry of mitochondrial lysates from WT and Sirt3 KO skeletal muscle revealed that a major target of Sirt3 deacetylation is the E1α subunit of PDH (PDH E1α). Sirt3 knockout in vivo and Sirt3 knockdown in myoblasts in vitro induced hyperacetylation of the PDH E1α subunit, altering its phosphorylation leading to suppressed PDH enzymatic activity. The inhibition of PDH activity resulting from reduced levels of Sirt3 induces a switch of skeletal muscle substrate utilization from carbohydrate oxidation toward lactate production and fatty acid utilization even in the fed state, contributing to a loss of metabolic flexibility. Thus, Sirt3 plays an important role in skeletal muscle mitochondrial substrate choice and metabolic flexibility in part by regulating PDH function through deacetylation.

Adipose depots possess unique developmental gene signatures.

We have previously demonstrated that subcutaneous and intra‐abdominal adipose tissue show different patterns of expression for developmental genes (Shox2, En1, Tbx15 Hoxa5, Hoxc8, and Hoxc9), and that the expression level of Tbx15 and Hoxa5 in humans correlated with the level of obesity and fat distribution. To further explore the role of these developmental genes in adipose tissue, we have characterized their expression in different adipose depots in mice, and studied their regulation in obesity and by fasting. Developmental and adipogenic gene expression was compared in two subcutaneous and three intra‐abdominal white adipose tissue (WAT) depots as well as brown adipose tissue (BAT) from lean or obese mice in a fed or fasting state. Each of these six adipose depots display a unique pattern of developmental gene expression, whereas expression of adipogenic transcription factors PPARγ2 C/EBPα, β, and Δ showed constant expression levels in all depots. Expression levels of developmental genes were similar in obese (ob/ob and high‐fat diet (HFD)) and lean mice in most depots. Fasting systematically decreased expression of Hoxc8, PPARγ2, and increased C/EBPΔ in both lean and ob/ob mice, but produced only variable changes in the expression of other developmental and adipogenic genes. These data indicate that each fat depot has a unique developmental gene expression signature, which is largely independent of nutritional state. This finding further supports a fundamental role of developmental genes in fat distribution and the development and/or function of specific adipose tissue depots.

Foxa2 Is Essential for Mouse Endometrial Gland Development and Fertility

During embryonic development, Foxa2 is required for the formation of the node and notochord, and ablation of this gene results in defects in gastrulation, neural tube patterning, and gut morphogenesis. Foxa2 has been shown to be expressed specifically in the glandular epithelium of the murine uterus. To study the uterine function of Foxa2, this gene was conditionally ablated in the mouse uterus by crossing mice with floxed Foxa2 alleles, Foxa2loxP/loxP, with the Pgrcre mouse model. Pgrcre/+ Foxa2loxP/loxP mice showed significantly reduced fertility. Analysis of the uterus on Day 5.5 of pregnancy showed disrupted blastocyst implantation. Pgrcre/+ Foxa2loxP/loxP mice also showed a severe impairment of the uterus to respond to the artificial induction of the decidual response. Morphological examination of the uteri of these mice showed a severe reduction in the number of endometrial glands. The loss of endometrial glands resulted in the reduction of leukemia inhibitory factor (Lif) expression. The lack of a decidual response could be partially rescued by an intrauterine injection of LIF before the initiation of the decidual response. This analysis demonstrates that Foxa2 regulates endometrial gland development and that mice with a loss of endometrial glands cannot support implantation in part due to the loss of LIF, which is a requisite for fertility in the mouse.

ASC-1, PAT2, and P2RX5 are cell surface markers for white, beige, and brown adipocytes

The cell surface markers ASC-1, PAT2, and P2RX5 can be used to mark and identify brown, beige, and white adipocytes in both rodents and humans. Fat Cells Gain New Identities There’s “good fat” and there’s “bad fat.” Good fat is considered to be brown adipose tissue (BAT), which burns calories. Bad fat can be white adipose tissue (WAT), which stores lipids as energy and, in excess, contributes to obesity. When brown fat cells, or adipocytes, develop within white fat, they are called “beige.” Sorting out these different adipocyte subtypes within the human body has been challenging but will be important in uncovering the underlying mechanisms for obesity and its comorbidities, such as type 2 diabetes. To this end, Ussar and colleagues have now identified three new surface markers of white, beige, and brown fat cells. These markers—ASC-1, PAT2, and P2RX5—were first selected in silico, then confirmed in mouse WAT and BAT, and lastly verified in human adipose tissue biopsies. ASC-1, PAT2, and P2RX5 are located in the plasma membrane of adipocytes, thus making them prime targets for imaging fat locations within the body and for directing therapeutics toward particular fat depots. White, beige, and brown adipocytes are developmentally and functionally distinct but often occur mixed together within individual depots. To target white, beige, and brown adipocytes for diagnostic or therapeutic purposes, a better understanding of the cell surface properties of these cell types is essential. Using a combination of in silico, in vitro, and in vivo methods, we have identified three new cell surface markers of adipose cell types. The amino acid transporter ASC-1 is a white adipocyte–specific cell surface protein, with little or no expression in brown adipocytes, whereas the amino acid transporter PAT2 and the purinergic receptor P2RX5 are cell surface markers expressed in classical brown and beige adipocytes in mice. These markers also selectively mark brown/beige and white adipocytes in human tissue. Thus, ASC-1, PAT2, and P2RX5 are membrane surface proteins that may serve as tools to identify and target white and brown/beige adipocytes for therapeutic purposes.