Jaideep Moitra

Accelerated Tumor Formation in a Fatless Mouse with Type 2 Diabetes and Inflammation

Epidemiologic studies show a positive association between obesity and cancer risk. In addition to increased body adiposity and secretion of fat-derived hormones, obesity is also linked to insulin resistance, type 2 diabetes, and chronic inflammation. We used the fatless A-ZIP/F-1 transgenic mouse to dissociate the relative role of each of these underlying factors in the development of cancer. These mice are unique in that they do not have white fat but do develop type 2 diabetes. In two cancer models, the classic two-stage skin carcinogenesis protocol and the C3(1)/T-Ag transgenic mouse mammary tumor model, A-ZIP/F-1 mice displayed higher tumor incidence, tumor multiplicity, and decreased tumor latency than wild-type mice. We examined circulating levels of adipokines, growth factors, and cytokines. As expected, adipokines (i.e., leptin, adiponectin, and resistin) were undetectable or found at very low levels in the blood of fatless mice. However, insulin, insulin-like growth factor-I, growth hormone, vascular endothelial growth factor, and proinflammatory Th2 cytokines, such as interleukin (IL)-1beta, IL-4, and IL-6, were elevated in A-ZIP/F-1 mice. Additionally, we examined multiple phosphorylated proteins (i.e., protein kinase B/Akt and ErbB2/HER-2 kinase) associated with cancer development. Results show that many of these phosphorylated proteins were activated specifically in the A-ZIP/F-1 skin but not in the wild-type skin. These findings suggest that adipokines are not required for the promotion of tumor development and thus contradict the epidemiologic data linking obesity to carcinogenesis. We postulate that insulin resistance and inflammation are responsible for the positive correlation with cancer observed in A-ZIP/F-1 mice.

Transgenic Mice Lacking White Fat: Models for Understanding Human Lipoatrophic Diabetes

ABSTRACT: The human disease lipoatrophic (or lipodystrophic) diabetes is a rare syndrome in which a deficiency of adipose tissue is associated with Type 2 diabetes. This disease is an interesting contrast to the usual situation in which diabetes is associated with obesity, an excess of fat. Aside from obesity, patients with lipodystrophic diabetes have the other features associated with Metabolic Syndrome X, including hypertension and dyslipidemia. The contrast between diabetes with a lack of fat and diabetes with an excess of fat provides an opportunity to study the mechanisms causing Type 2 diabetes and its complications. Recently, three laboratories have produced transgenic mice that are deficient in white adipose tissue. These mice have insulin resistance and other features of lipoatrophic diabetes, and are a faithful model for the human disease. Here we review the different murine models of fat ablation and compare the murine and human diseases, addressing the questions: Is the lack of fat causative of the diabetes, and if so by what mechanism? How could the other clinical features be explained mechanistically? And finally, what can be gleaned about insight into treatment options?

Adipose tissue: a vital in vivo role in mammary gland development but not differentiation.

Development and differentiation of the mammary gland occurs by means of critical stromal‐epithelial interactions. Although many studies have attempted to understand these complex interactions, it has been difficult to demonstrate the essential role of adipose tissue in the development and function of the mammary gland. By using the A‐ZIP/F‐1 transgenic mice lacking in white adipose tissue (WAT), we have studied the role of adipocytes in mammary gland development and differentiation. In the absence of WAT, rudimentary mammary anlagen form but are unable to grow and branch normally, resulting in a few, short, severely distended ducts. However, during pregnancy, a tremendous amount of epithelial cell division and alveolar cell formation occurs even in the absence of adipocytes, illustrating that adipose tissue is not required for mammary gland differentiation. Mammary gland transplantation revealed that epithelial cells from these transgenic mice possess the potential for normal growth and differentiation when placed into a normal stromal environment. These experiments clearly demonstrate that the absence of adipocytes in the mammary gland results in disruption of stromal‐epithelial interactions that prevent normal mammary gland development. The rudimentary epithelial anlage, however, contain mammary stem cells, which are fully capable of alveolar differentiation. Published 2002 Wiley‐Liss, Inc.

Activator Protein-1 Activity Regulates Epithelial Tumor Cell Identity

To examine the consequences of inhibiting activator protein-1 (AP-1) transcription factors in skin, transgenic mice were generated, which use the tetracycline system to conditionally express A-FOS, a dominant negative that inhibits AP-1 DNA binding. Older mice develop mild alopecia and hyperplasia of sebaceous glands, particularly around the eyes. When A-FOS was expressed during chemical-induced skin carcinogenesis, mice do not develop characteristic benign and malignant squamous lesions but instead develop benign sebaceous adenomas containing a signature mutation in the H-ras proto-oncogene. Inhibiting AP-1 activity after tumor formation caused squamous tumors to transdifferentiate into sebaceous tumors. Furthermore, reactivating AP-1 in sebaceous tumors results in a reciprocal transdifferentiation into squamous tumors. In both cases of transdifferentiation, individual cells express molecular markers for both cell types, indicating individual tumor cells have the capacity to express multiple lineages. Molecular characterization of cultured keratinocytes and tumor material indicates that AP-1 regulates the balance between the wnt/beta-catenin and hedgehog signaling pathways that determine squamous and sebaceous lineages, respectively. Chromatin immunoprecipitation analysis indicates that c-Jun binds several wnt promoters, which are misregulated by A-FOS expression, suggesting that members of the wnt pathway can be a primary targets of AP-1 transcriptional regulation. Thus, AP-1 activity regulates tumor cell lineage and is essential to maintain the squamous tumor cell identity.

Suppression of the C/EBP family of transcription factors in adipose tissue causes lipodystrophy

Adipose-specific inactivation of both AP-1 and CCAAT-enhancer-binding protein (C/EBP) families of B-ZIP transcription factors in transgenic mice causes severe lipoatrophy. To evaluate whether inactivation of only C/EBP members was critical for lipoatrophy, A-C/EBP, a dominant-negative protein that specifically inhibits the DNA binding of the C/EBP members, was expressed in adipose tissue. For the first 2 weeks after birth, aP2-A-C/EBP mice had no white adipose tissue (WAT), drastically reduced brown adipose tissue (BAT), and exhibited marked hepatic steatosis, hyperinsulinemia, and hyperlipidemia. However, WAT appeared during the third week, coinciding with significantly improved metabolic functioning. In adults, BAT remained reduced, causing cold intolerance. At 30 weeks, the aP2-A-C/EBP mice had only 35% reduced WAT, with clear morphological signs of lipodystrophy in subcutaneous fat. Circulating leptin and adiponectin levels were less than the wild-type levels, and these mice exhibited impaired triglyceride clearance. Insulin resistance, glucose intolerance, and reduced free fatty acid release in response to β3-adrenergic agonist suggest improper functioning of the residual WAT. Gene expression analysis of inguinal WAT identified reduced mRNA levels of several enzymes involved in fatty acid synthesis and glucose metabolism that are known C/EBPα transcriptional targets. There were increased levels for genes involved in inflammation and muscle differentiation. However, when dermal fibroblasts from aP2-A-C/EBP mice were differentiated into adipocytes in tissue culture, muscle markers were elevated more than the inflammatory markers. These results demonstrate that the C/EBP family is essential for adipose tissue development during the early postnatal period, the regulation of glucose and lipid homeostasis in adults, and the suppression of the muscle lineage.

Inhibition of CREB Function in Mouse Epidermis Reduces Papilloma Formation

We used a double transgenic tetracycline system to conditionally express A-CREB, a dominant negative protein that prevents the DNA binding and function of cAMP-responsive element binding protein (CREB) family members, in mouse basal epidermis using the keratin 5 promoter. There was no phenotype in the adult. However, following a 7,12-dimethylbenz(a)anthracene (DMBA)/phorbol-12-myristate-13-acetate two-stage skin carcinogenesis experiment, A-CREB–expressing epidermis develop 5-fold fewer papillomas than wild-type controls. However, A-CREB expression one month after DMBA treatment does not prevent papilloma formation, suggesting that CREB functions at an early stage of papilloma formation. Oncogenic H-Ras genes with A→T mutations in codon 61 were found in wild-type skin but not in A-CREB–expressing skin 2 days after DMBA treatment, suggesting that A-CREB either prevents DMBA mutagenesis or kills oncogenic H-Ras cells. In primary keratinocyte cultures, A-CREB expression induced apoptosis of v-RasHa–infected cells and suppressed the expression of cell cycle proteins cyclin B1 and cyclin D1. These results suggest that inhibiting CREB function is a valuable cancer prevention strategy.(Mol Cancer Res 2009;7(5):654–64)

A search for candidate genes for lipodystrophy, obesity and diabetes via gene expression analysis of A-ZIP/F-1 mice

Genome scans for diabetes have identified many regions of the human genome that correlate with the disease state. To identify candidate genes for type 2 diabetes, we examined the transgenic A-ZIP/F-1 mouse. This mouse model has no white fat, resulting in abnormal levels of glucose, insulin, and leptin, making the A-ZIP/F-1 mice a good model for lipodystrophy and insulin resistance. We used cDNA-based microarrays to find differentially expressed genes in four tissues of these mice. We examined these results in the context of human linkage scans for lipodystrophy, obesity, and type 2 diabetes. We combined 199 known human orthologs of the misregulated mouse genes with 33 published human genome scans on a genome map. Integrating expression data with human linkage results permitted us to suggest and prioritize candidate genes for lipodystrophy and related disorders. These genes include a cluster of 3 S100A genes on chromosome 1 and SLPI1 on chromosome 20.