Section: Rotations

Postdoctoral Position Available

Michael Czech, Ph.D.

Academic Role: Professor

Faculty Appointment(s) In:
   Program in Molecular Medicine

Joint Faculty In:
   Biochemistry and Molecular Pharmacology

Other Affiliation(s):
   Center for AIDS Research
   Interdisciplinary Graduate Program

Rotation Projects

There are several available rotation projects in the laboratory for graduate students. Examples are outlined below:

Oral delivery of siRNA as a therapeutic strategy for types 1 and 2 diabetes

The autoimmune reactions that cause destruction of beta cells and type 1 diabetes are driven by autoreactive T cells in association with innate inflammatory cells (macrophages and dendritic cells). Paradoxically, recent research indicates these latter cells in the immature state also play key roles in establishing immunological tolerance, through presentation of antigens to T cells within a "tolerogenic" context. One of the most promising concepts that has emerged from these studies is that induction of immunological tolerance to beta cell auto-antigens might prevent or even alleviate the disease.  Immunological tolerance has many forms and mechanisms, but a major paradigm is the generation of regulatory T cells (T-regs) by tolerogenic macrophages or dendritic cells that suppress the cascade of events leading to auto-reactivity and beta cell apoptosis. These events appear to take place in the local regions of the islets and nearby lymph nodes. Further, the gut-associated lymphatic system represents a key site where induction of immunological tolerance may be critically important. This collaborative research project with Gary Ostroff's laboratory is based on our discovery that siRNA encapsulated within micron-sized, hollow shells of beta 1,3-D-glucan can be delivered orally to mice to target dendritic cells and macrophages in the gut. This technology advance enables us to integrate two approaches that might promote immunological tolerance to prevent or alleviate type 1 diabetes: 1) tolerize the gut immune system to beta cell antigens through targeted antigen delivery to dendritic cells and macrophages in the gut; 2) orally deliver RNAi to silence genes that control the secretion of proinflammatory cytokines, co-stimulatory receptors or "maturity" factors in dendritic cells and macrophages during the early stages of beta cell antigen presentation to induce T-reg and hypo-responsive T cells. Type 2 diabetes in obesity is also associated with an inflammation phenotype-in this case within adipose tissue. Thus, we have also initiated experiments to target macrophages in obese mouse models of type 2 diabetes with orally delivered siRNA directed against genes that control inflammation. Rotation projects in the Czech laboratory applying oral delivery of encapsulated siRNA to mouse models of both types 1 and 2 diabetes are now available to graduate students.

Lipid Droplet Proteins and their Roles in Fat Deposition in Obesity and Diabetes

Fat homeostasis is perturbed in type II diabetes and obesity and is a major factor associated with pathogenesis of metabolic diseases. High concentrations of circulating fatty acids and triglyceride, observed in both obesity and lipodystrophy, are thought to cause muscle insulin resistance and decreased glucose tolerance. Adipocytes could protect muscle and liver from these deleterious effects of fatty acids by their large capacity to esterify them into triglyceride, and to sequester large amounts of the triglyceride within lipid droplets.  We have recently identified a set of novel proteins, Cidea and fat specific protein 27 (FSP27; also known as Cidec), which are associated with lipid droplets in adipocytes and their expression can enhance the accumulation of triglycerides in adipocytes both in vivo and in vitro (JBC 282: 34213-8, 2006; PNAS 105: 7833-8, 2008; Acta Physiologica 192: 103-115, 2008). Strikingly, Cidea and FSP27 expression was higher in insulin sensitive vs. resistant obese humans. Our studies indicate that Cidea and FSP27 define a novel, highly regulated pathway of triglyceride accumulation in mouse and human white adipose tissue and their expression is associated with insulin sensitivity in humans (JCI 118: 2693-6, 2008; Nature Reviews MCB 9, 367-77, 2008). Rotation projects in the laboratory are designed to address the mechanisms by which Cidea and FSP27 associate with lipid droplets and regulate triglyceride deposition. Currently, these projects utilize adipocytes in culture or from mice and human patients.  Lipid droplets and associated proteins are analyzed by live cell microscopy and by cell and molecular biology techniques.

Insulin and Map4k4 Signaling and Adipogenesis Regulation

Insulin resistance in muscle is a major syndrome in obese humans that contributes to the onset of type 2 diabetes. The development of obesity coincides with substantial infiltration of macrophages into adipose tissue, which is associated with increased expression of inflammatory cytokines such as TNFa; (Nat Rev Mol Cell Biol. 5: 367-377, 2008).  The nuclear receptor peroxisome proliferator-activated receptor   g(PPAR g ) is a ligand-dependent transcription factor that acts as a primary regulator of adipogenesis and controls adipocyte metabolism and insulin action. Increased expression of TNFa; in adipose tissue of obese subjects potently suppresses the expression of PPARg; and attenuates adipocyte functions.

We screened cultured adipocytes with siRNA directed against over two hundred protein kinases for negative regulators of insulin-sensitive glucose transport.  Among several hits in this screen, we discovered the mitogen-activated protein kinase 4 (MAP4k4) as a protein kinase of the Ste20 family that exhibits potent inhibitory effects on insulin sensitivity and adipogenesis (Proc. Natl. Acad. Sci. USA 103: 2087, 2006). Remarkably, silencing Map4k4 caused an increase in the expression of peroxisome proliferator-activated receptor; g(PPARg ), along with a corresponding increase in the expression of GLUT4 and stimulation of insulin-mediated deoxyglucose uptake.  More recent studies have shown that MAP4K4 mediates the insulin resistance in muscle induced by TNF-a; (J Biol Chem. 282: 7783-7789, 2007).  Interestingly, TNF-a; both increases Map4k4 acute catalytic activity and Map4k4 expression in adipocytes through activation of TNF-a -receptor 1 (TNFR1), but not TNFR2. (Proc. Natl. Acad. Sci. USA 103: 2087, 2006; J Biol Chem., 282:19302-19312). An important question that is currently under investigation in our laboratory is what are the molecular mechanisms by which MAP4K4 upregulates PPAR  function? We found that RNAi-mediated silencing of MAP4K4 elevated the levels of both PPAR 1 and PPAR 2 proteins 2-3 fold in 3T3-L1 adipocytes without affecting PPAR  mRNA levels, suggesting that MAP4K4 regulates PPARg  at a post-transcriptional step.  Moreover, silencing MAP4K4 had no effect on PPARg; degradation. However, depletion of MAP4K4 significantly enhances [S-35]-incorporation into proteins, suggesting that silencing of MAP4K4 increases protein translation. Based on our preliminary results, it appears that MAP4K4 regulates PPARg; protein expression by inhibiting mTOR-mediated protein translation. Rotation projects in the laboratory are designed to identify the elements downstream of the MAP4K4 signaling pathways that mediate its effects on protein translation in cultured adipocytes.

Inflammation in Adipose Tissues of Obese Human Subjects

Obesity is an independent risk factor for multiple medical conditions.  Interestingly, not all obese individuals manifest obesity-related comorbidities, highlighting the importance of identifying specific genetic and biological factors that place a subset of obese individuals at the greatest risk for complications.  We are collaborating with Dr. Richard Perugini in the Department Surgery on this topic. With the goal of elucidating the molecular mechanisms of insulin resistance in obesity we compared differences in gene expression in intra-abdominal and subcutaneous adipose tissue from obese patients undergoing gastric bypass surgery.  Using Affymetrix GeneChip profiling we identified three gene sets (chemokine activity, chemokine receptor binding, g-protein couple receptor binding) that were upregulated in omental tissue from insulin resistant patients compared to BMI-matched insulin sensitive patients.  Differential expression of seven genes common to all three gene sets (CCL2, CCL3, CCL4, CCL8, CXCL2, CXCL10, IL8) was confirmed with RTqPCR and showed a clear association with insulin resistance.  The omental adipose tissue from insulin resistant subjects also demonstrated adipocyte hypertrophy and increased macrophage infiltration compared to omental tissue from insulin sensitive subjects.  These exciting results showing that elevated chemokine expression in omental adipose tissue is associated with insulin resistance and adipose inflammation in obese human subjects of equal BMI values raise several important questions: Which cell type is responsible for the chemokine production and secretion? Are other immune cells involved in this inflammatory process? Do serum levels of chemokines increase in the insulin resistant state? Does the inflammation in adipose tissue we observe cause insulin resistance in skeletal muscle in these patients? These questions can be solved utilizing a variety of techniques including fractionation of whole adipose tissue, immunohistochemistry, and serum analysis using Cytometric Bead Analysis. Rotation students will be able to pursue studies of this type in our laboratory.

Office: Suite 100
Phone: 508-856-2254
Fax: 508-856-1617
E-mail: Michael.Czech@umassmed.edu
Keywords: Signal Transduction, Cell Biology, Biochemistry

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Postdoctoral Position Available A postdoctoral position is available to study in this laboratory. Contact Dr. Czech for additional details.