William Donelan

Irisin Stimulates Browning of White Adipocytes Through Mitogen-Activated Protein Kinase p38 MAP Kinase and ERK MAP Kinase Signaling

The number and activity of brown adipocytes are linked to the ability of mammals to resist body fat accumulation. In some conditions, certain white adipose tissue (WAT) depots are readily convertible to a ‘‘brown-like’’ state, which is associated with weight loss. Irisin, a newly identified hormone, is secreted by skeletal muscles into circulation and promotes WAT “browning” with unknown mechanisms. In the current study, we demonstrated in mice that recombinant irisin decreased the body weight and improved glucose homeostasis. We further showed that irisin upregulated uncoupling protein-1 (UCP-1; a regulator of thermogenic capability of brown fat) expression. This effect was possibly mediated by irisin-induced phosphorylation of the p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal–related kinase (ERK) signaling pathways. Inhibition of the p38 MAPK by SB203580 and ERK by U0126 abolished the upregulatory effect of irisin on UCP-1. In addition, irisin also promoted the expression of betatrophin, another newly identified hormone that promotes pancreatic β-cell proliferation and improves glucose tolerance. In summary, our data suggest that irisin can potentially prevent obesity and associated type 2 diabetes by stimulating expression of WAT browning-specific genes via the p38 MAPK and ERK pathways.

Pancreatic duodenal homeobox 1 protein is a novel beta-cell-specific autoantigen for type I diabetes.

Pancreatic duodenal homeobox 1 (Pdx1) protein is a key transcription factor involved in the regulation of insulin gene expression that is expressed at high levels in the β-cells of the pancreatic islets. We asked whether Pdx1 is a target of anti-islet autoimmunity in type I diabetes (T1D). Pdx1 autoantibodies (PAAs) were detected in non-obese diabetic (NOD) mice using ELISA, western blotting, and radioimmunoprecipitation of [35S]-labeled insulinoma cell line-derived Pdx1 protein. PAAs were detected as early as at 5 weeks of age, and generally peaked before the onset of clinically overt diabetes in diabetes-prone female NOD mice. Levels declined substantially after the onset of diabetes. PAAs were not detected in the sera of NOD–scid, C57BL/6, or BALB/c mice. The titers of PAAs in NOD mouse sera were as high as 1/93 750 by ELISA. The fine specificity of PAAs was determined by western blotting using a series of truncated recombinant Pdx1 proteins. The immunodominant epitopes were located to the C-terminus of the Pdx1 (p200–283) in NOD mice. PAAs also were detected in sera from human T1D patients, but the major epitopes were localized to amino acids 159–200 as well as the same region (p200–283) recognized by PAAs from NOD mice. Using [3H]thymidine incorporation, the p83 fragment of Pdx1 specifically stimulated proliferation of splenic T cells from recent-onset diabetic NOD mice. The presence of PAAs in prediabetic NOD mice and human T1D patients, and Pdx1-specific T-cell proliferation in NOD mice provide a strong rationale for further investigation of the pathogenic role of immune responses against Pdx1 in T1D.

A Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK)-dependent Transcriptional Program Controls Activation of the Early Growth Response 1 (EGR1) Gene during Amino Acid Limitation

Background: Increased transcription by mammalian amino acid response (AAR) has been linked primarily to the GCN2/eIF2/ATF4 pathway. Results: Some cells contain a GCN2- and ATF4-independent AAR pathway that is MEK-dependent. Conclusion: A novel MEK pathway activates a specific subset of AA-responsive genes. Significance: The mammalian AAR network is more complex than previously thought, extending beyond the GCN2/eIF2/ATF4 pathway. Amino acid (AA) limitation in mammalian cells triggers a collection of signaling cascades jointly referred to as the AA response (AAR). In human HepG2 hepatocellular carcinoma, the early growth response 1 (EGR1) gene was induced by either AA deprivation or endoplasmic reticulum stress. AAR-dependent EGR1 activation was discovered to be independent of the well characterized GCN2-ATF4 pathway and instead dependent on MEK-ERK signaling, one of the MAPK pathways. ChIP showed that constitutively bound ELK1 at the EGR1 proximal promoter region was phosphorylated after AAR activation. Increased p-ELK1 binding was associated with increased de novo recruitment of RNA polymerase II to the EGR1 promoter. EGR1 transcription was not induced in HEK293T cells lacking endogenous MEK activity, but overexpression of exogenous constitutively active MEK in HEK293T cells resulted in increased basal and AAR-induced EGR1 expression. ChIP analysis of the human vascular endothelial growth factor A (VEGF-A) gene, a known EGR1-responsive gene, revealed moderate increases in AAR-induced EGR1 binding within the proximal promoter and highly inducible binding to a site within the first intron. Collectively, these data document a novel AA-activated MEK-ERK-ELK1 signaling mechanism.

Angiopoietin-like protein 8 (betatrophin) is a stress-response protein that down-regulates expression of adipocyte triglyceride lipase

AIM Atypical angiopoietin-like 8 (ANGPTL8), also known as betatrophin, is known to regulate lipid metabolism. However, its mechanism of action remains elusive. METHODS HepG2, 3T3-L1, and NIT-1 cells were cultured in amino acid-complete MEM or histidine-free MEM to detect ANGPTL8 expression. The three cell types were treated with or without recombinant ANGPTL8 to investigate its role in lipid metabolism. Hydrodynamic tail vein gene delivery was also used to examine the role of ANGPTL8 in mice. RESULTS ANGPTL8 is significantly up-regulated in amino acid-deprived cultured cells in vitro. The activation of ANGPTL8 gene transcription was mediated through the RAS/c-RAF/MAPK signaling pathway rather than the general GCN2/ATF4 pathways. ANGPTL8 activated the ERK signal transduction pathway in hepatocytes, adipocytes, and pancreatic β-cells, up-regulating early growth response transcription factor (Egr1) and down-regulating adipose triglyceride lipase (ATGL). CONCLUSION ANGPTL8 is a stress-response protein that regulates fat metabolism by suppressing ATGL expression, revealing a mechanistic connection between ANGPTL8 and lipid homeostasis in mammalian cells.

MAPK signaling triggers transcriptional induction of cFOS during amino acid limitation of HepG2 cells.

Amino acid (AA) deprivation in mammalian cells activates a collection of signaling cascades known as the AA response (AAR), which is characterized by transcriptional induction of stress-related genes, including FBJ murine osteosarcoma viral oncogene homolog (cFOS). The present study established that the signaling mechanism underlying the AA-dependent transcriptional regulation of the cFOS gene in HepG2 human hepatocellular carcinoma cells is independent of the classic GCN2-eIF2-ATF4 pathway. Instead, a RAS-RAF-MEK-ERK cascade mediates AAR signaling to the cFOS gene. Increased cFOS transcription is observed from 4-24 h after AAR-activation, exhibiting little or no overlap with the rapid and transient increase triggered by the well-known serum response. Furthermore, serum is not required for the AA-responsiveness of the cFOS gene and no phosphorylation of promoter-bound serum response factor (SRF) is observed. The ERK-phosphorylated transcription factor E-twenty six-like (p-ELK1) is increased in its association with the cFOS promoter after activation of the AAR. This research identified cFOS as a target of the AAR and further highlights the importance of AA-responsive MAPK signaling in HepG2 cells.

Distinct Regulation of Hepatic Nuclear Factor 1α by NKX6.1 in Pancreatic Beta Cells

Hepatic nuclear factor 1α (HNF1α) is a key regulator of development and function in pancreatic beta cells and is specifically involved in regulation of glycolysis and glucose-stimulated insulin secretion. Abnormal expression of HNF1α leads to development of MODY3 (maturity-onset diabetes of the young 3). We report that NK6 homeodomain 1 (NKX6.1) binds to a cis-regulatory element in the HNF1α promoter and is a major regulator of this gene in beta cells. We identified an NKX6.1 recognition sequence in the distal region of the HNF1α promoter and demonstrated specific binding of NKX6.1 in beta cells by electrophoretic mobility shift and chromatin immunoprecipitation assays. Site-directed mutagenesis of the NKX6.1 core-binding sequence eliminated NKX6.1-mediated activation and substantially decreased activity of the HNF1α promoter in beta cells. Overexpression or small interfering RNA-mediated knockdown of the Nkx6.1 gene resulted in increased or diminished HNF1α gene expression, respectively, in beta cells. We conclude that NKX6.1 is a novel regulator of HNF1α in pancreatic beta cells. This novel regulatory mechanism for HNF1α in beta cells may provide new molecular targets for the diagnosis of MODY3.

Expression, purification, and characterization of recombinant human pancreatic duodenal homeobox-1 protein in Pichia pastoris

Pancreatic duodenal hemeobox-1 (PDX1) is essential for the development of the embryonic pancreas and plays a key role in pancreatic beta-cell differentiation, maturation, regeneration, and maintenance of normal pancreatic beta-cell insulin-producing function. Purified recombinant PDX1 (rPDX1) may be a useful tool for many research and clinical applications, however, using the Escherichia coli expression system has several drawbacks for producing quality PDX1 protein. To explore the yeast expression system for generating rPDX1 protein, the cDNA coding for the full-length human PDX1 gene was cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and western blotting analysis of culture medium from methanol-induced expression yeast clones demonstrated that the rPDX1 was secreted into the culture medium, had a molecular weight by SDS-PAGE of 50kDa, and was glycosylated. The predicted size of the mature unmodified PDX1 polypeptide is 31kDa, suggesting that eukaryotic post-translational modifications are the result of the increased molecular weight. The recombinant protein was purified to greater than 95% purity using a combined ammonium sulfate precipitation with heparin-agarose chromatography. Finally, 120mug of the protein was obtained in high purity from 1L of the culture supernatant. Bioactivity of the rPDX1 was confirmed by the ability to penetrate cell membranes and activation of an insulin-luciferase reporter gene. Our results suggest that the P. pastoris expression system can be used to produce a fully functional human rPDX1 for both research and clinical application.

Expression of recombinant human IL-4 in Pichia pastoris and relationship between its glycosylation and biological activity.

Secretory human interleukin 4 (hIL4) is an N-glycosylated pleiotropic cytokine. It is unknown if these N-linked glycans are required and essential for hIL4 protein stability, expression, secretion, and activity in vivo, and hIL4 expressed from Pichia pastoris yeast has not been tested to date. In this study, we successfully expressed human hIL4 in P. pastoris, the methylotrophic yeast, with a yield of 15.0mg/L. Using the site-directed mutagenesis technique, we made two mutant hIL4 cDNA clones (N38A and N105L) and subsequently expressed them in P. pastoris to analyze the relevant function of each N-glycosylation site on hIL4. Our results demonstrate that the glycosylation only occurs at position Asn38, but not Asn105. The glycosylated form of hIL4 unexpectedly has lower biological activity and lower stability when compared to its non-glycosylated form. The implications of this are discussed.

The reprogrammed pancreatic progenitor-like intermediate state of hepatic cells is more susceptible to pancreatic beta cell differentiation

Summary Induced pluripotent stem cells (iPSCs) hold great promise for cell therapy. However, their low efficiency of lineage-specific differentiation and tumorigenesis severely hinder clinical translation. We hypothesized that reprogramming of somatic cells into lineage-specific progenitor cells might allow for large-scale expansion, avoiding the tumorigenesis inherent with iPSCs and simultaneously facilitating lineage-specific differentiation. Here we aimed at reprogramming rat hepatic WB cells, using four Yamanaka factors, into pancreatic progenitor cells (PPCs) or intermediate (IM) cells that have characteristics of PPCs. IM clones were selected based on their specific morphology and alkaline phosphatase activity and stably passaged under defined culture conditions. IM cells did not have iPSC properties, could be stably expanded in large quantity, and expressed all 14 genes that are used to define the PPC developmental stage. Directed differentiation of IM and WB cells by Pdx1-Ngn3-MafA (PNM) into pancreatic beta-like cells revealed that the IM cells are more susceptible to directed beta cell differentiation because of their open chromatin configuration, as demonstrated by expression of key pancreatic beta cell genes, secretion of insulin in response to glucose stimulation, and easy access to exogenous PNM proteins at the rat insulin 1 and Pdx1 promoters. This notion that IM cells are superior to their parental cells is further supported by the epigenetic demonstration of accessibility of Pdx1 and insulin 1 promoters. In conclusion, we have developed a strategy to derive and expand PPC cells from hepatic WB cells using conventional cell reprogramming. This proof-of-principal study may offer a novel, safe and effective way to generate autologous pancreatic beta cells for cell therapy of diabetes.

Autoantigen-Specific Immunotherapy

The incidence of type 1 diabetes (T1D) is increasing dramatically and new treatment modalities are needed urgently. Arising from autoimmune destruction of the pancreatic ┚cells, T1D manifests itself when less than 10-20% of functional ┚-cells remain in the islets and is characterized as a disorder of glucose regulation due to the insufficient production of insulin. Insulin is a hormone secreted by pancreatic islet ┚-cells and its main function is to move blood sugar into cells where it is stored and later used for energy. In T1D, the ┚-cells are destroyed by the patients’ immune cells leading to limited or no insulin production. Without enough insulin, glucose accumulates in the bloodstream instead of entering the cells, causing symptoms of hyperglycemia. Chronic hyperglycemia can lead to many serious complications. Insulin replacement therapy is the current standard for treatment involving injection of recombinant insulin. Normal pancreatic insulin secretion is exquisitely sensitive to the minute-to-minute changes in blood glucose and glucose-stimulated insulin secretion (GSIS) and cannot be mimicked precisely by exogenous insulin injections. Thus, while insulin treatment successfully prolongs the life of affected individuals, it fails to prevent some of the serious complications of T1D that adversely affect quality of life and ultimately lead to significant morbidity and mortality. An alternative promising therapy is islet cell transplantation, but this suffers from several drawbacks including limited pancreatic islet tissue from cadaveric donors, the requirement for lifelong immunosuppression, and the increased risk of infection due to non-specific suppression of the immune system. To cure T1D, researchers are pursuing combined immunological and biological strategies for restoring ┚-cell mass. Strategies for increasing ┚-cell mass include promoting endogenous pancreatic ┚-cell regeneration, reprogramming non-pancreatic cells into insulin-producing cells (IPCs), and generating unlimited autologous pancreatic ┚-cells from induced pluripotent stem (iPS) cells of the T1D patients. At the same time, there is considerable interest in approaches for modulating and suppressing autoimmunity to pancreatic ┚-cells and surrogates of IPCs. An individual’s pancreatic ┚-cell mass is tightly regulated according to insulin demand, reflecting a balance between the rate of ┚-cell replication, regeneration/reprogramming, and the rate of ┚-cell apoptosis. Research efforts are focusing on how best to expand, reprogram, or generate ┚-cells or their surrogates from pancreatic stem/precursor cells, non-pancreatic adult/stem cells, or the iPS cells of T1D patients for ┚-cell replacement therapy. However, without preventing ongoing autoimmune