Islet Neogenesis Associated Protein (INGAP) induces the differentiation of an adult human pancreatic ductal cell line into insulin-expressing cells through stepwise activation of key transcription factors for embryonic beta cell development.

Regeneration of ß-cells in diabetic patients is an important goal of diabetes research. Islet Neogenesis Associated Protein (INGAP) was discovered in the partially duct-obstructed hamster pancreas. Its bioactive fragment, pentadecapeptide 104-118 (INGAP-P), has been shown to reverse diabetes in animal models and to improve glucose homeostasis in patients with diabetes in clinical trials. Further development of INGAP as a therapy for diabetes requires identification of target cells in the pancreas and characterization of the mechanisms of action. We hypothesized that adult human pancreatic ductal cells retain morphogenetic plasticity and can be induced by INGAP to undergo endocrine differentiation. To test this hypothesis, we treated the normal human pancreatic ductal cell line (HPDE) with either INGAP-P or full-length recombinant protein (rINGAP) for short-term periods. Our data show that this single drug treatment induces both proliferation and transdifferentiation of HPDE cells, the latter being characterized by the rapid sequential activation of endocrine developmental transcription factors Pdx-1, Ngn3, NeuroD, IA-1, and MafA and subsequently the expression of insulin at both the mRNA and the protein levels. After 7 days, C-peptide was detected in the supernatant of INGAP-treated cells, reflecting their ability to secrete insulin. The magnitude of differentiation was enhanced by embedding the cells in Matrigel, which led to islet-like cluster formation. The islet-like clusters cells stained positive for nuclear Pdx-1 and Glut 2 proteins, and were expressing Insulin mRNA. These new data suggest that human adult pancreatic ductal cells retain morphogenetic plasticity and demonstrate that a short exposure to INGAP triggers their differentiation into insulin-expressing cells in vitro. In the context of the urgent search for a regenerative and/or cellular therapy for diabetes, these results make INGAP a promising therapeutic candidate.

Japonicone A antagonizes the activity of TNF-α by directly targeting this cytokine and selectively disrupting its interaction with TNF receptor-1.

Anti-TNF biologics are effective therapies for various inflammatory diseases. Unfortunately, their clinical use is associated with an increased risk of infections. Selectively inhibiting TNF receptor-1 (TNFR1)-mediated signaling while preserving TNFR2 signaling may reduce inflammation yet maintain host immune response to pathogens. However, few small molecules that selectively target the TNF/TNFR system have been discovered. In the present study, we identified Japonicone A (Jap A), a nature compound derived from Inula japonica Thunb, as a novel TNF-α antagonist, as it reduced the TNF-α-mediated cytotoxicity on L929 cells and inhibited the binding of (125)I-labeled TNF-α to L929 cell surface. Furthermore, Jap A could directly bind to TNF-α rather than TNFR1 as determined by surface plasmon resonance. More importantly, Jap A could effectively inhibit the binding of TNF-α to TNFR1, while displaying only marginal inhibitory effects on that to TNFR2. Jap A also could block TNFR1-mediated signaling as it inhibited TNF-α-induced NF-κB activation in 293 cells. In addition, Jap A suppressed TNF-α-induced expressions of adhesion molecules (ICAM-1, VCAM-1) and chemokine (MCP-1) in the endothelial cells by blocking TNF-α-triggered multiple signaling pathways. Data from in vivo experiments demonstrated that Jap A protected mice from acute hepatitis induced by TNF-α/d-galactosamine, but did not compromise host antiviral immunity in adenovirus-infected mice. These results indicate that Jap A can directly target TNF-α, selectively disrupt its interaction with TNFR1, and antagonize its pro-inflammatory activities without compromising host defense against virus, thus emphasizing the potential of Jap A as an interesting lead compound for development of new anti-inflammatory drugs.

Mechanisms of action of islet neogenesis-associated protein: comparison of the full-length recombinant protein and a bioactive peptide.

Islet neogenesis-associated protein (INGAP) was discovered in the partially duct-obstructed hamster pancreas as a factor inducing formation of new duct-associated islets. A bioactive portion of INGAP, INGAP(104-118) peptide (INGAP-P), has been shown to have neogenic and insulin-potentiating activity in numerous studies, including recent phase 2 clinical trials that demonstrated improved glucose homeostasis in both type 1 and type 2 diabetic patients. Aiming to improve INGAP-P efficacy and to understand its mechanism of action, we cloned the full-length protein (rINGAP) and compared the signaling events induced by the protein and the peptide in RIN-m5F cells that respond to INGAP with an increase in proliferation. Here, we show that, although both rINGAP and INGAP-P signal via the Ras/Raf/ERK pathway, rINGAP is at least 100 times more efficient on a molar basis than INGAP-P. For either ligand, ERK1/2 activation appears to be pertussis toxin sensitive, suggesting involvement of a G protein-coupled receptor(s). However, there are clear differences between the peptide and the protein in interactions with the cell surface and in the downstream signaling. We demonstrate that fluorescent-labeled rINGAP is characterized by clustering on the membrane and by slow internalization (≤5 h), whereas INGAP-P does not cluster and is internalized within minutes. Signaling by rINGAP appears to involve Src, in contrast to INGAP-P, which appears to activate Akt in addition to the Ras/Raf/ERK1/2 pathway. Thus our data suggest that interactions of INGAP with the cell surface are important to consider for further development of INGAP as a pharmacotherapy for diabetes.

Berberine induces dendritic cell apoptosis and has therapeutic potential for rheumatoid arthritis.

OBJECTIVE: To investigate the effects of berberine on dendritic cell (DC) apoptosis and its potential as a therapeutic agent in rheumatoid arthritis (RA). METHODS: Bone marrow (BM)-derived myeloid DCs (MDCs) and plasmacytoid DCs (PDCs) were generated by culturing BM cells with granulocyte-macrophage colony-stimulating factor/interleukin-4 or flt3L, respectively. Splenic DCs, T cells, and B cells were purified using a magnetic-activated cell sorting system. In vitro apoptosis was assessed by annexin V/propidium iodide or Hoechst 33258 staining. The in vivo effects of berberine were examined in mice with collagen-induced arthritis (CIA). Immune responses against type II collagen (CII) were determined by assaying serum antibody levels, lymphocyte proliferation, and cytokine production. The proportions of DCs and apoptosis of different immune cell subsets in spleens and lymph nodes were analyzed by flow cytometry and immunohistochemistry after subset-specific surface marker labeling and TUNEL staining. RESULTS: Exposure of MDCs to berberine during BM cell differentiation reduced cell recovery by inducing apoptosis. Sensitivity to berberine-induced apoptosis was acquired starting on day 3 of DC differentiation, and mature DCs were more sensitive to berberine than immature DCs. Murine peritoneal macrophages, RAW 264.7 cells, and Jurkat cells were insensitive to berberine-induced apoptosis. Splenic DCs were more sensitive to berberine than T and B cells. Susceptibility of PDCs to berberine-induced apoptosis was similar to that of MDCs. In mice with CIA, berberine treatment ameliorated arthritis, suppressed CII-specific immune responses, and selectively increased the incidence of apoptosis in DCs within spleens and lymph nodes. CONCLUSION: These findings show that berberine selectively induces apoptosis in DCs. Berberine may thus represent a novel therapeutic agent for RA.

Production and characterization of the recombinant Islet Neogenesis Associated Protein (rINGAP).

Islet Neogenesis Associated Protein (INGAP) is implicated in pancreatic islet neogenesis. INGAP peptide, a pentadecapeptide comprising amino acids 104-118, reverses diabetes in rodents and improves glucose homeostasis in patients with diabetes. The mechanism of INGAP action is unknown, but such studies would benefit from the availability of the full-length recombinant protein (rINGAP). Here we report the production of rINGAP from 293-SF cells following lentiviral transduction, and its characterization by MALDI-TOF and Q-TOF Mass Spectrometry, and HPLC. Importantly, we show that rINGAP exhibits 100x the bioactivity of INGAP peptide on a molar basis in an in vitro assay of human islet regeneration.