Initiation of NALT Organogenesis Is Independent of the IL-7R, LTβR, and NIK Signaling Pathways but Requires the Id2 Gene and CD3−CD4+CD45+ Cells

AbstractInitiation of nasopharyngeal-associated lymphoid tissue (NALT) development is independent of the programmed cytokine cascade necessary for the formation of Peyer's patches (PP) and peripheral lymph nodes (PLN), a cytokine cascade which consists of IL-7R, LTα1β2/LTβR, and NIK. However, the subsequent organization of NALT seems to be controlled by these cytokine signaling cascades since the maturation of NALT structure is generally incomplete in those cytokine cascade-deficient mice. NALT as well as PP and PLN are completely absent in Id2−/− mice. NALT organogenesis is initiated following the adoptive transfer of CD3−CD4+CD45+ cells into Id2−/− mice, constituting direct evidence that CD3−CD4+CD45+ inducer cells can provide an IL-7R-, LTα1β2/LTβR-, and NIK-independent tissue organogenesis pathway for secondary lymphoid tissue development

The efficacy of incretin therapy in patients with type 2 diabetes undergoing hemodialysis

BACKGROUND: Although incretin therapy is clinically available in patients with type 2 diabetes undergoing hemodialysis, no study has yet examined whether incretin therapy is capable of maintaining glycemic control in this group of patients when switched from insulin therapy. In this study, we examined the efficacy of incretin therapy in patients with insulin-treated type 2 diabetes undergoing hemodialysis. METHODS: Ten type 2 diabetic patients undergoing hemodialysis received daily 0.3 mg liraglutide, 50 mg vildagliptin, and 6.25 mg alogliptin switched from insulin therapy on both the day of hemodialysis and the non-hemodialysis day. Blood glucose level was monitored by continuous glucose monitoring. After blood glucose control by insulin, patients were treated with three types of incretin therapy in a randomized crossover manner, with continuous glucose monitoring performed for each treatment. RESULTS: During treatment with incretin therapies, severe hyperglycemia and ketosis were not observed in any patients. Maximum blood glucose and mean blood glucose on the day of hemodialysis were significantly lower after treatment with liraglutide compared with treatment with alogliptin (p < 0.05), but not with vildagliptin. The standard deviation value, a marker of glucose fluctuation, on the non-hemodialysis day was significantly lower after treatment with liraglutide compared with treatment with insulin and alogliptin (p < 0.05), but not with vildagliptin. Furthermore, the duration of hyperglycemia was significantly shorter after treatment with liraglutide on both the hemodialysis and non-hemodialysis days compared with treatment with alogliptin (p < 0.05), but not with vildagliptin. CONCLUSIONS: The data presented here suggest that patients with type 2 diabetes undergoing hemodialysis and insulin therapy could be treated with incretin therapy in some cases

Combined Treatment with Exendin-4 and Metformin Attenuates Prostate Cancer Growth

Recently, the pleiotropic benefits of incretin-based therapy have been reported. We have previously reported that Exendin–4, a glucagon-like peptide–1 (GLP–1) receptor agonist, attenuates prostate cancer growth. Metformin is known for its anti-cancer effect. Here, we examined the anti-cancer effect of Exendin–4 and metformin using a prostate cancer model. experiments, LNCaP cells were transplanted subcutaneously into the flank region of athymic mice, which were then treated with Exendin–4 and/or metformin. TUNEL assay and immunohistochemistry were performed on tumors., Exendin–4 and metformin significantly decreased tumor size, and further significant tumor size reduction was observed after combined treatment. Immunohistochemistry on tumors revealed that the P504S and Ki67 expression decreased by Exendin–4 and/or metformin, and that metformin increased phospho-AMPK expression and the apoptotic cell number.These data suggest that Exendin–4 and metformin attenuated prostate cancer growth by inhibiting proliferation, and that metformin inhibited proliferation by inducing apoptosis. Combined treatment with Exendin–4 and metformin attenuated prostate cancer growth more than separate treatments

Combined treatment with DPP-4 inhibitor linagliptin and SGLT2 inhibitor empagliflozin attenuates neointima formation after vascular injury in diabetic mice

Incretin therapy has emerged as one of the most popular medications for type 2 diabetes. We have previously reported that the dipeptidyl peptidase-4 (DPP-4) inhibitor linagliptin attenuates neointima formation after vascular injury in non-diabetic mice. In the present study, we examined whether combined treatment with linagliptin and the sodium glucose cotransporter 2 (SGLT2) inhibitor empagliflozin attenuates neointima formation in diabetic mice after vascular injury. Diabetic db/db mice were treated with 3 mg/kg/day linagliptin and/or 30 mg/kg/day empagliflozin from 5 to 10 weeks of age. Body weight was significantly decreased by empagliflozin and the combined treatment. Blood glucose levels and glucose tolerance test results were significantly improved by empagliflozin and the combined treatment, but not by linagliptin. An insulin tolerance test suggested that linagliptin and empagliflozin did not improve insulin sensitivity. In a model of guidewire-induced femoral artery injury in diabetic mice, neointima formation was significantly decreased in mice subjected to combined treatment. In an in vitro assay using rat aortic smooth muscle cells (RASMC), 100, 500, or 1000 nM empagliflozin significantly decreased the RASMC number in a dose-dependent manner. A further significant reduction in RASMC proliferation was observed after combined treatment with 10 nM linagliptin and 100 nM empagliflozin. These data suggest that combined treatment with the DPP-4 inhibitor linagliptin and SGLT2 inhibitor empagliflozin attenuates neointima formation after vascular injury in diabetic mice in vivo and smooth muscle cell proliferation in vitro. Keywords: DPP-4I, SGLT2I, Neointima formation, VSMC proliferatio

Metformin induces apoptosis and attenuates cell proliferation in prostate cancer cells via AMPK activation.

<p>(A) LNCaP cells were plated on glass coverslips in Lab-Tek 2 well Chamber Slides. After incubation with 0.1 or 10 mM metformin for 24 h, or 1 unit/100 μL RQ1 DNase (positive control) for 10 min, apoptotic cells were detected with TUNEL staining. Images shown are representative of three independent experiments. (B) LNCaP cells maintained in media with 10% FBS were stimulated with saline (control), Ex–4 (10 nM), metformin (0.1 mM) or both Ex–4 (10 nM) and metformin (0.1 mM) for 24 h. Cell lysates were harvested and subjected to western blotting to assess phosphorylated AMPK and AMPK expression. Phosphorylated AMPK/AMPK protein levels (C) and phosphorylated AMPK/GAPDH protein levels (D) were quantified by densitometry. Data were calculated from three independent experiments and are shown as a ratio of the control (*<i>P</i> < 0.05, **<i>P</i> < 0.01 vs. control, ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.05 vs. Ex–4). (E) LNCaP cells were maintained in media supplemented with 10% FBS with Compound C (0.1μM; CC) or control vehicle (NT), and with or without Met (0.1mM). After 0, 24, 48 and 72h, the cells were harvested, and cell proliferation was analyzed by cell counting using a hemocytometer. Unpaired <i>t</i>-tests were performed to calculate statistical significance (*<i>P</i> < 0.05, **<i>P</i> < 0.01 vs. NT-control, ^#<i>P</i> < 0.05 vs. NT-Met 0.1 mM). (F) LNCaP cells were maintained in media supplemented with 10% FBS after transfection of 10 nM control siRNA (siCT) or siRNA for AMPKα1/2 (siAMPK) with or without Met (0.1 mM). After 0, 24, 48 and 72h, the cells were harvested, and cell proliferation was analyzed by cell counting using a hemocytometer. Unpaired <i>t</i>-tests were performed to calculate statistical significance (**<i>P</i> < 0.01 vs. siCT-control, ^##<i>P</i> < 0.01 vs. siCT-Met 0.1 mM). (G) LNCaP cells were plated at a density of 5000 cells/well in 96-well plates in media supplemented with 10% FBS and incubated with Compound C (0.1μM) (CC) or control vehicle (NT), and with or without Met (0.1mM) for 24 h. BrdU solution was added during the last 2 h, and cells were harvested for measurement of DNA synthesis using a microplate reader at 450–620 nm. Mean data are expressed as a ratio of the control cell proliferation. Unpaired <i>t</i>-tests were performed to calculate statistical significance (**<i>P</i> < 0.01 vs. Met (-) and Compound C (-), ^#<i>P</i> < 0.05 vs. Met (+) and Compound C (-)). (H) LNCaP cells were plated at a density of 5000 cells/well in 96-well plates in media supplemented with 10% FBS after transfection of 10nM control siRNA (siCT) or siRNA for AMPKɑ1/2 (siAMPK) with or without Met (0.1mM) for 24h. BrdU solution was added during the last 2 h, and cells were harvested for measurement of DNA synthesis using a microplate reader at 450–620 nm. Mean data are expressed as a ratio of the control cell proliferation. Unpaired <i>t</i>-tests were performed to calculate statistical significance (**<i>P</i> < 0.01 vs. Met (-) and siControl, ^##<i>P</i> < 0.01 vs. Met (+) and siControl).</p

Combined treatment with Exendin–4 and metformin additively attenuates prostate cancer growth <i>in vivo</i>.

<p>(A) Athymic CAnN.Cg-<i>Foxn1nu</i>/CrlCrlj mice (aged 6 weeks) were transplanted with 1×10⁶ LNCaP cells (passage 4–8) and treated with vehicle (n = 10), Ex–4 (300 pmol kg body weight⁻¹ day⁻¹; n = 9), metformin (met; 750 mg kg⁻¹ day⁻¹; n = 10), or a combined treatment of Ex–4 and metformin (n = 10). Tumors were imaged at 12 weeks of age. (B) Tumor volume was calculated with the modified ellipsoid formula. Unpaired <i>t-</i>tests were performed to calculate statistical significance (**<i>P</i> < 0.01 vs. control). In the mice without a mounding tumor, tumor volume was calculated as “zero”. (C) Tumor weight was measured on scales. Unpaired <i>t</i>-tests were performed to calculate statistical significance (**<i>P</i> < 0.01 vs. control; ^#<i>P</i> < 0.05 vs. Ex–4). In the mice without a mounding tumor, tumor weight was calculated as “zero”.</p