The Role of CD38 in the Pathogenesis of Cardiorenal Metabolic Disease and Aging, an Approach from Basic Research.

Aging is a major risk factor for the leading causes of mortality, and the incidence of age-related diseases including cardiovascular disease, kidney disease and metabolic disease increases with age. NAD+ is a classic coenzyme that exists in all species, and that plays a crucial role in oxidation-reduction reactions. It is also involved in the regulation of many cellular functions including inflammation, oxidative stress and differentiation. NAD+ declines with aging in various organs, and the reduction in NAD+ is possibly involved in the development of age-related cellular dysfunction in cardiorenal metabolic organs through the accumulation of inflammation and oxidative stress. Levels of NAD+ are regulated by the balance between its synthesis and degradation. CD38 is the main NAD+-degrading enzyme, and CD38 is activated in response to inflammation with aging, which is associated with the reduction in NAD+ levels. In this review, focusing on CD38, we discuss the role of CD38 in aging and the pathogenesis of age-related diseases, including cardiorenal metabolic disease.

Adenosine/adenosine type 1 receptor signaling pathway did not play dominant roles on the influence of sodium-glucose cotransporter 2 inhibitor in the kidney of bovine serum albumin-overloaded streptozotocin-induced diabetic mice.

AIMS/INTRODUCTION: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have been shown to display excellent renoprotective effects in diabetic kidney disease with macroalbuminuria/proteinuria. Regarding the renoprotective mechanism of SGLT2i, a sophisticated hypothesis was made by explaining the suppression of glomerular hypertension/hyperfiltration through the adenosine/adenosine type 1 receptor (A1R) signaling-mediated restoration of the tubuloglomerular feedback mechanism; however, how such A1R signaling is relevant for renoprotection by SGLT2i in diabetic kidney disease with proteinuria has not been elucidated. MATERIALS AND METHODS: Streptozotocin-induced diabetic CD-1 mice were injected with bovine serum albumin (BSA) and treated with SGLT2i in the presence/absence of A1R inhibitor administration. RESULTS: We found that the influences of SGLT2i are essentially independent of the activation of A1R signaling in the kidney of BSA-overloaded streptozotocin-induced diabetic mice. BSA-overloaded diabetic mice showed the trend of kidney damage with higher glomerular filtration rate (GFR) and the significant induction of fibrogenic genes, such as transforming growth factor-ß2 and collagen type III. SGLT2i TA-1887 suppressed diabetes-induced GFR in BSA-overloaded diabetic mice was associated with the significant suppression of transforming growth factor-ß2 and collagen type III; A1R-specific inhibitor 8-cyclopentyl-1,3-dipropylxanthine did not cancel the effects of TA-1887 on either GFR or associated gene levels. Both TA-1887 and 8-cyclopentyl-1,3-dipropylxanthine-treated BSA-overloaded diabetic mice showed suppressed glycated hemoglobin levels associated with the increased food intake. When analyzing the association among histological evaluation, GFR and potential fibrogenic gene levels, each group of mice showed distinct correlation patterns. CONCLUSIONS: A1R signaling activation was not the dominant mechanism on the influence of SGLT2i in the kidney of BSA-overloaded diabetic mice.

Dapagliflozin Restores Impaired Autophagy and Suppresses Inflammation in High Glucose-Treated HK-2 Cells.

Sodium-glucose cotransporter2 (SGLT2) inhibitors have a reno-protective effect in diabetic kidney disease. However, the detailed mechanism remains unclear. In this study, human proximal tubular cells (HK-2) were cultured in 5 mM glucose and 25 mM mannitol (control), 30 mM glucose (high glucose: HG), or HG and SGLT2 inhibitor, dapagliflozin-containing medium for 48 h. The autophagic flux was decreased, accompanied by the increased phosphorylation of S6 kinase ribosomal protein (p-S6RP) and the reduced phosphorylation of AMP-activated kinase (p-AMPK) expression in a HG condition. Compared to those of the control, dapagliflozin and SGLT2 knockdown ameliorated the HG-induced alterations of p-S6RP, p-AMPK, and autophagic flux. In addition, HG increased the nuclear translocation of nuclear factor-κB p65 (NF-κB) p65 and the cytoplasmic nucleotide-binding oligomerization domain-like receptor 3 (NLRP3), mature interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factorα (TNFα) expression. Dapagliflozin, SGLT2 knockdown, and NF-κB p65 knockdown reduced the extent of these HG-induced inflammatory alterations. The inhibitory effect of dapagliflozin on the increase in the HG-induced nuclear translocation of NF-κB p65 was abrogated by knocking down AMPK. These data indicated that in diabetic renal proximal tubular cells, dapagliflozin ameliorates: (1) HG-induced autophagic flux reduction, via increased AMPK activity and mTOR suppression; and (2) inflammatory alterations due to NF-κB pathway suppression.

Effect of Methionine Restriction on Aging: Its Relationship to Oxidative Stress.

Enhanced oxidative stress is closely related to aging and impaired metabolic health and is influenced by diet-derived nutrients and energy. Recent studies have shown that methionine restriction (MetR) is related to longevity and metabolic health in organisms from yeast to rodents. The effect of MetR on lifespan extension and metabolic health is mediated partially through a reduction in oxidative stress. Methionine metabolism is involved in the supply of methyl donors such as S-adenosyl-methionine (SAM), glutathione synthesis and polyamine metabolism. SAM, a methionine metabolite, activates mechanistic target of rapamycin complex 1 and suppresses autophagy; therefore, MetR can induce autophagy. In the process of glutathione synthesis in methionine metabolism, hydrogen sulfide (H2S) is produced through cystathionine-ß-synthase and cystathionine-γ-lyase; however, MetR can induce increased H2S production through this pathway. Similarly, MetR can increase the production of polyamines such as spermidine, which are involved in autophagy. In addition, MetR decreases oxidative stress by inhibiting reactive oxygen species production in mitochondria. Thus, MetR can attenuate oxidative stress through multiple mechanisms, consequently associating with lifespan extension and metabolic health. In this review, we summarize the current understanding of the effects of MetR on lifespan extension and metabolic health, focusing on the reduction in oxidative stress.

Sodium-glucose cotransporter 2 inhibitors in type 2 diabetes patients with renal function impairment slow the annual renal function decline, in a real clinical practice.

AIMS/INTRODUCTION: The aim of this study was to elucidate whether sodium-glucose cotransporter 2 inhibitors (SGLT2is) treatment has any renoprotective effect for type 2 diabetes mellitus patients with an estimated glomerular filtration rate (eGFR) of <60 mL/min/1.73 m2 in clinical practice. MATERIALS AND METHODS: We evaluated the annual eGFR slope in 85 type 2 diabetes mellitus patients with renal impairment, treated with SGLT2is ≥2 years. Each patient's eGFR was <60 mL/min/1.73 m2 at the start of SGLT2is therapy. The calculation of the annual change in eGFR for each patient was obtained by acquired eGFR data before and after 2 years of the initial SGLT2is administration, followed by analysis of the changes in the mean eGFR slope. RESULTS: The participants' mean age was 72.0 ± 9.4 years, and the mean eGFR was 47.1 ± 9.7 mL/min/1.73 m2 at the start of additional treatment with SGLT2is. The mean annual eGFR slope after SGLT2is administration (-0.11 ± 0.20 mL/min/1.73 m2 /year) was significantly slower than before SGLT2is administration (-2.93 ± 0.59 mL/min/1.73 m2 /year; P < 0.0001). Additionally, SGLT2is treatment slowed the annual decline of eGFR, independent of the levels of both the initial eGFR and albuminuria levels before SGLT2is therapy was started. In the patient groups who showed an annual eGFR decline of ≥3 and 1-3 mL/min/1.73 m2 , there was a significant slowing of the decline after SGLT2is therapy, compared with before the treatment (P < 0.001, respectively). CONCLUSIONS: SGLT2is administration slows the decline observed in the annual renal function in type 2 diabetes mellitus patients with eGFR of <60 mL/min/1.73 m2 in clinical practice.

Metformin Mitigates DPP-4 Inhibitor-Induced Breast Cancer Metastasis via Suppression of mTOR Signaling.

The biological influence of antidiabetic drugs on cancer cells and diabetic cancer patients has not yet been completely elucidated. We reported that a dipeptidyl peptidase (DPP)-4 inhibitor accelerates mammary cancer metastasis by inducing epithelial-mesenchymal transition (EMT) through the CXCL12/CXCR4/mTOR axis. Metformin has been shown to inhibit the mTOR signaling pathway. In this study, we investigated whether metformin mitigates breast cancer metastasis induced by a DPP-4 inhibitor via suppression of mTOR signaling. In cultured mouse mammary and human breast cancer cells, metformin suppressed DPP-4 inhibitor KR62436 (KR)-induced EMT and cell migration via suppression of the mTOR pathway associated with AMPK activation. For the in vivo study, metformin intervention was performed in an allograft 4T1 breast cancer model mouse with or without KR. We also analyzed mice transplanted with shRNA-mediated DPP-4 knockdown 4T1 cells. Treatment with metformin inhibited the lung metastasis of DPP-4-deficient 4T1 mammary tumor cells generated by either KR administration or DPP-4 knockdown. Immunostaining of primary tumors indicated that DPP-4 suppression promoted the expression of EMT-inducing transcription factor Snail through activation of the CXCR4-mediated mTOR/p70S6K pathway in an allograft breast cancer model; metformin abolished this alteration. Metformin treatment did not alter DPP-4-deficiency-induced expression of CXCL12 in either plasma or primary tumors. Our findings suggest that metformin may serve as an antimetastatic agent by mitigating the undesirable effects of DPP-4 inhibitors in patients with certain cancers. IMPLICATIONS: Metformin could combat the detrimental effects of DPP-4 inhibitor on breast cancer metastasis via mTOR suppression, suggesting the potential clinical relevance. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/1/61/F1.large.jpg.

Endothelial FGFR1 (Fibroblast Growth Factor Receptor 1) Deficiency Contributes Differential Fibrogenic Effects in Kidney and Heart of Diabetic Mice.

Endothelial-to-mesenchymal transition (EndMT) has been shown to contribute to organ fibrogenesis. We have reported that N-acetyl-seryl-aspartyl- lysyl-proline (AcSDKP) restored levels of diabetes mellitus-suppressed FGFR1 (fibroblast growth factor receptor 1), the endothelial receptor essential for combating EndMT. However, the molecular regulation and biological/pathological significance of the AcSDKP-FGFR1 relationship has not been elucidated yet. Here, we demonstrated that endothelial FGFR1 deficiency led to AcSDKP-resistant EndMT and severe fibrosis associated with EndMT-stimulated fibrogenic programming in neighboring cells. Diabetes mellitus induced severe kidney fibrosis in endothelial FGFR1-deficient mice (FGFR1fl/fl; VE-cadherin-Cre: FGFR1EKO) but not in control mice (FGFR1fl/fl); AcSDKP completely or partially suppressed kidney fibrosis in control or FGFR1EKO mice. Severe fibrosis was also induced in hearts of diabetic FGFR1EKO mice; however, AcSDKP had no effect on heart fibrosis in FGFR1EKO mice. AcSDKP also had no effect on EndMT in either kidney or heart but partially suppressed epithelial-to-mesenchymal transition in kidneys of diabetic FGFR1EKO mice. The medium from FGFR1-deficient endothelial cells stimulated TGFß (transforming growth factor ß)/Smad-dependent epithelial-to-mesenchymal transition in cultured human proximal tubule epithelial cell line, AcSDKP inhibited such epithelial-to-mesenchymal transition. These data demonstrated that endothelial FGFR1 is essential as an antifibrotic core molecule as the target of AcSDKP.

Supplementation with Red Wine Extract Increases Insulin Sensitivity and Peripheral Blood Mononuclear Sirt1 Expression in Nondiabetic Humans.

The aim of this study was to investigate the effects of dietary supplementation with a nonalcoholic red wine extract (RWE), including resveratrol and polyphenols, on insulin sensitivity and Sirt1 expression in nondiabetic humans. The present study was a single-arm, open-label and prospective study. Twelve subjects received supplementation with RWE, including 19.2 mg resveratrol and 136 mg polyphenols, daily for 8 weeks. After 8 weeks, metabolic parameters, including glucose/lipid metabolism and inflammatory markers, were evaluated. mRNA expression of Sirt1 was evaluated in isolated peripheral blood mononuclear cells (PBMNCs). Additionally, Sirt1 and phosphorylated AMP-activated kinase (p-AMPK) expression were evaluated in cultured human monocytes (THP-1 cells). Supplementation with RWE for 8 weeks decreased the homeostasis model assessment for insulin resistance (HOMA-IR), which indicates an increase in insulin sensitivity. Serum low-density lipoprotein-cholesterol (LDL-C), triglyceride (TG) and interleukin-6 (IL-6) were significantly decreased by RWE supplementation for 8 weeks. Additionally, Sirt1 mRNA expression in isolated PBMNCs was significantly increased after 8 weeks of RWE supplementation. Moreover, the rate of increase in Sirt1 expression was positively correlated with the rate of change in HOMA-IR. The administration of RWE increased Sirt1 and p-AMPK expression in cultured THP-1 cells. Supplementation with RWE improved metabolism, such as insulin sensitivity, lipid profile and inflammation, in humans. Additionally, RWE supplementation induced an increase in Sirt1 expression in PBMNCs, which may be associated with an improvement in insulin sensitivity.

Mechanism of Activation of Mechanistic Target of Rapamycin Complex 1 by Methionine.

Nutrients are closely involved in the regulation of lifespan and metabolic health. Cellular activities, such as the regulation of metabolism, growth, and aging, are mediated by a network of nutrients and nutrient-sensing pathways. Among the nutrient-sensing pathways, the mechanistic target of rapamycin complex 1 (mTORC1) acts as the central regulator of cellular functions, which include autophagy. Autophagy plays a significant role in the removal of protein aggregates and damaged or excess organelles, including mitochondria, to maintain intracellular homeostasis, which is involved in lifespan extension and cardiometabolic health. Moreover, dietary methionine restriction may have a beneficial effect on lifespan extension and metabolic health. In contrast, methionine may activate mTORC1 and suppress autophagy. As the mechanism of methionine sensing on mTORC1, SAMTOR was identified as a sensor of S-adenosyl methionine (SAM), a metabolite of methionine, in the cytoplasm. Conversely, methionine may activate the mTORC1 signaling pathway through the activation of phosphatase 2A (PP2A) because of increased methylation in response to intracellular SAM levels. In this review, we summarized the recent findings regarding the mechanism via which methionine activates mTORC1.

Manganese Superoxide Dismutase Dysfunction and the Pathogenesis of Kidney Disease.

The mitochondria are a major source of reactive oxygen species (ROS). Superoxide anion (O2 •-) is produced by the process of oxidative phosphorylation associated with glucose, amino acid, and fatty acid metabolism, resulting in the production of adenosine triphosphate (ATP) in the mitochondria. Excess production of reactive oxidants in the mitochondria, including O2 •-, and its by-product, peroxynitrite (ONOO-), which is generated by a reaction between O2 •- with nitric oxide (NO•), alters cellular function via oxidative modification of proteins, lipids, and nucleic acids. Mitochondria maintain an antioxidant enzyme system that eliminates excess ROS; manganese superoxide dismutase (Mn-SOD) is one of the major components of this system, as it catalyzes the first step involved in scavenging ROS. Reduced expression and/or the activity of Mn-SOD results in diminished mitochondrial antioxidant capacity; this can impair the overall health of the cell by altering mitochondrial function and may lead to the development and progression of kidney disease. Targeted therapeutic agents may protect mitochondrial proteins, including Mn-SOD against oxidative stress-induced dysfunction, and this may consequently lead to the protection of renal function. Here, we describe the biological function and regulation of Mn-SOD and review the significance of mitochondrial oxidative stress concerning the pathogenesis of kidney diseases, including chronic kidney disease (CKD) and acute kidney injury (AKI), with a focus on Mn-SOD dysfunction.

Stromal cell-derived factor 1 (SDF1) attenuates platelet-derived growth factor-B (PDGF-B)-induced vascular remodeling for adipose tissue expansion in obesity.

Platelet-derived growth factor-B (PDGF-B) is a main factor to promote adipose tissue angiogenesis, which is responsible for the tissue expansion in obesity. In this process, PDGF-B induces the dissociation of pericytes from blood vessels; however, its regulatory mechanism remains unclear. In the present study, we found that stromal cell-derived factor 1 (SDF1) plays an essential role in this regulatory mechanism. SDF1 mRNA was increased in epididymal white adipose tissue (eWAT) of obese mice. Ex vivo pharmacological analyses using cultured adipose tissue demonstrated that physiological concentrations (1-100 pg/mL) of SDF1 inhibited the PDGF-B-induced pericyte dissociation from vessels via two cognate SDF1 receptors, CXCR4 and CXCR7. In contrast, higher concentrations (> 1 ng/mL) of SDF1 alone caused the dissociation of pericytes via CXCR4, and this effect disappeared in the cultured tissues from PDGF receptor ß (PDGFRß) knockout mice. To investigate the role of SDF1 in angiogenesis in vivo, the effects of anagliptin, an inhibitor of dipeptidyl peptidase 4 (DPP4) that degrades SDF1, were examined in mice fed a high-fat diet. Anagliptin increased the SDF1 levels in the serum and eWAT. These changes were associated with a reduction of pericyte dissociation and fat accumulation in eWAT. AMD3100, a CXCR4 antagonist, cancelled these anagliptin effects. In flow-cytometry analysis, anagliptin increased and decreased the PDGF-B expression in endothelial cells and macrophages, respectively, whereas anagliptin reduced the PDGFRß expression in pericytes of eWAT. These results suggest that SDF1 negatively regulates the adipose tissue angiogenesis in obesity by altering the reactivity of pericytes to PDGF-B.

CD38 inhibition by apigenin ameliorates mitochondrial oxidative stress through restoration of the intracellular NAD+/NADH ratio and Sirt3 activity in renal tubular cells in diabetic rats.

Mitochondrial oxidative stress is a significant contributor to the pathogenesis of diabetic kidney disease (DKD). We previously showed that mitochondrial oxidative stress in the kidneys of Zucker diabetic fatty rats is associated with a decreased intracellular NAD+/NADH ratio and NAD+-dependent deacetylase Sirt3 activity, and increased expression of the NAD+-degrading enzyme CD38. In this study, we used a CD38 inhibitor, apigenin, to investigate the role of CD38 in DKD. Apigenin significantly reduced renal injuries, including tubulointerstitial fibrosis, tubular cell damage, and pro-inflammatory gene expression in diabetic rats. In addition, apigenin down-regulated CD38 expression, and increased the intracellular NAD+/NADH ratio and Sirt3-mediated mitochondrial antioxidative enzyme activity in the kidneys of diabetic rats. In vitro, inhibition of CD38 activity by apigenin or CD38 knockdown increased the NAD+/NADH ratio and Sirt3 activity in renal proximal tubular HK-2 cells cultured under high-glucose conditions. Together, these results demonstrate that by inhibiting the Sirt3 activity and increasing mitochondrial oxidative stress in renal tubular cells, CD38 plays a crucial role in the pathogenesis of DKD.

CD-1db/db mice: A novel type 2 diabetic mouse model with progressive kidney fibrosis.

AIMS/INTRODUCTION: To establish novel therapies to combat diabetic kidney disease, a human disease-relevant animal model is essential. However, a type 2 diabetic mouse model presenting progressive kidney fibrosis has not yet been established. Kidneys of streptozotocin-induced diabetic CD-1 mice showed severe fibrosis compared with other backgrounds of mice associated with the suppression of antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline. The BKS background (BKSdb / db ) is often utilized for diabetic kidney disease research; the kidney fibrosis in the BKSdb / db phenotype is minimal. MATERIALS AND METHODS: We generated CD-1db / db mice by backcrossing the db gene into the CD-1 background, and analyzed phenotypic differences compared with BKSdb / db and CD-1db / m mice. RESULTS: Male CD-1db / db mice appeared to have elevated blood glucose levels compared with those of BKSdb / db mice. Fasting insulin levels declined in CD-1db / db mice. Plasma cystatin C levels tended to be elevated in CD-1db / db mice from 16 to 24 weeks-of-age. Male CD-1db / db mice showed significantly progressive kidney and heart fibrosis from 16 to 24 weeks-of-age when compared with that of age-matched BKSdb / db mice. The gene expression profile showed fibrogenic program-associated genes in male CD-1db / db mice. Male CD-1db / db mice displayed significantly lower urine antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline when compared to that of BKSdb / db at 24 weeks-of-age. The gene expression of prolyl oligopeptidase, the enzyme essential for antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline production from thymosin ß4, was significantly lower in the CD-1 mice. Thymosin ß4 levels were also lower in CD-1 mice. CONCLUSIONS: These results suggest that CD-1db / db mice are a novel type 2 diabetic mouse model with progressive kidney and heart fibrosis.

Methionine abrogates the renoprotective effect of a low-protein diet against diabetic kidney disease in obese rats with type 2 diabetes.

Dietary interventions, including a low-protein diet (LPD) and methionine (Met) restriction, have shown longevity, anti-aging and metabolic health effects. We previously reported that the LPD has a renoprotective effect against diabetic kidney disease (DKD) in rats with type 2 diabetes and obesity. However, it is unclear whether the beneficial effect of the LPD is mediated by low-Met intake or how Met is related to the pathogenesis for DKD. We herein show that the addition of Met with the LPD abrogates the beneficial effects induced by the LPD such as anti-oxidative stress, anti-inflammation and anti-fibrosis, in diabetic kidney. Additionally, the increased levels of S-adenosylmethionine (SAM) in renal tubular cells, which are associated with the reduced expression of glycine N-methyltransferase (Gnmt) and non-restricted Met intake, contributes to the activation of mechanistic target of rapamycin complex 1 (mTORC1) and impaired autophagy, in diabetic kidney. Moreover, starvation-induced autophagy was suppressed in renal cortex of Gnmt null mice and amino acid free-induced autophagy was also suppressed by administration of SAM in cultured HK-2 cells. A LPD could exert a renoprotective effect through the suppression of mTORC1 and restoration of autophagy, which is associated with reduced levels of SAM due to low-Met intake, in diabetic kidney.

Pro-inflammatory macrophages coupled with glycolysis remodel adipose vasculature by producing platelet-derived growth factor-B in obesity.

Adipose tissue macrophages (ATMs) play a central role in tissue remodeling and homeostasis. However, whether ATMs promote adipose angiogenesis in obesity remains unclear. We examined the impact of ATMs deletion on adipose angiogenesis and tissue expansion in the epididymal white adipose tissue (eWAT) of high-fat diet (HFD)-fed mice by using liposome-encapsulated clodronate. We further elucidated the induction mechanisms of platelet-derived growth factor (PDGF)-B in macrophages in response to obesity-associated metabolic stresses, since it plays a significant role in the regulation of pericyte behavior for the initiation of neoangiogenesis during tissue expansion. ATM depletion prevented adipose tissue expansion in HFD-fed mice by inhibiting pericyte detachment from vessels, resulting in less vasculature in eWAT. The lipopolysaccharide (LPS) stimulation and high glucose concentration augmented glucose incorporation and glycolytic capacity with the induction of Pdgfb mRNA. This effect was mediated through extracellular signal-regulated kinase (ERK) among mitogen-activated protein kinases coupled with glycolysis in RAW264.7 macrophages. The Pdgfb induction system was distinct from that of inflammatory cytokines mediated by mechanistic target of rapamycin complex 1 (mTORC1) and NFκB signaling. Thus, obesity-associated hyperglycemia and chronic inflammation fuels ERK signaling coupled with glycolysis in pro-inflammatory macrophages, which contribute to the expansion of eWAT through PDGF-B-dependent vascular remodeling.

Deficiency in Dipeptidyl Peptidase-4 Promotes Chemoresistance through the CXCL12/CXCR4/mTOR/TGFß Signaling Pathway in Breast Cancer Cells.

Dipeptidyl peptidase (DPP)-4, a molecular target of DPP-4 inhibitors, which are type 2 diabetes drugs, is expressed in a variety of cell types, tissues and organs. DPP-4 has been shown to be involved in cancer biology, and we have recently shown that a DPP-4 inhibitor promoted the epithelial mesenchymal transition (EMT) in breast cancer cells. The EMT is known to associate with chemotherapy resistance via the induction of ATP-binding cassette (ABC) transporters in cancer cells. Here, we demonstrated that deficiency in DPP-4 promoted chemotherapy resistance via the CXCL12/CXCR4/mTOR axis, activating the TGFß signaling pathway via the expression of ABC transporters. DPP-4 inhibition enhanced ABC transporters in vivo and in vitro. Doxorubicin (DOX) further induced ABC transporters in DPP-4-deficient 4T1 cells, and the induction of ABC transporters was suppressed by either the CXCR4 inhibitor AMD3100, the mTOR inhibitor rapamycin or a neutralizing TGFß (1, 2 and 3) antibody(N-TGFß). Knockdown of snail, an EMT-inducible transcription factor, suppressed ABC transporter levels in DOX-treated DPP-4-deficient 4T1 cells. In an allograft mouse model, however, the effects of DOX in either primary tumor or metastasis were not statistically different between control and DPP-4-kd 4T1. Taken together, our findings suggest that DPP-4 inhibitors potentiate chemotherapy resistance via the induction of ABC transporters by the CXCL12/CXCR4/mTOR/TGFß signaling pathway in breast cancer cells.

Endothelial autophagy deficiency induces IL6 - dependent endothelial mesenchymal transition and organ fibrosis.

Macroautophagy/autophagy plays a vital role in the homeostasis of diverse cell types. Vascular endothelial cells contribute to vascular health and play a unique role in vascular biology. Here, we demonstrated that autophagy defects in endothelial cells induced IL6 (interleukin 6)-dependent endothelial-to-mesenchymal transition (EndMT) and organ fibrosis with metabolic defects in mice. Inhibition of autophagy, either by a specific inhibitor or small interfering RNA (siRNA) for ATG5 (autophagy related 5), in human microvascular endothelial cells (HMVECs) induced EndMT. The IL6 level was significantly higher in ATG5 siRNA-transfected HMVECs culture medium compared with the control HMVECs culture medium, and neutralization of IL6 by a specific antibody completely inhibited EndMT in ATG5 siRNA-transfected HMVECs. Similar to the in vitro data, endothelial-specific atg5 knockout mice (Atg5 Endo; Cdh5-Cre Atg5 flox/flox mice) displayed both EndMT-associated kidney and heart fibrosis when compared to littermate controls. The plasma level of IL6 was higher in Atg5 Endo compared to that of control mice, and fibrosis was accelerated in Atg5 Endo treated with a HFD; neutralization of IL6 by a specific antibody inhibited EndMT and fibrosis in HFD-fed Atg5 Endo associated with the amelioration of metabolic defects. These results revealed the essential role of autophagy in endothelial cell integrity and revealed that the disruption of endothelial autophagy could lead to significant pathological IL6-dependent EndMT and organ fibrosis. Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; EndMT: endothelial-to-mesenchymal transition; HES: hematoxylin and eosin stain; HFD: high-fat diet; HMVECs: human microvascular endothelial cells; IFNG: interferon gamma; IL6: interleukin 6; MTS: Masson's trichrome staining; NFD: normal-fat diet; siRNA: small interfering RNA; SMAD3: SMAD family member 3; TGFB: transforming growth factor ß; TNF: tumor necrosis factor; VEGFA: vascular endothelial growth factor A.

Dipeptidyl peptidase-4 plays a pathogenic role in BSA-induced kidney injury in diabetic mice.

Diabetic kidney disease (DKD) is appeared to be higher risk of declining kidney function compared to non-diabetic kidney disease with same magnitude of albuminuria. Epithelial-mesenchymal transition (EMT) program of tubular epithelial cells (TECs) could be important for the production of the extracellular matrix in the kidney. Caveolin-1 (CAV1), dipeptidyl peptidase-4 (DPP-4) and integrin ß1 have shown to be involved in EMT program. Here, we found diabetic kidney is prone for albuminuria-induced TECs damage and DPP-4 plays a vital role in such parenchymal damages in diabetic mice. The bovine serum albumin (BSA) injection induced severe TECs damage and altered expression levels of DPP-4, integrin ß1, CAV1, and EMT programs including relevant microRNAs in type 1 diabetic CD-1 mice when compared to non-diabetic mice; teneligliptin (TENE) ameliorated these alterations. TENE suppressed the close proximity among DPP-4, integrin ß1 and CAV1 in a culture of HK-2 cells. These findings suggest that DPP-4 inhibition can be relevant for combating proteinuric DKD by targeting the EMT program induced by the crosstalk among DPP-4, integrin ß1 and CAV1.

N-Acetyl-seryl-aspartyl-lysyl-proline is a potential biomarker of renal function in normoalbuminuric diabetic patients with eGFR ≥ 30 ml/min/1.73 m2.

BACKGROUND: A biomarker, by which we can predict alterations of renal function in normoalbuminuric diabetic patients, is not available. Here, we report that endogenous anti-fibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) represents a potential biomarker to predict alterations in eGFR in normoalbuminuric diabetic patients. METHODS: We analyzed 21 normoalbuminuric diabetic patients with eGFR ≥ 30 ml/min/1.73 m2 and measured AcSDKP levels in first morning void urine. We divided patients into two groups based on the median values: low or high urinary AcSDKP groups (uAcSDKP/Crlow or uAcSDKP/Crhigh). At baseline, no significant differences in sex, age, HbA1c, BMI, serum creatinine levels, etc., were observed between the two groups. RESULTS: During ~ 4 years, the alteration in eGFR [ΔeGFRop (ΔeGFR observational periods)] was significantly stable in uAcSDKP/Crhigh group compared with uAcSDKP/Crlow group over time (P = 0.003, χ2 = 8.58). We also evaluated urine kidney injury molecule-1 (uKim-1) levels and found that ΔeGFRop was also stable in low uKim-1 group compared with high uKim-1 group over time (P = 0.004, χ2 = 8.38). Patients who fulfilled the criteria for both uAcSDKP/Crhigh and uKim-1low exhibited stable ΔeGFRop (P < 0.001, χ2 = 30.4) when compared to the remaining patients. Plasma AcSDKP (P = 0.015, χ2 = 5.94) and urine ß2-microglobulin (P = 0.038, χ2 = 4.31) also display weak but significant predictor of ΔeGFRop as well. CONCLUSION: AcSDKP represents a potentially useful biomarker to predict alterations in the renal function of patients with diabetes presenting normoalbuminuria.

Inhibition of Dipeptidyl Peptidase-4 Accelerates Epithelial-Mesenchymal Transition and Breast Cancer Metastasis via the CXCL12/CXCR4/mTOR Axis.

Dipeptidyl peptidase (DPP)-4 is a multifunctional glycoprotein involved in various biological and pathologic processes. DPP-4 has been widely recognized as a therapeutic target for type 2 diabetes mellitus but is also implicated in the development of human malignancies. Here, we show that inhibition of DPP-4 accelerates breast cancer metastasis via induction of CXCL12/CXCR4, which activates mTOR to promote epithelial-mesenchymal transition (EMT). In cultured cells, DPP-4 knockdown induced EMT and cell migration. Treatment with the DPP-4 inhibitor KR62436 (KR) promoted primary tumor growth and lung metastasis in a 4T1 tumor allograft mouse model; DPP-4 knockdown in 4T1 cells displayed similar phenotypes in vivo and in vitro. KR treatment enhanced the levels of CXCL12/CXCR4 and phosphorylated mTOR, which were associated with the induction of EMT in metastatic cancer cells. KR-induced EMT in cancer cells was inhibited by treatment with the CXCR4 inhibitor AMD3100 or the mTOR inhibitor rapamycin, and AMD3100 suppressed KR-induced metastasis in vivo. Our findings suggest that DPP-4 plays a significant role in cancer biology and that inhibition of DPP-4 promotes cancer metastasis via induction of the CXCL12/CXCR4/mTOR/EMT axis. SIGNIFICANCE: These findings reveal that inhibition of DPP-4 increases the metastatic potential of breast cancer. This is especially important given the potential use of DPP-4 inhibition as a therapeutic strategy for type 2 diabetes.