Canagliflozin regulates metabolic reprogramming in diabetic kidney disease by inducing fasting-like and aestivation-like metabolic patterns.

AIMS/HYPOTHESIS: Sodium-glucose co-transporter 2 (SGLT2) inhibitors (SGLT2i) are antihyperglycaemic drugs that protect the kidneys of individuals with type 2 diabetes mellitus. However, the underlying mechanisms mediating the renal benefits of SGLT2i are not fully understood. Considering the fuel switches that occur during therapeutic SGLT2 inhibition, we hypothesised that SGLT2i induce fasting-like and aestivation-like metabolic patterns, both of which contribute to the regulation of metabolic reprogramming in diabetic kidney disease (DKD). METHODS: Untargeted and targeted metabolomics assays were performed on plasma samples from participants with type 2 diabetes and kidney disease (n=35, 11 women) receiving canagliflozin (CANA) 100 mg/day at baseline and 12 week follow-up. Next, a systematic snapshot of the effect of CANA on key metabolites and pathways in the kidney was obtained using db/db mice. Moreover, the effects of glycine supplementation in db/db mice and human proximal tubular epithelial cells (human kidney-2 [HK-2]) cells were studied. RESULTS: Treatment of DKD patients with CANA for 12 weeks significantly reduced HbA1c from a median (interquartile range 25-75%) of 49.0 (44.0-57.0) mmol/mol (7.9%, [7.10-9.20%]) to 42.2 (39.7-47.7) mmol/mol (6.8%, [6.40-7.70%]), and reduced urinary albumin/creatinine ratio from 67.8 (45.9-159.0) mg/mmol to 47.0 (26.0-93.6) mg/mmol. The untargeted metabolomics assay showed downregulated glycolysis and upregulated fatty acid oxidation. The targeted metabolomics assay revealed significant upregulation of glycine. The kidneys of db/db mice undergo significant metabolic reprogramming, with changes in sugar, lipid and amino acid metabolism; CANA regulated the metabolic reprogramming in the kidneys of db/db mice. In particular, the pathways for glycine, serine and threonine metabolism, as well as the metabolite of glycine, were significantly upregulated in CANA-treated kidneys. Glycine supplementation ameliorated renal lesions in db/db mice by inhibiting food intake, improving insulin sensitivity and reducing blood glucose levels. Glycine supplementation improved apoptosis of human proximal tubule cells via the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway. CONCLUSIONS/INTERPRETATION: In conclusion, our study shows that CANA ameliorates DKD by inducing fasting-like and aestivation-like metabolic patterns. Furthermore, DKD was ameliorated by glycine supplementation, and the beneficial effects of glycine were probably due to the activation of the AMPK/mTOR pathway.

Long noncoding RNA MIR22HG promotes Leydig cell apoptosis by acting as a competing endogenous RNA for microRNA-125a-5p that targets N-Myc downstream-regulated gene 2 in late-onset hypogonadism.

Leydig cells (LCs) apoptosis is responsible for the deficiency of serum testosterone in Late-onset hypogonadism (LOH), while its specific mechanism is still unknown. This study focuses on the role of long noncoding RNA (lncRNA) MIR22HG in LC apoptosis and aims to elaborate its regulatory mechanism. MIR22HG was up-regulated in the testicular tissues of mice with LOH and H2O2-treated TM3 cells (mouse Leydig cell line). Interference of MIR22HG ameliorated cell apoptosis and upregulated miR-125a-5p expression in H2O2-treated TM3 cells. Then, the interaction between MIR22HG and miR-125a-5p was confirmed with RIP and RNA pull-down assay. Further study showed that miR-125a-5p downregulated N-Myc downstream-regulated gene 2 (NDRG2) expression by targeting its 3'-UTR of mRNA. What's more, MIR22HG overexpression aggravated cell apoptosis and reduced testosterone production in TM3 cells via miR-125a-5p/NDRG2 pathway. MIR22HG knockdown elevated testosterone levels in LOH mice. In conclusion, MIR22HG up-regulated NDRG2 expression through targeting miR-125a-5p, thus promoting LC apoptosis in LOH.

LncRNA FENDRR promotes apoptosis of Leydig cells in late-onset hypogonadism by facilitating the degradation of Nrf2.

This study aimed to investigate the role of lncRNA FENDRR in apoptosis of Leydig cells and the further mechanism. The apoptosis of Leydig cells (TM3 cell line) was induced by H2O2-treatment and detected by flow cytometry. The function of FENDRR was determined by in vitro and in vivo silencing experiments. The mechanism of FENDRR in regulating the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) was assessed by RNA immunoprecipitation, RNA pull-down, and ubiquitination assays. FENDRR expression was up-regulated in H2O2-treated TM3 cells. Knockdown of FENDRR augmented Nrf2 and HO-1 protein levels and testosterone production in H2O2-treated TM3 cells, whereas the apoptosis rate and caspase 3 activity were decreased. Mechanically, FENDRR bound to Nrf2 and promoted its ubiquitination and degradation. Nrf2 overexpression reversed the effects FENDRR overexpression on apoptosis, caspase 3 activity, and testosterone concentration in H2O2-treated TM3 cells. The in vivo experiments showed that FENDRR silence increased serum testosterone level and improved testosterone-related anti-depression behaviors of late-onset hypogonadism (LOH) mice. Our findings suggested that FENDRR could promote apoptosis of Leydig cells in LOH partly through facilitating Nrf2 degradation.

Linc00210 enhances the malignancy of thyroid cancer cells by modulating miR-195-5p/IGF1R/Akt axis.

Emerging evidence has indicated that long noncoding RNA (lncRNAs) play crucial roles in regulating thyroid cancer (TC) development. Linc00210 is a newly identified lncRNA which plays an oncogenic role in hepatocellular carcinoma and nasopharyngeal carcinoma, but whether Linc00210 can modulate the development of TC remains elusive. Here, we found that Linc00210 expression was upregulated in TC tissues compared to the matched noncancerous tissues. Overexpression of Linc00210 augmented the proliferation, migration, and invasion of TC cells. Mechanistically, Linc00210 served as a sponge for miR-195-5p, thereby counteracting its ability in downregulating the expression of IGF1R and the activation of PI3K/Akt signaling. Moreover, inhibition of Linc00210 suppressed the growth of TC cells in nude mice. Our findings for the first time uncovered the oncogenic property of Linc00210 in TC.

High-fat diet triggers obesity-related early infiltration of macrophages into adipose tissue and transient reduction of blood monocyte count.

Infiltration of adipose tissue macrophages (ATMs) is a typical feature of obesity, and circulating immune cells may indicate immune cell accumulation. However, it remains unclear whether this is true in the early stages of obesity. This study aimed to define the role of blood monocytes in obesity and the relationship between blood monocytes and ATMs in early-stage obesity. Two groups of male C57BL/6 J mice were fed on a 60 % high-fat diet (HFD) or a 10 % fat normal diet (ND), respectively, and monitored at 1, 2, 3, 7, and 12 weeks. Populations of circulating blood monocytes (CD11b + CD115+), ATMs (F4/80+CD11b+), and their subtypes were collected and analyzed using flow cytometry and immunofluorescence. Some cytokines (TNF-a, IL-1ß) and chemokines (CCL2, CCL7) were also analyzed by real-time PCR. HFD induced obesity, dramatic fat expansion, and accumulation of ATMs in mice after 12 weeks. However, an acute and transient reduction of circulating monocyte count, elevated expression of CD11c in ly6clow monocytes, and concurrent infiltration of ATMs into visceral adipose tissues (VAT) were observed as early as 1 week after initiating HFD. Further, HFD-induced changes in VAT, but not blood monocyte count, were partially reversed upon reverting to ND for 6 weeks. An acute but transient reduction of blood monocyte count was observed at the early stages of HFD feeding, which might be related to early infiltration of macrophages into adipose tissues. We believe that blood monocytes could be targeted as a new obesity treatment following additional studies.

Melanocortin 4 Receptor Activation Attenuates Mitochondrial Dysfunction in Skeletal Muscle of Diabetic Rats.

A previous study has confirmed that the central melanocortin system was able to mediate skeletal muscle AMP-activated protein kinase (AMPK) activation in mice fed a high-fat diet, while activation of the AMPK signaling pathway significantly induced mitochondrial biogenesis. Our hypothesis was that melanocortin 4 receptor (MC4R) was involved in the development of skeletal muscle injury in diabetic rats. In this study, we treated diabetic rats intracerebroventricularly with MC4R agonist R027-3225 or antagonist SHU9119, respectively. Then, we measured the production of reactive oxygen species (ROS), the levels of malondialdehyde (MDA) and glutathione (GSH), the mitochondrial DNA (mtDNA) content and mitochondrial biogenesis, and the protein levels of p-AMPK, AMPK, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), sirtuin 1 (SIRT1), and manganese superoxide dismutase (MnSOD) in the skeletal muscle of diabetic rats. The results showed that there was significant skeletal muscle injury in the diabetic rats along with serious oxidative stress and decreased mitochondrial biogenesis. Treatment with R027-3225 reduced oxidative stress and induced mitochondrial biogenesis in skeletal muscle, and also activated the AMPK-SIRT1-PGC-1α signaling pathway. However, diabetic rats injected with MC4R antagonist SHU9119 showed an aggravated oxidative stress and mitochondrial dysfunction in skeletal muscle. In conclusion, our results revealed that MC4R activation was able to attenuate oxidative stress and mitochondrial dysfunction in skeletal muscle induced by diabetes partially through activating the AMPK-SIRT1-PGC-1α signaling pathway. J. Cell. Biochem. 118: 4072-4079, 2017. © 2017 Wiley Periodicals, Inc.

SIRT1 overexpression in skeletal muscle in vivo induces increased insulin sensitivity and enhanced complex I but not complex II–V functions in individual subsarcolemmal and intermyofibrillar mitochondria

SIRT1 is known to improve insulin resistance (IR), but whether this effect is direct or not is still unclear, and this question has not been addressed in vivo in the skeletal muscle. Therefore, we sought to test if acute overexpression of SIRT1 in skeletal muscle of high-fat diet (HFD) rats in vivo would affect subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial complexes I–V activities and antioxidant enzymes thereby improving insulin action. In vivo electrotransfer was used to overexpress SIRT1 in the skeletal muscle of rats fed HFD for 12 weeks. Skeletal muscle insulin sensitivity and downstream effects of SIRT1 on AMPK, SIRT3, and mitochondrial biogenesis were studied. Citrate synthase (CS), complexes I–V, oxidative stress, and antioxidant levels were assessed in SS and IMF mitochondria. HFD rats showed skeletal muscle IR as well as decreasedSIRT1 and SIRT3 expressions, mitochondrial DNA (mtDNA), and mitochondrial biogenesis (p < 0.05). SS and IMF mitochondria displayed lower CS, complexes I–V, and antioxidant enzyme activities (p < 0.05). By contrast, moderate (~2.5 folds) SIRT1 overexpression attenuated HFD-induced skeletal muscle IR. This improvement was associated with increased AMPK, PGC-1alfa, SIRT3, and mtDNA expressions as well as SS and IMF mitochondrial CS and complexes I–V activities. Importantly, SIRT1 overexpression largely restored antioxidant enzyme activities and enhanced complex I but not complexes II–V functions in individual SS and IMF mitochondria. This study suggests that SIRT1 overexpression improved IR at least partly by targeting complex I functions of SS and IMF mitochondria through the activation of SIRT1 and SIRT3

SIRT1 overexpression in skeletal muscle in vivo induces increased insulin sensitivity and enhanced complex I but not complex II-V functions in individual subsarcolemmal and intermyofibrillar mitochondria.

SIRT1 is known to improve insulin resistance (IR), but whether this effect is direct or not is still unclear, and this question has not been addressed in vivo in the skeletal muscle. Therefore, we sought to test if acute overexpression of SIRT1 in skeletal muscle of high-fat diet (HFD) rats in vivo would affect subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial complexes I-V activities and antioxidant enzymes thereby improving insulin action. In vivo electrotransfer was used to overexpress SIRT1 in the skeletal muscle of rats fed HFD for 12 weeks. Skeletal muscle insulin sensitivity and downstream effects of SIRT1 on AMPK, SIRT3, and mitochondrial biogenesis were studied. Citrate synthase (CS), complexes I-V, oxidative stress, and antioxidant levels were assessed in SS and IMF mitochondria. HFD rats showed skeletal muscle IR as well as decreased SIRT1 and SIRT3 expressions, mitochondrial DNA (mtDNA), and mitochondrial biogenesis (p < 0.05). SS and IMF mitochondria displayed lower CS, complexes I-V, and antioxidant enzyme activities (p < 0.05). By contrast, moderate (~2.5 folds) SIRT1 overexpression attenuated HFD-induced skeletal muscle IR. This improvement was associated with increased AMPK, PGC-1α, SIRT3, and mtDNA expressions as well as SS and IMF mitochondrial CS and complexes I-V activities. Importantly, SIRT1 overexpression largely restored antioxidant enzyme activities and enhanced complex I but not complexes II-V functions in individual SS and IMF mitochondria. This study suggests that SIRT1 overexpression improved IR at least partly by targeting complex I functions of SS and IMF mitochondria through the activation of SIRT1 and SIRT3.

Myeloid differentiation factor 88 promotes cisplatin chemoresistance in ovarian cancer.

To investigate regulatory effects and associated mechanisms of myeloid differentiation factor 88 (MyD88) on cisplatin chemoresistance in human ovarian cancer (OC) cells. The expression of MyD88 in SKOV3/DDP cells was restrained by short interfering RNA (siRNA) of MyD88. Cell viability and proliferation in the presence or absence of cisplatin were detected by Cell Counting Kit-8 (CCK-8). The expression of p-Akt protein, X-linked inhibitor of apoptosis protein (XIAP), and multidrug resistance protein 1 (MRP1) was detected by Western blot analysis. After Protein Kinase B (PKB/Akt) signal pathway was inhibited by the p-Akt inhibitor (LY294002) and the expression of MRP1 was restrained by siRNA of MRP1, CCK-8 was used to examine the cell proliferation after treatment with cisplatin. After the expression of MyD88 in SKOV3/DDP cells was restrained, the cell proliferation was inhibited, the cisplatin resistance decreased and the half maximal inhibitory concentration (IC50) reduced to 38 % of the control group (P < 0.01). The increased expression of p-Akt, XIAP, and MRP1 in SKOV3/DDP cells after cisplatin treatment was also repressed by the downregulation of MyD88. Furthermore, the inhibition of PKB/Akt signal pathway or expression of MRP1 both could decrease the cisplatin resistance of SKOV3/DDP cells and the IC50 decreased to 75 and 53 % of the control group (P < 0.01, P < 0.05), respectively. MyD88 promoted cisplatin chemoresistance in human OC cells through activating PKB/Akt signal pathway, and enhancing the expression of XIAP and MRP1. MyD88 might be a new target of the novel pharmacological treatments for cisplatin-resistant OC.

[Study on five point mutations in mitochondrial DNA in patients with Leber's hereditary optic neuropathy].

OBJECTIVE: To analyze the relationship between the primary mutation at np11778 and the secondary mutations at np9804, np13708, np13730, np15257 in three Chinese pedigrees with Leber's hereditary optic neuropathy (LHON) and to detect the effects of the mutations on LHON. METHODS: Thirty-seven maternal individuals from three LHON pedigrees and forty-seven normal controls were involved in this study. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing were used to detect the mutations in mitochondrial DNA (mtDNA). RESULTS: All patients and their maternal relatives had the np11778 mtDNA primary mutation. None had the secondary mutations at np9804, np13708 and np13730 and np15257. DNA sequencing of the PCR fragment revealed six new point mutations at np13759, np13928, np13942, np15301, np15323 and np15326. CONCLUSION: All three Chinese pedigrees with LHON had the mtDNA11778 primary mutation. The frequency of mutation at np13759 in Chinese patients with LHON is higher than that in normal Chinese controls. These findings indicate that np13759 is a new secondary mutation of LHON in Chinese.