PiT2 is an inorganic phosphate (Pi) transporter whose mutations are linked to primary familial brain calcification (PFBC). PiT2 mainly consists of two ProDom (PD) domains and a large intracellular loop region (loop7). The PD domains are crucial for the Pi transport, but the role of PiT2-loop7 remains unclear. In PFBC patients, mutations in PiT2-loop7 are mainly nonsense or frameshift mutations that probably cause PFBC due to C-PD1131 deletion. To date, six missense mutations have been identified in PiT2-loop7; however, the mechanisms by which these mutations cause PFBC are poorly understood. Here, we found that the p.T390A and p.S434W mutations in PiT2-loop7 decreased the Pi transport activity and cell surface levels of PiT2. Furthermore, we showed that these two mutations attenuated its membrane localization by affecting adenosine monophosphate-activated protein kinase (AMPK)- or protein kinase B (AKT)-mediated PiT2 phosphorylation. In contrast, the p.S121C and p.S601W mutations in the PD domains did not affect PiT2 phosphorylation but rather impaired its substrate-binding abilities. These results suggested that missense mutations in PiT2-loop7 can cause Pi dyshomeostasis by affecting the phosphorylation-regulated cell-surface localization of PiT2. This study helps understand the pathogenesis of PFBC caused by PiT2-loop7 missense mutations and indicates that increasing the phosphorylation levels of PiT2-loop7 could be a promising strategy for developing PFBC therapies.
Humans ; Cell Membrane ; Mutation, Missense ; Phosphates/metabolism* ; Sodium-Phosphate Cotransporter Proteins, Type III/genetics*

Metformin has robust glucose-lowering effects and multiple benefits beyond hypoglycemic effects. It can also be used in combination with various hypoglycemic drugs and is cost effective. In the absence of the strong indications of glucagon like peptide-1 receptor agonist (GLP-1RA) or sodium glucose cotransporter 2 inhibitor (SGLT2i) for cardiorenal protection, metformin should be used as the first-line pharmacological treatment for newly diagnosed type 2 diabetes and the basic drug for the combined treatment of hypoglycemic drugs. Metformin does not increase the risk of liver and kidney function damage, but patients with renal dysfunction should adjust the dosage of metformin based on estimated glomerular filtration rate (eGFR) levels. Moreover, the correct use of metformin does not increase the risk of lactic acidosis. Because long-term use of metformin is associated with a decrease in vitamin B₁₂ levels, patients with insufficient intake or absorption of vitamin B₁₂ should be regularly monitored and appropriately supplemented with vitamin B₁₂. In view of the new progress made in the basic and clinical research related to metformin, the consensus updating expert group updated the consensus on the basis of the Expert Consensus on the Clinical Application of Metformin (2018 Edition).
Humans ; Consensus ; Diabetes Mellitus, Type 2/complications* ; Hypoglycemic Agents ; Metformin/therapeutic use* ; Sodium-Glucose Transporter 2 Inhibitors/therapeutic use* ; Vitamins/therapeutic use* ; China

Humans ; Diabetes Mellitus, Type 2/drug therapy* ; Sodium-Glucose Transporter 2 Inhibitors/therapeutic use* ; Arrhythmias, Cardiac/etiology* ; Glucose ; Sodium

Humans ; Diabetes Mellitus, Type 2/drug therapy* ; Sodium-Glucose Transporter 2 Inhibitors/therapeutic use* ; Arrhythmias, Cardiac/etiology* ; Glucose ; Sodium

Objective: To investigate the expression of cation chloride cotransporter (NKCC1/KCC2) in the neurons from cerebral lesions of children with focal cortical dysplasia (FCD) type Ⅱ, to provide a morphological basis for revealing the possible mechanism of epilepsy. Methods: Eight cases of FCD type Ⅱ diagnosed at Beijing Haidian Hospital, Beijing, China and 12 cases diagnosed at Xuanwu Hospital, Capital Medical University, Beijing, China from February 2017 to December 2019 were included. The expression of NKCC1 and KCC2 in FCD type Ⅱa and FCD type Ⅱb was detected using immunohistochemistry and double immunohistochemical stains. The average optical density of NKCC1 in dysmorphic neurons and normal neurons was also determined using immunohistochemical staining in FCD type Ⅱa (10 cases). Results: The patients were all younger than 14 years of age. Ten cases were classified as FCD type IIa, and 10 cases as FCD type Ⅱb. NKCC1 was expressed in the cytoplasm of normal cerebral cortex neurons and KCC2 expressed on cell membranes. In dysmorphic neurons of FCD type Ⅱa, expression of NKCC1 increased, which was statistically higher than that of normal neurons (P<0.01). Aberrant expression of KCC2 in dysmorphic neurons was also noted in the cytoplasm. In the FCD Ⅱb type, the expression pattern of NKCC1/KCC2 in dysmorphic neurons was the same as that of FCD type Ⅱa. The aberrant expression of NKCC1 in balloon cells was negative or weakly positive on the cell membrane, while the aberrant expression of KCC2 was absent. Conclusions: The expression pattern of NKCC1/KCC2 in dysmorphic neurons and balloon cells is completely different from that of normal neurons. The NKCC1/KCC2 protein-expression changes may affect the transmembrane chloride flow of neurons, modify the effect of inhibitory neurotransmitters γ-aminobutyric acid and increase neuronal excitability. These effects may be related to the occurrence of clinical epileptic symptoms.
Child ; Humans ; Brain/pathology* ; Cations/metabolism* ; Chlorides/metabolism* ; Epilepsy/metabolism* ; Malformations of Cortical Development, Group I/metabolism* ; Solute Carrier Family 12, Member 2/metabolism* ; Symporters/metabolism*

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular mortality in patients with diabetes mellitus but the protective mechanism remains elusive. Here we demonstrated that the SGLT2 inhibitor, Empagliflozin (EMPA), suppresses cardiomyocytes autosis (autophagic cell death) to confer cardioprotective effects. Using myocardial infarction (MI) mouse models with and without diabetes mellitus, EMPA treatment significantly reduced infarct size, and myocardial fibrosis, thereby leading to improved cardiac function and survival. In the context of ischemia and nutritional glucose deprivation where autosis is already highly stimulated, EMPA directly inhibits the activity of the Na⁺/H⁺ exchanger 1 (NHE1) in the cardiomyocytes to regulate excessive autophagy. Knockdown of NHE1 significantly rescued glucose deprivation-induced autosis. In contrast, overexpression of NHE1 aggravated the cardiomyocytes death in response to starvation, which was effectively rescued by EMPA treatment. Furthermore, in vitro and in vivo analysis of NHE1 and Beclin 1 knockout mice validated that EMPA's cardioprotective effects are at least in part through downregulation of autophagic flux. These findings provide new insights for drug development, specifically targeting NHE1 and autosis for ventricular remodeling and heart failure after MI in both diabetic and non-diabetic patients.
Animals ; Diabetes Mellitus ; Diabetes Mellitus, Type 2/drug therapy* ; Glucose ; Humans ; Mice ; Myocardial Infarction/metabolism* ; Sodium-Glucose Transporter 2 Inhibitors/therapeutic use* ; Ventricular Remodeling

Diabetes Mellitus, Type 2 ; Humans ; Myocardial Infarction/drug therapy* ; Sodium-Glucose Transporter 2 Inhibitors/therapeutic use*

OBJECTIVE: To investigate the effects of co-expression of sodium iodide symporter (NIS) reporter gene on the proliferation and cytotoxic activity of chimeric antigen receptor (CAR)-T cells in vitro. METHODS: T cells expressing CD19 CAR (CAR-T cells), NIS reporter gene (NIS-T cells), and both (NIS-CAR-T cells) were prepared by lentiviral infection. The transfection rates of NIS and CAR were determined by flow cytometry, and the cell proliferation rate was assessed using CCK-8 assay at 24, 48 and 72 h of routine cell culture. The T cells were co-cultured with Nalm6 tumor cells at the effector-target ratios of 1∶2, 1∶1, 2∶1 and 4∶1 for 24, 48 and 72 h, and the cytotoxicity of CAR-T cells to the tumor cells was evaluated using lactate dehydrogenase (LDH) assay. ELISA was used to detect the release of IFN-γ and TNF-β in the co-culture supernatant, and the function of NIS was detected with iodine uptake test. RESULTS: The CAR transfection rate was 91.91% in CAR-T cells and 99.41% in NIS-CAR-T cells; the NIS transfection rate was 47.83% in NIS-T cells and 50.24% in NIS- CAR-T cells. No significant difference in the proliferation rate was observed between CAR-T and NIS-CAR-T cells cultured for 24, 48 or 72 h (P> 0.05). In the co-cultures with different effector-target ratios, the tumor cell killing rate was significantly higher in CAR-T group than in NIS-CAR-T group at 24 h (P < 0.05), but no significant difference was observed between the two groups at 48 h or 72 h (P>0.05). Higher IFN-γ and TNF-β release levels were detected in both CAR-T and NIS-CAR-T groups than in the control group (P < 0.05). NIS-T cells and NIS-CAR-T cells showed similar capacity of specific iodine uptake (P>0.05), which was significantly higher than that in the control T cells (P < 0.05). CONCLUSION The co-expression of the NIS reporter gene does not affect CAR expression, proliferation or tumor cell-killing ability of CAR-T cells.
Antineoplastic Agents ; Cell Line, Tumor ; Cell Proliferation ; Iodine ; Lymphotoxin-alpha ; Receptors, Chimeric Antigen ; Symporters ; T-Lymphocytes

To investigate the effect of Rehmanniae Radix on depression-like behavior and monoamine neurotransmitters of chronic unpredictable mild stress(CUMS) model rats. CUMS combined with isolated feeding was used to induce the depression model of rats. The depression-like behavior of rats was evaluated by sucrose preference test, open field test, and forced swim test. Hematoxylin-Eosin(HE) staining was used to investigate the pathological changes of neurons in the CA1 and CA3 area of hippocampus. Ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS) was used to detect the contents of 5-hydroxytryptamine(5-HT), 5-hydroxyindoleacetic acid(5-HIAA), dopamine(DA), 3,4-dihydroxyphenylacetic acid(DOPAC), homovanillic acid(HVA), norepinephrine(NE), and 3-methoxy-4-hydroxyphenyl glycol(MHPG) in rats. Western blot was used to detect the protein expressions of tryptophan hydroxylase 2(TPH2), serotonin transporter(SERT), and monoamine oxidase A(MAO-A) in the hippocampus of rats. Compared with the normal group, depressive-like behavior of rats was obvious in the model group. The arrangements of neurons in the CA1 and CA3 area of hippocampus were loose and disorderly. The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in the hippocampal area were decreased(P<0.01). The protein expression of TPH2 was decreased(P<0.01), but those of SERT and MAO-A were increased(P<0.01). In the Rehmanniae Radix groups with 1.8 g·kg~(-1) and 7.2 g·kg~(-1), the depression-like behavior of CUMS rats and pathological changes of neurons in CA1, CA3 area of hippocampus were improved. The protein expression of TPH2(P<0.05, P<0.01) was increased, and those of SERT and MAO-A were down-regulated(P<0.05, P<0.01). The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in hippocampus were increased(P<0.05, P<0.01). The changes in DA, DOPAC, HVA, DA/(DOPAC +HVA), NE, DHPG, and NE/DHPG were not statistically significant. The results suggested that Rehmanniae Radix improved depression-like behavior of CUMS rats, and the mechanism might be related to the regulation of synthesis, transportation, and metabolism of 5-HT neurotransmitter in the hippocampus.
3,4-Dihydroxyphenylacetic Acid/pharmacology* ; Animals ; Antidepressive Agents/therapeutic use* ; Chromatography, Liquid ; Depression/drug therapy* ; Disease Models, Animal ; Dopamine ; Eosine Yellowish-(YS)/pharmacology* ; Hematoxylin/pharmacology* ; Hippocampus/metabolism* ; Homovanillic Acid/pharmacology* ; Hydroxyindoleacetic Acid/metabolism* ; Methoxyhydroxyphenylglycol/pharmacology* ; Monoamine Oxidase/metabolism* ; Neurotransmitter Agents/metabolism* ; Norepinephrine/pharmacology* ; Plant Extracts ; Rats ; Rehmannia/chemistry* ; Serotonin/metabolism* ; Serotonin Plasma Membrane Transport Proteins/pharmacology* ; Stress, Psychological/metabolism* ; Tandem Mass Spectrometry ; Tryptophan Hydroxylase/metabolism*

BACKGROUND: Recent cardiovascular outcome trials (CVOTs) changed the therapeutic strategy of guidelines for type 2 diabetes. We compared the characteristics of patients from real-world hospital settings with those of participants in recent pragmatic randomized trials. METHODS: This electronic medical record (EMR)-based retrospective observational study investigated the data of patients with diabetes from inpatient and outpatient settings in West China Hospital of Sichuan University from January 1, 2011, to June 30, 2019. We identified patients meeting the inclusion criteria of a pragmatic randomized trial (EMPA-REG OUTCOME) based on EMRs and compared their baseline characteristics with those of the trial participants. The cutoff for the clinical significance of each characteristic was set as its minimal clinically important difference based on expert consultation. RESULTS: We included 48,257 inpatients and 36,857 outpatients with diabetes and found that 8389 (17.4%) inpatients and 2646 (7.2%) outpatients met the inclusion criteria for the EMPA-REG OUTCOME trial. Compared with the trial population, the real-world inpatients meeting the eligibility criteria of the EMPA-REG OUTCOME had similar age, blood pressure, and lipid profiles but comprised of fewer males, metformin users, anti-hypertensive drug users, and aspirin users, and had a lower body mass index. The group of outpatients meeting the eligibility criteria had fewer males, similar age, fewer metformin users, fewer insulin users, fewer anti-hypertensive drug users, and fewer aspirin users compared with the trial population. CONCLUSIONS The trial population in EMPA-REG OUTCOME represents only a small portion of patients with diabetes from the inpatient and outpatient departments of a Chinese tertiary medical center. Evidence localization in different clinical settings and validation are essential to enabling extrapolation of the results from CVOTs in patients with diabetes to Chinese clinical practice.
Benzhydryl Compounds ; Canagliflozin ; Cardiovascular Diseases ; China ; Diabetes Mellitus, Type 2/drug therapy* ; Glucosides ; Humans ; Male ; Sodium-Glucose Transporter 2 Inhibitors ; Tertiary Care Centers