Sortilin-induced lipid accumulation and atherogenesis are suppressed by HNF1b SUMOylation promoted by flavone of Polygonatum odoratum / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

This study aims to investigate the impact of hepatocyte nuclear factor 1β (HNF1b) on macrophage sortilin-mediated lipid metabolism and aortic atherosclerosis and explore the role of the flavone of Polygonatum odoratum (PAOA-flavone)-promoted small ubiquitin-related modifier (SUMO) modification in the atheroprotective efficacy of HNF1b. HNF1b was predicted to be a transcriptional regulator of sortilin expression via bioinformatics, dual-luciferase reporter gene assay, and chromatin immunoprecipitation. HNF1b overexpression decreased sortilin expression and cellular lipid contents in THP-1 macrophages, leading to a depression in atherosclerotic plaque formation in low-density lipoprotein (LDL) receptor-deficient (LDLR-/-) mice. Multiple SUMO1-modified sites were identified on the HNF1b protein and co-immunoprecipitation confirmed its SUMO1 modification. The SUMOylation of HNF1b protein enhanced the HNF1b-inhibited effect on sortilin expression and reduced lipid contents in macrophages. PAOA-flavone treatment promoted SUMO-activating enzyme subunit 1 (SAE1) expression and SAE1-catalyzed SUMOylation of the HNF1b protein, which prevented sortilin-mediated lipid accumulation in macrophages and the formation of atherosclerotic plaques in apolipoprotein E-deficient (ApoE-/-) mice. Interference with SAE1 abrogated the improvement in lipid metabolism in macrophage cells and atheroprotective efficacy in vivo upon PAOA-flavone administration. In summary, HNF1b transcriptionally suppressed sortilin expression and macrophage lipid accumulation to inhibit aortic lipid deposition and the development of atherosclerosis. This anti-atherosclerotic effect was enhanced by PAOA-flavone-facilitated, SAE1-catalyzed SUMOylation of the HNF1b protein.

Baicalin protects against 17α-ethinylestradiol-induced cholestasis via Sirt1/HNF-1α/FXR pathway / 中国药理学与毒理学杂志

OBJECTIVE Baicalin is a major flavonoid component of Scutellaria baicalensis, and has been used in the treatment of liver diseases for many years. However, the role of baicalin in estrogen-induced cholestasis (EIC) remains to be elucidated. This present study explored the protective effect of baicalin against estrogen-induced liver injury and further elucidated the mechanisms involved both in vivo and in vitro. METHODS We conducted a series of experiments using 17α-ethinylestradiol (EE) induced cholestatic rats and cultured HepG2 cells. Serum, bile, and liver samples were collected for biochemical and histological analyses. Bile acid composition in liver was analyzed by LC-MS/MS. The mechanisms underlying the hepatoprotective of baicalin were investigated by RT-PCR, Western blotting analyses and immunohistochemistry. RESULTS Baicalin showed obvious hepatoprotective effects in EIC rats by reducing serum bio?markers and increasing the bile flow rate, as well as by alleviating liver histology and restoring the abnormal composition of hepatic bile acids (BAs). In addition, baicalin protected against EE induced liver injury by up-regulation of the expres?sion of hepatic efflux transporters and down-regulation of hepatic uptake transporters. Furthermore, baicalin increased the expression of hepatic BA synthase (CYP27A1) and metabolic enzymes (Bal, Baat and Sult2a1) in EIC rats. We showed that baicalin significantly inhibited hepatic inflammatory responses in EIC rats through reducing elevated levels of TNF-α, IL-1β, IL-6 and NF-κB. Finally, we confirmed that baicalin maintains BA homeostasis and alleviates inflamma?tion through Sirt1/HNF-1α/FXR signaling pathway. CONCLUSION Baicalin protects against estrogen-induced cholestatic liver injury, and the underlying mechanism involved is related to activation of the Sirt1/HNF-1α/FXR signaling pathway.

Effect of Shuangyu Tiaozhi Decoction on HNF1α/PCSK9/LDLR Signaling Pathway in Liver of Hypercholesterolemic Rats / 中国实验方剂学杂志

Objective:To observe the expression of hepatocyte nuclear factor 1<italic>α</italic> （HNF1<italic>α</italic>）， proprotein convertase subtilisin/kexin type 9 （PCSK9） and low-density lipoprotein cholesterol （LDLR） in hypercholesterolemia rat liver， and investigate the mechanism of Shuangyu Tiaozhi Decoction regulating cholesterol metabolism and attenuating hypercholesterolemia. Method:After providing a high-fat diet for 4 weeks， 40 SD rats were selected， 8 of which were randomly selected as normal group and fed a normal diet， and the remaining 32 rats were fed a high-fat diet. The rats successfully established as hypercholesterolemic model， were randomized into 4 groups： model group， low dose of Shuangyu Tiaozhi decoction group （7.8 g·kg^-1）， high dose of Shuangyu Tiaozhi decoction group （15.6 g·kg^-1）， and simvastatin group （4 mg·kg^-1）， with 8 rats in each group. The drugs were continuously given for 8 weeks. Serum total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， high-density lipoprotein cholesterol （HDL-C） were measured. The pathomorphological changes in liver were observed by hematoxylin and eosin （HE） staining. The immunohistochemistry was used to detect PCSK9 and LDLR expression in liver. The mRNA and protein expression levels of HNF1<italic>α</italic>， PCSK9 and LDLR were determined by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. Result:Compared with normal group， the TC， TG， LDL-C levels in model group were significantly increased （<italic>P</italic><0.01）， the morphology showed obvious liver steatosis. The mRNA and protein expression of HNF1<italic>α</italic> and PCSK9 were increased （<italic>P</italic><0.05）， the mRNA and protein expression of LDLR was decreased （<italic>P</italic><0.05）. Compared with model group， the serum TC， TG， LDL-C levels were significantly lowered in the Shuangyu Tiaozhi decoction high-dose group （<italic>P</italic><0.01）， the serum TC， LDL-C levels were significantly lowered in the Shuangyu Tiaozhi decoction low-dose group and simvastatin group （<italic>P</italic><0.05，<italic>P</italic><0.01）， while no significant effect was observed on the serum HDL-C levels in each treatment group. The liver steatosis decreased in each treatment group. The mRNA and protein expression of HNF1<italic>α</italic> was obviously decreased in each treatment group （<italic>P</italic><0.05，<italic>P</italic><0.01）， the mRNA and protein expression of PCSK9 was obviously decreased in Shuangyu Tiaozhi decoction low and high-dose groups （<italic>P</italic><0.05，<italic>P</italic><0.01）， the mRNA expression of PCSK9 was significantly increased in the simvastatin group （<italic>P</italic><0.01）， while the protein expression showed a downward trend. The LDLR mRNA levels were significantly increased in each treatment group （<italic>P</italic><0.01）， the LDLR protein expression was significantly increased in Shuangyu Tiaozhi high-dose group （<italic>P</italic><0.01）， and showed an upward trend in Shuangyu Tiaozhi low-dose group and simvastatin group. Results of immunohistochemistry showed PCSK9 expression was weakly positive， the expression of LDLR was strongly positive in each treatment group. The therapeutic effect of Shuangyu Tiaozhi decoction high-dose group was more remarkable than simvastatin group， while there was no obvious difference between the Shuangyu Tiaozhi decoction low-dose group and simvastatin group. Conclusion:Shuangyu Tiaozhi decoction may reduce the blood lipid levels through HNF1<italic>α</italic>/PCSK9/LDLR signaling pathway， play an active role on regulating cholesterol metabolism and alleviating high-fat diet-induced hypercholesterolemia.

MODY probability calculator for GCK and HNF1A screening in a multiethnic background population

ABSTRACT Objective We aimed to identify the frequency of monogenic diabetes, which is poorly studied in multiethnic populations, due to GCK or HNF1A mutations in patients with suggestive clinical characteristics from the Brazilian population, as well as investigate if the MODY probability calculator (MPC) could help patients with their selection. Subjects and methods Inclusion criteria were patients with DM diagnosed before 35 years; body mass index < 30 kg/m2; negative autoantibodies; and family history of DM in two or more generations. We sequenced HNF1A in 27 patients and GCK in seven subjects with asymptomatic mild fasting hyperglycemia. In addition, we calculated MODY probability with MPC. Results We identified 11 mutations in 34 patients (32.3%). We found three novel mutations. In the GCK group, six cases had mutations (85.7%), and their MODY probability on MPC was higher than 50%. In the HNF1A group, five of 27 individuals had mutations (18.5%). The MPC was higher than 75% in 11 subjects (including all five cases with HNF1A mutations). Conclusion Approximately one third of the studied patients have GCK or HNF1A mutations. Inclusion criteria included efficiency in detecting patients with GCK mutations but not for HNF1A mutations (< 20%). MPC was helpful in narrowing the number of candidates for HNF1A screening.

Inhibition of miR-802 on insulin secretion by targeting Hnf1B / 中国药科大学学报

@#To investigate the effect of miR-802 on insulin secretion by islet β cells and its mechanism, miR-802 was overexpressed or knocked down in primary islet cells and Min6 cells via transfecting miR-802 mimic and miR-802 inhibitor, respectively. The effect of miR-802 on insulin secretion was detected by ELISA. The target gene of miR-802 was confirmed by miRNA target gene database prediction, luciferase report and Western blot. The function recovery experiment was carried out to clarify the mechanism of miR-802 regulating β cell secretion of insulin. The results showed that overexpression of miR-802 in islet primary cells and Min6 cells inhibited insulin secretion. qPCR and Western blot showed that miR-802 inhibited insulin secretion by inhibiting the transcription and translation of the target gene, hepatocyte nuclear factor 1β(Hnf1B).

Searching for mutations in the HNF1B gene in a Brazilian cohort with renal cysts and hyperglycemia

ABSTRACT Objective To verify the presence of variants in HNF1B in a sample of the Brazilian population selected according to the presence of renal cysts associated with hyperglycemia. Subjects and methods We evaluated 28 unrelated patients with clinical suspicion of HNF1B mutation because of the concomitant presence of diabetes mellitus (DM) or prediabetes and renal cysts. Genotyping was accomplished using Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA). In positive cases, available relatives were recruited. Results We found two patients with HNF1B mutations. The first presented the variant p.Pro328Leufs*48(c.983delC) and had DM, renal cysts, and hypomagnesemia. The second presented a heterozygous whole gene deletion in HNF1B, DM, renal cysts, body and tail pancreatic agenesis, and hypomagnesemia; this alteration was also found in his two siblings and his father. Conclusion The recruitment of suspected cases of HNF1B gene mutations in Brazilians due to hyperglycemia and renal cysts presents two positive cases. Our cases contribute to the annotation of clinical and biochemical phenotypes of this rare form of maturity-onset diabetes of the young (MODY).

Clinical phenotypes of hepatocyte nuclear factor 1 homeobox b-associated disease / 中华儿科杂志

Objective@#Hepatocyte nuclear factor 1 homeobox b (HNF1B) -associated disease is an autosomal dominant inherited disorder with a variable, multi-systemic phenotype. In China, five adult probands and one child proband with HNF1B-associated disease had been reported, whereas few fetuses are described. The aims of this retrospective study were to understand about the clinical manifestations of HNF1B-associated disease and to further improve the recognition of this disorder.@*Method@#Four patients (3 males, 1 female) and three fetuses with HNF1B mutations were included in this study. They were admitted to our hospital from January 2013 to March 2017. HNF1B mutations were detected using targeted next generation sequencing and quantitative real-time PCR or Sanger sequencing. HNF1B heterozygous deletion of exons 1-9 was found in 4 patients and 2 fetuses, and HNF1B heterozygous missense mutation in 1 fetus. These two mutations had been reported. Two patients and 1 fetus had de novo mutations. Results of renal ultrasonography with or without magnetic resonance imaging, biochemical investigations, urine routine examination and other necessary investigations in 7 cases were analyzed.@*Result@#Three patients were Han Chinese ethnicity, and one patient was Mongolian. In patients 1 and 4, abnormal fetal kidneys were discovered by routine ultrasonography, and the age at first feature identified in Patients 2 and 3 were 13 years and 28 years. Patient 3 had normal renal function and the remainder had reduced glomerular filtration rate. In addition, patient 4 presented with nephrotic syndrome and glycosuria, patient 2 with early onset hyperparathyroidism and renal osteodystrophy, and patient 3 with diabetes mellitus. All the 4 patients had renal structural abnormalities including bilateral multiple renal cysts, dysplasia and hyperechogenic kidneys. Only patient 3 had a positive family history of renal diseases, the remainder had a negative family history of renal diseases. In 3 fetuses, prenatal ultrasound anomalies were detected during the second trimester. These 3 fetuses had hyperechogenic kidneys with or without renal cysts. Polyhydramnios was detected in only one of the 3 fetuses. Two of the 3 fetuses had a positive family history of renal diseases.@*Conclusion@#Clinical phenotypes of HNF1B-related disease are heterogeneous, renal malformations clearly appear to be the most common manifestation, multiple renal cysts are characteristic, and patients can progress to impaired kidney function during childhood; HNF1B mutation is a differential diagnosis of fetal hyperechogenic kidneys or multiple renal cysts.

Diagnostic value of HNF-1βin the diagnosis of ovarian clear cell carcinoma / 临床与实验病理学杂志

Purpose To study the expression of HNF-1β in different type ovarian carcinoma and to explore the diagnostic value of HNF-1β in the diagnosis of ovarian clear cell carcinoma. Methods Immunohistochemical EnVision method was used to detect the ex-pression of HNF-1βin 27 clear cell carcinomas, 35 high-grade serous carcinomas, 21 endometrioid adenocarcinoma, 10 mucious carci-nomas, 13 metastatic Krukenberg tumors, and 2 transitional carcinomas. Results All of ovarian clear cell carcinomas variably ex-pressed HNF-1β and the positive rate of HNF-1β in ovarian cleat cell carcinoma was 85. 2%. Of 21 ovarian serous carcinomas, the nuclear positive of HNF-1β was 4 and HNF-1βpositive rate was 2. 9%. There was 5 HNF-1βexpression in endometrioid adenocarci-noma and the positive rate of HNF-1β was 23. 8%. The positive rate of HNF-1β in mucious carcinoma and metastatic Krukenberg tumor was 60. 0% and 53. 8%, respectively. No HNF-1βexpression was seen in transtional cell carcinoma. The sensitivity and speci-ficity of HNF-1β in the diagnosis of ovarian clear cell carcinoma was 85. 2% and 76. 5%. Conclusions HNF-1β may be a higher sensitive marker for the diagnosis of ovarian clear cell carcinoma. The diffuse and strong positive of HNF-1βmay have higher specific for diagnosis of ovarian clear cell carcinoma.

The Renal Cysts and Diabetes (RCAD) Syndrome in a Child with Deletion of the Hepatocyte Nuclear Factor-1β Gene.

The authors describe a 14-yr-old boy who presented with non-ketotic hyperglycemia, elevated serum creatinine levels and deranged liver function. There was no microalbuminuria or proteinuria. He also had mild mental retardation with learning difficulties. Ultrasonography of the abdomen revealed multiple renal cysts of varying sizes in both the kidneys. Dosage analysis of the hepatocyte nuclear factor (HNF)-1β gene by multiplex ligation-dependent probe amplification (MLPA) detected a heterozygous whole gene deletion (p.Met1_Trp557del). This finding is consistent with the diagnosis of renal cysts and diabetes (RCAD) syndrome. This is the first case of the RCAD syndrome reported in an Indian patient. Pediatricians need to be aware of this entity whenever renal disease is seen in a diabetic child in the absence of microalbuminuria or proteinuria.

The Na+/glucose cotransporters: from genes to therapy

Glucose enters eukaryotic cells via two types of membrane-associated carrier proteins, the Na+/glucose cotransporters (SGLT) and the facilitative glucose transporters (GLUT). The SGLT family consists of six members. Among them, the SGLT1 and SGLT2 proteins, encoded by the solute carrier genes SLC5A1 and SLC5A2, respectively, are believed to be the most important ones and have been extensively explored in studies focusing on glucose fluxes under both physiological and pathological conditions. This review considers the regulation of the expression of the SGLT promoted by protein kinases and transcription factors, as well as the alterations determined by diets of different compositions and by pathologies such as diabetes. It also considers congenital defects of sugar metabolism caused by aberrant expression of the SGLT1 in glucose-galactose malabsorption and the SGLT2 in familial renal glycosuria. Finally, it covers some pharmacological compounds that are being currently studied focusing on the interest of controlling glycemia by antagonizing SGLT in renal and intestinal tissues.

Expressions of hepatocyte nuclear factor -1α and hepatocyte nuclear factor -4α in human hepatocellular carcinoma / 解剖学报

Objective To investigate the expression of hepatocyte nuclear factor-1α (HNF-1α) and hepatocyte nuclear factor-4α (HNF-4α) in human hepatocellular carcinoma (HCC) and explore the function of HNF-1α and HNF-4α during HCC carcinogenesis and development. Methods Twenty-six specimens of hepatocellular carcinoma were collected. The expressions of HNF-1α and HNF-4α in HCC tissues and adjacent non-cancerous tissues were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry staining. Results The mRNA levels of HNF-1α and HNF-4α were significantly lower in HCC tissues than that in adjacent non-cancerous tissues (0.818±0.371 vs. 0.383±0.102 for HNF-1α, P＜0.05;0.846±0.384 vs. 0.397±0.105 for HNF-4α, P＜0.05).The positive rates of HNF-1α and HNF-4α protein were significantly lower in HCC tissues than in adjacent non-cancerous tissues (92.3% vs. 42.3% for HNF-1α, P＜0.05;96.2% vs. 50.0% for HNF-4α, P＜0.05). The mRNA and protein expressions of HNF-1α and HNF-4α were correlated with tumor differentiation (P＜0.05). There was a negative correlation between HNF-1α and HNF-4α mRNA expressions in HCC tissues.Conclusion The expressions of HNF-1α and HNF-4α are down-regulated in HCC, which might be related to carcinogenesis and development of HCC.

Construction of a mutation due to a fourteen base-pair insertion in HNF-1α gene causing maturity-onset diabetes of the young (MODY) in Thai patients.

Objective: The aim of this study is to generate a mutation causing maturity-onset diabetes of the young (MODY) in Thai patients by insertion of a fourteen base-pair (bp) into a HNF-1a gene using a modified site-directed ligase-independent mutagenesis (SLIM) method. Methods: Two pairs of long- and short-tailed primers were designed to amplify a plasmid construct containing a HNF-1a and to insert a 14-bp at a desired position. Long-tailed primers contained the overhanging 14-nucleotide (nt) insert at their termini which were complementary to each other. Polymerase chain reactions (PCR) were performed in two separated tubes using different pairs of primers. After amplifications, PCR products from both tubes were pooled together, denatured and then re-annealed to allow formation of double stranded DNA molecules containing the 14-bp insert within HNF-1a. The pooled and reannealed PCR products without ligation were transformed into competent E.coli cells to generate a ligated recombinant plasmid with a 14-bp insertion. Results: Five of 14 bacterial colonies contained the desired recombinant plasmid with a 14-bp insertion within HNF-1a The efficiency of the method for generation of recombinant plasmid was about 36 percent. Conclusion: This method is simple and rapid to insert a long stretch of nucleotides into a plasmid construct containing a gene of interest at a desired position. A recombinant plasmid containing an insertion mutation in a HNF-1a gene was successfully generated, allowing an opportunity to perform functional study of the mutated gene.

Mutation Screening of HNF-1alpha Gene in Korean Women with Gestational Diabetes Mellitus / 당뇨병

BACKGROUNDS: Gestational diabetes mellitus (GDM) is defined as glucose intolerance with onset or first detection during pregnancy and mostly caused by insulin resistance and beta-cell dysfunction like type 2 diabetes. However, autoimmune or monogenic diabetes can contribute to GDM. Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes characterized by an early age of onset and an autosomal dominant pattern of inheritance. Most MODY cases are attributable to mutations in HNF-1alpha gene, also known as MODY3. We investigated whether mutations in HNF-1alpha gene are present in Korean women with GDM. METHODS: A total of 96 Korean women with GDM who have a family history of DM were screened for mutations in the HNF-1alpha gene. We evaluated the clinical characteristics of GDM women with HNF-1alpha gene mutations. RESULTS: Five of 96 patients (5.2%) were found to have a mutation in HNF-1alpha gene. Four of those (-23C > G, 833G > A (Arg278Gln), 923C > T, IVS5 + 106A > G) were novel and one (-124G > C) in promoter region was reported in previous study. The mean age of GDM women with mutations of HNF-1alpha gene was 34 years. Four women with MODY3 gene mutations required insulin therapy during pregnancy. GDM women with MODY3 gene mutations appeared to be decreased insulin secretion (HOMA-%B) than those without mutations. CONCLUSIONS: We have found the existence of MODY3 as well as novel HNF-1alpha gene mutations in Korean women with GDM.

The Prevalence of Maturity Onset Diabetes of the Young(MODY) 3 in Children with Type 2 Diabetes Mellitus / 소아과

PURPOSE: Maturity-onset diabetes of the young(MODY) is a subtype of type 2 diabetes defined by autosomal dominant mode of inheritance, onset of diabetes usually before the age of 25 yrs, and a primary defect in the function of the beta cells of the pancreas. MODY3 is known as the most common form and is caused by mutations in hepatocyte nuclear factor(HNF)-1alpha. We examined the prevalence of MODY3 in children with type 2 diabetes mellitus(DM). METHODS: Children with type 2 DM(N=17) and their family members with type 2 DM(N=5) were enrolled. Inclusion criteria for the children were fasting C-peptide and postprandial C-peptide more than 1.0 ng/mL and 1.5 ng/mL respectively, familial type 2 DM in at least two generations, and body mass index(BMI)(kg/m(2)) less than 95th percentile. Genomic DNA was extracted from blood samples. We analyzed HNF-1alpha for mutation by DNA microarray method and direct sequencing. RESULTS: We found one case with a mutation of the promoter region of HNF-1alpha(5'-ctaGGCTAGTGGGGTTTTGCGGGGGCAGTGGGTGCAAGG-3') in one child's family member among 22 children and adult subjects with type 2 DM. CONCLUSION: Although we found a mutation of HNF-1alpha in an adult family member with type 2 DM, we did not find this mutation in a child with type 2 DM. The further investigation of MODY in children, including other types, is required.

HNF1 and/or HNF3 may contribute to the tissue specific expression of glucokinase gene

A possible role of hepatocyte nuclear factor 1 (HNF1) or HNF3, a predominant trans-acting factors of hepatic or pancreatic beta-cells, was examined on the tissue specific interdependent expression of glucokinase (GK) in liver, H4IIE, HepG2, HIT-T15 and MIN6 cell line. The tissues or cell lines known to express GK showed abundant levels of HNF1 and HNF3 mRNA as observed in liver, H4IIE, HepG2, HIT-T15 and MIN6 cells, whereas they were not detected in brain, heart, NIH 3T3, HeLa cells. The promoter of glucokinase contains several HNF3 consensus sequences and are well conserved in human, mouse and rat. Transfection of the glucokinase promotor linked with luciferase reporter to liver or pancreatic beta cell lines showed high interacting activities with HNF1 and HNF3, whereas minimal activities were detected in the cells expressing very low levels of HNFs. The binding of HNF1 or HNF3 to the GK promoter genes was confirmed by electrophoretic mobility shift assay (EMSA). From these data, we propose that the expression of HNF1 and/or HNF3 may, in part, contribute to the tissue specific expression of GK.

The polymorphism in hepatic nuclear factor-1? gene and associated plasma insulin concentration / 中国糖尿病杂志

Objective To observe the role of the mild changes in hepatic nuclear factor (HNF)-1? gene in the pathogenesis of type 2 diabetes mellitus. Methods With PCR, denatured gradient gel electrophoresis and DNA sequencing,the genomic DNAs from the blood of type 2 diabetic patients and normal control were analyzed to find the changes in HNF-1? gene. These changes were added up in-groups and compared with plasma insulin concentration. Results Code 459 CTG changed to TTC,codes 489 AGC changed to AAC in the 7th exon;no 7G changed to A in the 7th intron. The 3 point changes were of one kind of polymorphism. Its frequency to normal genotype was 61: 39 in normal control, 56: 44 in type 2 diabetes mellitus. Homozygotes of the polymorphism in the group of type 2 diabetes mellitus had slightly lower plasma insulin concentration, but t-test result was P