A Journey to Understand Glucose Homeostasis: Starting from Rat Glucose Transporter Type 2 Promoter Cloning to Hyperglycemia

My professional journey to understand the glucose homeostasis began in the 1990s, starting from cloning of the promoter region of glucose transporter type 2 (GLUT2) gene that led us to establish research foundation of my group. When I was a graduate student, I simply thought that hyperglycemia, a typical clinical manifestation of type 2 diabetes mellitus (T2DM), could be caused by a defect in the glucose transport system in the body. Thus, if a molecular mechanism controlling glucose transport system could be understood, treatment of T2DM could be possible. In the early 70s, hyperglycemia was thought to develop primarily due to a defect in the muscle and adipose tissue; thus, muscle/adipose tissue type glucose transporter (GLUT4) became a major research interest in the diabetology. However, glucose utilization occurs not only in muscle/adipose tissue but also in liver and brain. Thus, I was interested in the hepatic glucose transport system, where glucose storage and release are the most actively occurring.

Role of transcription factor acetylation in the regulation of metabolic homeostasis

Post-translational modifications (PTMs) of transcription factors play a crucial role in regulating metabolic homeostasis. These modifications include phosphorylation, methylation, acetylation, ubiquitination, SUMOylation, and O-GlcNAcylation. Recent studies have shed light on the importance of lysine acetylation at nonhistone proteins including transcription factors. Acetylation of transcription factors affects subcellular distribution, DNA affinity, stability, transcriptional activity, and current investigations are aiming to further expand our understanding of the role of lysine acetylation of transcription factors. In this review, we summarize recent studies that provide new insights into the role of protein lysine-acetylation in the transcriptional regulation of metabolic homeostasis.

Modulation of the Transcriptional Activity of Peroxisome Proliferator-Activated Receptor Gamma by Protein-Protein Interactions and Post-Translational Modifications

Peroxisome proliferator-activated receptor gamma (PPARgamma) belongs to a nuclear receptor superfamily; members of which play key roles in the control of body metabolism principally by acting on adipose tissue. Ligands of PPARgamma, such as thiazolidinediones, are widely used in the treatment of metabolic syndromes and type 2 diabetes mellitus (T2DM). Although these drugs have potential benefits in the treatment of T2DM, they also cause unwanted side effects. Thus, understanding the molecular mechanisms governing the transcriptional activity of PPARgamma is of prime importance in the development of new selective drugs or drugs with fewer side effects. Recent advancements in molecular biology have made it possible to obtain a deeper understanding of the role of PPARgamma in body homeostasis. The transcriptional activity of PPARgamma is subject to regulation either by interacting proteins or by modification of the protein itself. New interacting partners of PPARgamma with new functions are being unveiled. In addition, post-translational modification by various cellular signals contributes to fine-tuning of the transcriptional activities of PPARgamma. In this review, we will summarize recent advancements in our understanding of the post-translational modifications of, and proteins interacting with, PPARgamma, both of which affect its transcriptional activities in relation to adipogenesis.

The functional role of the CARM1-SNF5 complex and its associated HMT activity in transcriptional activation by thyroid hormone receptor

We have investigated the function and mechanisms of the CARM1-SNF5 complex in T3-dependent transcriptional activation. Using specific small interfering RNAs (siRNA) to knock down coactivators in HeLa alpha2 cells, we found that coactivator associated arginine methyltransferase 1 (CARM1) and SWI/SNF complex component 5 (SNF5) are important for T3-dependent transcriptional activation. The CARM1- SWI/SNF chromatin remodeling complex serves as a mechanism for the rapid reversal of H3-K9 methylation. Importantly, siRNA treatment against CARM1 and/or SNF5 increased the recruitment of HMTase G9a to the type 1 deiodinase (D1) promoter even with T3. Knocking- down either CARM1 or SNF5 also inhibited the down- regulation of histone macroH2A, which is correlated with transcriptional activation. Finally, knocking down CARM1 and SNF5 by siRNA impaired the association of these coactivators to the D1 promoter, suggesting functional importance of CARM1- SNF5 complex in T3-dependent transcriptional activation.

Identification and characterization of peroxisome proliferator response element in the mouse GLUT2 promoter

In the present study, we show that the expression of type 2 glucose transporter isoform (GLUT2) could be regulated by PPAR-gamma in the liver. Rosiglitazone, PPAR-gamma agonist, activated the GLUT2 mRNA level in the primary cultured hepatocytes and Alexander cells, when these cells were transfected with PPAR-gamma/RXR-alpha. We have localized the peroxisome proliferator response element in the mouse GLUT2 promoter by serial deletion studies and site-directed mutagenesis. Chromatin immunoprecipitation assay using ob/ob mice also showed that PPAR-gamma rather than PPAR-alpha binds to the -197/-184 region of GLUT2 promoter. Taken together, liver GLUT2 may be a direct target of PPAR-gamma ligand contributing to glucose transport into liver in a condition when PAPR-gamma expression is increased as in type 2 diabetes or in severe obesity.

C/EBP binding activity to site F of the rat GLUT2 glucose transporter gene promoter is attenuated by c-Jun in vitro

The expression of the GLUT2 glucose transporter gene in liver is suppressed in cultured hepatoma cell lines and primary cultured hepatocytes. Earlier report showed that CCAAT/enhancer binding protein (C/EBP) regulates the promoter activity of the rat GLUT2 glucose transporter gene in liver cells. C/EBPa and C/EBPb activated the promoter activity by binding to at least two regions of the promoter and one of the C/EBP binding sites, named as site F, also has the AP-1 binding consensus. In this study, we investigated whether the AP-1 can influence on C/EBP binding to this site. The addition of recombinant c-Jun protein with liver extract caused the attenuation of C/EBP binding to site F with the appearance of a new shifted band. The shifted band was competed out with the addition of unlabeled AP-1 consensus oligonucleotide, indicating that c-Jun also can bind to site F. Another C/EBP site on GLUT2 promoter, site H, did not bind AP-1. Analysis of the DNA-protein complex revealed that C/EBP and c-Jun bind to site F in mutually exclusive manner rather than form heterodimeric complex with each other. From these results, it is suggested that the transcriptional activation of C/EBP may be influenced by c-Jun protein in certain status of the liver cells, such as acute phase response, as well as hepatocarcinogenesis.

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.

Protein Transplantation : Its Significance in Biology and Medicine

No abstract available.

Molecular Genetic Analyses of Charcot-Marie-Tooth Disease Type 1A in Korean

BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A) is an autosomal dominant inherited demyelinating peripheral neuropathy characterized by progressive distal muscular atrophy and marked slowing of nerve conduction velocities. A 1.5 Mb DNA duplication within chromosome 17p11.2-p12 has been reported. This disease appears to be caused by an altered copy number of the PMP-22 gene within the critical region. METHODS: DNA analysis was carried out for 158 persons from 40 unrelated families. PCR was done by D17S122 and D17S261. The DNA of the patients was ana-lyzed to detect three alleles for the presence of duplication. RESULTS: CMT1A duplication was found in 7 families (64%) of the patients with CMT1 by D17S122, but not by D17S261. CONCLUSIONS: We have found seven families of Charcot-Marie-Tooth disease type 1A with chromosome 17p11.2-p12 duplication by D17S122. We recommend the screening test by D17S122 for the detection of CMT1A in Korean because genetic analysis done by D17S261 was not informative.

Molecular Genetic Analyses of Charcot-Marie-Tooth Disease Type 1A in Korean

BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A) is an autosomal dominant inherited demyelinating peripheral neuropathy characterized by progressive distal muscular atrophy and marked slowing of nerve conduction velocities. A 1.5 Mb DNA duplication within chromosome 17p11.2-p12 has been reported. This disease appears to be caused by an altered copy number of the PMP-22 gene within the critical region. METHODS: DNA analysis was carried out for 158 persons from 40 unrelated families. PCR was done by D17S122 and D17S261. The DNA of the patients was ana-lyzed to detect three alleles for the presence of duplication. RESULTS: CMT1A duplication was found in 7 families (64%) of the patients with CMT1 by D17S122, but not by D17S261. CONCLUSIONS: We have found seven families of Charcot-Marie-Tooth disease type 1A with chromosome 17p11.2-p12 duplication by D17S122. We recommend the screening test by D17S122 for the detection of CMT1A in Korean because genetic analysis done by D17S261 was not informative.

KAL Gene and GnRH Receptor Gene Analysis in Patients with Kallmann's Syndrome / 대한내분비학회지

BACKGROUND: Kallmann's syndrome is related to the defect in migration of olfactory neuron and GnRH neuron from the olfactory placode to the brain and it represents hypogonadism with anosmia or hyposmia. There are 3 modes of transmission in Kallmann's syndrome: X-linked, autosomal recessive and autosomal dominant. X-linked form is the most common. KAL gene is responsible for the X-linked form of Kallmann's syndrome and it had been localized to Xp22.3. The intron-exon organization had been determined and KAL gene mutation had also been identified in familial Kallmann's syndrome and it is very rare and shows heterogeneity. Furthermore, in the sporadic cases, KAL gene mutation is more rare. METHODS: In order to investigate the KAL gene mutation and the regulation of the gene expression in Kallmann's syndrome, we examined genomic DNA of 35 patients with sporadic Kallmann's syndrome. In the exon 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 of KAL gene and 1, 2, 3 of GnRH receptor gene, the mutations were analyzed by PCR-based DNA sequencing. RESULTS: In our study, the mutation of KAL gene and GnRH receptor gene was not identified in the studied exons that were known as preferable sites of the mutation. CONCLUSION: The mutation of KAL gene and GnRH receptor gene is rare, and it might be needed to investigate mutations in other genes or in other part of the KAL gene such as intron or promoter region.

A mechanism of differential expression of GLUT2 in hepatocyte and pancreatic beta-cell line

DNase I footprinting assay using liver nuclear extracts revealed six protected regions between nucleotide -600 and +110 and hence named Box I-VI. Upstream promoter element (UPE), a DNA element playing crucial role in transcriptional control of the tissue specific expression of pancreatic beta-cell, has been detected within the proximal region of rat GLUT2 promoter. This region is included in Box VI. The protein-DNA interaction in this region (Box VI) was confirmed by mobility shift assay using liver nuclear extracts. Deletion of the region between -585 bp and -146 bp resulted in dramatic changes in promoter activity when they were expressed in liver and beta-cell derived cell line. When -585/-146 construct was expressed in liver, the activity was decreased to 46%, whereas the activity in beta-cell line, HIT-T15 cell, was increased by 84% when compared to -146/+190 construct. These opposing phenomena can be explained by the fact that beta-cell specifically expresses the UPE binding protein. Assuming that there may be Box VI-binding protein playing negative roles both in hepatocyte and beta-cell, and that the protein acts as a negative regulator of GLUT2 gene, the UPE binding protein in the beta-cell may overcome the inhibition by binding to the protein.

A Case of Henoch-Schonlein Purpura with Epididymitis

Small cell neuroendocrine carcinoma of the uterine cervix is a distinct subtype of cervical cancer that appears analogous to oat cell carcinoma and carcinoid tumors of the lung. It has been assumed to be derived from the neural crest via argyrophilic cells in the normal endocervix. We have recently encountered a case of small cell neuroendocrine carcinoma of the uterine cervix coexisting with adenocarcinoma which was argyrophil negative. A 66-year-old multiparous woman was admitted because of vaginal bleeding for 2 months. Cervicovaginal smear revealed several scattered clusters and sheets of monotonous small cells with some peripheral palisading in the background of hemorrhage and necrosis. Radical hysterectomy specimen revealed an ulcerofungating tumor on endocervical canal which was composed of two components. Major component of the tumor was made up of monomorphic population of small oval-shaped tumor cells arranged in sheets and partly in acinar structures or trabecular fashion. Other component was adenocarcinoma, endocervical well-differentiated type. Argyrophilia was present on the Grimelius stain and immunohistochemical studies revealed diffuse positivity to neuron-specific enolase and carcinoembryonic antigen. Electron microscopic examination showed clusters of small round to oval cells, which had a few well-formed desmosomes and several membrane-bound, dense-core neuro- sectetory granules.

Antisense GLUT1 RNA suppresses the transforming phenotypes of NIH 3T3 cells transformed by N-Ras

An antisense approach was attempted to investigate the role of antisense GLUT1 RNA in suppressing tumor cell phenotypes using N-ras-transformed NIH 3T3 cells. The established cell line transformed by ras showed typical biological characteristics of cancer cells, such as increased glucose transport, GLUT1 mRNA contents, and the ability to form colonies on the soft agar. In this system, the plasmids (pMAM-GLUT1(rev)) which can transcribe the antisense GLUT1 RNA were transfected and the accompanying changes in the phenotypes of the ras-transformed cells were observed. The expression of antisense GLUT1 RNA by induction with dexamethasone reduced the glucose transport by 30% (1.97 +/- 0.13 nmoles) after 4 min incubation when compared to the non-induction group of transformed cell (2.85 +/- 0.19 nmoles). Also, the number of colonies sized over 50 microns on the soft agar was reduced significantly in the antisense RNA expressing group compared to non-induction group. These results suggest that the expression of antisense GLUT1 RNA reduced the glucose transport and transforming potential in soft agar possibly by hybridization with GLUT1 mRNA in N-ras-transformed NIH 3T3 cells.

Cloning and expression of rat liver type glucose transporter and translocation by insulin in Chinese hamster ovary cells

The 5'- and 3'-side half of liver type glucose transporter (GLUT2) cDNA was amplified from total RNA or mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified 5'-side fragment of GLUT2 cDNA was inserted into pGEM4Z and named pGLGT1, and the 3'-side fragment of GLUT2 cDNA was inserted into the HindIII site of pGLGT1 to construct pGLGT2 which contains an entire open reading frame of GLUT2 cDNA. The GLUT2 cDNA in pGLGT2 was transferred to an eukaryotic expression vector (pMAM) to construct pMLGT, which was expressed in the insulin-sensitive Chinese hamster ovary (CHO) cells. Western blot analysis showed that the GLUT2 gene in pMLGT was expressed in the transfected CHO cells successfully. The GLUT2 content in the plasma membrane fraction of insulin-treated CHO cells expressing GLUT2 increased 3.8-fold compared to that of the control group. This result suggests that GLUT2, which is not subjected to translocation by insulin in the cells of its major distribution, can be translocated if it is expressed in the suitable cells sensitive to insulin action.

A study on the regulation of translocation of glucose transporters during hepatocarcinogenesis induced by 3'-Me DAB

The mechanism of glucose transported (GT) expression on the plasma membranes of hepatoma cells in rats induced by 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) was studied. Cytochalasin B binding to plasma membrane fractions from control and 3'-MeDAB group in the absence of cold cytochalasin B showed 9,825 +/- 925 and 30,165 +/- 625 dpm/mg membrane protein. Scatchard plot analysis showed that the GTs present on the plasma membrane fractions in control and 3'-Me DAB groups were 5.0 and 16.0 pmol/mg membrane protein and their Kd values were 151 and 157 nM, respectively. These results suggest that the numbers of GTs in plasma membrane were increased in the 3'-Me DAB group compared to the control group. In contrast, the amounts of GTs in low density microsomal (LDM) fractions measured by a photoaffinity labeling technique using [3H]-cytochalasin B were 31,207 and 11,702 dpm/mg protein in the control and 3'-Me DAB group, respectively. These results suggest that GTs were translocated from LDM to plasma membranes during carcinogenesis. To confirm these results by an independent method 10% SDS-polyacrylamide gel electrophoresis was carried out. Gel slice No. 13 corresponding to MW of 45 kDa from plasma membrane fractions showed increased radioactivities in the 3'-Me DAB group compared to the control group. However, LDM fractions of the 3'-Me DAB group showed decreased radioactivities compared to the control group. Western blot analysis using anti-human RBC GT antibody present in the plasma membranes and LDM fractions from control and 3'-Me DAB groups did not show any significant difference, indicating low cross-reactivity between them. These results indicate that increased glucose transport seems to be more likely due to reciprocal redistribution of GTs between plasma membrane and LDM fractions.

Anticancer effect of liposome incorporated with methotrexate and antibody against tumor specific surface antigen of rat hepatoma

antibody against tumor specific surface membrane protein was produced by immunizing a New Zealand White rabbit with antigen (66 kDa) prepared from the plasma membrane of rat hepatoma induced by feeding a diet containing 3'-methyl-4-dimethylaminoazobenzene, and was purified by protein A-Sepharose 6MB affinity chromatography. The purified antibody was incorporated into liposomes by a reverse phase evaporation vesicle method in order to prepare a tumor specific anticancer drug carrier. The effect of the antibody against tumor specific antigen was evaluated by comparing the inhibition of DNA synthesis in hepatoma cells with different preparations of methotrexate. Methotrexate encapsulated into liposome showed a stronger inhibitory effect on DNA synthesis (1.4-1.7 times) than free methotrexate. Liposomes having the antibody showed stronger inhibitory effect (3.1 times) on DNA synthesis than free methotrexate group in hepatic nodular area. From these results, it is concluded that tumor specific antibody inserted into liposomal membrane would be recognized by surface antigens which were expressed on the plasma surface membrane of rat hepatoma cells and thereby increase the carrying efficiency of drugs to the target cells. This could be useful in cancer chemotherapy.

Anticancer effect of liposome incorporated with methotrexate and antibody against tumor specific surface antigen of rat hepatoma

antibody against tumor specific surface membrane protein was produced by immunizing a New Zealand White rabbit with antigen (66 kDa) prepared from the plasma membrane of rat hepatoma induced by feeding a diet containing 3'-methyl-4-dimethylaminoazobenzene, and was purified by protein A-Sepharose 6MB affinity chromatography. The purified antibody was incorporated into liposomes by a reverse phase evaporation vesicle method in order to prepare a tumor specific anticancer drug carrier. The effect of the antibody against tumor specific antigen was evaluated by comparing the inhibition of DNA synthesis in hepatoma cells with different preparations of methotrexate. Methotrexate encapsulated into liposome showed a stronger inhibitory effect on DNA synthesis (1.4-1.7 times) than free methotrexate. Liposomes having the antibody showed stronger inhibitory effect (3.1 times) on DNA synthesis than free methotrexate group in hepatic nodular area. From these results, it is concluded that tumor specific antibody inserted into liposomal membrane would be recognized by surface antigens which were expressed on the plasma surface membrane of rat hepatoma cells and thereby increase the carrying efficiency of drugs to the target cells. This could be useful in cancer chemotherapy.

A Study of the Regulation of the Glucose Transporter in the Plasma Membranes of Hepatoma Cells Induced by 3'-Me DAB

5'-nucelotidase and glucose-6-phosphatase are liver plasma and microsomal membranes markers and their respective activities were determined. In the liver homogenate, the activities of 5'-nucleotidase were 0.58 +/- 0.08 and 0.29 +/- 0.07 micromols/mg protein/10min in the control and 3'-methyl-4-dimethyl aminoazobenzene (3'-Me DAB) groups respectively. The enzyme activities m the partially purified plasma membranes were 2.15 +/- 0.25 and 1.31 +/- 0.23 micromols/mg protein/10min in the control and 3'-Me DAB groups respectively. The glucose-6-phosphatase activities in the homogenates of the control and 3'-Me DAB groups were 0.23 +/- 0.10, and 0.45 +/- 0.25 micromols/mg protein/10min, and in the microsomal fraction, 1.14 +/- 0.32, and 0.63 +/- 0.11 micromols/mg protein/10min, respectively, The concentrations of glucose carrier in the plasma membranes from the control and 3'-Me DAB group were 25, and 35 pmols/mg membrane protein, respectively, and the Ka values for cytochalsin B in each group were 5.20 X 109. and 5.14 X 109ml/mol, respectively. However in the microsomal fraction, no significant differences of glucose carrier were found between the control and 3'-Me DAB groups from the DEAE Sephadex A-50 ion exchange chromatography, fractions I and ll were obtained. Electrophoretic analysis of fraction I revealed a major protein band with a molecular weight of 45,000 and minor bands with MWs of 50,000, 55,000 and 15,000. Following AcA 34 gel filtration, a major protein band with a MW of 45,000 was obtained. From these results, it can be concluded that the glucose carrier protein was increased on plasma membrane of hepatoma induced by 3'-Me DAB, and the carrier protein showed similar molecular weight to other glucose carrier found in the RBC, muscle cells and adipocyte.