Inhibition of fibrotic changes in infrapatellar fat pad alleviates persistent pain and articular cartilage degeneration in monoiodoacetic acid-induced rat arthritis model.

OBJECTIVE: We have reported that fibrotic changes in infrapatellar fat pad (IFP) after acute joint inflammation are closely associated with persistent pain in rats. In this study, to examine the effects of anti-fibrotic treatment on persistent pain, we used C-type natriuretic peptides (CNP) at the recovery phase after acute joint inflammation. DESIGN: Thirty-two male Wistar rats were used in this study. Monoiodoacetic acid (MIA) was injected intra-articularly to induce IFP fibrosis and persistent pain. CNP was injected after acute inflammatory phase in the same knee joint. Time-course pain-avoidance behavior tests and histological analyses were performed to examine the effects of CNP. RESULTS: Histological evaluations indicated that intra-articular injection of CNP inhibited fibrotic changes in IFP after acute inflammation. Incapacitance tests indicated that MIA injection into rat knee joint quickly decreased the percent weight on ipsilateral limb. In the vehicle group, the decrease was maintained up to day 28, suggesting that pain persistence occurred after acute inflammation (Day 0/Day 28, Est Dif -8.15, CI -10.78∼-5.53, Linear mixed-effect model). In contrast, the pain was alleviated in the CNP group after day 14 (Day0/Day 14, -0.51, -2.62-1.59). In addition, we observed significant improvement in the degree of articular cartilage degeneration at day 14 in the CNP group (OARSI score: vehicle 16.14 ± 4.37 vs CNP 6.87 ± 3.44, P < 0.01; Wilcoxon rank sum test). CONCLUSION: Fibrotic changes in IFP may play important roles in both persistent pain and articular cartilage degeneration.

Definition of a Critical Size Osteochondral Knee Defect and its Negative Effect on the Surrounding Articular Cartilage in the Rat.

BACKGROUND: Joint trauma is predisposing to the incidence of osteoarthritis (OA) of the knee. There is a limited knowledge on the impact of posttraumatic osteochondral defects on the whole joint. This study was designed to define a critical size osteochondral defect in the knee of rats and to investigate a possible association between osteochondral defects and degeneration of the surrounding joint surface. METHODS: Cylindrical osteochondral defects of different sizes were created in the knee joint of rats. The natural course of these lesions was studied by macroscopic observation, histology, and immunohistochemistry. Gene expression of the articular cartilage surrounding the defects in vivo and of articular chondrocytes cultured in vitro in IL1ß and fibroblast growth factor 2 (FGF2) supplemented media was evaluated by quantitative polymerase chain reaction (qPCR). RESULTS: In defects of 0.9 mm diameter, spontaneous joint surface healing was observed but also upward advancing of the subchondral bone plate at 16 weeks. Larger 1.4 mm diameter defects were critical size, not resulting in successful healing at any time point. Importantly, the articular cartilage surrounding the defects expressed FGF2 and IL1ß, but not ACAN and Col2. Chondrocytes cultured in IL1ß and FGF2 supplemented media lost the natural fibroblast growth factor receptors - FGFr1/FGFr3 balance and showed decreased viability. CONCLUSIONS: A critical size osteochondral defect was defined as 1.4 mm in diameter in rat. Subchondral bone plate advancement occured rapidly. The articular cartilage surrounding osteochondral defects showed catabolic activity with expression of IL1ß, FGF2 and a disturbed FGFr1/FGFr3 balance, potentially initiating a process of early osteoarthritic disease.

A Distinct Subpopulation of Bone Marrow Mesenchymal Stem Cells, Muse Cells, Directly Commit to the Replacement of Liver Components.

Genotyping graft livers by short tandem repeats after human living-donor liver transplantation (n = 20) revealed the presence of recipient or chimeric genotype cases in hepatocytes (6 of 17, 35.3%), sinusoidal cells (18 of 18, 100%), cholangiocytes (15 of 17, 88.2%) and cells in the periportal areas (7 of 8, 87.5%), suggesting extrahepatic cell involvement in liver regeneration. Regarding extrahepatic origin, bone marrow mesenchymal stem cells (BM-MSCs) have been suggested to contribute to liver regeneration but compose a heterogeneous population. We focused on a more specific subpopulation (1-2% of BM-MSCs), called multilineage-differentiating stress-enduring (Muse) cells, for their ability to differentiate into liver-lineage cells and repair tissue. We generated a physical partial hepatectomy model in immunodeficient mice and injected green fluorescent protein (GFP)-labeled human BM-MSC Muse cells intravenously (n = 20). Immunohistochemistry, fluorescence in situ hybridization and species-specific polymerase chain reaction revealed that they integrated into regenerating areas and expressed liver progenitor markers during the early phase and then differentiated spontaneously into major liver components, including hepatocytes (≈74.3% of GFP-positive integrated Muse cells), cholangiocytes (≈17.7%), sinusoidal endothelial cells (≈2.0%), and Kupffer cells (≈6.0%). In contrast, the remaining cells in the BM-MSCs were not detected in the liver for up to 4 weeks. These results suggest that Muse cells are the predominant population of BM-MSCs that are capable of replacing major liver components during liver regeneration.

Non-Hodgkin lymphoma of the female genital tract mimicking primary gynecological tumors: a single-center series of 3 cases.

INTRODUCTION: Malignant lymphoma of the female genital tract is quite rare and its presentation may resemble that of other, more common tumors, causing confusion for clinicians. CASE HISTORY: The authors report three patients with a non-Hodgkin lymphoma (NHL) involving the female genital tract: two cases involved the ovary and one involved the uterus. In all patients, the genital tract was the initial site of clinical presentation of a B cell lymphoma. One patient was diagnosed postoperatively and subsequently received chemotherapy; the other two patients were diagnosed by imaging-guided biopsy and were successfully managed by chemotherapy without resection surgery. Two patients were alive, without evidence of disease, and one patient was alive with disease at their most recent follow-up visit. CONCLUSION: The authors' experience emphasizes that lymphoma should be in the differential diagnosis of pelvic gynecological malignancies, and its clinical, biological, and radiological signs must be actively sought. Imaging-guided biopsy should be performed to avoid unnecessary surgery.

Therapy-related myelodysplastic syndrome and acute myeloid leukemia following chemotherapy (paclitaxel and carboplatin) and radiation therapy in ovarian cancer: a case report.

In recent years, the incidence of therapy-related myelodysplastic syndrome (t-MDS) and therapy-related acute myeloid leukemia (t-AML) that occur during chemotherapy for ovarian cancer has increased. While alkylating agents and topoisomerase II inhibitors are particularly mutagenic and have strong leukemogenic potential, paclitaxel and combination chemotherapy/radiation therapy also appear to induce t-MDS. The present authors report a case of t-MDS that developed during chemotherapy and radiation therapy for ovarian cancer. The patient was a 75-year-old woman who received six courses of cyclophosphamide/doxorubicin/cisplatin (CAP) therapy after initial surgery for Stage IIIc grade ovarian cancer in 1995. Beginning in February 2005, the patient experienced multiple recurrences due to sternal metastasis. Chemotherapy, including paclitaxel and carboplatin (TC), was administered intermittently and was combined with radiation therapy to a sternal metastatic lesion. Pancytopenia was observed in December 2008, and she was diagnosed with t-MDS (WHO subtype, refractory cytopenias with multilineage dysplasia [RCMD]): the time from first chemotherapy to t-MDS onset was 106 months. Without evidence of blast crisis, the recurrent lesions continued to grow and caused multiple cerebral infarctions, from which she eventually died. The cumulative doses of paclitaxel and carboplatin administered to this patient were 1,968 mg and 6,480 mg, respectively.

Perivascular epithelial cell tumor arising from polypoid adenomyoma: a case report.

The present report describes a rare case of a uterine perivascular epithelioid cell tumor (PEComa) arising from a polypoid adenomyoma. The patient, a 44-year-old woman with tuberous sclerosis, was incidentally found to have a uterine mass with malignant-appearing features on a computed tomography (CT) scan. Pathological examination of the hysterectomy specimen demonstrated that the tumor was composed of pale, spindle-shaped, epithelioid tumor cells which were positive for SMA and HMB-45. These findings were consistent with a PEComa arising from a polypoid adenomyoma.

Surgical treatment outcomes of serious chronic tubo-ovarian abscess: a single-center series of 20 cases.

In recent years, Shimane University Hospital has begun to see patients with pelvic inflammatory disease (PID) which has become severe and chronic after insufficient conservative treatment in primary or secondary medical care facilities. Serious chronic tubo-ovarian abscess (TOA) is complicated by intraperitoneal inflammatory adhesions to surrounding organs, so that it is difficult to determine the original anatomical position of organs at surgery. Forcible synechotomy can result in damage to the adhering organs and insufficient drainage after surgery can cause recurrence of inflammation. In order to increase the chances for a successful surgical treatment, careful preparation, such as preoperative administration of antibiotics and ureteral stent insertion are necessary. In addition, the chances for recurrence of inflammation can be lessened by thorough intraperitoneal irrigation and insertion of a drainage tube.

Inferior Healing Rate in Isolated Meniscal Repair than that in Meniscal Repair with Concomitant ACL Reconstruction Evaluated with MRI.

Introduction: Isolated meniscal repair has been suggested as one of the contributing factors in unhealed meniscal repair. The purpose of this study was to compare the healing rate between isolated meniscal repair and meniscal repair with concomitant anterior cruciate ligament reconstruction (ACLR) using a standardised assessment method after propensity score matching. Materials and methods: Accuracy of the Crues' grading system for meniscal healing was validated using second-look arthroscopy as the reference standard in 17 patients. Propensity score matching (one-to-one) was performed between 26 patients who underwent isolated meniscal repair and 98 patients who underwent meniscal repair with concomitant ACLR. Patients were matched for sex, age, side and zone of the meniscal repair, and number of sutures. Healing rates at one year which were evaluated with magnetic resonance imaging (MRI) were compared between the two groups. Results: The sensitivity and specificity of the Crues' grading system on multiple plane MRI for meniscal healing were 100% and 83.3%, respectively. Both the isolated meniscal repair group and the meniscal repair with concomitant ACLR group included 21 patients after propensity score matching. Baseline characteristics did not differ significantly between the two groups. The healing rate was significantly lower in the isolated meniscal repairs group (14.3%) than in the meniscal repair concomitant with ACLR group (47.6%, P=0.04). Conclusion: The healing rate for isolated meniscal repair using a standardised MRI assessment method was inferior to that of meniscal repair with concomitant ACLR after propensity score matching.

Fatty acid synthase expression associated with NAC1 is a potential therapeutic target in ovarian clear cell carcinomas.

BACKGROUND: This study examined the clinical significance of NAC1 and the expression level of its potential downstream target fatty acid synthase (FASN) in ovarian clear cell carcinomas (OCCCs), and evaluated the NAC1/FASN pathway as a potential therapeutic target. METHODS: NAC1 and FASN expression and NACC1 gene amplification were assessed in ovarian cancers by immunohistochemistry, fluorescence in situ hybridisation, and clinical data collected by a retrospective chart review. C75, a FASN inhibitor, was used to assess whether this pathway represented a therapeutic target in OCCC. RESULTS: High NAC1 expression was most frequent in clear cell tumours (40.0%:24/60). NACC1 gene amplification was identified in none of the 58 OCCCs. The frequency of NACC1 gene amplification was significantly higher in the high-grade serous histology than in the clear cell histology (P<0.01). NAC1 expression was significantly correlated with FASN expression in both OCCC samples and OCCC cell lines. Either high NAC1 expression or high FASN expression significantly correlated with shorter progression-free and overall survival (P=0.002 and 0.0048). NAC1 overexpression stimulated FASN expression, and NAC1 silencing using siRNA decreased FASN expression in OCCC cell lines. Profound growth inhibition was observed in C75-treated carcinoma cells with FASN overexpression when compared with the response in carcinoma cells without FASN expression. CONCLUSION: These findings indicate that NAC1/FASN overexpression is critical to the growth and survival of a subset of OCCC. The FASN silencing by the C75-induced phenotypes depends on the expression status of the targeted cell line. Therefore, NAC1/FASN pathway-targeted therapy may benefit selected OCCC patients.

Enlarged Virchow's node as an initial complaint of serous ovarian adenocarcinoma.

BACKGROUND: An enlarged Virchow's node or left supraclavicular lymph node is a classic precursor to the diagnosis of metastatic cancer, usually originating from an abdominal organ. It is rarely found in ovarian carcinoma. CASE REPORT: A 49-year-old woman presented a painless mass in her left supraclavicular fossa. A histopathological examination of the same mass was consistent with a serous adenocarcinoma of ovarian origin. The patient was initially asymptomatic, even with the disease in an advanced stage. Left supraclavicular adenopathy has not been previously reported as a presenting complaint of ovarian carcinoma. CONCLUSION: Ovarian carcinoma in contemporary with a Virchow's node is an isolated finding.

Incidence and risk factors of falling in ambulatory patients with rheumatoid arthritis: a prospective 1-year study.

UNLABELLED: A prospective 1-year study showed that fall incidence was 50% in women with rheumatoid arthritis. Multivariate analysis identified swollen joint count, use of antihypertensives or diuretics, one-leg standing time, and sway area measured by stabilometer as significant parameters associated with falls. INTRODUCTION: Patients with rheumatoid arthritis (RA) may be at increased risk of falling because they frequently experience muscle weakness and stiff or painful joints. The aim of this study was to use a prospective design to determine the incidence of falls and their risk factors in women with RA. METHODS: Eighty-four women aged 50 and over who had RA were enrolled. The mean age was 64.1 years. We evaluated postural stability, physical performance related to falls, disease activity, muscle volume, and bone density. The occurrence of falls was assessed every month for 1 year. Among 84 patients, 80 completed a 1-year observation. RESULTS: Forty patients (50.0%) reported one or more falls, and two of them (5.0%) had fractures during the follow-up period. The fall group had more swollen joints and took more antihypertensives and/or diuretics. The fall group also had lower postural stability and tended to have reduced physical performance. The one-leg standing time was shorter, and the step-up-and-down test score was lower in the fall group. The sway area was larger in the fall group. DISCUSSION: Multiple logistic regression analysis identified that number of swollen joints, use of antihypertensives or diuretics, shorter time standing on one foot, and the sway area were the most significant parameters associated with falls. CONCLUSION: We concluded that fall rates in RA patients were higher than in the general population and that balance impairment or side effects of drugs may play a role in increasing the risk of falls.

Enhanced inhibitory effects of TBT chloride on the development of F1 rats.

Neurotoxicity is one of the major effects of tributyltin (TBT). The effects on the next generation of F(1) rats exposed to TBT via the placenta and their dams' milk may be stronger than those on adults. Pregnant Wister rats were exposed to TBT at 0 and 125 ppm in their food. Half of the female F(1) rats in both groups were exposed to TBT at 125 ppm in their food from 9 to 15 weeks of age. Female F(1) rats were divided into the following groups: the control-control (CC) group, with no exposure; the TBT-control (TC) group, exposed to TBT via the placenta and their dams' milk; the control-TBT (CT) group, exposed to TBT via their food from 9 to 15 weeks of age; and the TBT-TBT (TT) group, exposed to TBT via the placenta, their dams' milk, and their food (n = 10/group). After administration, an open-field test and prepulse inhibition (PPI) test were performed at 15 weeks of age. The mean body weights of the TC and TT groups were significantly lower than that of the CC group from 9 to 15 weeks of age. The mean relative thymus weight of the TC and TT groups was significantly lower than that of the CC group. In the open-field test, a marked decrease in the total locomotion distance was observed in the TT group. The mean values in the TT and TC groups were significantly lower than that in the CC group. For the locomotion distance between 15 and 20 min, the mean values in the CT, TC, and TT groups were significantly lower than that in the CC group. The mean locomotor distance between 25 and 30 min in the TT group was significantly lower than that in the CC and TC groups. The mean values of instances of wall rearing in the TC, CT, and TT groups were significantly lower than that in the CC group. The mean value of face washing or body washing in the TT group was significantly lower than that in the CT group. There were no significant differences in indexes of the PPI test. Exposure to TBT via the placenta and their dams' milk inhibited the development of F(1) rats, which continued after weaning. Inhibition of the rats' activity induced by exposure to TBT via the placenta and their dams' milk and/or via their food was suggested. The effects were most evident in the TT group.

Measurement of human trabecular bone by novel ultrasonic bone densitometry based on fast and slow waves.

SUMMARY: Two longitudinal transmitted waves, fast and slow waves, were observed by employing a new quantitative ultrasound (QUS) method. The trabecular bone measurements generated by this method reflect three-dimensional structural information, and the new QUS parameters were able to identify vertebral fractures. INTRODUCTION: The aims were to identify new quantitative ultrasound (QUS) parameters that based on new QUS method reflecting not only bone volume but also the microstructures of trabecular bone ex vivo and to observe how much they predict fracture risk in vivo. METHODS: Ex vivo measurement: Three human femoral heads were used for the experiment. Attenuation of the slow wave, attenuation of the fast wave, speed of the slow wave, speed of the fast wave (SOFW), bone mass density of trabecular bone, and elastic modulus of the trabecular bone (EMTb) of each specimen were obtained using a new QUS method and compared with three-dimensional structural parameters measured by micro-computed tomography. In vivo measurement: Eighty-nine volunteers were enrolled, and the bone status in the distal radius was measured using a new QUS method. These parameters were compared with data evaluated by peripheral quantitative computed tomography and dual X-ray absorptiometry. RESULTS: Ex vivo measurement: SOFW and EMTb showed correlations with the parameter of trabecular anisotropy. In vivo measurement: The new QUS parameters were able to identify vertebral fractures. CONCLUSION: The newly developed QUS technique reflects the three-dimensional structure and is a promising method to evaluate fracture risk.

Recent trends in the incidence and lifetime risk of hip fracture in Tottori, Japan.

SUMMARY: Hip fracture incidence from 2004 to 2006 in the Tottori prefecture of Japan was investigated and compared with previously reported rates. The age- and gender-specific incidence of hip fracture in the Tottori prefecture has not plateaued, as has been reported for populations in Northern Europe or North America. INTRODUCTION: Recent data from Northern Europe and North America indicate that the incidence of hip fracture has plateaued, whereas most reports from Asia indicate that the incidence is increasing. The aims of this study were to investigate the recent incidence of hip fracture in the Tottori prefecture, Japan, and to compare it with previous reports. METHODS: All hip fractures in patients aged 35 years and older occurring between 2004 and 2006 were surveyed in all of the hospitals from the Tottori prefecture. The age- and gender-specific incidence rates were then calculated. Using these and previously reported data, the estimated number of hip fracture patients was determined using the age- and gender-specific incidence rates in each year from 1986 to 2006. RESULTS: The survey identified 851, 906, and 1,059 patients aged 35 years and older, in 2004, 2005, and 2006 respectively. The residual lifetime risk of hip fracture for individuals at 50 years of age was estimated to be 5.6% for men and 20.0% for women. The estimated number of patients from 1986 to 2006 showed a significant increase over time for both genders. CONCLUSIONS: The age- and gender-specific incidence of hip fracture in the Tottori prefecture, Japan has not plateaued for either gender.

Reciprocal role of ERK and NF-kappaB pathways in survival and activation of osteoclasts.

To examine the role of mitogen-activated protein kinase and nuclear factor kappa B (NF-kappaB) pathways on osteoclast survival and activation, we constructed adenovirus vectors carrying various mutants of signaling molecules: dominant negative Ras (Ras(DN)), constitutively active MEK1 (MEK(CA)), dominant negative IkappaB kinase 2 (IKK(DN)), and constitutively active IKK2 (IKK(CA)). Inhibiting ERK activity by Ras(DN) overexpression rapidly induced the apoptosis of osteoclast-like cells (OCLs) formed in vitro, whereas ERK activation after the introduction of MEK(CA) remarkably lengthened their survival by preventing spontaneous apoptosis. Neither inhibition nor activation of ERK affected the bone-resorbing activity of OCLs. Inhibition of NF-kappaB pathway with IKK(DN) virus suppressed the pit-forming activity of OCLs and NF-kappaB activation by IKK(CA) expression upregulated it without affecting their survival. Interleukin 1alpha (IL-1alpha) strongly induced ERK activation as well as NF-kappaB activation. Ras(DN) virus partially inhibited ERK activation, and OCL survival promoted by IL-1alpha. Inhibiting NF-kappaB activation by IKK(DN) virus significantly suppressed the pit-forming activity enhanced by IL-1alpha. These results indicate that ERK and NF-kappaB regulate different aspects of osteoclast activation: ERK is responsible for osteoclast survival, whereas NF-kappaB regulates osteoclast activation for bone resorption.

Advancing osteochondral tissue engineering: bone morphogenetic protein, transforming growth factor, and fibroblast growth factor signaling drive ordered differentiation of periosteal cells resulting in stable cartilage and bone formation in vivo.

BACKGROUND: Chondrogenic mesenchymal stem cells (MSCs) have not yet been used to address the clinical demands of large osteochondral joint surface defects. In this study, self-assembling tissue intermediates (TIs) derived from human periosteum-derived stem/progenitor cells (hPDCs) were generated and validated for stable cartilage formation in vivo using two different animal models. METHODS: hPDCs were aggregated and cultured in the presence of a novel growth factor (GF) cocktail comprising of transforming growth factor (TGF)-ß1, bone morphogenetic protein (BMP)2, growth differentiation factor (GDF)5, BMP6, and fibroblast growth factor (FGF)2. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to study in vitro differentiation. Aggregates were then implanted ectopically in nude mice and orthotopically in critical-size osteochondral defects in nude rats and evaluated by microcomputed tomography (µCT) and immunohistochemistry. RESULTS: Gene expression analysis after 28 days of in vitro culture revealed the expression of early and late chondrogenic markers and a significant upregulation of NOGGIN as compared to human articular chondrocytes (hACs). Histological examination revealed a bilayered structure comprising of chondrocytes at different stages of maturity. Ectopically, TIs generated both bone and mineralized cartilage at 8 weeks after implantation. Osteochondral defects treated with TIs displayed glycosaminoglycan (GAG) production, type-II collagen, and lubricin expression. Immunostaining for human nuclei protein suggested that hPDCs contributed to both subchondral bone and articular cartilage repair. CONCLUSION: Our data indicate that in vitro derived osteochondral-like tissues can be generated from hPDCs, which are capable of producing bone and cartilage ectopically and behave orthotopically as osteochondral units.

Expression of the GLUT1 glucose transporter increases thymidine uptake in Chinese hamster ovary cells at low glucose concentrations.

An increase in expression of the GLUT1 glucose transporter gene has been observed to be associated with an increase in glucose transport activity upon oncogenic transformation of the cells. Increased expression of this glucose transporter isoform has been also observed in fetal tissues. To investigate the consequences of this phenomenon on cellular metabolism and cell growth, an expression vector containing the GLUT1 glucose transporter complementary DNA was transfected into Chinese hamster ovary cells. Overexpression of this glucose transporter isoform resulted in an increase in not only glucose uptake and utilization but also thymidine uptake when cells were exposed to glucose-deficient conditions. This increase in glucose metabolism and DNA synthesis may play an important role on the growth and/or survival of cancer cells and fetal tissues.

Insulin prevents cardiomyocytes from oxidative stress-induced apoptosis through activation of PI3 kinase/Akt.

BACKGROUND: Loss of cardiomyocytes by apoptosis is proposed to cause heart failure. Reactive oxygen species induce apoptosis in many types of cells including cardiomyocytes. Because insulin has been reported to have protective effects, we examined whether insulin prevents cardiomyocytes from oxidative stress-induced apoptotic death. METHODS AND RESULTS: Cultured cardiomyocytes of neonatal rats were stimulated by hydrogen peroxide (H(2)O(2)). Apoptosis was evaluated by means of the TUNEL method and DNA laddering. Incubation with 100 micromol/L H(2)O(2) for 24 hours increased the number of TUNEL-positive cardiac myocytes (control, approximately 4% versus H(2)O(2), approximately 23%). Pretreatment with 10(-)(6) mol/L insulin significantly decreased the number of H(2)O(2)-induced TUNEL-positive cardiac myocytes (approximately 12%) and DNA fragmentation induced by H(2)O(2). Pretreatment with a specific phosphatidylinositol 3 kinase (PI3K) inhibitor, wortmannin, and overexpression of dominant negative mutant of PI3K abolished the cytoprotective effect of insulin. Insulin strongly activated both PI3K and the putative downstream effector AKT: Moreover, a proapoptotic protein, BAD:, was significantly phosphorylated and inactivated by insulin through PI3K. CONCLUSIONS: These results suggest that insulin protects cardiomyocytes from oxidative stress-induced apoptosis through the PI3K pathway.

Peptide-based radioimmunoassay specific for GLUT1 glucose transporter.

A radioimmunoassay for the GLUT1 glucose transporter was developed with a synthesized peptide based on the sequence of the cDNA for GLUT1. A peptide corresponding to the COOH-terminal domain of the GLUT1 glucose transporter (Thr-Pro-Glu-Glu-Leu-Phe-His-Pro-Leu-Gly-Ala-Asp-Ser-Gln-Val) was synthesized and conjugated to keyhole limpet hemocyanin through the NH2-terminal of the peptide. An antibody was raised against this complex and affinity purified with the immobilized peptide. A second peptide, with tyrosine residue added to the NH2-terminal of the above peptide, was synthesized and used as a standard and iodinated for preparation of the radioactive ligand. The assay is highly reproducible, sensitive, and specific for the COOH-terminal domain of the GLUT1 glucose transporter. It has no cross-reactivity with the other glucose-transporter isoforms GLUT2 and GLUT4. Furthermore, the results obtained with this radioimmunoassay on the number of glucose transporters in human erythrocytes were in good agreement with previous studies based on cytochalasin B binding, suggesting that this radioimmunoassay is able to quantify the number of glucose transporters. The assay is completed within 4 h and can be used for simultaneous measurement of GLUT1 in many samples. In addition, it can be applied to the measurement of GLUT1 in several types of tissue.

Upregulation of GLUT2 mRNA by glucose, mannose, and fructose in isolated rat hepatocytes.

Previously, demonstrated that GLUT2 mRNA and protein are increased in liver of streptozocin-induced diabetic rats. To examine the mechanisms whereby GLUT2 mRNA is regulated, we cultured isolated hepatocytes in the absence and presence of various concentrations of glucose. Culture of hepatocytes in high glucose concentration (27.8 mM) for 20 h induced a 3.2-fold increase in GLUT2 mRNA levels compared with hepatocytes cultured without D-glucose. Interestingly, D-mannose and D-fructose could substitute for D-glucose to elevate the GLUT2 mRNA level, whereas 3-O-methyl-D-glucose, 2-deoxy-D-glucose, and sucrose, which were not metabolized or taken up by the cells, were without effect. Insulin had no significant effect on GLUT2 mRNA levels in hepatocytes in the presence or absence of D-glucose. Therefore, the regulation of the GLUT2 gene by D-glucose in hepatocytes is contrary to that reported for GLUT1 and GLUT4 genes, which are downregulated by D-glucose. These results also suggest that the elevated GLUT2 mRNA level observed in diabetic rat liver is due to the high blood glucose concentration rather than to insulin deficiency.