Intrinsic Factor-vitamin B12 Research Articles

Trans-pacific multicenter collaborative study of minimally invasive proximal versus total gastrectomy for proximal gastric and gastroesophageal junction cancers

BackgroundThe current standard operation for proximal gastric and gastroesophageal junction (P/GEJ) cancers with limited esophageal extension is total gastrectomy (TG). TG is associated with impaired appetite and weight loss due to the loss of gastric functions such as production of ghrelin and with anemia due to intrinsic factor loss and vitamin B12 malabsorption. Theoretically, proximal gastrectomy (PG) can mitigate these problems by preserving gastric function. However, PG with direct esophagogastric reconstruction is associated with severe postoperative reflux, delayed gastric emptying, and poor quality of life (QoL). Minimally invasive PG (MIPG) with antireflux techniques has been increasingly performed by experts but is technically demanding owing to its complexity. Moreover, the actual advantages of MIPG over minimally invasive TG (MITG) with regards to postoperative QoL are unknown. Our overall objective of this study is to determine the short-term QoL benefits of MIPG. Our central hypotheses are that MIPG is safe and that patients have improved appetite after MIPG with effective antireflux techniques, which leads to an overall QoL improvement when compared with MITG.MethodsEnrollment of a total of 60 patients in this prospective survey-collection study is expected. Procedures (MITG versus MIPG, antireflux techniques for MIPG [double-tract reconstruction versus the double-flap technique]) will be chosen based on surgeon and/or patient preference. Randomization is not considered feasible because patients often have strong preferences regarding MITG and MIPG. The primary outcome is appetite level (reported on a 0-10 scale) at 3 months after surgery. With an expected 30 patients per cohort (MITG versus MIPG), this study will have 80% power to detect a one-point difference in appetite level. Patient-reported outcomes will be longitudinally collected (including questions about appetite and reflux), and specific QoL items, body weight, body mass index and ghrelin, albumin, and hemoglobin levels will be compared.DiscussionSurgeons from the US, Japan, and South Korea formed this collaboration with the agreement that the surgical approach to P/GEJ cancers is an internationally important but controversial topic that requires immediate action. At the completion of the proposed research, our expected outcome is the establishment of the benefit and safety of MIPG.Trial registrationThis trial was registered with Clinical Trials Reporting Program Registration under the registration number NCI-2022–00267 on January 11, 2022, as well as with ClinicalTrials.gov under the registration number NCT05205343 on January 11, 2022.

Vitamin B-related Gene Polymorphisms and Cardiovascular Disease.

Hyperhomocysteinemia is an independent risk factor for atherosclerosis, even in early childhood. A mutation in genes that code homocysteine metabolism enzymes or deficiency of specific vitamin cofactors may cause hyperhomocysteinemia. Vitamin B complex has been correlated with serum homocysteine levels. Any abnormality in its metabolism or nutritional deficiency may lead to hyperhomocysteinemia. Both vitamin B complex and homocysteine levels are partly genetically determined. Specifically, the most studied polymorphism is 677T-C in exon 5 of the 5,10- methylenetetrahydrofolate reductase (MTHFR) gene, which plays an important role in folate's metabolism. This polymorphism has been shown to be correlated with hypertension and cardiovascular disease. Polymorphisms in methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L) gene have also been correlated with increased risk for coronary artery disease. Other common serious polymorphisms regard the area with high linkage disequilibrium, including the neuroblastoma breakpoint family, NBPF3 gene, and ~ 12-50 kb upstream of the tissue nonspecific alkaline phosphatase gene. Finally, the polymorphisms which have been mostly associated with vitamin B12 concentration are the rs11254363 polymorphism at intron 52 of the intrinsic factor vitamin B12 receptor of the CUBN and the rs526934 polymorphism at intron 8 of transcobalamin I. To sum up, several polymorphisms have already been associated with vitamin B complexes and therefore homocysteine level, highlighting the complex nature of vitamin B genetics.

Generation of Porcine Ileum Through Spring-Mediated Mechanical Distraction

IntroductionShort bowel syndrome is a devastating gastrointestinal disorder in which decreased bowel length results in inadequate absorption causing nutritional deficiencies. Current treatment options are accompanied by significant morbidity. We have proposed spring-mediated distraction enterogenesis as a method to lengthen bowel with success seen in porcine jejunum. We hypothesize that spring-mediated distraction enterogenesis can be demonstrated in porcine ileum with preservation of ileal structure and function. Materials and methodsLaparotomy was performed on juvenile female mini-Yucatan pigs and a gelatin-encapsulated compressed nitinol spring was inserted into the ileal lumen and affixed proximally and distally. A control segment distal to the spring segment was marked with sutures. Postoperatively, pigs were placed on a liquid diet and euthanized on postoperative day 7. Spring and control segments were measured and processed for immunohistochemistry to evaluate for the presence of vitamin B12–intrinsic factor cotransporter, chromogranin A-producing cells, and 5-HT producing cells. ResultsAll seven pigs survived to postoperative day 7 with no adverse effects. On average, pigs gained 84.3 ± 66.4 g/d. Spring segments lengthened 1.5 ± 0.7 cm with a relative lengthening by 128% ± 56%, which was statistically significant when compared to control (P < 0.01). The average density of chromogranin-A cells in control compared to spring segments was not significantly changed (2.9 ± 1.1 cells/mm versus 3.2 ± 1.2 cells/mm, P = 0.17). Both vitamin B12–intrinsic factor cotransporter and 5-HT producing cells were present in both control and lengthened ileum. ConclusionsIntraluminal nitinol springs significantly lengthened porcine ileum. The increase in density of enteroendocrine cells may indicate enhanced endocrine function of the lengthened ileum.

Metformin-induced vitamin B12 deficiency can cause or worsen distal symmetrical, autonomic and cardiac neuropathy in the patient with diabetes.

Metformin blocks the absorption of vitamin B12 through a mechanism that has not been established but could be because of interference with the calcium-dependent binding of the intrinsic factor vitamin B12 complex to the cubam receptor in the terminal ileum. The subsequent deficiency of vitamin B12 may cause or accelerate distal symmetrical and autonomic neuropathy in the patient with diabetes. Several observational studies and meta-analyses have reported a significant association between metformin utilization and vitamin B12 deficiency. Prospective studies have shown that not only do metformin utilizers have lower vitamin B12 levels but they also have higher frequencies of distal symmetrical polyneuropathy and autonomic neuropathy (including cardiac denervation, which is associated with increased incidences of cardiac arrhythmias, cardiac events and mortality). Therefore, periodic monitoring of vitamin B12 is recommended in all patients who utilize metformin, particularly if metformin has been used for over 5 years at which stage hepatic stores of vitamin B12 would probably be depleted. Factors that accelerate the loss of hepatic vitamin B12 stores are proton pump inhibitors, bariatric surgery, being elderly and having an increased turnover of red blood cells. If serum vitamin B12 levels are borderline, measurement of methylmalonic acid and homocysteine levels can detect vitamin B12 deficiency at its earliest stage. Therapies include prophylactic calcium and vitamin B12 supplements, metformin withdrawal, replenishing vitamin B12 stores with intramuscular or oral vitamin B12 therapy and regular monitoring of vitamin B12 levels and vitamin B12 supplements if metformin continues to be utilized. With adequate vitamin B12 replacement, while symptoms of neuropathy may or may not improve, objective findings of neuropathy stabilize but do not improve.

Cubilin, the intrinsic factor-vitamin B12 receptor.

Cubilin (CUBN), the intrinsic factor-vitamin B12 receptor is a large endocytic protein involved in various physiological functions: vitamin B12 uptake in the gut; reabsorption of albumin and maturation of vitamin D in the kidney; nutrient delivery during embryonic development. Cubilin is an atypical receptor, peripherally associated to the plasma membrane. The transmembrane proteins amnionless (AMN) and Lrp2/Megalin are the currently known molecular partners contributing to plasma membrane transport and internalization of Cubilin. The role of Cubilin/Amn complex in the handling of vitamin B12 in health and disease has extensively been studied and so is the role of the Cubilin-Lrp2 tandem in renal pathophysiology. Accumulating evidence strongly supports a role of Cubilin in some developmental defects including impaired closure of the neural tube. Are these defects primarily caused by the dysfunction of a specific Cubilin ligand or are they secondary to impaired vitamin B12 or protein uptake? We will present the established Cubilin functions, discuss the developmental data and provide an overview of the emerging implications of Cubilin in the field of cardiovascular disease and cancer pathogenesis.

Profiling immuno-metabolic mediators of vitamin B12 deficiency among metformin-treated type 2 diabetic patients in Ghana.

The association between prolong metformin usage and B12 deficiency has been documented. However, the prevalence estimates of metformin-induced vitamin B12 deficiency showed substantial disparity among studies due to varied study definitions of vitamin B12 deficiency. Metformin blocks the calcium dependent absorption of the vitamin B12-Intrinsic Factor complex at the terminal ileum. Lack of intrinsic factor due to the presence of auto-antibodies to parietal cells (IFA) could lead to vitamin B12 deficiency and subsequently cause peripheral neuropathy. We investigated the prevalence of vitamin B12 deficiency using more sensitive, combined markers of vitamin B12 status (4cB12) and the immuno-biochemical mediators of vitamin B12 deficiency. In this observational study, 200 consecutive consenting metformin-treated T2DM patients, aged 35 and above, attending the diabetic clinic at KATH were recruited. Vitamin B12 deficiency was classified based on the Fedosov age-normalized wellness quotient. Anthropometric measurement was taken as well as blood samples for immunological and biochemical mediators. Peripheral neuropathy was assessed using the Michigan Neuropathy Screening Instrument (MNSI). Statistical analysis was performed using the R Language for Statistical Computing. Using the combined indicator (4cB12), the prevalence of metformin induced vitamin B12 deficiency was 40.5% whilst the prevalence of MNSI-Q and MNSI-PE diabetic neuropathy was 32.5% and 6.5% respectively. Participants with vitamin B12 deficiency had significantly higher levels of IFA, GPA, TNF-α, TC, LDL and albumin compared to those with normal vitamin B12 levels (p < 0.05). Correlation analysis revealed a statistically significant negative association between 4cB12 and the immunological markers [IFA (rs = -0.301, p<0.0001), GPA (rs = -0.244, p = 0.001), TNF-α (rs = -0.242, p = 0.001) and IL-6 (rs = -0.145, p = 0.041)]. Likewise, 4cB12 was negatively associated with TC (rs = -0.203, p = 0.004) and LDL (rs = -0.222, p = 0.002) but positively correlated with HDL (rs = 0.196, p = 0.005). Vitamin B12 deficiency and diabetic neuropathy are very high among metformin-treated T2DM patients and it is associated with increased GPA, IFA, TNF-α and cardiometabolic risk factors (higher LDL and TC and lower HDL). Upon verification of these findings in a prospective case-control study, it may be beneficial to include periodic measurement of Vitamin B12 using the more sensitive combined indicators (4cB 12) in the management of patients with T2DM treated with metformin in Ghana.

Robotic Proximal Gastrectomy with Double-Tract Reconstruction for Gastroesophageal Junction Cancer

The current standard surgical procedure for proximal gastric and gastroesophageal junction (P/GEJ) cancers with limited esophageal involvement is total gastrectomy (TG). TG is associated with impaired appetite and weight loss due to decreased levels of ghrelin (a “hunger hormone” secreted by the stomach) and with anemia due to intrinsic factor loss and vitamin B12 malabsorption. Theoretically, proximal gastrectomy (PG) with an anti-reflux technique such as double-tract reconstruction (DTR) can improve quality of life (QoL) by preserving gastric function.1 A recent Japanese prospective GEJ adenocarcinoma study reported a low incidence of lymph node metastases at peripyloric stations,2 indicating the oncological safety of PG for GEJ adenocarcinoma regardless of tumor stage. As a result, PG is increasingly performed in South Korea and Japan, although the QoL benefit of PG over TG remains unknown.3, 4 We have performed PG with DTR in select cases with satisfying short-term outcomes. In this video, we introduce our technique for robotic PG with DTR. The presented case is a 75-year-old woman with GEJ adenocarcinoma that showed an excellent response to preoperative chemoradiation therapy. The patient underwent robotic PG with DTR. Fluorescent sentinel lymphatic mapping was performed by injecting indocyanine green solution (total of 2 ml, at four quadrants around the tumor at submucosal space) via endoscopy at the beginning of the operation. It showed absence of sentinel lymphatic flow to peripyloric lymph nodes, which were thus considered safe to preserve. Pathologic examination confirmed a complete response. The patient’s recovery was favorable, and she reported satisfaction with her QoL and good appetite, though some intermittent bloating after eating. PG with DTR has theoretical disadvantages including incomplete lymph node removal, which may result in recurrence; therefore, PG should be carefully performed for P/GEJ cancers with low risk of perigastric lymph node metastases, such as cT1 tumors or GEJ tumors with limited gastric involvement.2 In addition, delayed gastric emptying of the remnant stomach can cause upper gastrointestinal symptoms such as reflux and bloating. The QoL benefits of PG with DTR must be demonstrated before encouraging its use in the USA and other countries. International collaboration is warranted to test the benefits and safety of PG, and the effective use of sentinel lymphatic mapping, to standardize the surgical care of patients with P/GEJ cancers.

Intrinsic factor autoantibodies by luminescent immuno-precipitation system in patients with corpus atrophic gastritis

BackgroundCorpus atrophic gastritis (CAG) may lead to intrinsic factor (IF) deficiency and vitamin B12 malabsorption. Intrinsic factor autoantibodies (IFA) are considered markers of pernicious anemia, but their clinical utility in CAG has not been evaluated. This study aimed to assess IFA in CAG patients and controls using a luciferase immunoprecipitation system (LIPS). MethodsRecombinant nanoluciferase-tagged IF secreted from transfected Expi293F cells was used as antigen in an IFA-LIPS assay. IFA IgG were measured in sera from subjects undergoing gastroscopy and biopsy (updated Sydney system) mainly for anemia (57%) or dyspepsia (34%). This cohort comprised 105 patients with histologically-proven-CAG (cases: median age 64 years, 68% females) and 110 subjects with suspected CAG that were histologically negative (controls: median age 67 years, 54% females). Cut-off values were selected by Q-Q-plot analysis (negative: <2.5 arbitrary units). ResultsIFA levels were higher in cases than in controls (Mann-Whitney:p < 10−5). The ROC-AUC was 0.67 (95%CI 0.60–0.73, p < 0.0001). The IFA LIPS sensitivity and specificity for CAG were 32% (95% CI 24–42) and 95% (95% CI 90–99). This diagnostic performance remained similar after stratification for the presence/absence of anemia, dyspepsia or vitamin B12 deficiency. IFA levels were higher in females compared with males (p = 0.0127). In females aged <65 years, IFA-positives were more prevalent than in males (43.5% vs 6.6%, p = 0.011). ConclusionsThe IFA-LIPS assay discriminated between CAG patients and controls showing a good specificity (95%) at the cost of sensitivity (32%). IFA-positivity occurred independently from anemia and vitamin B12 deficiency, but was more frequent in younger females. IFA testing should be considered in patients at high clinical suspicion of CAG.

Cubilin, the Intrinsic Factor-Vitamin B12 Receptor in Development and Disease

Gp280/Intrinsic factor-vitamin B12 receptor/Cubilin (CUBN) is a large endocytic receptor serving multiple functions in vitamin B12 homeostasis, renal reabsorption of protein or toxic substances including albumin, vitamin D-binding protein or cadmium. Cubilin is a peripheral membrane protein consisting of 8 Epidermal Growth Factor (EGF)-like repeats and 27 CUB (defined as Complement C1r/C1s, Uegf, BMP1) domains. This structurally unique protein interacts with at least two molecular partners, Amnionless (AMN) and Lrp2/Megalin. AMN is involved in appropriate plasma membrane transport of Cubilin whereas Lrp2 is essential for efficient internalization of Cubilin and its ligands. Observations gleaned from animal models with Cubn deficiency or human diseases demonstrate the importance of this protein. In this review addressed to basic research and medical scientists, we summarize currently available data on Cubilin and its implication in renal and intestinal biology. We also discuss the role of Cubilin as a modulator of Fgf8 signaling during embryonic development and propose that the Cubilin-Fgf8 interaction may be relevant in human pathology, including in cancer progression, heart or neural tube defects. We finally provide experimental elements suggesting that some aspects of Cubilin physiology might be relevant in drug design.

Effect of diabetes mellitus on vitamin B12, pepsinogen and gastric intrinsic factor levels in rats

Background: The factor leading to low Vitamin B12 status in patients with diabetes mellitus (DM) is still controversial. Objectives: The main objective of this study was to explore DM-induced changes in selected biological markers of Vitamin B12 in rats. Materials and Methods: DM was induced in rats by intraperitoneal administration of streptozotocin at 60 mg/kg body weight. The localisation and ultrastructure of pepsinogen (PEP)-containing cells were investigated using immunohistochemistry and transmission electron microscopy. In addition, enzyme-linked immunoassay was used to determine the gastric and serum levels of Vitamin B12, intrinsic factor (IF) and PEP-1. Results: The number of PEP-1-immunopositive cell was significantly (P

Loss of Cubilin, the intrinsic factor-vitamin B12 receptor, impairs visceral endoderm endocytosis and endodermal patterning in the mouse

The visceral endoderm is a polarized epithelial monolayer necessary for early embryonic development in rodents. A key feature of this epithelium is an active endocytosis and degradation of maternal nutrients, in addition to being the source of various signaling molecules or inhibitors required for the differentiation and patterning of adjacent embryonic tissues. Endocytosis across the visceral endoderm epithelium involves specific cell surface receptors and an extensive sub-membrane vesicular system with numerous apical vacuoles/lysosomes. We previously reported that Cubilin, the endocytic receptor for intrinsic factor-vitamin B12, albumin and apolipoproteinA-I/HDL allows maternal nutrient uptake by the visceral endoderm. In the present study, we show that the germline ablation of Cubilin impairs endodermal and mesodermal patterning, and results in developmental arrest at gastrulation. Notably, visceral endoderm dispersal is impeded in Cubilin null embryos. We further confirm the essential role of Cubilin in nutrient internalization by the early visceral endoderm and highlight its involvement in the formation of apical vacuoles. Our results reveal essential roles for Cubilin in early embryonic development, and suggest that in addition to its nutritive function, Cubilin sustains signaling pathways involved in embryonic differentiation and patterning.

Imerslund-Grasbeck syndrome presenting as pancytopenia: A rare case report

Imerslund-Grasbeck syndrome or selective Vitamin B12 malabsorption with proteinuria or megaloblastic anemia 1 is a rare autosomal recessive disorder. The main feature of this condition is megaloblastic anemia due to Vitamin B12 deficiency with or without persistent proteinuria (with no signs of kidney disease). The disease appears from 4 months up to several years after birth. The cause is a defect in the receptor of the Vitamin B12-intrinsic factor complex of the ileal enterocytes. Management includes lifelong parenteral Vitamin B12, and with this, the patients remain healthy for decades. However, the proteinuria persists. We present a 14-year-old male child suffering from this syndrome presenting to us with pancytopenia due to Vitamin B12 deficiency and proteinuria with normal kidneys. There is hardly any case report of this syndrome from North India.

Autoimmune thyroid diseases (AITD) and mental illness: An evaluation of parietal cell antibodies, intrinsic factor antibodies, vitamin B12 and psychiatric symptoms in patients with AITD and Non-AITD controls

Autoimmune thyroid diseases (AITD) and mental illness: An evaluation of parietal cell antibodies, intrinsic factor antibodies, vitamin B12 and psychiatric symptoms in patients with AITD and Non-AITD controls

Detailed investigations of proximal tubular function in Imerslund-Gräsbeck syndrome

BackgroundImerslund-Gräsbeck Syndrome (IGS) is a rare genetic disorder characterised by juvenile megaloblastic anaemia. IGS is caused by mutations in either of the genes encoding the intestinal intrinsic factor-vitamin B12 receptor complex, cubam. The cubam receptor proteins cubilin and amnionless are both expressed in the small intestine as well as the proximal tubules of the kidney and exhibit an interdependent relationship for post-translational processing and trafficking. In the proximal tubules cubilin is involved in the reabsorption of several filtered plasma proteins including vitamin carriers and lipoproteins. Consistent with this, low-molecular-weight proteinuria has been observed in most patients with IGS. The aim of this study was to characterise novel disease-causing mutations and correlate novel and previously reported mutations with the presence of low-molecular-weight proteinuria.MethodsGenetic screening was performed by direct sequencing of the CUBN and AMN genes and novel identified mutations were characterised by in silico and/or in vitro investigations. Urinary protein excretion was analysed by immunoblotting and high-resolution gel electrophoresis of collected urines from patients and healthy controls to determine renal phenotype.ResultsGenetic characterisation of nine IGS patients identified two novel AMN frameshift mutations alongside a frequently reported AMN splice site mutation and two CUBN missense mutations; one novel and one previously reported in Finnish patients. The novel AMN mutations were predicted to result in functionally null AMN alleles with no cell-surface expression of cubilin. Also, the novel CUBN missense mutation was predicted to affect structural integrity of the IF-B12 binding site of cubilin and hereby most likely cubilin cell-surface expression. Analysis of urinary protein excretion in the patients and 20 healthy controls revealed increased urinary excretion of cubilin ligands including apolipoprotein A-I, transferrin, vitamin D-binding protein, and albumin. This was, however, only observed in patients where plasma membrane expression of cubilin was predicted to be perturbed.ConclusionsIn the present study, mutational characterisation of nine IGS patients coupled with analyses of urinary protein excretion provide additional evidence for a correlation between mutation type and presence of the characteristic low-molecular-weight proteinuria.

Cubilin expression is monoallelic and epigenetically augmented via PPARs

BackgroundCubilin is an endocytic receptor that is necessary for renal and intestinal absorption of a range of ligands. Endocytosis mediated by cubilin and its co-receptor megalin is the principal mechanism for proximal tubule reabsorption of proteins from the glomerular filtrate. Cubilin is also required for intestinal endocytosis of intrinsic factor-vitamin B12 complex. Despite its importance, little is known about the regulation of cubilin expression.ResultsHere we show that cubilin expression is under epigenetic regulation by at least two processes. The first process involves inactivation of expression of one of the cubilin alleles. This monoallelic expression state could not be transformed to biallelic by inhibiting DNA methylation or histone deacetylation. The second process involves transcriptional regulation of cubilin by peroxisome proliferator-activated receptor (PPAR) transcription factors that are themselves regulated by DNA methylation and histone deacetylation. This is supported by findings that inhibitors of DNA methylation and histone deacetylation, 5Aza and TSA, increase cubilin mRNA and protein in renal and intestinal cell lines. Not only was the expression of PPARα and γ inducible by 5Aza and TSA, but the positive effects of TSA and 5Aza on cubilin expression were also dependent on both increased PPAR transcription and activation. Additionally, 5Aza and TSA had similar effects on the expression of the cubilin co-receptor, megalin.ConclusionsTogether, these findings reveal that cubilin and megalin mRNA expression is under epigenetic control and thus point to new avenues for overcoming pathological suppression of these genes through targeting of epigenetic regulatory processes.

Gastric intrinsic factor deficiency with combined GIF heterozygous mutations and FUT2 secretor variant

Several genome-wide association studies (GWAS) have identified a strong association between serum vitamin B12 and fucosyltransferase 2 (FUT2), a gene associated with susceptibility to Helicobacter pylori infection. Hazra etal. conducted a meta-analysis of three GWAS and found three additional loci in MUT, CUBN and TCN1. Other GWAS conducted in Italy and China confirmed the association for FUT2 gene. Alpha-2-fucosyltransferase (FUT2) catalyzes fucose addition to form H-type antigens in exocrine secretions. FUT2 non-secretor variant produces no secretion of H-type antigens and is associated with high-plasma vitamin B12 levels. This association was explained by the influence of FUT2 on H. pylori, which is a risk factor of gastritis, a main cause of vitamin B12 impaired absorption. However, we recently showed that H. pylori serology had no influence on FUT2 association with vitamin B12, in a large sample population, suggesting the involvement of an alternative mechanism. GIF is another gene associated with plasma levels of vitamin B12 and gastric intrinsic factor (GIF) is a fucosylated protein needed for B12 absorption. Inherited GIF deficiency produces B12 deficiency unrelated with gastritis. We report 2 families with heterozygous GIF mutation, 290T>C, M97T, with decreased binding affinity of GIF for vitamin B12 and one family with heterozygous GIF mutation 435_437delGAA, K145_N146delinsN and no B12 binding activity of mutated GIF. All cases with vitamin B12 deficit carried the FUT2 rs601338 secretor variant. Ulex europeus binding to GIF was influenced by FUT2 genotypes and GIF concentration was lower, in gastric juice from control subjects with the secretor genotype. GIF290C allele was reported in 5 European cases and no Africans among 1282 ambulatory subjects and was associated with low plasma vitamin B12 and anaemia in the single case bearing the FUT2 secretor variant. We concluded that FUT2 secretor variant worsens B12 status in cases with heterozygous GIF mutations by impairing GIF secretion, independently from H. pylori-related gastritis.

Amnionless Expression in the Mouse Testis and Leydig Cells and Its Change in Leydig Cell Hyperplasia.

In testis, vitamin B12 (cobalmin)-deficiency resulted in atrophy of seminiferous tubules and aplasia of sperms and spermatids. Deficiency in vitamin B12 decreased in methionine synthase activity. Vitamin B12 involves in DNA synthesis as it participates in methionine synthase followed by thymidine synthesis. Amnionless (Amn), a transmembrane protein directs endocytosis of cubilin with its ligand, contributing intrinsic factor-vitamin B12 absorption. Expressional profile from data base revealed that Amn is considerably expressed in kidney, small intestine, placenta, and testis in mouse. In an effort to understand vitamin B12 transport to the testicular cells, we analyzed the Amn gene expression in the developing mouse testis and Leydig cells. In the meantime, change of Amn expression in Leydig cell hyperplasia by flutamide an antiandrogen was examined in testis. In mouse testis, Amn mRNA levels were low until 14 days post partum (pp), markedly increased at puberty onwards. In the interstitium, Amn mRNA levels were low until 14 days pp, markedly increased together with 3β-hydroxysteroid dehydrogenase type VI mRNA at puberty (28 days pp), and further increased at 56 days pp. AMN immunoreactivity was found weakly in Sertoli cells throughout the testis development. Early spermatocytes showed strong AMN immunoreactivity at 14 days pp onwards, suggesting that AMN participates in the early meiosis. In Leydig cells, AMN immunoreactivity was not found until 14 days pp but strongly expressed at 28 days pp onwards. Quantitative image analysis revealed that AMN immunoreactivity in Leydig cells was increased by 2.8 fold between 28 and 56 days pp. Mean size of AMN-positive Leydig cells was increased by 1.8 fold between 14 and 28 days pp and 1.5 fold between 28 and 56 days pp. This suggests positive relationship between AMN expression and functional differentiation of adult Leydig cells. In adult male mice, oral administration of flutamide (100 mg/kg/day) for 10 days induced a Leydig cell hyperplasia in mouse testis in which Amn mRNA and AMN immunoreactivity were increased in Leydig cells, suggesting that AMN may participate in the pathogenesis of Leydig cell hyperplasia. In adult Leydig cells primary culture, flutamide (100 nM) increased Amn mRNA expression, suggesting that Amn gene expression is negatively coupled with androgen receptor activation. In conclusion, AMN may participate in the meiosis of early spermatocytes and functional differentiation of adult Leydig cells via mediating the vitamin B12 transport in mouse testis. In addition, AMN may participate in the Leydig cell hyperplasia via promoting cell proliferation in Leydig cell via stimulating DNA synthesis. This is the first report on the AMN expression in male germ cells and somatic cells in mammalian testis. (poster)

Report of a cubilin deficient patient

BackgroundCubilin is an important receptor involved in intestinal absorption of vitamin B12 and reabsorption of proteins from the glomerular ultrafiltrate. Murine studies furthermore suggest that both cubilin and the receptor partner amnionless are essential for normal embryonic development. Cubilin function is affected in Imerslund‐Gräsbeck syndrome (IGS) patients. IGS is a rare, autosomal recessive disorder characterized by a selective, intestinal, intrinsic factor‐vitamin B12 malabsorption and is often accompanied by an additional proteinuria.AimTo investigate the role of cubilin in renal protein handling in a patient with cubilin deficiency.ResultsA novel homozygous null mutation of the CUBN gene was identified in a conserved donor splice site in a male IGS patient. No cubilin was detected in a renal biopsy and an abnormal, cytoplasmic distribution of the receptor partner amnionless was also observed. Immunoblotting of patient urine, accordingly, showed selective tubular proteinuria with increased excretion of cubilin ligands.ConclusionsThis clearly demonstrates a central role of cubilin in handling of filtered proteins in the human kidney and demonstrates the interdependent relationship of cubilin and amnionless in man. This patient furthermore, presented with no additional developmental abnormalities suggesting that cubilin is not essential for human embryonic development.

A Patient with Cubilin Deficiency

To the Editor: Imerslund–Grasbeck syndrome, or megaloblastic anemia 1, is a rare autosomal recessive disorder characterized by selective intestinal malabsorption of intrinsic factor–vitamin B12; it...

Cubilin Is Essential for Albumin Reabsorption in the Renal Proximal Tubule

Receptor-mediated endocytosis is responsible for protein reabsorption in the proximal tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that proximal tubule cells did not localize amnionless to the plasma membrane in the absence of cubilin, indicating a mutual dependency of cubilin and amnionless to form a functional membrane receptor complex. The cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, cubilin-deficient mice exhibited markedly decreased uptake of albumin by proximal tubule cells and resultant albuminuria. Inactivation of both megalin and cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds cubilin and megalin. In addition, we observed cubilin-independent reabsorption of the "specific" cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, cubilin is essential for its reabsorption by proximal tubule cells, and megalin drives internalization of cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases.