Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line.

In pancreatic beta-cells, the high Km glucose transporter GLUT2 catalyzes the first step in glucose-induced insulin secretion by glucose uptake. Expression of the transporter has been reported to be modulated by glucose either at the protein or mRNA levels. In this study we used the differentiated insulinoma cell line INS-1 which expresses high levels of GLUT2 and show that the expression of GLUT2 is regulated by glucose at the transcriptional level. By run-on transcription assays we showed that glucose induced GLUT2 gene transcription 3-4-fold in INS-1 cells which was paralleled by a 1.7-2.3-fold increase in cytoplasmic GLUT2 mRNA levels. To determine whether glucose regulatory sequences were present in the promoter region of GLUT2, we cloned and characterized a 1.4-kilobase region of mouse genomic DNA located 5' of the translation initiation site. By RNase protection assays and primer extension, we determined that multiple transcription initiation sites were present at positions -55, -64, and -115 from the first coding ATG and which were identified in liver, intestine, kidney, and beta-cells mRNAs. Plasmids were constructed with the mouse promoter region linked to the reporter gene chloramphenicol acetyltransferase (CAT), and transiently and stably transfected in the INS-1 cells. Glucose induced a concentration-dependent increase in CAT activity which reached a maximum of 3.6-fold at 20 mM glucose. Similar CAT constructs made of the human GLUT2 promoter region and the CAT gene displayed the same glucose-dependent increase in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence identity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common sequences.

Replacement therapy for iron deficiency improves exercise capacity and quality of life in patients with cyanotic congenital heart disease and/or the Eisenmenger syndrome

La teràpia suplementària de ferro millora la capacitat d’exercici i la qualitat de vida en malalts amb una cardiopatia congènita cianòtica i/ o síndrome d’Eisenmenger El dèficit de ferro és una troballa comú en la cardiopatia congènita cianòtica, i pot ser la causa d’una reducció en la capacitat d’exercici. Actualment, està indicada la reposició dels dipòsits de ferro en aquest grup de malalts, éssent les evidències científiques escasses. En el present treball investiguem la seguretat i eficàcia del tractament amb ferro en malalts amb una cardiopatia congènita cianòtica. Per tal motiu, vint-i-cinc malalts amb una cardiopatia congenita cianòtica i dèficit de ferro van ser inclosos de forma prospectiva entre Agost del 2008 i Gener del 2009. El tractament utilitzat fou fumarat ferròs oral, fins a una dosi màxima de 200 mg tres vegades al dia. En l’anàlisi basal i als tres mesos de seguiment es va utilitzar el test de qualitat de vida “CAMPHOR”, el test de la marxa dels 6 minuts i la prova d’esforç amb consum d’oxigen. L’edat mitja fou 39.9+/-10.9 anys, 80% dones. Catorze malalts tenien la síndrome d’Eisenmenger, sis una malaltia cianòtica complexa i cinc circulació de Fontan. Cap d’ells va haver d'interrompre el tractament degut a efectes adversos. Després de tres mesos de tractament, l’hemoglobina (19.0+/-2.9g/dL a 20.4+/-2.7g/dL, p&0.001), ferritina (13.3+/-4.7mug/L a 54.1+/-24.2mug/L, p&0.001) i saturació de transferrina (17.8+/-9.6% a 34.8+/-23.4%, p&0.001) van augmentar significativament. També hi va haver una millora significativa en la puntuació del test de qualitat de vida (20.7+/-10.9 a 16.2+/-10.4, p=0.001) i el test de la marxa (371.7+/-84.7m a 402.8.0+/-74.9m, p=0.001). No es van evidenciar canvis significatius en els valors de consum d’oxigen (40.7+/-9.2% a 43.8+/-12.4%, p=0.15). En definitiva, la teràpia suplementària amb ferro en els malats amb una cardiopatia congènita cianòtica i dèficit de ferro és segura i millora la qualitat de vida i la capacitat funcional. En aquest grup de malalts, per tant, és aconsellable identificar el dèficit de ferro i restaurar-ne els seus dipòsits.

Blunted glucose-induced thermogenesis in 'overweight' patients: a factor contributing to relapse of obesity.

Glucose-induced thermogenesis was studied in 12 overweight patients (9F and 3M) before (mean body weight +/- s.e.m. 83 +/- 2 kg) and after weight loss (68 +/- 2 kg), and in eight of the same patients following relapse of body weight gain (84 +/- 5 kg). Expressed as a percentage of the energy content of the 100 g oral glucose load, glucose-induced thermogenesis was lower in the overweight before weight loss (6.5 +/- 0.5 per cent, P less than 0.05), after weight loss (3.9 +/- 0.6 per cent, P less than 0.01) and after weight regain (6.3 +/- 0.9 per cent, P less than 0.05) than in a group of lean control subjects, matched for sex and age (8.3 +/- 0.5 per cent). Basal energy expenditure was lower after weight reduction than before (1.16 +/- 0.04 vs 1.41 +/- 0.08 kcal/min, P less than 0.01). In the formerly overweight patients, the combined effect of a decreased basal energy expenditure and an attenuation of glucose induced thermogenesis resulted in a postprandial energy expenditure which was markedly lower than in the overweight state (P less than 0.001). Following relapse of obesity, glucose-induced thermogenesis remained attenuated compared to control subjects. These results suggest that a lowered basal energy expenditure and a reduced glucose-induced thermogenesis contribute to the positive energy balance which results in relapse of body weight gain after cessation of a hypocaloric diet.

Discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (GIP) receptor: the impact on the pancreatic beta cell responses and functional expression studies in Chinese hamster fibroblast cells.

The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are insulinotropic factors released from the small intestine to the blood stream in response to oral glucose ingestion. The insulinotropic effect of GLP-1 is maintained in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas, for unknown reasons, the effect of GIP is diminished or lacking. We defined the exon-intron boundaries of the human GIP receptor, made a mutational analysis of the gene and identified two amino acid substitutions, A207 V and E354Q. In an association study of 227 Caucasian Type II diabetic patients and 224 matched glucose tolerant control subjects, the allelic frequency of the A207 V polymorphism was 1.1% in Type II diabetic patients and 0.7% in control subjects (p = 0.48), whereas the allelic frequency of the codon 354 polymorphism was 24.9% in Type II diabetic patients versus 23.2% in control subjects. Interestingly, the glucose tolerant subjects (6% of the population) who were homozygous for the codon 354 variant had on average a 14% decrease in fasting serum C-peptide concentration (p = 0.01) and an 11% decrease in the same variable 30 min after an oral glucose load (p = 0.03) compared with subjects with the wild-type receptor. Investigation of the function of the two GIP receptor variants in Chinese hamster fibroblasts showed, however, that the GIP-induced cAMP formation and the binding of GIP to cells expressing the variant receptors were not different from the findings in cells expressing the wildtype GIP receptor. In conclusion, amino acid variants in the GIP receptor are not associated with random Type II diabetes in patients of Danish Caucasian origin or with altered GIP binding and GIP-induced cAMP production when stably transfected in Chinese hamster fibroblasts. The finding of an association between homozygosity for the codon 354 variant and reduced fasting and post oral glucose tolerance test (OGTT) serum C-peptide concentrations, however, calls for further investigations and could suggest that GIP even in the fasting state regulates the beta-cell secretory response.

Glucose and leptin induce apoptosis in human beta-cells and impair glucose-stimulated insulin secretion through activation of c-Jun N-terminal kinases.

c-Jun N-terminal kinases (SAPK/JNKs) are activated by inflammatory cytokines, and JNK signaling is involved in insulin resistance and beta-cell secretory function and survival. Chronic high glucose concentrations and leptin induce interleukin-1beta (IL-1beta) secretion from pancreatic islets, an event that is possibly causal in promoting beta-cell dysfunction and death. The present study provides evidence that chronically elevated concentrations of leptin and glucose induce beta-cell apoptosis through activation of the JNK pathway in human islets and in insulinoma (INS 832/13) cells. JNK inhibition by the dominant inhibitor JNK-binding domain of IB1/JIP-1 (JNKi) reduced JNK activity and apoptosis induced by leptin and glucose. Exposure of human islets to leptin and high glucose concentrations leads to a decrease of glucose-induced insulin secretion, which was partly restored by JNKi. We detected an interplay between the JNK cascade and the caspase 1/IL-1beta-converting enzyme in human islets. The caspase 1 gene, which contains a potential activating protein-1 binding site, was up-regulated in pancreatic sections and in isolated islets from type 2 diabetic patients. Similarly, cultured human islets exposed to high glucose- and leptin-induced caspase 1 and JNK inhibition prevented this up-regulation. Therefore, JNK inhibition may protect beta-cells from the deleterious effects of high glucose and leptin in diabetes.

Sensing of glucose in the brain.

The brain, and in particular the hypothalamus and brainstem, have been recognized for decades as important centers for the homeostatic control of feeding, energy expenditure, and glucose homeostasis. These structures contain neurons and neuronal circuits that may be directly or indirectly activated or inhibited by glucose, lipids, or amino acids. The detection by neurons of these nutrient cues may become deregulated, and possibly cause metabolic diseases such as obesity and diabetes. Thus, there is a major interest in identifying these neurons, how they respond to nutrients, the neuronal circuits they form, and the physiological function they control. Here I will review some aspects of glucose sensing by the brain. The brain is responsive to both hyperglycemia and hypoglycemia, and the glucose sensing cells involved are distributed in several anatomical sites that are connected to each other. These eventually control the activity of the sympathetic or parasympathetic nervous system, which regulates the function of peripheral organs such as liver, white and brown fat, muscle, and pancreatic islets alpha and beta cells. There is now evidence for an extreme diversity in the sensing mechanisms used, and these will be reviewed.

Effects of colostrum feeding and glucocorticoid administration on insulin-dependent glucose metabolism in neonatal calves

Colostrum feeding and glucocorticoid administration affect glucose metabolism and insulin release in calves. We have tested the hypothesis that dexamethasone as well as colostrum feeding influence insulin-dependent glucose metabolism in neonatal calves using the euglycemic-hyperinsulinemic clamp technique. Newborn calves were fed either colostrum or a milk-based formula (n=14 per group) and in each feeding group, half of the calves were treated with dexamethasone (30 microg/[kg body weight per day]). Preprandial blood samples were taken on days 1, 2, and 4. On day 5, insulin was infused for 3h and plasma glucose concentrations were kept at 5 mmol/L+/-10%. Clamps were combined with [(13)C]-bicarbonate and [6,6-(2)H]-glucose infusions for 5.5h (i.e., from -150 to 180 min, relative to insulin infusion) to determine glucose turnover, glucose appearance rate (Ra), endogenous glucose production (eGP), and gluconeogenesis before and at the end of the clamp. After the clamp liver biopsies were taken to measure mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC). Dexamethasone increased plasma glucose, insulin, and glucagon concentrations in the pre-clamp period thus necessitating a reduction in the rate of glucose infusion to maintain euglycemia during the clamp. Glucose turnover and Ra increased during the clamp and were lower at the end of the clamp in dexamethasone-treated calves. Dexamethasone treatment did not affect basal gluconeogenesis or eGP. At the end of the clamp, dexamethasone reduced eGP and PC mRNA levels, whereas mitochondrial PEPCK mRNA levels increased. In conclusion, insulin increased glucose turnover and dexamethasone impaired insulin-dependent glucose metabolism, and this was independent of different feeding.

Trypanosoma cruzi: inhibition of alpha-hydroxyacid dehydrogenase isozyme II by N-allyl and N-propyl oxamates and their effects on intact epimastigotes

N-allyl (NAOx) and N-propyl (NPOx) oxamates were designed as inhibitors of alpha-hydroxyacid dehydrogenase (HADH) isozyme II from Trypanosoma cruzi. The kinetic studies showed that NAOx and NPOx were competitive inhibitors of HADH-isozyme II (Ki = 72 µM, IC50 = 0.33 mM and 70 µM, IC50 = 0.32 mM, respectively). The attachment of the allylic and propylic chains to nitrogen of the competitive inhibitor oxamate (Ki = 0.91 mM, IC50 = 4.25 mM), increased 12.6 and 13-folds respectively, the affinity for T. cruzi HADH-isozyme II. NAOx and NPOx were selective inhibitors of HADH-isozyme II, because other T. cruzi dehydrogenases were not inhibited by these substances. Since HADH-isozyme II participates in the energy metabolism of T. cruzi, a trypanocidal effect can be expected with these inhibitors. However, we were not able to detect any trypanocidal activity with these oxamates. When the corresponding ethyl esters of N-allyl (Et-NAOx) and N-propyl (Et-NPOx) oxamates were tested as a possible trypanocidal prodrugs, in comparison with nifurtimox and benznidazole, the expected trypanocidal effects were obtained.

A single high dose of oral vitamin D3 is insufficient to correct a deficiency in a rheumatologic population

Introduction Vitamin D plays a major role in bone metabolismand neuromuscular function. Supplementation with vitamin D iseffective to reduce the risk of fall and of fracture. However adherenceto oral daily vitamin D is low. Screening and correcting vitamin Dinsufficiency in a rheumatologic population could improve bothmorbidity and quality of life. After determining the prevalence ofvitamin D deficiency in this population, we evaluated if supplementationwith a single high dose of oral 25-OH vitamin D3 wassufficient to correct this abnormality.Methods During one month (November 2009), levels of 25-OHvitamin D were systematically determined in our rheumatology outpatientclinic and classified in: vitamin D deficiency (< 10 μg/l),vitamin D insufficiency (10 to 30 μg/l) or normal vitamin D (> 30 μg/l).Patients with insufficiency or deficiency received respectively a singlehigh dose of 300'000 IU or 600'000 IU oral vitamin D3. In addition,all patients with osteoporosis were prescribed daily supplement ofcalcium (1 g) and vitamin D (800 IU). 25-OH vitamin D levels werereevaluated after 3 months.Results Vitamin D levels were initially determined in 292 patients(mean age 53, 211 women, 87 % Caucasian). 77 % had inflammatoryrheumatologic disease (IRD), 20 % osteoporosis (OP) and 12 %degenerative disease (DD). Vitamin D deficiency was present in 20(6.8 %), while 225 (77.1 %) had insufficiency. Of the 245 patientswith levels < 30μg/l, a new determination of vitamin D level wasavailable in 173 (71 %) at 3 months.Conclusion Vitamin D insufficiency is highly prevalent in ourrheumatologic population (84 %), and is not adequately correctedby a single high dose of oral vitamin D3 in > 50 % of the patientswith IRD and DD. In patients with OP, despite association of asingle high dose with daily oral vitamin D supplementation, 40 %of patients are still deficient when reevaluated at 3 months.

A rice cis-natural antisense RNA acts as a translational enhancer for its cognate mRNA and contributes to phosphate homeostasis and plant fitness.

cis-natural antisense transcripts (cis-NATs) are widespread in plants and are often associated with downregulation of their associated sense genes. We found that a cis-NAT positively regulates the level of a protein critical for phosphate homeostasis in rice (Oryza sativa). PHOSPHATE1;2 (PHO1;2), a gene involved in phosphate loading into the xylem in rice, and its associated cis-NATPHO1;2 are both controlled by promoters active in the vascular cylinder of roots and leaves. While the PHO1;2 promoter is unresponsive to the plant phosphate status, the cis-NATPHO1;2 promoter is strongly upregulated under phosphate deficiency. Expression of both cis-NATPHO1;2 and the PHO1;2 protein increased in phosphate-deficient plants, while the PHO1;2 mRNA level remained stable. Downregulation of cis-NATPHO1;2 expression by RNA interference resulted in a decrease in PHO1;2 protein, impaired the transfer of phosphate from root to shoot, and decreased seed yield. Constitutive overexpression of NATPHO1;2 in trans led to a strong increase of PHO1;2, even under phosphate-sufficient conditions. Under all conditions, no changes occurred in the level of expression, sequence, or nuclear export of PHO1;2 mRNA. However, expression of cis-NATPHO1;2 was associated with a shift of both PHO1;2 and cis-NATPHO1;2 toward the polysomes. These findings reveal an unexpected role for cis-NATPHO1;2 in promoting PHO1;2 translation and affecting phosphate homeostasis and plant fitness.

3-M syndrome associated with growth hormone deficiency: 18 year follow-up of a patient.

3-M syndrome is a rare autosomal recessive disorder that causes short stature, unusual facial features and skeletal abnormalities. Mutations in the CUL7, OBSL1 and CCDC8 genes could be responsible for 3-M syndrome.Here we describe the growth and evolution of dismorphic features of an Italian boy with 3-M syndrome and growth hormone deficiency (GHD) from birth until adulthood. He was born full term with a very low birth weight (2400 g=-3.36 standard deviation score, SDS) and length (40.0 cm =-6.53 SDS). At birth he presented with a broad, fleshy nose with anteverted nostrils, thick and patulous lips, a square chin, curvilinear shaped eyebrows without synophrys, short thorax and long slender bones. Then, during childhood tall vertebral bodies, hip dislocation, transverse chest groove, winged scapulae and hyperextensible joints became more evident and the diagnosis of 3-M syndrome was made; this was also confirmed by the finding of a homozygous deletion in exon 18 of the CUL7 gene, which has not been previously described.The patient also exhibited severe GHD (GH <5 ng/ml) and from the age of 18 months was treated with rhGH. Notwithstanding the early start of therapy and good compliance, his growth rate was always very low, except for the first two years of treatment and he achieved a final height of 132 cm (-6.42 SDS).

Analysis of teichoic acid biosynthesis regulation reveals that the extracytoplasmic function sigma factor sigmaM is induced by phosphate depletion in Bacillus subtilis W23.

The expression of the Bacillus subtilis W23 tar genes specifying the biosynthesis of the major wall teichoic acid, the poly(ribitol phosphate), was studied under phosphate limitation using lacZ reporter fusions. Three different regulation patterns can be deduced from these beta-galactosidase activity data: (i) tarD and tarL gene expression is downregulated under phosphate starvation; (ii) tarA and, to a minor extent, tarB expression after an initial decrease unexpectedly increases; and (iii) tarO is not influenced by phosphate concentration. To dissect the tarA regulatory pattern, its two promoters were analysed under phosphate limitation: The P(tarA)-ext promoter is repressed under phosphate starvation by the PhoPR two-component system, whereas, under the same conditions, the P(tarA)-int promoter is upregulated by the action of an extracytoplasmic function (ECF) sigma factor, sigma(M). In contrast to strain 168, sigma(M) is activated in strain W23 in phosphate-depleted conditions, a phenomenon indirectly dependent on PhoPR, the two-component regulatory system responsible for the adaptation to phosphate starvation. These results provide further evidence for the role of sigma(M) in cell-wall stress response, and suggest that impairment of cell-wall structure is the signal activating this ECF sigma factor.

The transcription factor PHR1 plays a key role in the regulation of sulfate shoot-to-root flux upon phosphate starvation in Arabidopsis.

Background: Sulfate and phosphate are both vital macronutrients required for plant growth and development. Despite evidence for interaction between sulfate and phosphate homeostasis, no transcriptional factor has yet been identified in higher plants that affects, at the gene expression and physiological levels, the response to both elements. This work was aimed at examining whether PHR1, a transcription factor previously shown to participate in the regulation of genes involved in phosphate homeostasis, also contributed to the regulation and activity of genes involved in sulfate inter-organ transport. Results: Among the genes implicated in sulfate transport in Arabidopsis thaliana, SULTR1;3 and SULTR3;4 showed up-regulation of transcripts in plants grown under phosphate-deficient conditions. The promoter of SULTR1;3 contains a motif that is potentially recognizable by PHR1. Using the phr1 mutant, we showed that SULTR1;3 up regulation following phosphate deficiency was dependent on PHR1. Furthermore, transcript up regulation was found in phosphate-deficient shoots of the phr1 mutant for SULTR2;1 and SULTR3;4, indicating that PHR1 played both a positive and negative role on the expression of genes encoding sulfate transporters. Importantly, both phr1 and sultr1;3 mutants displayed a reduction in their sulfate shoot-to-root transfer capacity compared to wild-type plants under phosphate-deficient conditions. Conclusions: This study reveals that PHR1 plays an important role in sulfate inter-organ transport, in particular on the regulation of the SULTR1;3 gene and its impact on shoot-to-root sulfate transport in phosphate-deficient plants. PHR1 thus contributes to the homeostasis of both sulfate and phosphate in plants under phosphate deficiency. Such a function is also conserved in Chlamydomonas reinhardtii via the PHR1 ortholog PSR1.

Iron deficiency and anaemia in bariatric surgical patients: causes, diagnosis and proper management

Obesity-induced chronic inflammation leads to activation of the immune system that causes alterations of iron homeostasis including hypoferraemia, iron-restricted erythropoiesis, and finally mild-to-moderate anaemia. Thus, preoperative anaemia and iron deficiency are common among obese patients scheduled for bariatric surgery (BS). Assessment of patients should include a complete haematological and biochemical laboratory work-up, including measurement of iron stores, vitamin B12 and folate. In addition, gastrointestinal evaluation is recommended for most patients with iron-deficiency anaemia. On the other hand, BS is a long-lasting inflammatory stimulus in itself and entails a reduction of the gastric capacity and/or exclusion from the gastrointestinal tract which impair nutrients absorption, including dietary iron. Chronic gastrointestinal blood loss and iron-losingenteropathy may also contribute to iron deficiency after BS. Perioperative anaemia has been linked to increased postoperative morbidity and mortality and decreased quality of life after major surgery, whereas treatment of perioperative anaemia, and even haematinic deficiency without anaemia, has been shown to improve patient outcomes and quality of life. However, long-term follow-up data in regard to prevalence, severity, and causes of anaemia after BS are mostly absent. Iron supplements should be administered to patients after BS, but compliance with oral iron is no good. In addition, once iron deficiency has developed, it may prove refractory to oral treatment. In these situations, IV iron (which can circumvent the iron blockade at enterocytes and macrophages) has emerged as a safe and effective alternative for perioperative anaemia management. Monitoring should continue indefinitely even after the initial iron repletion and anaemia resolution, and maintenance IV iron treatment should be provided as required. New IV preparations, such ferric carboxymaltose, are safe, easy to use and up to 1000 mg can be given in a single session, thus providing an excellent tool to avoid or treat iron deficiency in this patient population.

Steady-state brain glucose transport kinetics re-evaluated with a four-state conformational model.

Glucose supply from blood to brain occurs through facilitative transporter proteins. A near linear relation between brain and plasma glucose has been experimentally determined and described by a reversible model of enzyme kinetics. A conformational four-state exchange model accounting for trans-acceleration and asymmetry of the carrier was included in a recently developed multi-compartmental model of glucose transport. Based on this model, we demonstrate that brain glucose (G(brain)) as function of plasma glucose (G(plasma)) can be described by a single analytical equation namely comprising three kinetic compartments: blood, endothelial cells and brain. Transport was described by four parameters: apparent half saturation constant K(t), apparent maximum rate constant T(max), glucose consumption rate CMR(glc), and the iso-inhibition constant K(ii) that suggests G(brain) as inhibitor of the isomerisation of the unloaded carrier. Previous published data, where G(brain) was quantified as a function of plasma glucose by either biochemical methods or NMR spectroscopy, were used to determine the aforementioned kinetic parameters. Glucose transport was characterized by K(t) ranging from 1.5 to 3.5 mM, T(max)/CMR(glc) from 4.6 to 5.6, and K(ii) from 51 to 149 mM. It was noteworthy that K(t) was on the order of a few mM, as previously determined from the reversible model. The conformational four-state exchange model of glucose transport into the brain includes both efflux and transport inhibition by G(brain), predicting that G(brain) eventually approaches a maximum concentration. However, since K(ii) largely exceeds G(plasma), iso-inhibition is unlikely to be of substantial importance for plasma glucose below 25 mM. As a consequence, the reversible model can account for most experimental observations under euglycaemia and moderate cases of hypo- and hyperglycaemia.