Glucose transporters in the mammalian blood cells

Glucose is the main source of metabolic energy for various cellular functions, and thus plays a central role in supporting intermediary metabolism and cellular homeostasis. Since plasma membrane is impermeable to glucose, its cellular uptake is mediated by two distinct processes via specific glucose transporter proteins that belong to the family of solute carriers (SLC); the SLC2 family members, GLUTs (glucose transporters), are sodium-independent facilitators of the glucose transport, whereas the SLC5 family members, SGLTs (sodium and glucose transporters) mediate the secondary-active sodium- glucose cotransport. Until now, 14 GLUTs and 12 SGLTs isoforms have been identified in humans of which 5 GLUTs and none SGLTs were detected in the mammalian blood cells. Detailed physiological function, precise mechanism of transport, substrates affinity, exact three-dimensional structures, and a precise tissue distribution of most GLUTs in various mammalian organs, including blood, have been poorly explored. In this review we will focus on GLUTs in the mammalian blood cells, where the data on their expression and functional roles are contradictory or largely missing. Since many GLUTs are associated with diabetes, and are up-regulated in cancers, it is undoubtedly important to further investigate GLUTs expression in different organs/tissues, including the blood cells. Understanding the complexity of glucose homeostasis that includes knowledge about tissue distribution and function of GLUTs, as well as the signaling pathways that regulate glucose metabolism, may help to develop new therapeutic strategies to target specific diseases, such as diabetes mellitus, some autoimmunity diseases, and cancer

Glucose transporters in the mammalian blood cells

Glucose is the main source of metabolic energy for various cellular functions, and thus plays a central role in supporting intermediary metabolism and cellular homeostasis. Since plasma membrane is impermeable to glucose, its cellular uptake is mediated by two distinct processes via specific glucose transporter proteins that belong to the family of solute carriers (SLC); the SLC2 family members, GLUTs (glucose transporters), are sodium-independent facilitators of the glucose transport, whereas the SLC5 family members, SGLTs (sodium and glucose transporters) mediate the secondary-active sodium- glucose cotransport. Until now, 14 GLUTs and 12 SGLTs isoforms have been identified in humans of which 5 GLUTs and none SGLTs were detected in the mammalian blood cells. Detailed physiological function, precise mechanism of transport, substrates affinity, exact three-dimensional structures, and a precise tissue distribution of most GLUTs in various mammalian organs, including blood, have been poorly explored. In this review we will focus on GLUTs in the mammalian blood cells, where the data on their expression and functional roles are contradictory or largely missing. Since many GLUTs are associated with diabetes, and are up-regulated in cancers, it is undoubtedly important to further investigate GLUTs expression in different organs/tissues, including the blood cells. Understanding the complexity of glucose homeostasis that includes knowledge about tissue distribution and function of GLUTs, as well as the signaling pathways that regulate glucose metabolism, may help to develop new therapeutic strategies to target specific diseases, such as diabetes mellitus, some autoimmunity diseases, and cancer

Comparison of Three RT-PCR Based Methods for Relative Quantification of mRNA

Comparison of three RT-PCR based methods: semi-quantitative, competitive and real--time RT-PCR for relative quantification of mRNA is presented. Aminopeptidase N expressed on human promyeloid HL-60 cell line, at basal and activated state, served as a model for comparison. HL-60 cells were stimulated with IFN- (6 ng/mL) for 72 h at 37 oC, total cellular RNA was isolated, reverse transcribed to cDNA and semi-quantitative, competitive and real-time RT-PCR were performed to obtain the relative levels of mRNA for aminopeptidase N. The data obtained showed that all three RT-PCR based methods gave reliable and comparable results, i.e. approximately twofold increase of aminopeptidase N mRNA on IFN- stimulated HL-60 cells. Thus, in spite of rapid advances made in the area of real-time RT-PCR, end-point RT-PCR such as competitive and semi-quantitative RT- -PCR, although laborious and time consuming, may still remain useful techniques for relative mRNA quantification when small number of samples are to be analyzed

Prijenosnici natrija i glukoze: nove mete ciljanih terapija u liječenju raka?

Glucose, the key source of metabolic energy, is imported into cells by two categories of transporters: 1) facilitative glucose transporters (GLUTs) and 2) secondary active sodium-glucose cotransporters (SGLTs). Cancer cells have an increased demand for glucose uptake and utilisation compared to normal cells. Previous studies have demonstrated the overexpression of GLUTs, mainly GLUT1, in many cancer types. As the current standard positron emission tomography (PET) tracer 2-deoxy-2-(18F)fluoro-D-glucose (2-FDG) for imaging tumour cells via GLUT1 lacks in sensitivity and specificity, it may soon be replaced by the newly designed, highly sensitive and specific SGLT tracer α-methyl-4-(F-18)fluoro-4-deoxy-Dglucopyranoside (Me-4FDG) in clinical detection and tumour staging. This tracer has recently demonstrated the functional activity of SGLT in pancreatic, prostate, and brain cancers. The mRNA and protein expression of SGLTs have also been reported in colon/colorectal, lung, ovarian, head, neck, and oral squamous carcinomas. So far, SGLTs have been poorly investigated in cancer, and their protein expression and localisation are often controversial due to a lack of specific SGLT antibodies. In this review, we describe current knowledge concerning SGLT1 and SGLT2 (over)expression in various cancer types. The findings of SGLTs in malignant cells may help in developing novel cancer therapies with SGLT2 or SGLT1/SGLT2 inhibitors already used in diabetes mellitus treatment.Glukoza, glavni izvor metaboličke energije, ulazi u stanicu na dva načina: 1) olakšanom difuzijom pomoću prijenosnika glukoze GLUT i 2) sekundarno aktivnim prijenosom pomoću prijenosnika natrija i glukoze SGLT. Stanice raka imaju povećani unos glukoze u usporedbi s normalnim stanicama. Prethodna istraživanja pokazala su povećanu ekspresiju prijenosnika GLUT, uglavnom GLUT1, u mnogim tipovima raka. Radiofarmaceutik (engl. tracer) 2-deoksi-2-(18F) fluoro-D-glukoza (2-FDG), koji se koristi za detekciju tumorskih stanica putem GLUT1, nije dovoljno osjetljiv i specifičan. Uskoro bi mogao biti zamijenjen α-metil-4-(F-18) fluoro-4-deoksi-D-glukopiranozidom (Me-4FDG), novim i visoko osjetljivim, i specifičnim SGLT-radiofarmaceutikom u kliničkoj detekciji i određivanju stadija tumora. Tim je radiofarmaceutikom nedavno dokazana funkcionalna aktivnost prijenosnika SGLT u raku gušterače, prostate i mozga. Ekspresija mRNA i proteina SGLT također je pronađena u raku debelog crijeva, pluća, jajnika, glave, vrata i pločastih stanica usne šupljine. Prijenosnici SGLT nedovoljno su istraženi u raku, a njihova ekspresija i lokalizacija često su oprečne zbog nedostatka specifičnih SGLT-protutijela. U ovom preglednom radu opisujemo trenutačna znanja o povećanoj ekspresiji prijenosnika SGLT1 i SGLT2 u različitim tipovima raka. Spoznaje o ekspresiji i/ili lokalizaciji prijenosnika SGLT u malignim stanicama pomoći će u razvoju novih terapija u liječenju raka korištenjem već poznatih antidijabetika, SGLT2 ili SGLT1/SGLT2 inhibitora

Are mice, rats, and rabbits good models for physiological, pharmacological and toxicological studies in humans?

In the mammalian kidneys, handling of various organic compounds is mediated by multispecific organic anion and cation transporters localized in the luminal and contraluminal cell membrane domains of specific nephron segments, largely in proximal tubules. These transporters are responsible for cellular uptake and/or elimination of endogenous and xenobiotic organic compounds, including various anionic and cationic drugs, thus contributing to their reabsorption and/or secretion along the nephron. Recent studies have indicated a pivotal role of these transporters in drug resistance, drug-drug interactions, and drug-induced nephrotoxicity, whereas the presence of disfunctional transporters due to truncated isoforms or point mutations can cause genetic diseases. In rat, mouse and rabbit nephrons, a number of these transporters exhibit sex differences in their protein and/or mRNA expression. In comparison with the expression in rodents and rabbits, in the human nephrons some transporters are absent, some exhibit different localization in the cell membrane domains, and none exhibit the sex-dependent expression. Species differences in some transporters have been further demonstrated concerning substrate selectivity, distribution in cells along the nephron, levels of mRNA and/or protein expression, sensitivity to inhibitors, and regulation. Overall these differences in the mammalian kidneys indicate that: a) data on the membrane transporters-related functions in one species can not simply be regarded as relevant for other species, and b) many physiological, pharmacological, and toxicological findings related to organic anion and cation transport and transporters in rodents and rabbits do not reflect the situation in humans

Comparison of Three RT-PCR Based Methods for Relative Quantification of mRNA

Comparison of three RT-PCR based methods: semi-quantitative, competitive and real--time RT-PCR for relative quantification of mRNA is presented. Aminopeptidase N expressed on human promyeloid HL-60 cell line, at basal and activated state, served as a model for comparison. HL-60 cells were stimulated with IFN- (6 ng/mL) for 72 h at 37 oC, total cellular RNA was isolated, reverse transcribed to cDNA and semi-quantitative, competitive and real-time RT-PCR were performed to obtain the relative levels of mRNA for aminopeptidase N. The data obtained showed that all three RT-PCR based methods gave reliable and comparable results, i.e. approximately twofold increase of aminopeptidase N mRNA on IFN- stimulated HL-60 cells. Thus, in spite of rapid advances made in the area of real-time RT-PCR, end-point RT-PCR such as competitive and semi-quantitative RT- -PCR, although laborious and time consuming, may still remain useful techniques for relative mRNA quantification when small number of samples are to be analyzed

Oksalat – od okoliša do bubrežnih kamenaca

Oxalate urolithiasis (nephrolithiasis) is the most frequent type of kidney stone disease. Epidemiological research has shown that urolithiasis is approximately twice as common in men as in women, but the underlying mechanism of this sex-related prevalence is unclear. Oxalate in the organism partially originate from food (exogenous oxalate) and largely as a metabolic end-product from numerous precursors generated mainly in the liver (endogenous oxalate). Oxalate concentrations in plasma and urine can be modified by various foodstuffs, which can interact in positively or negatively by affecting oxalate absorption, excretion, and/or its metabolic pathways. Oxalate is mostly removed from blood by kidneys and partially via bile and intestinal excretion. In the kidneys, after reaching certain conditions, such as high tubular concentration and damaged integrity of the tubule epithelium, oxalate can precipitate and initiate the formation of stones. Recent studies have indicated the importance of the SoLute Carrier 26 (SLC26) family of membrane transporters for handling oxalate. Two members of this family [Sulfate Anion Transporter 1 (SAT-1; SLC26A1) and Chloride/Formate EXchanger (CFEX; SLC26A6)] may contribute to oxalate transport in the intestine, liver, and kidneys. Malfunction or absence of SAT-1 or CFEX has been associated with hyperoxaluria and urolithiasis. However, numerous questions regarding their roles in oxalate transport in the respective organs and male-prevalent urolithiasis, as well as the role of sex hormones in the expression of these transporters at the level of mRNA and protein, still remain to be answered.Oksalatna urolitijaza (nefrolitijaza) najučestaliji je tip bolesti bubrežnih kamenaca. Rezultati epidemioloških istraživanja pokazali su da je urolitijaza približno dvostruko učestalija u muškaraca nego u žena, ali osnovni mehanizam nastanka ove spolno-ovisne prevalencije nije razjašnjen. Oksalat u organizmu dijelom potječe iz hrane (egzogeni oksalat), a glavninom nastaje kao konačni produkt metabolizma raznih preteča u jetrima (endogeni oksalat). Na koncentraciju oksalata u plazmi i urinu utječu razne tvari iz hrane, koje mogu pozitivno ili negativno djelovati na apsorpciju, metaboličke puteve i/ili izlučivanje oksalata. Oksalat se iz organizma izlučuje u manjem obimu putem žuči u crijevo, a glavninom bubrezima. U bubrezima, pri odgovarajućim uvjetima kao što su visoka koncentracija oksalata i oštećenje epitela bubrežnih kanalića, oksalat može precipitirati i time potaknuti stvaranje kamenaca. Rezultati novih istraživanja upućuju na važnost membranskih prijenosnika otopljenih tvari (SoLute Carriers) iz obitelji 26 (SLC26) za prijenos oksalata u specifičnim organima. Smatra se da dva člana ove obitelji: prijenosnik sulfatnog aniona (Sulfate Anion Transporter 1; SAT-1; SLC26A1) i izmjenjivač klora i mravlje kiseline (Chloride/Formate EXchanger; CFEX; SLC26A6), imaju značajnu ulogu u prijenosu oksalata u crijevima, jetrima i bubrezima; hiperoksalurija i nefrolitijaza utvrđeni su pri slaboj aktivnosti ili nedostatku SAT-1 i CFEX proteina. Međutim, još uvijek postoje brojne nejasnoće glede prijenosa oksalata u navedenim organima, mehanizma nastanka spolnih razlika u nefrolitijazi i utjecaja spolnih hormona na ekspresiju proteina i mRNA za navedene prijenosnike

Expression and immunolocalization of metallothioneins MT1, MT2 and MT3 in rat nephron

Rodent kidneys exhibit three isoforms of metallothioneins (MTs), MT1, MT2 and MT3, with poorly characterized localization along the nephron. Here we studied in adult male Wistar rats the renal expression of MTs mRNA by end-point RT-PCR and MT proteins by immunochemical methods The expression pattern of MT1 mRNA was cortex (CO)>outer stripe (OS)=inner stripe (IS)=inner medulla (IM), of MT2 mRNA was IM>CO>IS=OS, and of MT3 mRNA was IM>CO=OS=IM. MT1/2-antibody stained with heterogeneous intensity the cell cytoplasm and nuclei in proximal tubule (PT) and thin ascending limb, whereas MT3-antibody stained weakly the cell cytoplasm in various cortical tubules and strongly the nuclei in all nephron segments. However, the isolated nuclei exhibited an absence of MT1/2 and presence of MT3 protein. In MT1/2-positive PT cells, the intracellular staining appeared diffuse or bipolar, but the isolated brush-border, basolateral and endosomal membranes were devoid of MT1/2 proteins. In the lumen of some PT profiles, the heterogeneously sized MT1/2-rich vesicles were observed, with the limiting membrane positive for NHE3, but negative for V-ATPase, CAIV, and megalin, whereas their interior was positive for CAII and negative for cytoskeleton. They seem to be pinched off from the luminal membrane of MT1/2-rich cells, as also indicated by transmission electron microscopy. We conclude that in male rats, MTs are heterogeneously abundant in the cell cytoplasm and/or nuclei along the nephron. The MT1/2-rich vesicles in the tubule lumen may represent a source of urine MT and membranous material, whereas MT3 in nuclei may handle zink and locally-produced reactive oxygen species

In female rats, ethylene glycol treatment elevates protein expression of hepatic and renal oxalate transporter sat-1 (Slc26a1) without inducing hyperoxaluria

Aim To investigate whether the sex-dependent expression of hepatic and renal oxalate transporter sat-1 (Slc26a1) changes in a rat model of ethylene glycol (EG)-induced hyperoxaluria. Methods Rats were given tap water (12 males and 12 females; controls) or EG (12 males and 12 females; 0.75% v/v in tap water) for one month. Oxaluric state was confirmed by biochemical parameters in blood plasma, urine, and tissues. Expression of sat-1 and rate-limiting enzymes of oxalate synthesis, alcohol dehydrogenase 1 (Adh1) and hydroxy-acid oxidase 1 (Hao1), was determined by immunocytochemistry (protein) and/or real time reverse transcription polymerase chain reaction (mRNA). Results EG-treated males had significantly higher (in μmol/L; mean ± standard deviation) plasma (59.7 ± 27.2 vs 12.9 ± 4.1, P < 0.001) and urine (3716 ± 1726 vs 241 ± 204, P < 0.001) oxalate levels, and more abundant oxalate crystaluria than controls, while the liver and kidney sat-1 protein and mRNA expression did not differ significantly between these groups. EG-treated females, in comparison with controls had significantly higher (in μmol/L) serum oxalate levels (18.8 ± 2.9 vs 11.6 ± 4.9, P < 0.001), unchanged urine oxalate levels, low oxalate crystaluria, and significantly higher expression (in relative fluorescence units) of the liver (1.59 ± 0.61 vs 0.56 ± 0.39, P = 0.006) and kidney (1.77 ± 0.42 vs 0.69 ± 0.27, P < 0.001) sat-1 protein, but not mRNA. The mRNA expression of Adh1 was femaledominant and that of Hao1 male-dominant, but both were unaffected by EG treatment. Conclusions An increased expression of hepatic and renal oxalate transporting protein sat-1 in EG-treated female rats could protect from hyperoxaluria and oxalate urolithiasis