Overexpression of glucose transporters in rat mesangial cells cultured in a normal glucose milieu mimics the diabetic phenotype.

An environment of high glucose concentration stimulates the synthesis of extracellular matrix (ECM) in mesangial cell (MC) cultures. This may result from a similar increase in intracellular glucose concentration. We theorized that increased uptake, rather than glucose concentration per se is the major determinant of exaggerated ECM formation. To test this, we compared the effects of 35 mM glucose on ECM synthesis in normal MCs with those of 8 mM glucose in the same cells overexpressing the glucose transporter GLUT1 (MCGT1). Increasing medium glucose from 8 to 35 mM caused normal MCs to increase total collagen synthesis and catabolism, with a net 81-90% increase in accumulation. MCs transduced with the human GLUT1 gene (MCGT1) grown in 8 mM glucose had a 10-fold greater GLUT1 protein expression and a 1.9, 2.1, and 2.5-fold increase in cell myo-inositol, lactate production, and cell sorbitol content, respectively, as compared to control MCs transduced with bacterial beta-galactosidase (MCLacZ). MCGT1 also demonstrated increased glucose uptake (5-fold) and increased net utilization (43-fold), and greater synthesis of individual ECM components than MCLacZ. In addition, total collagen synthesis and catabolism were also enhanced with a net collagen accumulation 111-118% greater than controls. Thus, glucose transport activity is an important modulator of ECM formation by MCs; the presence of high extracellular glucose concentrations is not necessarily required for the stimulation of matrix synthesis.

D-glucose stimulates mesangial cell GLUT1 expression and basal and IGF-I-sensitive glucose uptake in rat mesangial cells: implications for diabetic nephropathy.

The complications of diabetes arise in part from abnormally high cellular glucose uptake and metabolism. To determine whether altered glucose transporter expression may be involved in the pathogenesis of diabetic nephropathy, we investigated the effects of elevated extracellular glucose concentrations on facilitative glucose transporter (GLUT) expression in rat mesangial cells. GLUT1 was the only transporter isoform detected. Cells exposed to 20 mmol/l glucose medium for 3 days demonstrated increases in GLUT1 mRNA (134%, P < 0.002), GLUT1 protein (68%, P < 0.02), and V(max) (50%, P < 0.05) for uptake of the glucose analog [3H]2-deoxyglucose (3H2-DOG), when compared to cells chronically adapted to physiologic glucose concentrations (8 mmol/l). The increase in GLUT1 protein was sustained at 3 months, the latest time point tested (77% above control, P < 0.01). In contrast, hypertonic mannitol had no effect on GLUT1 protein levels. Insulin-like growth factor I (IGF-I; 30 ng/ml) increased the uptake of 3H2-DOG by 28% in 8 mmol/l glucose-treated cells (P < 0.05) and by 75% in cells switched to 20 mmol/l glucose for 3 days (P < 0.005). These increases in 3H2-DOG uptake occurred despite a lack of effect of IGF-I on GLUT1 protein levels (P > 0.5 vs. control). Therefore, hyperglycemia and IGF-I treatment both lead to increases in mesangial cell glucose uptake, and hyperglycemia induces increased GLUT1 expression, which can directly lead to the pathological changes of diabetic nephropathy. The effects of high glucose and of IGF-I to stimulate 3H2-DOG uptake also appear to be additive.

Dementia without Alzheimer pathology.

We studied five demented patients who, on neuropathologic examination, had cell loss and Lewy bodies in substantia nigra and locus ceruleus and few Alzheimer-type changes. The nucleus basalis had minimal cell loss in three patients and was not available in two. The lesions in the substantia nigra and locus ceruleus were unlikely to account for the dementia, and other structural or biochemical derangements, probably cortical but possibly subcortical, must also have been present but not visible at the light microscopic level.

Glucose-specific regulation of aldose reductase in human retinal pigment epithelial cells in vitro.

PURPOSE: To test the hypothesis that pathophysiological levels of glucose regulate aldose reductase (AR2) gene expression, protein production, and activity in human retinal pigment epithelial (RPE) cells in vitro. METHODS: Primary cultures of human RPE cells were grown for up to 72 hours in media supplemented with various concentrations of glucose (5, 20, or 75 mM), or in 5 mM glucose containing media supplemented with one of the following: galactose, the transported but nonmetabolized glucose analogue 3-O-methylglucose (3-OMG), or the impermeant hexitol mannitol-so that the final hexose concentrations were equimolar to those of the various glucose concentrations used. Changes in the transcript levels for AR2 mRNA, AR2 protein content, and AR2 enzyme activity were determined. RPE glucose utilization and lactate production were determined in media containing 5 and 20 mM glucose. RESULTS: Glucose utilization and lactate production increased 4.8-fold and 4.4-fold, respectively, when RPE cells were grown in media containing 20 mM versus 5 mM glucose. Glucose was more effective than any other hexose in the induction of AR2 mRNA or increased AR2 protein expression. When RPE cells were grown in media containing 20 mM mannitol, 3-OMG, or galactose they had lower levels of AR2 mRNA expression than when cells were grown in medium containing 5 mM glucose. RPE cells grown in medium supplemented with 20 or 75 mM galactose did not show a greater increase in AR2 protein expression than cells grown in medium containing 5 mM glucose. Hyperosmotic induction of AR2 mRNA was the same in medium containing 75 mM glucose or 75 mM mannitol, but was at least 50% lower when RPE cells were grown in 75 mM galactose or 3-OMG. CONCLUSIONS. These data indicate that elevations in ambient glucose result in greater metabolism of glucose through glycolysis and polyol metabolism. Induction of AR2 was greatest when RPE cells were grown in pathophysiological concentrations of glucose. Hyperosmolar stress is not a necessary determinant of AR2 mRNA, AR2 protein, or AR2 protein activity in cells that form the outer blood-retinal barrier. Increased facilitative glucose transport or glucose metabolism appears to be requisite for glucose-specific and nonosmotic regulation of AR2 in the RPE cell in vitro.

Identification of a novel form of renal glucosuria with overexcretion of arginine, carnosine, and taurine.

Glucosuria occurs in diabetes mellitus, generalized proximal tubular dysfunction of Fanconi's syndrome, glucose-galactose malabsorption syndrome, and primary renal glucosuria. Patients with primary renal glucosuria have normal blood glucose levels, normal oral glucose tolerance test results, and persistent glucosuria that may approach the filtered load of glucose in the most severe cases. The primary defect is proposed to be in the sodium-glucose cotransporter type-2 (SGLT2) located in the apical membrane of S1 segment proximal renal tubule cells. Primary renal glucosuria is classified as types A, B, or O based on the characteristics of the transport defect. The magnitude of glucosuria has varied from 20 to 150 g of glucose excreted in 24 hours. Described inheritance patterns have included both autosomal dominant and autosomal recessive mechanisms. Some cases have been associated with selective aminoaciduria, distinctly unlike the generalized aminoaciduria seen in Fanconi's syndrome. We report the first case of primary renal glucosuria with selective overexcretion of arginine, carnosine, and taurine. This case may represent a genetic defect unique from the abnormalities in previously described cases of primary renal glucosuria with different amino acid excretion patterns. Future investigations could determine whether the syndrome involves a defect in the SGLT2 gene.

Glucose transporters of the glomerulus and the implications for diabetic nephropathy.

Several glucose transporters have recently been identified in glomeruli, and in cultured glomerular cells. These include the facilitative glucose transporter isoforms GLUTs 1, 3 and 4, and sodium-glucose cotransport activity with characteristics of SGLT1. GLUTs 1, 3 and 4 are all high affinity, low capacity, facilitative glucose transporters which typically would be saturated at or near physiologic glucose concentrations. The SGLT transporter of mesangial cells is also a high affinity transporter which similarly could be saturated under normal glucose conditions. This suggests that in order for mesangial cells to take up excessive quantities of glucose in diabetes, changes in glucose transporter expression, translocation or activity may be required. Accordingly, recent investigations discovered positive-feedback regulation of the mesangial cell GLUT1 transporter by glucose, and a regulatory role for GLUT1 in glucose metabolism and extracellular matrix synthesis. Future investigations of glucose transporters in the pathogenesis of diabetic renal disease will now likely proceed in multiple directions, including but not limited to: (1) examination of their regulation by growth factors implicated in diabetic nephropathy, and the resultant effects on ECM synthesis; (2) determination of the mechanisms by which GLUT1 regulates the expression of aldose reductase, PKC, GLUT1, and other genes in the mesangial cell; and (3) Suppression of glucose transporters in attempts to prevent high glucose-induced diabetic glomerulosclerosis.

Altered vitamin D metabolism in type II diabetic mouse glomeruli may provide protection from diabetic nephropathy.

The db/db mouse develops features of type II diabetes mellitus as the result of impaired signaling through its abnormal leptin receptor. In spite of accurate metabolic features of diabetes, renal disease manifestations in these mice are not as severe as in humans suggesting the presence of protective genes. There is a growing body of evidence in humans for the relevance of vitamin D in diabetes. Here we followed a large cohort of db/db mice and their non-diabetic db/+ littermates. Transcriptional profiling revealed significant upregulation of 23 genes involved in Ca2+ homeostasis and vitamin D metabolism in db/db glomeruli relative to db/+ glomeruli. Increased glomerular expression of vitamin D3 1alpha-hydroxylase, vitamin D binding protein, calbindins D9K and D28K, and calcyclin mRNA was confirmed by quantitative reverse transcription-polymerase chain reaction in 20-, 36-, and 52-week-old db/db glomeruli. Although vitamin D3 1alpha-hydroxylase protein was primarily expressed and upregulated in db/db renal tubules, it was also expressed in glomerular podocytes in vivo. Serum 1,25-dihydroxyvitamin D3 and urinary Ca2+ excretion were increased >3-fold in db/db mice compared to db/+ mice. Cultured glomerular podocytes had mRNA for vitamin D3 1alpha-hydroxylase, vitamin D receptor, and calbindin D28K, each of which was increased in high glucose conditions. High glucose also led to enhanced production of fibronectin and collagen IV protein, which was blocked by 1,25-dihydroxyvitamin D3. These results show that vitamin D metabolism is altered in db/db mice leading to metabolic and transcriptional effects. The podocyte is affected by paracrine and potentially autocrine effects of vitamin D, which may explain why db/db mice are resistant to progressive diabetic nephropathy.

Methylamine and polyol responses to salt loading in renal inner medulla.

Methylamines and polyhydric alcohols (polyols) are major organic osmolytes of the mammalian renal inner medulla and have generally been noted to change in parallel with urine osmolality. In the present study, responses of inner medullary methylamines and polyols to 5 days of salt loading were investigated. Salt loading increased plasma sodium concentration and induced a saline diuresis that resulted in a significantly lower urine osmolality (Uosmol) in salt-loaded rats (1,246 mosmol) compared with controls (2,147 mosmol). Analysis of inner medullary organic osmolytes using 1H-NMR spectroscopy and biochemical assays indicated no significant change in total methylamines, total polyols, or total osmolytes with salt loading. However, there were marked changes in individual organic osmolytes. Renal inner medullary glycerophosphorylcholine (GPC) was 41% lower in salt-loaded rats, and was the only organic osmolyte that changed in parallel with Uosmol, which was 42% lower in this group. In contrast, glycine betaine (betaine) and sorbitol contents were elevated by 286% and 33%, respectively, with salt loading, and myo-inositol (inositol) was unchanged. These findings indicate selective renal inner medullary osmolyte responses to salt loading with only GPC varying directly with changes in urine osmolality.

Upregulation of inositol transport mediates inositol accumulation in hyperosmolar brain cells.

Attempts to understand brain volume regulation have been greatly hampered by the structural complexity of the mammalian central nervous system, indicating a need for the investigation of cultured brain cell lines whose behavior reflects that observed in situ. We demonstrate here that rat C6 glioma cells exhibit a pattern of hyperosmolar volume regulation qualitatively similar to that of the intact brain. Chronic (2-6 days) acclimation of C6 cells to high NaCl media (440 or 590 mosM) resulted in a 46-133 mM increase in cellular inositol, a known major brain osmolyte. C6 cells exposed acutely to 440 mosM medium shrank abruptly and then underwent a complete regulatory volume increase (RVI) within 4 h. Inositol levels began to increase after 10 h of hyperosmolar stress and reached maximal values by 24 h, suggesting that RVI is initially mediated by inorganic ion uptake. [3H]inositol uptake measurements revealed a sevenfold stimulation of phlorizin-inhibitable inositol transport in hyperosmotic cells. The enhancement of inositol transport paralleled the rise in cellular inositol content. Phlorizin reduced inositol accumulation in hyperosmolar cells by 44%. Our studies provide the first demonstration of RVI and organic osmolyte accumulation in a cultured brain cell line.

Glucose transporters control gene expression of aldose reductase, PKCalpha, and GLUT1 in mesangial cells in vitro.

The process linking increased glucose utilization and activation of metabolic pathways leading to end-organ damage from diabetes is not known. We have previously described rat mesangial cells that were transduced to constitutively express the facilitative glucose transporter 1 (GLUT1, MCGT1 cells) or bacterial beta-galactosidase (MCLacZ, control cells). Glucose transport was rate limiting for extracellular matrix production in the MCGT1 cells. In the present work, we investigated the effect of GLUT1 overexpression in mesangial cells on aldose reductase (AR), protein kinase Calpha (PKCalpha), and native GLUT1 transcript levels, to determine whether changes in GLUT1 alone could regulate their expression in the absence of high extracellular glucose concentrations. MCGT1 cells grown in normal (8 mM) or elevated (20 mM) glucose had elevated abundance of AR, PKCalpha, and the native GLUT1 transcripts compared with control cells. AR protein levels, AR activity, sorbitol production, and PKCalpha protein content were also greater in the MCGT1 cells than in control cells grown in the same media. This is the first report of the concomitant activation of AR, PKCalpha, and GLUT1 genes by enhanced GLUT1 expression. We conclude that increased GLUT1 expression leads to a positive feedback of greater GLUT1 expression, increased AR expression and activity with polyol accumulation, and increased total and active PKCalpha protein levels, which leads to detrimental stimulation of matrix protein synthesis by diabetic mesangial cells.

Methylamines and polyols in kidney, urinary bladder, urine, liver, brain, and plasma. An analysis using 1H nuclear magnetic resonance spectroscopy.

Methylamines and polyols are known to behave as organic osmolytes in the adaptation of many cells to hyperosmolar conditions. Using 1H nuclear magnetic resonance spectroscopy to analyze perchloric acid extracts we have examined several tissues in the rat for the presence of these compounds. Methylamines such as glycerophosphorylcholine, choline and betaine were observed in the renal inner medulla, urinary bladder, urine, liver, brain, and plasma. Myoinositol was relatively abundant in the renal inner medulla and brain whereas sorbitol was detected only in the inner medulla. A variety of unidentified compounds was also detected in each tissue. Although these methylamines and polyols are known to respond to osmotic changes in the renal inner medulla, their responses in other tissues remain to be investigated.

Characterization of the major brain osmolytes that accumulate in salt-loaded rats.

Previous studies demonstrated an accumulation of "idiogenic osmoles" in the brain with chronic salt loading. Amino acids are known to constitute a portion of these solutes, but the balance of the solutes has yet to be fully characterized. In the present study, 1H-nuclear magnetic resonance (NMR) spectroscopy and biochemical assays of rat brain were used to identify and quantify changes in organic solutes in two different animal models of hypernatremia: hypertonic salt loading and water deprivation. Five days of salt loading increased plasma sodium concentration (PNa) to 165 meq/l and 3 days of water deprivation increased PNa to 151 meq/l, compared with 141 meq/l in controls. Amino acids, methylamines, and polyols were all significantly higher in salt-loaded animals compared with controls. Specifically, higher contents of glutamine (+65%), glutamate (+27%), myo-inositol (+36%), phosphocreatine + creatine (PCr + Cr) (32%), glycerophosphorylcholine (GPC) (+75%), and choline (+114%) were observed. Sorbitol and betaine, osmolytes known to accumulate in the hypertonic inner medulla, were present in low amounts in the brain and were unchanged with salt loading. In contrast to the results with salt loading, no accumulation of brain organic solutes was detected after 3 days of water deprivation. Based on these findings, we propose that amino acids, methylamines, and polyols function as osmoregulatory solutes in the brains of salt-loaded rats in a manner similar to that observed in other biological systems, whereas 3 days of water deprivation is an insufficient stimulus for their accumulation.

Modulation of osmolytes in MDCK cells by solutes, inhibitors, and vasopressin.

MDCK cells accumulate organic osmolytes in response to hyperosmotic NaCl-supplemented medium. We examined time course and inhibitor sensitivity of myo-inositol, sorbitol, and glycerophosphorylcholine (GPC) accumulation in MDCK cells exposed to hyperosmotic NaCl-, D-glucose-, or mannitol-supplemented media. In NaCl medium, cells preferentially accumulated inositol and GPC. In comparison, in glucose medium cells preferentially accumulated sorbitol and GPC. Inositol demonstrated a late (72-96 h) accumulation in glucose medium, although less than in NaCl medium. Mannitol medium did not significantly stimulate accumulation of any of these three osmolytes at 24 h, suggesting that hyperosmolality alone is not sufficient stimulus for their accumulation in this time frame. GPC accumulation was very rapid in glucose medium, and fell to the level induced by NaCl medium at 96 h (approximately 50 nmol/mg protein). Inositol and sorbitol accumulated more gradually, each reaching greater than 400 nmol/mg protein after 96 h. Sorbitol was still accumulating at 96 h, whereas inositol plateaued at 72-96 h. Phlorizin or sorbinil blocked accumulation of inositol or sorbitol, respectively. Sorbitol and GPC accumulation in glucose medium were partially inhibited in absence of serum or in presence of 1 microM vasopressin. Thus NaCl and glucose appear to stimulate specific cellular mechanisms responsible for accumulation of inositol, sorbitol, and GPC in MDCK cells. This accumulation is also modulated by constituents of serum.

Organic osmolytes in inner medulla of Brattleboro rat: effects of ADH and dehydration.

Inner medullary methylamine [glycerophosphorylcholine (GPC) and glycine betaine (betaine)] and polyol [sorbitol and myo-inositol (inositol)] osmolytes were measured in water-restricted and antidiuretic hormone (ADH)-infused Brattleboro (DI) rats. Compared with DI rats allowed water ad libitum, rats dehydrated for 3 days had higher urinary osmolality (Uosmol) (812 vs. 239 mosmol/kgH2O) and plasma osmolality (Posmol) (333 vs. 296 mosmol/kgH2O). Dehydration reduced betaine content (36 vs. 66 nmol/mg protein) but had no significant effect on GPC, sorbitol, or inositol. In separate protocols, DI rats, allowed water ad libitum, were infused for either 3 or 12 days with either ADH in saline (+ADH) or saline alone (-ADH). Compared with -ADH controls, 3- or 12-day ADH-infused rats were antidiuretic (Uosmol, 1,000-1,300 mosmol/kgH2O) but not dehydrated (Posmol, 297-300 mosmol/kgH2O). Three days of ADH infusion caused an increase in GPC (340%), betaine (80%), and sorbitol (248%) but not in inositol. After 12 days of ADH, further increases were observed in GPC (730%) and sorbitol (870%); inositol was also elevated (170%), whereas betaine was unchanged. Consequently, the total osmolyte content was significantly higher in +ADH than in -ADH [449 vs. 256 (3 days) and 778 vs. 199 (12 day) nmol/mg protein], whereas total osmolyte levels in dehydrated and control rats were similar (222 vs. 219 nmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of major organic osmolytes in rat renal inner medulla in dehydration.

Osmotically active organic solutes, osmolytes, exist at high concentrations in the renal inner medulla; however, their modulation during antidiuresis remains largely undefined. Renal osmolyte levels were measured by nuclear magnetic resonance spectroscopy and biochemical assays in perchloric acid extracts from normal and dehydrated (3 days) rats. Dehydration increased urine osmolality from 1,503 to 3,748 mosmol/kg and inner medullary urea content from 2,036 to 4,405 nmol/mg protein. In addition, inner medullary trimethylamines [glycerophosphorylcholine (GPC) and betaine] and polyhydric alcohols (inositol and sorbitol) significantly increased by 95 and 78%, respectively. Ninhydrin-positive substances (amino acids), although abundant, were unchanged. Renal cortex also contained GPC, betaine, and inositol but only inositol increased with dehydration. Analysis of correlations among inner medullary osmolytes showed that only GPC was consistently elevated by dehydration and was not directly correlated with the other osmolytes. In contrast, betaine and inositol contents were linearly related to each other and both tended to rise only when sorbitol content was unchanged. In conclusion, the major osmolytes in the rat renal inner medulla can increase during antidiuresis but they are regulated in a complex manner.

Antisense GLUT-1 protects mesangial cells from glucose induction of GLUT-1 and fibronectin expression.

A stable clone of rat mesangial cells expressing antisense GLUT-1 (i.e., MCGT1AS cells) was developed to protect them from high glucose exposure. GLUT-1 protein was reduced 50%, and the 2-deoxy-[(3)H]glucose uptake rate was reduced 33% in MCGT1AS. MCLacZ control cells and MCGT1 GLUT-1-overexpressing cells were used for comparisons. In MCLacZ, 20 mM D-glucose increased GLUT-1 transcription 90% vs. no increase in MCGT1AS. Glucose (8 mM) and 12 mM xylitol [a hexose monophosphate (HMP) shunt substrate] did not stimulate GLUT-1 transcription. An 87% replacement of the standard 8 mM D-glucose with 3-O-methylglucose reduced GLUT-1 transcription 80%. D-Glucose (20 mM) increased fibronectin mRNA and protein by 47 and 100%, respectively, in MCLacZ vs. no increases in MCGT1AS. Fibronectin synthesis was elevated 48% in MCGT1 and reduced 44% in MCGT1AS. We conclude that 1) transcription of GLUT-1 in response to D-glucose depends on glucose metabolism, although not through the HMP shunt, and 2) antisense GLUT-1 treatment of mesangial cells blocks D-glucose-induced GLUT-1 and fibronectin expression, thereby demonstrating a protective effect that could be beneficial in the setting of diabetes.