Rexhep Rexhepaj

Na+-d-glucose Cotransporter SGLT1 is Pivotal for Intestinal Glucose Absorption and Glucose-Dependent Incretin Secretion

To clarify the physiological role of Na+-d-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1−/− mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1−/− mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1−/− mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose–free diet. In wild-type mice, passage of d-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1−/− mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2.

Vitamin D-Rich Diet in Mice Modulates Erythrocyte Survival.

Background/Aims: Epidemiological evidence suggests that vitamin D deficiency is associated with anemia. The potent metabolite 1,25(OH)2 vitamin D3 [1,25(OH)2D3] activates various signaling cascades regulating a myriad of cellular functions including suicidal cell death or apoptosis. Suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Stimulation of eryptosis may limit lifespan of circulating erythrocytes and thus cause anemia. In the present study, we explored the effect of a high vitamin D diet (10,000 I.U. vitamin D for 14 days) in mice on eryptosis. Methods: Plasma concentrations of erythropoietin were estimated using an immunoassay kit, blood count using an electronic hematology particle counter, relative reticulocyte numbers using Retic-COUNT® reagent, PS exposure at the cell surface from annexin V binding, cell volume from forward scatter, and cytosolic Ca2+ ([Ca2+]i) from Fluo3-fluorescence in FACS analysis. Results: Vitamin D treatment decreased mean corpuscular volume, reticulocyte count, and plasma erythropoietin levels. Vitamin D treatment slightly but significantly decreased forward scatter but did not significantly modify spontaneous PS exposure and [Ca2+]i of freshly drawn erythrocytes. Vitamin D treatment augmented the stimulation of PS exposure and cell shrinkage following exposure to hyperosmotic shock (addition of 550 mM sucrose) or energy depletion (glucose removal) without significantly modifying [Ca2+]i. Conclusions: The present observations point to a subtle effect of exogenous vitamin D supplementation on erythrocyte survival.

Hyperaldosteronism in Klotho-deficient mice

Klotho is a membrane protein participating in the inhibitory effect of FGF23 on the formation of 1,25-dihydroxyvitamin-D(3) [1,25(OH)(2)D(3)]. It participates in the regulation of renal tubular phosphate reabsorption and stimulates renal tubular Ca(2+) reabsorption. Klotho hypomorphic mice (klotho(hm)) suffer from severe growth deficit, rapid aging, and early death, events largely reversed by a vitamin D-deficient diet. The present study explored the role of Klotho deficiency in mineral and electrolyte metabolism. To this end, klotho(hm) mice and wild-type mice (klotho(+/+)) were subjected to a normal (D(+)) or vitamin D-deficient (D(-)) diet or to a vitamin D-deficient diet for 4 wk and then to a normal diet (D(-/+)). At the age of 8 wk, body weight was significantly lower in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice, klotho(hm)D(-) mice, and klotho(hm)D(-/+) mice. Plasma concentrations of 1,25(OH)(2)D(3,) adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH), and aldosterone were significantly higher in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice. Plasma volume was significantly smaller in klotho(hm)D(-/+) mice, and plasma urea, Ca(2+), phosphate and Na(+), but not K(+) concentrations were significantly higher in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice. The differences were partially abrogated by a vitamin D-deficient diet. Moreover, the hyperaldosteronism was partially reversed by Ca(2+)-deficient diet. Ussing chamber experiments revealed a marked increase in amiloride-sensitive current across the colonic epithelium, pointing to enhanced epithelial sodium channel (ENaC) activity. A salt-deficient diet tended to decrease and a salt-rich diet significantly increased the life span of klotho(hm)D(+) mice. In conclusion, the present observation disclose that the excessive formation of 1,25(OH)(2)D(3) in Klotho-deficient mice results in extracellular volume depletion, which significantly contributes to the shortening of life span.

Stimulation of electrogenic glucose transport by glycogen synthase kinase 3.

Glycogen synthase kinase 3 GSK3β participates in a wide variety of functions including regulation of glucose metabolism. It is ubiquitously expressed including epithelial tissues. However, whether GSK3β participates in the regulation of epithelial transport is not known. The present study thus explored whether GSK3β influences the Na+-coupled transport of glucose. To this end, SGLT1 was expressed in Xenopus oocytes with or without GSK3β and glucose-induced current (Ig) determined by dual electrode voltage clamp. In Xenopus oocytes expressing SGLT1 but not in water-injected oocytes glucose induced an inwardly directed Ig, which was significantly enhanced by coexpression of GSK3β. According to chemiluminescence and confocal microscopy, GSK3β increased the SGLT1 protein abundance in the oocyte cell membrane. To explore whether GSK3β sensitivity of SGLT1 participates in the regulation of electrogenic intestinal glucose transport, Ussing chamber experiments were performed in intestinal segments from gene-targeted knockin mice with mutated and thus PKB/SGK-resistant GSK3α,β (gsk3KI), in which the serine of the PKB/SGK phosphorylation site was replaced by alanine, and from wild type mice (gsk3WT). The glucose-induced current was significantly larger in gsk3KI than in gsk3WT mice. The present observations reveal a novel function of GSK3, i.e. the stimulation of Na+-coupled glucose transport.

APC sensitive gastric acid secretion.

Adenomatous polyposis coli (APC) is a tumor suppressor gene inactivated in familial adenomatous polyposis and sporadic colorectal cancer. Mice carrying a loss-of-function mutation in the apc gene (apc^Min/+) spontaneously develop gastrointestinal tumors. APC fosters degradation of β-catenin, which in turn upregulates the serum- and glucocorticoid-inducible kinase SGK1. SGK1 stimulates KCNQ1, which is required for luminal K⁺ recycling and thus for gastric acid secretion. BCECF-fluorescence was utilized to determine gastric acid secretion in isolated gastric glands from apc^Min/+ mice and their wild type littermates (apc^+/+). Western blotting was employed to analyse β-catenin and SGK1 expression and immunohistochemistry to determine KCNQ1 protein abundance. β-catenin and SGK1 expression were enhanced in apc^Min/+ mice. Cytosolic pH was similar in apc^Min/+ mice and apc^+/+ mice. Na⁺-independent pH recovery following an ammonium pulse (ΔpH/min), which reflects H⁺/K⁺ ATPase activity, was, however, significantly faster in apc^Min/+ mice than in apc^+/+mice. In both genotypes ΔpH/min was abolished in the presence of H⁺/K⁺ ATPase inhibitor omeprazole (100 μM). Treatment of apc^Min/+ and apc^+/+mice with 5 μM forskolin 15 minutes prior to the experiment or increase in local K⁺-concentrations to 35 mM (replacing Na⁺/NMDG) significantly increased ΔpH/min and abrogated the differences between genotypes. The increase of ΔpH/min in apc^Min/+mice required SGK1, as it was abolished by additional knockout of SGK1 (apc^Min/+/sgk1^-/-). In conclusion, basal gastric acid secretion is significantly enhanced in apc^Min/+mice, pointing to a role of APC in the regulation of gastric acid secretion. The effect of APC requires H⁺/K⁺ ATPase activity and is at least partially due to SGK1-dependent upregulation of KCNQ1.

Role of maternal glucocorticoid inducible kinase SGK1 in fetal programming of blood pressure in response to prenatal diet

Maternal stress and malnutrition modify intrauterine fetal development with impact on postnatal blood pressure, nutrient, water, and electrolyte metabolism. The present study explored the possible involvement of maternal serum- and glucocorticoid-inducible kinase (SGK)-1 in fetal programming of blood pressure. To this end, wild-type (sgk1(+/+)) male mice were mated with SGK1 knockout (sgk1(-/-)) female mice, and sgk1(-/-) males with sgk1(+/+) females, resulting in both cases in heterozygotic (sgk1(-/+)) offspring. Following prenatal protein restriction, the offspring of sgk1(+/+) mothers gained weight significantly slower and had significantly higher blood pressure after birth. Moreover, a sexual dimorphism was apparent in fasting blood glucose and plasma corticosterone concentrations, with higher levels in female offspring. In contrast, prenatal protein restriction of sgk1(-/-) mothers had no significant effect on postnatal weight gain, blood pressure, plasma glucose concentration, or corticosterone levels, irrespective of offspring sex. Plasma aldosterone concentration, urinary flow rates, and urinary excretions of Na(+) and K(+) were not significantly modified by either maternal genotype or nutritional manipulation. In conclusion, maternal signals mediated by SGK1 may play a decisive role in fetal programming of hypertension induced by prenatal protein restriction.

Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice

The phosphoinositide‐dependent kinase PDK1 activates the serum‐ and glucocorticoid‐inducible kinase isoforms SGK1, SGK2, and SGK3 and protein kinase B, which in turn are known to up‐regulate a variety of sodium‐coupled transporters. The present study was performed to explore the role of PDK1 in amino acid transport. As mice completely lacking functional PDK1 are not viable, mice expressing 10–25% of PDK1 (pdk1hm) were compared with their wild‐type (WT) littermates (pdk1wt). Body weight was significantly less in pdk1hm than in pdk1wtmice. Despite lower body weight of pdk1hmmice, food and water intake were similar in pdk1hm and pdk1wtmice. According to Ussing chamber experiments, electrogenic transport of phenylalanine, cysteine, glutamine, proline, leucine, and tryptophan was significantly smaller in jejunum of pdk1hmmice than in pdk1wtmice. Similarly, electrogenic transport of phenylalanine, glutamine, and proline was significantly decreased in isolated perfused proximal tubules of pdk1hmmice. The urinary excretion of proline, valine, guanidinoacetate, methionine, phenylalanine, citrulline, glutamine/glutamate, and tryptophan was significantly larger in pdk1hm than in pdk1wtmice. According to immunoblotting of brush border membrane proteins prepared from kidney, expression of the Na‐dependent neutral amino acid transporter B0AT1 (SLC6A19), the glutamate transporter EAAC1/ EAAT3 (SLC1A1), and the transporter for cationic amino acids and cystine b0,+AT (SLC7A9) was decreased but the Na/proline cotransporter SIT (SLC6A20) was increased in pdk1hm mice. In conclusion, reduction of functional PDK1 leads to impairment of intestinal absorption and renal reabsorption of amino acids. The combined intestinal and renal loss of amino acids may contribute to the growth defect of PDK1deficient mice.—Rexhepaj, R., Grahammer, F., Völkl, H., Remy, C., Wagner, C. A., Sandulache, D., Artunc, F., Henke, G., Nammi, S., Capasso, G., Alessi, D. R., Lang, F. Reduced intestinal and renal amino acid transport in PDK1 hypomorphic mice. FASEB J. 20, 2214–2222 (2006)

PI3 kinase and PDK1 in the regulation of the electrogenic intestinal dipeptide transport.

The phosphoinositol 3 kinase (PI3K) and the phosphoinositide dependent kinase (PDK1) stimulate the serum and glucocorticoid inducible kinase (SGK) and protein kinase B (PKB/Akt) isoforms, kinases stimulating a variety of transporters. Most recently, SGK1 was shown to stimulate the peptide transporters PepT1 and PepT2, and to mediate the glucocorticoid stimulation of PepT1. Basal electrogenic intestinal peptide transport was, however, not dependent on the presence of SGK1. The present study explored whether basal electrogenic intestinal peptide transport is dependent on PI3K or PDK1. To this end, peptide transport in intestinal segments was determined utilizing Ussing chamber analysis. Cytosolic pH (pHi) was determined by BCECF fluorescence. The luminal addition of 5 mM dipeptide gly-gly induced a current (Ip) across intestinal segments. Ip was significantly decreased in the presence of PI3 kinase inhibitors Wortmannin (1 µM) or LY294002 (50 µM). Exposure of isolated intestinal cells to 5 mM gly-gly was followed by cytosolic acidification (ΔpHi), which was significantly blunted by Wortmannin and by LY294002. Both, Ip and ΔpHi were significantly smaller in PDK1 hypomorphic mice (pdk1flfl) than in their wild type littermates (pdk1wt). In conclusion, PI3K and PDK1 participate in the regulation of basal peptide transport.

Decreased intestinal glucose transport in the sgk3-knockout mouse

Xenopus oocyte coexpression experiments revealed the capacity of the serum- and glucocorticoid-inducible kinase isoform 3 (SGK3) to up-regulate a variety of transport systems including the sodium-dependent glucose transporter SGLT1. The present study explored the functional significance of SGK3-dependent regulation of intestinal transport. To this end, experiments were performed in gene targeted mice lacking functional sgk3 (sgk3−/−) and their wild type littermates (sgk3+/+). Oral food intake and fecal dry weight were significantly larger in sgk3−/− than in sgk3+/+ mice. Glucose-induced current (Ig) in Ussing chamber as a measure of Na+ coupled glucose transport was significantly smaller in sgk3−/− than in sgk3+/+ mouse jejunal segments. Fasting plasma glucose concentrations were significantly lower in sgk3−/− than in sgk3+/+ mice. Intestinal electrogenic transport of phenylalanine, cysteine, glutamine and proline were not significantly different between sgk3−/− and sgk3+/+ mice. In conclusion, SGK3 is required for adequate intestinal Na+ coupled glucose transport and impaired glucose absorption may contribute to delayed growth and decreased plasma glucose concentrations of SGK3 deficient mice. The hypoglycemia might lead to enhanced food intake to compensate for impaired intestinal absorption.

Hyperaldosteronism, hypervolemia, and increased blood pressure in mice expressing defective APC

Adenomatous polyposis coli (APC) fosters degradation of beta-catenin, a multifunctional protein upregulating the serum- and glucocorticoid-inducible kinase (SGK1). SGK1 regulates a wide variety of renal transport processes. The present study explored the possibility that APC influences renal function. To this end, metabolic cage experiments were performed in mice carrying a loss-of-function mutation in the APC gene (apc(Min/+)), their wild-type littermates (apc(+/+)), and apc(Min/+) mice lacking functional SGK1 (apc(Min/+)/sgk1(-/-)). As a result, mean body weight, food intake, fluid intake, salt appetite, urinary flow, as well as plasma Na(+) and K(+) concentrations were similar in apc(Min/+) mice, apc(+/+) mice, and apc(Min/+)/sgk1(-/-) mice. Glomerular filtration rate and absolute renal Na(+) excretion were decreased, and fractional urinary K(+) excretion was enhanced in apc(Min/+) mice. The antinatriuresis, but not the hypofiltration and kaliuresis was partially reversed by additional lack of SGK1. Plasma corticosterone and aldosterone concentrations were significantly enhanced in apc(Min/+) mice. While the plasma corticosterone concentration was similar in apc(+/+) mice and apc(Min/+)/sgk1(-/-) mice, plasma aldosterone was even higher in apc(Min/+)/sgk1(-/-) mice than in apc(Min/+) mice. The hyperaldosteronism of apc(Min/+) mice was paralleled by significantly elevated plasma volume and blood pressure. The experiments reveal an influence of defective APC on adrenal hormone release and renal function, effects partially but not completely explained by APC dependence of SGK1 expression.