Mutations in the gene encoding for the Na ϩ -glucose co-transporter SGLT2 (SLC5A2) associate with familial renal glucosuria, but the role of SGLT2 in the kidney is incompletely understood. Here, we determined the localization of SGLT2 in the mouse kidney and generated and characterized SGLT2-deficient mice. In wild-type (WT) mice, immunohistochemistry localized SGLT2 to the brush border membrane of the early proximal tubule. Sglt2 Ϫ/Ϫ mice had glucosuria, polyuria, and increased food and fluid intake without differences in plasma glucose concentrations, GFR, or urinary excretion of other proximal tubular substrates (including amino acids) compared with WT mice. SGLT2 deficiency did not associate with volume depletion, suggested by similar body weight, BP, and hematocrit; however, plasma renin concentrations were modestly higher and plasma aldosterone levels were lower in Sglt2mice. Whole-kidney clearance studies showed that fractional glucose reabsorption was significantly lower in Sglt2 Ϫ/Ϫ mice compared with WT mice and varied in Sglt2 Ϫ/Ϫ mice between 10 and 60%, inversely with the amount of filtered glucose. Free-flow micropuncture revealed that for early proximal collections, 78 Ϯ 6% of the filtered glucose was reabsorbed in WT mice compared with no reabsorption in Sglt2 Ϫ/Ϫ mice. For late proximal collections, fractional glucose reabsorption was 93 Ϯ 1% in WT and 21 Ϯ 6% in Sglt2 Ϫ/Ϫ mice, respectively. These results demonstrate that SGLT2 mediates glucose reabsorption in the early proximal tubule and most of the glucose reabsorption by the kidney, overall. This mouse model mimics and explains the glucosuric phenotype of individuals carrying SLC5A2 mutations. 22: 104 -112, 201122: 104 -112, . doi: 10.1681 Glucose is the main source of energy in eukaryotic organisms. The homeostasis of glucose is maintained by intestinal glucose absorption and the coordinated regulation of hepatic and renal glucose production, as well as tissue consumption of glucose. As a consequence of renal glomerular filtration, approximately 180 g/d glucose enter the tubular system of the kidneys in a healthy individual with normoglycemia, which is equivalent to approximately one third of the total energy consumed by the human body. Glucose in urine, however, is absent or at very low concentrations in healthy adults (range 0.03 to 0.30 g/d) as a result of near complete reabsorption along the nephron segments, primarily in the proximal tubule. The genes encoding transporter proteins participating in renal J Am Soc Nephrol

The Na-glucose cotransporter SGLT2 mediates high-capacity glucose uptake in the early proximal tubule and SGLT2 inhibitors are developed as new antidiabetic drugs. We used gene-targeted Sglt2 knockout (Sglt2(-/-)) mice to elucidate the contribution of SGLT2 to blood glucose control, glomerular hyperfiltration, kidney growth, and markers of renal growth and injury at 5 wk and 4.5 mo after induction of low-dose streptozotocin (STZ) diabetes. The absence of SGLT2 did not affect renal mRNA expression of glucose transporters SGLT1, NaGLT1, GLUT1, or GLUT2 in response to STZ. Application of STZ increased blood glucose levels to a lesser extent in Sglt2(-/-) vs. wild-type (WT) mice (∼300 vs. 470 mg/dl) but increased glucosuria and food and fluid intake to similar levels in both genotypes. Lack of SGLT2 prevented STZ-induced glomerular hyperfiltration but not the increase in kidney weight. Knockout of SGLT2 attenuated the STZ-induced renal accumulation of p62/sequestosome, an indicator of impaired autophagy, but did not attenuate the rise in renal expression of markers of kidney growth (p27 and proliferating cell nuclear antigen), oxidative stress (NADPH oxidases 2 and 4 and heme oxygenase-1), inflammation (interleukin-6 and monocyte chemoattractant protein-1), fibrosis (fibronectin and Sirius red-sensitive tubulointerstitial collagen accumulation), or injury (renal/urinary neutrophil gelatinase-associated lipocalin). SGLT2 deficiency did not induce ascending urinary tract infection in nondiabetic or diabetic mice. The results indicate that SGLT2 is a determinant of hyperglycemia and glomerular hyperfiltration in STZ-induced diabetes mellitus but is not critical for the induction of renal growth and markers of renal injury, inflammation, and fibrosis.

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Large collections of knockout organisms facilitate the elucidation of gene functions. Here we used retroviral insertion or homologous recombination to disrupt 472 genes encoding secreted and membrane proteins in mice, providing a resource for studying a large fraction of this important class of drug target. The knockout mice were subjected to a systematic phenotypic screen designed to uncover alterations in embryonic development, metabolism, the immune system, the nervous system and the cardiovascular system. The majority of knockout lines exhibited altered phenotypes in at least one of these therapeutic areas. To our knowledge, a comprehensive phenotypic assessment of a large number of mouse mutants generated by a gene-specific approach has not been described previously.

.-In the kidney, the sodiumglucose cotransporters SGLT2 and SGLT1 are thought to account for Ͼ90 and ϳ3% of fractional glucose reabsorption (FGR), respectively. However, euglycemic humans treated with an SGLT2 inhibitor maintain an FGR of 40 -50%, mimicking values in Sglt2 knockout mice. Here, we show that oral gavage with a selective SGLT2 inhibitor (SGLT2-I) dose dependently increased urinary glucose excretion (UGE) in wild-type (WT) mice. The dose-response curve was shifted leftward and the maximum response doubled in Sglt1 knockout (Sglt1Ϫ/Ϫ) mice. Treatment in diet with the SGLT2-I for 3 wk maintained 1.5-to 2-fold higher urine glucose/creatinine ratios in Sglt1Ϫ/Ϫ vs. WT mice, associated with a temporarily greater reduction in blood glucose in Sglt1Ϫ/Ϫ vs. WT after 24 h (Ϫ33 vs. Ϫ11%). Subsequent inulin clearance studies under anesthesia revealed free plasma concentrations of the SGLT2-I (corresponding to early proximal concentration) close to the reported IC50 for SGLT2 in mice, which were associated with FGR of 64 Ϯ 2% in WT and 17 Ϯ 2% in Sglt1Ϫ/Ϫ. Additional intraperitoneal application of the SGLT2-I (maximum effective dose in metabolic cages) increased free plasma concentrations ϳ10-fold and reduced FGR to 44 Ϯ 3% in WT and to Ϫ1 Ϯ 3% in Sglt1Ϫ/Ϫ. The absence of renal glucose reabsorption was confirmed in male and female Sglt1/Sglt2 double knockout mice. In conclusion, SGLT2 and SGLT1 account for renal glucose reabsorption in euglycemia, with 97 and 3% being reabsorbed by SGLT2 and SGLT1, respectively. When SGLT2 is fully inhibited by SGLT2-I, the increase in SGLT1-mediated glucose reabsorption explains why only 50 -60% of filtered glucose is excreted. proximal tubule; glucose reabsorption; glucose transport; sodium glucose cotransport inhibitor; diabetes mellitus ABOUT 180 G OF GLUCOSE ARE filtered daily by the kidney and enter the renal tubular system in a healthy normoglycemic subject. Glucose in urine is absent or at very low concentrations in healthy adults due to near complete reabsorption along the nephron segments, primarily in the proximal tubule. The renal Na ϩ -glucose cotransporter SGLT2 (SLC5A2) is localized to the early proximal tubule and thought to mediate the bulk of tubular glucose uptake across the apical membrane of the kidney (14,17,20). Studies in mice lacking Sglt2 demonstrated that SGLT2 mediates all glucose reabsorption in the early proximal tubule and most of overall glucose reabsorption by the kidney (17). In comparison, low-capacity SGLT1 (SLC5A1) "cleans up" most of the remaining luminal glucose in further distal parts of the proximal tubule (1,3,14,20). Studies in mice lacking Sglt1 (Sglt1Ϫ/Ϫ) revealed a fractional glucose excretion of 3% compared with 0.2% in wild-type (WT) (3). Whether glucose transporters other than SGLT2 and SGLT1 contribute in a measurable extent to renal glucose reabsorption across the luminal membrane of the renal epithelia has never been tested. Potential candidates include a lowaffinity Na ϩ -D-glucose cotransporter cloned from the rat named NaGL...

SummaryDysfunction and loss of insulin-producing pancreatic β cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38δ display improved glucose tolerance due to enhanced insulin secretion from pancreatic β cells. Deletion of p38δ results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38δ catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38δ null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated β cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38δ-deficient islets and glucose tolerance in p38δ null mice as well as their susceptibility to oxidative stress. In conclusion, the p38δ-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic β cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.

The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover blindness therapies. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells’ functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1−/− mice. We also find decreased photoreceptor-specific phosphatiydylcholine containing very long chain polyunsaturated fatty acids and severely-attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1−/− mice. RPE-rich eyecup cultures from AdipoR1−/− reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation, and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity.

The murine gene for adipocyte P2 encodes an adipocyte-specific member of the family of intracellular lipid binding proteins. The region upstream from the start of transcription of this gene has been found to contain binding sites for the transcription factors c-jun/c-fos and C/EBP (CCAAT/enhancer binding protein) and several short sequence elements found in other adipocyte gene promoters, termed fat-specific elements. To identify DNA sequences that were responsible for the high level of transcription of the gene for adipocyte P2 in vivo, we made a series of transgenic mice containing 168 base pairs (bp), 247 bp, 1.7 kilobases (kb), and 5.4 kb of 5' flanking sequence linked to the bacterial gene chloramphenicol acetyltransferase. Although plasmids containing only 168 bp of 5' sequence including the C/EBP and AP-1 (activation protein 1) binding sites were expressed well in cultured adipocytes, high levels of chloramphenicol acetyltransferase activity in the adipose tissue oftransgenic mice were not observed until the 5' flanking region was extended to kb -5.4. An enhancer mapping between kb -4.9 and kb -5.4 upstream from the start of transcription was identified by transfection of further deletions into cultured adipocytes. This enhancer, when linked to a bp -63 promoter fragment from the gene for adipocyte P2, directed very high level chloramphenicol acetyltransferase expression specifically to adipose tissue in transgenic mice. These results identify a functional adipose-specific enhancer and indicate that it is the major determinant of tissue specificity of the gene for adipocyte P2. These results also demonstrate that the proximal-promoter binding sites for AP-1 and C/EBP are not sufficient or necessary to give adipose-tissue-specific expression in vivo, though they may play an important role in the response of this promoter to glucocorticoids.The major role of the adipocyte in higher eukaryotes is the storage of nutritional energy in the form of triglycerides. Disordered gene expression in the adipocyte may result in pathological conditions such as lipodystrophy and obesity, the latter of which contributes heavily to morbidity and mortality through associated cardiovascular disorders and diabetes. In addition, the ability to genetically alter the expression of adipocyte genes and thus control the fatness of feed animals remains an important goal of agricultural research. Thus, the regulation of adipocyte gene expression is of interest for biological, medical, and agricultural reasons.