Generation of SGLT5-deficient mice and their fructose and mannose uptake by renal BBMV s.

<p>(A) Schematic representation of the strategy for targeting the <i>Slc5a10</i> gene. A targeting vector was constructed by inserting a neomycin resistant (<i>neo</i>) gene cassette to disrupt exons 3–6 of the <i>Slc5a10</i> genomic locus on a BAC genomic clone. Arrows indicate PCR primers for genotyping. (B) A representative result of genotyping the offspring obtained by intercrossing heterozygous-deficient mice. Wild type and null alleles are detected as signals of 900 bp and 350 bp, respectively. <i>Wt</i>: Wild type mice, <i>He</i>: Heterozygous null mutant, <i>Ho</i>: Homozygous null mutant. (C) Sodium-dependent uptake of fructose and (D) mannose in BBMVs of WT mice (+/+) and SGLT5-deficient mice (−/−). (E) Sodium-independent uptake of fructose and (F) mannose in BBMVs of WT mice (+/+) and SGLT5-deficient mice (−/−). Data are presented as means ± S.D. Data are derived from 3 independent experiments.</p

Influence of the long-term consumption of fructose on tissue weight and lipid metabolism.

<p>(A) Plasma triglyceride levels of WT mice (+/+) and SGLT5-deficient mice (−/−). (B) Plasma total cholesterol levels. (C) Weight of epididymal fat. (D) Weight of the liver. (E) Hepatic triglyceride levels. (F) Histopathological analysis of the liver sections. Two sections per mouse were stained with Sudan III. Representative images are shown (scale bar, 50 µm). Data are presented as means ± S.E.M (<i>n</i> = 8–10). * <i>P</i><0.05, *** <i>P</i><0.001 versus WT mice given 30% fructose water. # <i>P</i><0.05, ## <i>P</i><0.01, ### <i>P</i><0.001 versus respective plain water controls.</p

Gene expression analysis. Quantitative RT-PCR was performed with total RNA isolated from (A) the liver and (B) the kidney of WT mice (+/+) and SGLT5-deficient mice (−/−) given plain water or high fructose (HF) water.

<p><i>MAPK1</i> was used as an internal control. Data are presented as means ± S.E.M. <i>n</i> = 3 (A). <i>n</i> = 8–10 (B).</p

Food and water intake in WT (+/+) mice and SGLT5-deficient mice (−/−). Daily intake of

<p>(<b>A</b>) <b>food and</b> (<b>B</b>) <b>water of mice at 17 weeks of age.</b> (C) Calculated daily energy intake. Data are presented as means ± S.E.M (n = 8–10). ### P<0.001 versus respective plain water control.</p

Oral glucose tolerance test with WT mice (+/+) and SGLT5-deficient mice (−/−) given plain water or fructose water (HF).

<p>Fasted 21-week-old male mice received an oral dose of glucose (2 g/kg). Plasma glucose levels were determined at the indicated time points. Data are presented as means ± S.E.M (<i>n</i> = 8–10). ### <i>P</i><0.001 versus respective water controls by analysis of covariance (ANCOVA).</p

SGLT5 distribution and fructose uptake.

<p>(A) Tissue distribution of mouse SGLT5 and (B) SGLT5-mediated fructose uptake in COS-7 cells. Data are presented as means ± S.D. Data are derived from 3 independent experiments.</p

Effect of high fructose consumption in WT mice and SGLT5-deficient mice.

<p>(A) Plasma glucose levels of WT mice (+/+) and SGLT5-deficient mice (−/−) were measured every 2 weeks. <i>HF</i>: Mice given water containing high fructose. (B) Growth curves of WT mice and SGLT5-deficient mice. (C) Plasma samples were collected after 6 h fasting at 21 weeks of age, and immunoreactive insulin (<i>IRI</i>) was determined. (D) Plasma fructose concentrations measured in plasma samples collected under anesthesia after 3 h fasting. <i>Open circles</i> represent individual data. (E and F) WT mice and SGLT5-deficient mice given plain water or fructose water were maintained in metabolic cages and 24-h urine samples were collected. Urinary fructose excretion was calculated by multiplying urinary fructose concentration by the amount of urine. Data are presented as means ± S.E.M (<i>n</i> = 8–10). *** <i>P</i><0.001 versus WT mice given 30% fructose water. # <i>P</i><0.05, ## <i>P</i><0.01, ### <i>P</i><0.001 versus respective plain water controls. +++ <i>P</i><0.001 versus WT mice given plain water.</p

Microarray mRNA expression analysis of phase I and II metabolic enzymes levels in chimeric mice.

<p>mRNA expression levels of phase I and II expressed in human livers in (A and B) uPA/SCID and #1C2 homozygous mice, (C and D) #1C2 homo- and hemizygous mice, and (E and F) uPA/SCID and #1C2 mice were plotted. Phase I metabolic enzymes are CYP1A1, 1A2, 1B1, 2A6, 26A1, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7, 3A43, 39A1, 4A11, 4F2, 4F3, 7A1, 7B1, 8B1. Phase II metabolic enzymes are UGT1A6, 2A1, 2B15, 2B17, 2B4, SULT1A1, 1A3, 1B1, 1E1, 2A1, GSTA1, A4, M1, M2, M3, M4, P1, T1, Z1, and NAT1, 2. The solid and dotted lines represent unity and 4-fold differences, respectively. All values were within the 4-fold difference range.</p

Changes in h-alb concentration and body weight in chimeric mice up to 4 months of age.

<p>(A) h-alb levels in uPA/SCID chimeric mice were the highest among the 3 types of chimeric mice, and those of #1C2 homozygous mice were higher than those of hemizygous mice by 6 weeks of age. H-alb levels in uPA/SCID and #1C2 homozygous mice reached a plateau by 10 and 11 weeks of age, respectively, whereas those in #1C2 hemizygous mice continued to increase up to 17 weeks of age. (B) Body weight of #1C2 hemizygous mice was the highest, followed by #1C2 homozygous mice, and then uPA/SCID chimeric mice. (C) RI and h-alb concentrations plots showed similar correlation curves in uPA/SCID and #1C2 homo- and hemizygous chimeric mice.</p

Histopathological findings in chimeric mouse livers and kidneys.

<p>H&E staining of left lateral lobes of (A) uPA/SCID chimeric mice, (B) #1C2 homozygous and (C) #1C2 hemizygous chimeric mice, and magnifications of the rectangular parts (D) on the left side of A and (E) on the right side of A, (F) in B, and (G) in C are shown. h-heps with a clear cytoplasm and lipid droplets occupied most areas of the liver sections (A-G). Arrows show (D) degenerating m-heps and (E) hyperplastic m-hep nodules in uPA/SCID chimeric mice. M-heps with eosinophilic cytoplasm of various sizes are shown by arrows in (F) #1C2 homozygous and (G) hemizygous chimeric mice. (H) The left lateral lobe of a #1C2 hemizygous chimeric mouse was immunostained with anti-hCK8/18 antibodies. H-heps were brown-colored, and (I) an area of rectangle was magnified. m, m-heps, h, h-heps. Kidney sections in (J, M) uPA/SCID, (K, N) #1C2 homozygote and (L, O) hemizygous chimeric mice were stained with H&E. Enlarged glomeruli and glomerulosclerosis were observed in uPA/SCID mice (J, M). No pathological findings were observed in (K, N) #1C2 homozygote and (L, O) hemizygous chimeric mouse kidneys. (M), (N) and (O) were high magnification of (J), (K) and (L), respectively. Bar, 100 μm in D-G, I and J-O. Bar, 1 mm in A-C and H.</p