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The mechanism of apical [Na.sup.+]-dependent [H.sup.+] extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH ([pH.sub.i]) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal [Na.sup.+] stimulated similar rates of [pH.sub.i] recovery in cells at the base of distal colonic crypts of wild-type or [Na.sup.+]/[H.sup.+] exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 [micro]M HOE694 (NHE2 inhibitor) blocked 68-75% of the [pH.sub.i] recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 [micro]M), or the bicarbonate transport inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 20 [micro]M), inhibited [pH.sub.i] recovery in NHE2-null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial [Na.sup.+] leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of [Na.sup.+]-dependent [pH.sub.i] recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive [pH.sub.i] recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a [Na.sup.+]-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters. S1c9a2; S1c9a3; BCECF; SNARF-5F; intracellular pH; laser scanning confocal microscopy; colon; [Na.sup.+]/[H.sup.+] exchanger

The mechanism of apical Na+-dependent H+ extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH (pHi) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal Na+ stimulated similar rates of pHi recovery in cells at the base of distal colonic crypts of wild-type or Na+/H+ exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 µM HOE694 (NHE2 inhibitor) blocked 68-75% of the pHi recovery rate, whereas NHE2-nuIl crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 µM), or the bicarbonate transport inhibitor 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-(N-ethyl-N- isopropyl)amiloride (EIPA; 20 µM), inhibited pHi recovery in NHE2- null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial Na+ leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of Na+-dependent pHi recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive pHi recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a Na+-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters. [ABSTRACT FROM AUTHOR]

Excess plasma membrane cholesterol-mediated cellular insulin resistance has been shown to result from increased hexosamine biosynthesis pathway (HBP) O-GlcNAcylation of the transcription factor Sp1 in 3T3-L1 adipocytes and L6 skeletal muscle myotubes. Here we tested the causal relationship between excess skeletal muscle membrane (SMM) cholesterol and insulin resistance, as well as probed the potential HBP/Sp1 link, in mice fed a Western-style high-fat diet for one week or in mice with targeted skeletal muscle overexpression of glucosamine-fructose aminotransferase (GFAT, the rate-limiting enzyme of the HBP). HF feeding increased SMM cholesterol and impaired GLUT4 regulation, glucose tolerance and insulin sensitivity within one week. These early skeletal muscle and systemic metabolic abnormalities were fully prevented in HF-fed mice treated during the one-week diet challenge with two subcutaneous injections of the cholesterol-depleting agent methyl-β-cyclodextrin. Consistent with in vitro data, an HBP-mediated Sp1 cholesterolgenic response contributing to SMM cholesterol accumulation, the transcription of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, was increased in skeletal muscle from HF-fed mice. Furthermore, treating HF-fed mice during the one-week HF-feeding challenge with mithramycin, an agent that blocks Sp1 binding to DNA, prevented the SMM cholesterol buildup, glucose intolerance, and insulin resistance. Skeletal muscle GFAT overexpression also caused accumulation of SMM cholesterol, glucose intolerance, and insulin resistance.These data suggest that early skeletal muscle insulin resistance in the setting of HF feeding are a consequence of a HBP-mediated Sp1 cholesterolgenic transcriptional response and that targeting this pathway has therapeutic potential for preventing insulin resistance and the development of T2D. Disclosure: B.A. Grice: None. J.D. Covert: None. A.M. Kreilach: None. M. Thornburg: None. L. Tackett: None. D. McClain: None. J.S. Elmendorf: None. Funding: American Diabetes Association (1-15-BS-053 to J.S.E.); National Institutes of Health (R01DK114222-01A1, UL1TR001108T32, DK064466, T32GM077229, P30DK097512); Indiana University-Purdue University; Indianapolis Research Support Funds [ABSTRACT FROM AUTHOR]

Laboratory facilities use many varieties of contact bedding, including wood chips, paper products, and corncob, each with its own advantages and disadvantages. Corncob bedding, for example, is often used because of its high absorbency, ability to minimize detectable ammonia, and low cost. However, observations that mice eat the corncob lead to concerns that its use can interfere with dietary studies. We evaluated the effect of corncob bedding on feed conversion (change in body weight relative to the apparent number of kcal consumed over 7 d) in mice. Four groups of mice (6 to 12 per group) were housed in an individually ventilated caging system: (1) low-fat diet housed on recycled paper bedding, (2) low-fat diet housed on corncob bedding, (3) high-fat diet housed on recycled paper bedding, and (4) high-fat diet housed on corncob bedding. After 4 wk of the high-fat diet, feed conversion and percentage body weight change both were lower in corncob-bedded mice compared with paper-bedded mice. Low-fat-fed mice on corncob bedding versus paper bedding did not show statistically significant differences in feed conversion or change in percentage body weight. Average apparent daily feed consumption did not differ among the 4 groups. In conclusion, these data suggest that corncob bedding reduces the efficiency of feed conversion in mice fed a high-fat diet and that other bedding choices should be favored in these models.