Acute interactions between intestinal sugar and calcium transport in vitro

Abstract

Fructose consumption by Americans has increased markedly, whereas Ca2+ intake has decreased below recommended levels. Because fructose metabolism decreases enterocyte ATP concentrations, we tested the hypothesis that luminal fructose acutely reduces active, diet-inducible Ca2+ transport in the small intestine. We confirmed that the decrease in ATP concentrations was indeed greater in fructose- compared with glucose-incubated mucosal homogenates from wild-type and was prevented in fructose-incubated homogenates from ketohexokinase (KHK)−/− mice. We then induced active Ca2+ transport by chronically feeding wild-type, fructose transporter glucose transporter 5 (GLUT5)−/−, as well as KHK−/− mice a low Ca2+ diet and measured transepithelial Ca2+ transport in everted duodenal sacs incubated in solutions containing glucose, fructose, or their nonmetabolizable analogs. The diet-induced increase in active Ca2+ transport was proportional to dramatic increases in expression of the Ca2+-selective channel transient receptor potential vanilloid family calcium channel 6 as well as of the Ca2+-binding protein 9k (CaBP9k) but not that of the voltage-dependent L-type channel Ca(v)1.3. Crypt-villus distribution of CaBP9k seems heterogeneous, but low Ca2+ diets induce expression in more cells. In contrast, KHK distribution is homogeneous, suggesting that fructose metabolism can occur in all enterocytes. Diet-induced Ca2+ transport was not enhanced by addition of the enterocyte fuel glutamine and was always greater in sacs of wild-type, GLUT5−/−, and KHK−/− mice incubated with fructose or nonmetabolizable sugars than those incubated with glucose. Thus duodenal Ca2+ transport is not affected by fructose and enterocyte ATP concentrations but instead may decrease with glucose metabolism, as Ca2+ transport remains high with 3- O-methylglucose that is also transported by sodium-glucose cotransporter 1 but cannot be metabolized.

Fructose-induced increases in expression of intestinal fructolytic and gluconeogenic genes are regulated by GLUT5 and KHK

“…We have previously shown that KHK distribution tended to be homogeneous among enterocytes along the crypt-villus axis (50). In renal proximal tubules, KHK expression was greater in the medulla than in the cortex (38).…”

“…The cDNA was generated using 5 g of purified RNA by RT-PCR using a thermal cycler (i-Cycler; Bio-Rad, Hercules, CA) with a Super Script III Reverse Transcriptase kit (Invitrogen). Real-time PCR using Mx3000P (Stratagene, La Jolla, CA) was used to analyze cDNA as described in our earlier work (50). The Roche primer design software (http://www.rocheapplied-science.com) was used to design various primers that were purchased from Integrated DNA Technologies (IDT, Coralville, IA).…”

“…Elongation factor 1␣ (Ef1␣) was used as a housekeeping gene to correct for potential variability in loading. Previously published primer sequences for EF1␣ and KHK are listed in Tharabenjasin et al (50). Primers for the following genes are as follows (forward, reverse, annealing temperature): aldolase-B: 5=-AATAGGGACCAGCCCATTCT-3=, 5=-CTTCCAGCCTTGCTATCCAA-3=, 60°C; triokinase: 5=-GTGTTT-GCCTCTCCTCCTGT, 5=-AGCCGATCCCCAGTGTAGTT-3=, 56°C; fructose-1,6-bisphosphatase (F-1,6-P 2): 5=-TATACCCCGCCAACAA-GAAA-3=, 5=-AAGCTATGGGGTTGACTCA-3=, 58°C; glucose-6-phosphatase (G-6-Pase): 5=-GGCTCACTTTCCCCATCAGG-3=, 5=-ATCCAAGTGCGAAACCAAACAG-3=, 54°C; hexokinase I: 5=-ATGTGTGTGCCACTCCAGAC-3=, 5=-GACCCGGAAGTTTGTT-CCTC-3=, 54°C.…”

“…If indeed glyceraldehyde is not the inducer, then a genetic model with aldolase-B deletion will provide a clearer conclusion and indicate that, if aldolase-B is not required, then the signal is likely a reduction in ATP levels or an increase in ADP as well as fructose 1-phosphate concentrations. KHKdependent phosphorylation of fructose in the intestine and liver is known to markedly reduce cytosolic ATP concentrations as well as reduce ATP-to-ADP ratios (19,50). The intracellular location of KHK suggests that fructose may be metabolized mainly near the basolateral pole.…”

“…KHK-C is strongly expressed in mouse and rat enterocytes, hepatocytes, and renal proximal tubule cells (15,50), whereas KHK-A is expressed in many other tissues, but levels are vanishingly low. Like those of GLUT5 and GLUT2 (16), the intestinal-and hepatic-specific activities of KHK, aldolase-B, and triokinase increase with dietary fructose (29).…”