Establishment of a quantitative in vivo method for estimating adipose tissue volumes and the effects of dietary soy sauce oil on adipogenesis in medaka, Oryzias latipes.

Adipose tissue, which is conserved in higher eukaryotes, plays central roles in controlling the body's energy balance, including excess energy storage and energy expenditure during starvation. In adipogenesis, intranuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ) is a key molecule, and PPARγ agonists can promote adipogenesis. Many studies on the in vitro screening of PPARγ agonists with compounds derived from various materials have been reported; however, in vivo assays for quick examination of these feeding effects have not been established. In this study, we developed a technique using a lipophilic fluorescent reagent, Nile red to quantitatively estimate the adipose tissue volumes by using Japanese rice fish, medaka (Oryzias latipes) and studied effects of dietary soy sauce oil (SSO), which is a discarded by-product from Japanese traditional food and is known to have PPARγ-agonistic activity, on adipogenesis. We found that SSO feeding increased the adipose tissue volumes, and the expression levels of adipogenesis-related genes increased in these medaka larvae. These results suggest that SSO feeding increases the adipose tissue volumes through adipogenesis promotion by PPARγ-agonistic activity in medaka, and medaka is a powerful model for studying adipogenesis. Furthermore, our study also demonstrates the availability of SSO as a dietary additive for farmed fish.

Dietary fructose inhibits intestinal calcium absorption and induces vitamin D insufficiency in CKD.

Renal disease leads to perturbations in calcium and phosphate homeostasis and vitamin D metabolism. Dietary fructose aggravates chronic kidney disease (CKD), but whether it also worsens CKD-induced derangements in calcium and phosphate homeostasis is unknown. Here, we fed rats diets containing 60% glucose or fructose for 1 mo beginning 6 wk after 5/6 nephrectomy or sham operation. Nephrectomized rats had markedly greater kidney weight, blood urea nitrogen, and serum levels of creatinine, phosphate, and calcium-phosphate product; dietary fructose significantly exacerbated all of these outcomes. Expression and activity of intestinal phosphate transporter, which did not change after nephrectomy or dietary fructose, did not correlate with hyperphosphatemia in 5/6-nephrectomized rats. Intestinal transport of calcium, however, decreased with dietary fructose, probably because of fructose-mediated downregulation of calbindin 9k. Serum calcium levels, however, were unaffected by nephrectomy and diet. Finally, only 5/6-nephrectomized rats that received dietary fructose demonstrated marked reductions in 25-hydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(3) levels, despite upregulation of 1alpha-hydroxylase. In summary, excess dietary fructose inhibits intestinal calcium absorption, induces marked vitamin D insufficiency in CKD, and exacerbates other classical symptoms of the disease. Future studies should evaluate the relevance of monitoring fructose consumption in patients with CKD.

Molecular and conventional responses of large rainbow trout to dietary phosphorus restriction.

The dietary phosphorus (P) requirement for large fish is difficult to estimate because of insensitivities of known P status indicators. We examined dietary P requirement of large rainbow trout (mean body weight 278 g) using recently identified P-responsive genes (mRNA abundances) as well as conventional serum P and bone P. Fish were fed six diets (varied P contents), and the tissues of intestine, pyloric caeca (PC), kidney, serum and bone were collected at varying time intervals. Serum P responded clearly to dietary P by day 2, but the estimated P requirement based on this variable changed as feeding duration continued. Bone P did not respond clearly until week 7. Among P-responsive genes studied, Na/Pi cotransporter in PC (PC-NaPi) was the most sensitive, and responded in 2 days. Fish-to-fish (within treatment) variance was larger in mRNA than in serum P and bone P levels. Estimated dietary P requirements (%P in dry diet) were 0.45 (based on serum P), 0.45 (based on bone P), 0.36 (based on PC-NaPi), 0.33 (based on intestinal NaPi), 0.71 (based on renal NaPi), and 0.33 (based on mitochondrial Pi carrier). This study is the first to evaluate the potential of genomic approaches in determining nutrient requirements of fish.

Dietary acidification enhances phosphorus digestibility but decreases H+/K+-ATPase expression in rainbow trout.

Oxynticopeptic cells of fish stomach are thought to secrete less acid than the specialized parietal cells of mammalian stomach. Gastric acidity, however, has not been directly compared between fish and mammals. We therefore fed rainbow trout and rats the same meal, and found that the lowest postprandial pH of trout stomach was 2.7, which was only transiently sustained for 1 h, whereas that of rat stomach was 1.3, which was sustained for 3 h. Postprandial pH of the small intestine was slightly higher in trout (approximately 8.0) than in rats (approximately 7.6), but pH of the large intestine was similar (approximately 8.0). Addition of acids to fish feeds, in an attempt to aid the weak acidity of fish stomach, has been known to improve phosphorus digestibility, but its physiological effect on fish stomach is not known. Exogenous acids did improve phosphorus digestibility but also decreased steady-state mRNA expression of trout H(+)/K(+)-ATPase (ATP4A, the proton pump) as well as Na(+)/bicarbonate cotransporter (NBC), and had no effect on gastrin-like mRNA and somastostatin (SST) mRNA abundance. Gastrin-like mRNA and SST-2 mRNA were equally distributed between corpus and antrum. ATP4A mRNA and NBC mRNA were in the corpus, whereas SST-1 mRNA was in the antrum. Trout gastrin-like EST had modest homology to halibut and pufferfish gastrin, whereas trout ATP4A mRNA had > or = 95% amino acid homology with mammalian, Xenopus and flounder ATP4A. Although ATP4A seems highly conserved among vertebrates, gastric acidity is much less in trout than in rats, explaining the low digestibility of bone phosphorus, abundant in fish diets. Dietary acidification does not reduce acidity enough to markedly improve phosphorus digestibility, perhaps because exogenous acids may inhibit endogenous acid production.

Effluent profile of commercially used low-phosphorus fish feeds.

Excess phosphorus (P) in aquaculture feeds contributes to the eutrophication of natural waters. While commercially available low-P (LP) fish feeds have been developed, there is uncertainty about their potential to reduce effluent P while maintaining fish growth relative to regular P (RP) feeds. We therefore simulated commercial aquaculture conditions and fed for 55 days rainbow trout (approximately 190 kg/raceway, n = 3 raceways/diet) RP (1.4% total P) and LP (1.0%) feeds then determined effluent P levels, fish growth, and feed costs. Excretions of fecal-P and soluble-P, but not particulate-P, in effluents were greater in RP than in LP ponds. Fish growth, bone-P and plasma-P were similar between diets, demonstrating that LP feeds can lower effluent P levels without compromising growth. Costs were 0.97 dollars/kg fish production for LP feeds, and 0.74 dollars/kg for RP. Because feed is the largest variable cost in commercial aquaculture, the use of LP feeds can significantly increase production costs.

Dissolved oxygen and dietary phosphorus modulate utilization and effluent partitioning of phosphorus in rainbow trout (Oncorhynchus mykiss) aquaculture.

Phosphorus (P) is the limiting nutrient in freshwater primary production, and excessive levels cause premature eutrophication. P levels in aquaculture effluents are now tightly regulated. Increasing our understanding of waste P partitioning into soluble, particulate, and settleable fractions is important in the management of effluent P. When water supply is limited, dissolved oxygen concentration (DO) decreases below the optimum levels. Therefore, we studied effects of DO (6 and 10mg/L) and dietary P (0.7 and 1.0% P) on rainbow trout growth, P utilization, and effluent P partitioning. Biomass increased by 40% after 3 weeks. DO at 10mg/L significantly increased fish growth and feed efficiency, and increased the amount of P in the soluble fraction of the effluent. Soluble effluent P was greater in fish fed 1.0% P. DO increases fish growth and modulates P partitioning in aquaculture effluent.

Contributions of different NaPi cotransporter isoforms to dietary regulation of P transport in the pyloric caeca and intestine of rainbow trout.

The anatomical proximity and embryological relationship of the pyloric caeca (PC) and small intestine of rainbow trout has led to the frequent assumption, on little evidence, that they have the same enzymes and transporters. In trout, the PC is an important absorptive organ for dietary nutrients, but its role in dietary P absorption has not been reported. We found that apical inorganic phosphate (Pi) transport in PC comprises carrier-mediated and diffusive components. Carrier-mediated uptake was energy- and temperature-dependent, competitively inhibited and Na(+)-independent, and greater than the Na(+)-dependent intestinal uptake. Pi uptake in PC was pH-sensitive in the presence of Na(+). Despite the active Pi transport system in PC, high postprandial luminal Pi concentrations ( approximately 20 mmol l(-1)) indicate that diffusive uptake represents approximately 92% of total Pi uptake in PC of fed fish. The nucleotide sequence of a sodium-phosphate cotransporter (NaPi-II) isoform isolated from PC was approximately 8% different from the intestinal NaPi cotransporter. PC-NaPi mRNA was abundant in PC but rare in the intestine, whereas intestinal NaPi mRNA was abundant in the intestine but scarce in PC. Dietary P restriction reduced serum and bone P concentrations, increased intestine-type, but not PC-type, NaPi mRNA in PC, and increased Pi uptake in intestine but not in PC. Intestine-type NaPi expression may be useful for predicting dietary P deficiency.

Dietary phosphorus-responsive genes in the intestine, pyloric ceca, and kidney of rainbow trout.

Identification of phosphorus (P)-responsive genes is important in diagnosing the adequacy of dietary P intake well before clinical symptoms arise. The mRNA abundance of selected genes was determined in the intestine, pyloric ceca, and kidney of rainbow trout fed low-P (LP) or sufficient-P (SP) diet for 2, 5, and 20 days. The LP-to-SP ratio (LP/SP) of mRNA abundance was used to evaluate the difference in gene expression between LP and SP fish, and to compare the response with bone and serum P, which are conventional indicators of P status. The LP/SP of intestinal, cecal, and renal type II sodium-phosphate cotransporter (NaPi-II) mRNA abundance changed from approximately 1-2 (day 2) to approximately 1.4-4 (day 5) and to approximately 2-10 (day 20). The LP/SP of renal NaPi-II, vitamin D 24-hydroxylase, and vitamin D receptor mRNA abundance correlated inversely with serum P on day 5 but not on day 2 and day 20. In another study, differentially expressed genes between LP and SP fish were examined by subtractive hybridization, confirmed by Northern blot, and evaluated by t-test and correlation with serum and bone P concentrations. About 30 genes were identified as dietary P responsive at day 20, including intestinal meprin and cysteinesulfinic acid decarboxylase, renal S100 calcium-binding protein and mitochondrial P(i) carrier, and cecal apolipoprotein E, somatomedin B-related protein, and NaPi-II. The LP/SP of mRNA abundance of renal mitochondrial P(i) carrier and intestinal cysteinesulfinic acid decarboxylase changed significantly by day 2, and intestinal meprin by day 5. Hence, these genes and NaPi-II are among the earliest steady-response genes capable of predicting P deficiency well before the onset of clinical deficiency.

In vivo fractional P(i) absorption and NaPi-II mRNA expression in rainbow trout are upregulated by dietary P restriction.

Mammalian type II sodium-phosphate cotransporter (NaPi-II) and inorganic phosphate uptake stimulator (PiUS) genes are upregulated by dietary phosphorus (P) restriction to increase intestinal and renal P transport, but little is known about NaPi-II and PiUS regulation in other vertebrates. We studied the 1). the tissue distribution and dietary regulation of NaPi-II, PiUS, and sodium-glucose cotransporter (SGLT1) mRNA and NaPi-II protein in juvenile rainbow trout (Oncorhynchus mykiss) and 2). effects of dietary P on intestinal Pi absorption in vivo. NaPi-II, PiUS, and SGLT1 mRNA were found in the proximal and distal intestine, pyloric ceca, and kidney. PiUS mRNA was also found in the heart, gill, blood, stomach, liver, skin, and muscle. Tissue distribution of NaPi-II protein correlated with that of NaPi-II mRNA except in gill ionocytes where NaPi-II antibodies recognized related epitopes. Chronic consumption of a low-P diet increased NaPi-II and PiUS but not SGLT1 mRNA abundance in the intestine and kidney. Unlike mammals, there was no detectable shift in tissue or cellular localization of NaPi-II protein in response to dietary P restriction. Regulation of NaPi and PiUS mRNA expression was observed only in fish grown under optimal aqueous oxygen concentrations. In vivo fractional absorption of Pi by the intestine decreased in fish fed high-P diets. Decreases in absorption were less pronounced in fish previously fed low-P diets, suggesting that diet history modulates acute regulation of P absorption. Regulation of dietary Pi absorption in vivo may involve a specific change in intestinal NaPi-II and PiUS gene expression.