Ronaldo P. Ferraris

Regulation of the fructose transporter GLUT5 in health and disease

Fructose is now such an important component of human diets that increasing attention is being focused on the fructose transporter GLUT5. In this review, we describe the regulation of GLUT5 not only in the intestine and testis, where it was first discovered, but also in the kidney, skeletal muscle, fat tissue, and brain where increasing numbers of cell types have been found to have GLUT5. GLUT5 expression levels and fructose uptake rates are also significantly affected by diabetes, hypertension, obesity, and inflammation and seem to be induced during carcinogenesis, particularly in the mammary glands. We end by highlighting research areas that should yield information needed to better understand the role of GLUT5 during normal development, metabolic disturbances, and cancer.

Phosphorus utilization in rainbow trout (Oncorhynchus mykiss) fed practical diets and its consequences on effluent phosphorus levels

Excessive dietary phosphorous (P) concentrations in effluents from aquaculture present a major environmental problem. We therefore studied the effect of dietary P and vitamin D3 on P utilization by rainbow trout-fed practical diets and on P concentrations in the soluble, particulate and settleable components of the effluent from fish tanks. Rainbow trout (average weight: 78 g, initial biomass: 13 kg in 0.7 m3 tanks) were fed for 11 weeks, practical diets that varied in total P, available P, and vitamin D3 concentrations. Soluble, particulate (10–200 μm) and settleable (>200 μm) P in the effluent were sampled every 0.5–6 h for 1–3 days in the third and eleventh weeks of the experiment. Trout in all diets more than doubled their weight after 11 weeks. Increasing the concentrations of available dietary P from 0.24% to 0.88% modestly enhanced growth rate. Feed conversion ratio (FCR) and biomass gain per gram P consumed decreased as dietary P concentrations increased. Carcass P, daily P gain, and plasma P concentrations were lower in fish fed with low P diets. Soluble P concentrations in the effluent peaked immediately after and again 4–6 h after feeding, and is a linear function of available dietary P. No soluble P would be produced during consumption of diets containing less than 0.22±0.02% available P. Above this dietary concentration, soluble P would be excreted at 6.9±0.4 mg/day/kg for each 0.1% increase in available dietary P. Particulate P concentrations in the effluent were independent of dietary P concentrations. Settleable, presumably fecal, P concentrations tended to increase with dietary P concentrations. In trout fed with low P (0.24% available P, 0.6% total P) diets, 60% of total dietary P were retained by the fish and the remaining 40% were excreted in the effluent as settleable P (20–30%) and particulate or soluble P (10–20%). In trout fed with high P (0.59–0.88% available P; 0.9–1.2% total P) diets, 30–55% of total dietary P was retained by fish, and the remaining 15–25% appeared in the effluent as settleable P, 20–55% as soluble P, and 5–10% as particulate P. Vitamin D3 did not affect fish growth nor effluent P levels. Physicochemical management of aquaculture effluents should consider the effect of diets on partitioning of effluent P, the peaks of soluble P concentration following feeding, and the contributions of particulate P to total P in the effluent. Increasing our understanding of how dietary P is utilized and is subsequently partitioned in the effluent can contribute significantly towards alleviating this important environmental and industry problem.

Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice

The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases Cdc42 and Rab8a are critical regulators of these processes in mice. Conditional ablation of Cdc42 in the mouse intestinal epithelium resulted in the formation of large intracellular vacuolar structures containing microvilli (microvillus inclusion bodies) in epithelial enterocytes, a phenotype reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells, and increased apoptosis. Cdc42 deficiency impaired Rab8a activation and its association with multiple effectors, and prevented trafficking of Rab8a vesicles to the midbody. This impeded cytokinesis, triggering crypt apoptosis and disrupting epithelial morphogenesis. Rab8a was also required for Cdc42-GTP activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data suggest that defects of the stem cell niche can cause MVID. This hypothesis represents a conceptual departure from the conventional view of this disease, which has focused on the affected enterocytes, and suggests stem cell-based approaches could be beneficial to infants with this often lethal condition.

The role of fructose transporters in diseases linked to excessive fructose intake

Fructose intake has increased dramatically since humans were hunter‐gatherers, probably outpacing the capacity of human evolution to make physiologically healthy adaptations. Epidemiological data indicate that this increasing trend continued until recently. Excessive intakes that chronically increase portal and peripheral blood fructose concentrations to >1 and 0.1 mm, respectively, are now associated with numerous diseases and syndromes. The role of the fructose transporters GLUT5 and GLUT2 in causing, contributing to or exacerbating these diseases is not well known. GLUT5 expression seems extremely low in neonatal intestines, and limited absorptive capacities for fructose may explain the high incidence of malabsorption in infants and cause problems in adults unable to upregulate GLUT5 levels to match fructose concentrations in the diet. GLUT5‐ and GLUT2‐mediated fructose effects on intestinal electrolyte transporters, hepatic uric acid metabolism, as well as renal and cardiomyocyte function, may play a role in fructose‐induced hypertension. Likewise, GLUT2 may contribute to the development of non‐alcoholic fatty liver disease by facilitating the uptake of fructose. Finally, GLUT5 may play a role in the atypical growth of certain cancers and fat tissues. We also highlight research areas that should yield information needed to better understand the role of these GLUTs in fructose‐induced diseases.

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

SUMMARY 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 (∼8.0) than in rats (∼7.6), but pH of the large intestine was similar (∼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 ≥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.

Dietary phosphorus regulates intestinal transport and plasma concentrations of phosphate in rainbow trout.

Abstract Intestinal inorganic phosphate transport and its regulation have not been studied in fish. In this study, we initially characterized the mechanisms of intestinal inorganic phosphate transport in rainbow trout (Oncorhynchus mykiss) then determined the effects of dietary phosphorus concentrations on intestinal inorganic phosphate uptake, plasma inorganic phosphate, and intestinal luminal inorganic phosphate concentrations. In 11-g trout, the saturable mechanism of brushborder inorganic phosphate uptake had a Kt=1.2 mmol l−1 and a Vmax=0.22 nmol mg−1 min−1, while the diffusive component had a Kd=0.012 min−1. Similar kinetic constants were obtained from 51-g trout, suggesting that development or size had little effect on transport. Tracer inorganic phosphate (1.18 mmol l−1) uptake was almost completely inhibited (>95%) by 20 mmol l−1 unlabeled inorganic phosphate. Inorganic phosphate uptake (0.2 mmol l−1) was strongly inhibited (˜75% inhibition) by phosphonoformic acid, a competitive inhibitor of mammalian inorganic phosphate transport, as well as by the absence of Na+ (˜90% inhibition). Northern blot and reverse transcription-polymerase chain reaction indicated that the intestinal inorganic phosphate transporter in trout is not related to the cloned Na+ inorganic phosphate-II transporter of winter flounder. Intestinal luminal and plasma inorganic phosphate concentrations each increased with dietary P concentrations. Intestinal inorganic phosphate, but not proline, absorption rates decreased with dietary phosphorus concentrations. As in mammals and birds, a Na-dependent inorganic phosphate carrier that is tightly regulated by diet is present in trout small intestine.

Regulation of rat intestinal GLUT2 mRNA abundance by luminal and systemic factors.

Fructose in the lumen of the small intestine is transported across the brush border membrane by GLUT5, then across the basolateral membrane by GLUT2, which also transports glucose. Diets containing high fructose (HF) specifically enhance intestinal GLUT5 expression in neonatal rats, but there is little information concerning the dietary regulation of GLUT2 expression during early development. In this study, we perfused for 1-4 h 100 mM fructose, glucose (HG), alpha-methylglucose, or mannitol solutions into the jejunum of anaesthetized 20-day-old rat pups. GLUT2 mRNA abundance increased only in HF- and HG-perfused intestines, an effect inhibited by actinomycin D but not by cycloheximide. Bypassed (Thiry-Vella) intestinal loops were constructed, then pups were fed either HF or low-carbohydrate diets for 5 days. GLUT2 mRNA abundance increased significantly in both bypassed and anastomosed intestines of Thiry-Vella pups fed HF. In contrast, GLUT5 mRNA abundance increased only in the anastomosed segment. In sham-operated pups, GLUT2 and GLUT5 mRNA abundance increased in both intestinal regions that corresponded to the bypassed and anastomosed regions of Thiry-Vella pups. SGLT1 mRNA abundance was independent of diet and intestinal region in both Thiry-Vella and sham-operated pups. Unlike GLUT5 expression, which is regulated at the level of transcription only by luminal fructose, GLUT2 mRNA expression is transcriptionally regulated by luminal fructose and glucose as well as by systemic factors released during their absorption.

Luminal fructose modulates fructose transport and GLUT-5 expression in small intestine of weaning rats

In neonatal rats, precocious introduction of dietary fructose significantly enhances brush-border fructose transport rates and GLUT-5 mRNA levels during early weaning. In this study, these rates and levels were more than two times higher in the anastomosed intestine compared with those in the bypassed loop of weaning pups that underwent Thiry-Vella surgery and consumed high-fructose (HF) diets. In Thiry-Vella pups fed fructose-free (NF) diets, uptake rates and mRNA levels in the anastomosed intestine were very low and similar to those in the bypassed loop. In sham-operated littermates, transport rates and mRNA levels were similar between intestinal regions that corresponded to anastomosed and bypassed loops in Thiry-Vella pups and were two to three times greater in pups fed HF than in those fed NF diet. In contrast, rates of brush-border glucose transport and levels of SGLT-1 and of GLUT-2 mRNA were independent of diet and were similar between bypassed and anastomosed regions. Changes in GLUT-5 expression did not follow a distinct diurnal rhythm. When pups were fed HF diet after 12 h of starvation to empty the intestinal lumen, fructose transport rates increased with feeding duration and reached a plateau 12-24 h after feeding; in contrast, GLUT-5 mRNA levels were highest within 4 h after arrival of chyme in the jejunum and then decreased gradually and returned to baseline levels 24 h later. In littermates fed NF diet, mRNA levels and uptake rates were each independent of feeding duration. Luminal, and not endocrine, signals regulate GLUT-5 expression in weaning pups.

Dietary Induction of Intestinal Fructose Absorption in Weaning Rats

ABSTRACT: The onset of developmentally induced changes in rat intestinal nutrient absorption is well known: brushborder glucose and fructose transporters appear during prenatal and postweaning periods, respectively. The onset of diet-induced regulation, however, is unknown. To test the hypothesis that intestinal glucose and fructose transport is regulated by diet during weaning and postweaning, we fed rats experimental diets containing high (65%) glucose, high fructose, high sucrose, or no carbohydrate. In 16-d-old rats, 6 d of dietary fructose but not glucose modestly increased fructose absorption in everted sleeves of small intestine (SI) over control (mother-fed with access to chow) rats (p = 0.02). In 21-d-old (age when sucrase is present) rats, dietary fructose and sucrose each dramatically enhanced (p = 0.004) fructose absorption over control rats and rats fed high glucose or carbohydrate-free diets. In 35− (postweaning) and 60-d-old rats, dietary fructose and sucrose, but not glucose, stimulated fructose absorption (p < 0.005) over rats fed a carbohydrate-free diet. In all age groups, intestinal glucose absorption was independent of diet (p ≥ 0.12), and experimental rats grew at the same rate as control rats. Absorption of fructose or glucose was 2–3 times greater in the proximal and middle than in the distal SI. Intestinal fructose, but not glucose, absorption can be induced by diet even during early weaning, and dietary fructose followed by sucrose is the most potent inducer. Thus, mechanisms of diet regulation can change ontogenetically, and early introduction of certain diets can induce appearance of certain nutrient transporters.

Effect of dietary phosphorus and vitamin D3 on phosphorus levels in effluent from the experimental culture of rainbow trout (Oncorhynchus mykiss)

Abstract Excessive phosphorus (P) levels in aquaculture effluents violate federally mandated limits and pose a serious threat to the freshwater environment. In rainbow trout culture, effluent P probably originates as fecal and metabolic waste product because assimilation of dietary P is relatively low. We therefore decreased dietary P and increased dietary vitamin D 3 levels, methods that enhance P assimilation in mammals, in purified and semi-purified trout diets, then monitored effluent P. Soluble effluent P reached a peak right after feeding and returned to baseline levels in between feeding times. The peak and average concentrations of soluble P in the effluent were mainly influenced by dietary P. Average P in fecal dry matter also decreased with dietary P. Neither dietary P nor vitamin D 3 under the conditions of the experiment had significant effects on whole body P content but P deposition (as a percentage of P intake) decreased with increased dietary P. The dietary combination of low P and high vitamin D 3 decreased soluble and fecal P levels in the effluent indicating a strategy whereby effluent P concentrations can be reduced by regulation of P metabolism.