Bowman‐Birk inhibitor affects pathways associated with energy metabolism in Drosophila melanogaster

Bowman‐Birk inhibitor (BBI) is toxic when fed to certain insects, including the fruit fly, Drosophila melanogaster. Dietary BBI has been demonstrated to slow growth and increase insect mortality by inhibiting the digestive enzymes trypsin and chymotrypsin, resulting in a reduced supply of amino acids. In mammals, BBI influences cellular energy metabolism. Therefore, we tested the hypothesis that dietary BBI affects energy‐associated pathways in the D. melanogaster midgut. Through microarray and metabolomic analyses, we show that dietary BBI affects energy utilization pathways in the midgut cells of D. melanogaster. In addition, ultrastructure studies indicate that microvilli are significantly shortened in BBI‐fed larvae. These data provide further insights into the complex cellular response of insects to dietary protease inhibitors.     

Dietary supplementation of soybean kunitz trypsin inhibitor reduces lipopolysaccharide-induced lethality in mouse model

We examined the modifying effects of a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk trypsin inhibitor (BBI), purified from soybean, as intraperitoneal (i.p.) injection and dietary supplements on bacterial lipopolysaccharide (LPS)-induced lethality in mice. We initially examined the suppressing effects of i.p. injection of KTI (50 mg/kg) and BBI (50 mg/kg) on LPS-induced lethality after i.p. injection of LPS. Furthermore, groups of female C57BL/6 were fed a basal diet (control group) or the basal diet supplemented with KTI (50 g/kg) or BBI (50 g/kg). Here, we show that i.p. and daily oral administration of KTI, but not BBI, caused a significant reduction of the LPS-induced lethality; that LPS significantly induced plasma TNF-alpha, IL-1beta, and IL-6 levels in mice after LPS challenge; that concomitant administration of KTI, but not BBI, inhibits the LPS-induced plasma levels of these cytokines; and that KTI, but not BBI, suppressed LPS-induced upregulation of cytokine expression through suppression of phosphorylation of three mitogen-activated protein (MAP) kinase pathways, ERK1/2, JNK, and p38, in peritoneal macrophages. These data allow us to speculate that i.p. injection and dietary supplementation of a soybean KTI may play a role as a potent anti-inflammatory agent by inhibiting activation of MAP kinases, leading to the suppression of cytokine expression.     

Expression of Cyp6g1 and Cyp12d1 in DDT resistant and susceptible strains of Drosophila melanogaster

The Rst(2)DDT locus (loci) in Drosophila is associated with the over-expression of two cytochrome P450 genes, Cyp6g1 and Cyp12d1. Using northern and western blot analysis we observed the expression pattern of these two genes in two DDT susceptible (Canton-S and 91-C) and three DDT resistant strains (Wisconsin, 91-R and Hikone-R). In Canton-S and 91-R, the CYP6G1 protein was constitutively expressed throughout development. In the Wisconsin strain, CYP6G1 was not expressed in third instar larvae unless the larvae are exposed to DDT. CYP12D1 protein was only expressed in adults. Cyp12d1 mRNA is induced in DDT resistant strains post-exposure to DDT and the expression patterns of Cyp12d1 mRNA varied across DDT resistant strains. Our data support the hypothesis that there is evolutionary plasticity in the expression patterns of P450s associated with metabolic pesticide resistance.     

A comparison of Frost expression among species and life stages of Drosophila

Frost (Fst) is a gene associated with cold exposure in Drosophila melanogaster. We used real-time PCR to assess whether cold exposure induces expression of Fst in 10 different life stages of D. melanogaster, and adults of seven other Drosophila species. We exposed groups of individuals to 0 °C (2 h), followed by 1 h recovery (22 °C). Frost was significantly upregulated in response to cold in eggs, third instar larvae, and 2- and 5-day-old male and female adults in D. melanogaster. Life stages in which cold did not upregulate Fst had high constitutive expression. Frost is located on the opposite strand of an intron of Diuretic hormone (DH), but cold exposure did not upregulate DH. Frost orthologues were identified in six other species within the Melanogaster group (Drosophila sechellia, Drosophila simulans, Drosophila yakuba, Drosophila erecta, Drosophila ananassae and Drosophila mauritiana). Frost orthologues were upregulated in response to cold exposure in both sexes in adults of all of these species. The predicted structure of a putative Frost consensus protein shows highly conserved tandem repeats of motifs involved in cell signalling (PEST and TRAF2), suggesting that Fst might encode an adaptor protein involved in acute stress or apoptosis signalling in vivo.     

A role for alcohol dehydrogenase in the Drosophila immune response?

In a recent study Drosophila larvae were injected with bacterial lipopolysaccharide (LPS) suspended in 1% ethanol and differentially induced protein fractions were identified. The levels of several proteins, including alcohol dehydrogenase (ADH), increased in LPS-treated flies and were labelled as immune response proteins. However, because control larvae were not injected with ethanol alone the identified proteins could represent a response to ethanol. Here, we injected Drosophila larvae with combinations of ethanol and LPS. While ADH activity increased in larvae receiving 1% ethanol, it was not increased after LPS injection. These results suggest that ADH plays no role in the Drosophila immune response, and that other proteins identified in the previous study may instead mediate ethanol tolerance in flies and other organisms.     

Influence of dietary protein on the anti-inflammatory and ulcerogenic effects and on the pharmacokinetics of phenylbutazone in rats.

Influence of dietary protein deficiency on the anti-inflammatory and ulcerogenic effects and on the kinetics of phenylbutazone was studied in male Sprague-Dawley rats fed ad libitum a 21% (control) or a 5% (low) protein diet for 3 weeks. A low protein diet fed to a decrease in body weight gain, plasma proteins, albumin, globulins, hepatic total and microsomal proteins and in cytochrome P-450. Phenylbutazone produced a greater ulcerogenic effect in rats fed a low protein diet than in control rats; its anti-inflammatory effect did not increase. Plasma t 1/2 of phenylbutazone was longer in protein-deficient rats than in control rats. Dietary protein deprivation led to a decrease in the plasma clearance and plasma protein binding of phenylbutazone but did not lead to a change in its bioavailability. No relationship between the severity of gastric ulceration and the concentration of phenylbutazone or oxyphenbutazone in the stomach tissue was found in any animal of the two groups. The increased susceptibility of protein-deficient rats to the ulcerogenic effect of phenylbutazone was reversible and was not observed when these animals were fed a control diet for 3 weeks. It is concluded that a dietary protein deficiency increases the ulcerogenic toxicity of phenylbutazone relative to its useful anti-inflammatory effects.     

Adaptation of Na+-H+ exchange in renal microvillus membrane vesicles. Role of dietary protein and uninephrectomy.

The ablation of renal mass and institution of a high protein diet both lead to renal cortical hypertrophy and increased glomerular filtration rate (GFR). We studied Na+ transport in rat microvillus membrane vesicles isolated from uninephrectomized or sham operated rats fed 6% (low), 24% (standard), or 40% (high) protein diets. The feeding of high protein, as compared with low protein, was associated with a 50% increase in rates of pH-stimulated 22Na+ transport in isolated vesicles from sham and uninephrectomized animals. Values for the standard protein diet were intermediate to values for high and low protein. At each level of dietary protein intake, vesicular Na+ transport was greater in the uninephrectomized than in sham rats. The high protein diet was also associated with increased vesicular 22Na+ flux inhibitable by 1 mM amiloride. Increases in total and amiloride sensitive flux were also noted in the absence of a pH gradient. Conductive Na+ and H+ transport were not altered, nor were sodium-glucose and sodium-alanine cotransport. Kinetic studies revealed evidence for an increased Vmax of Na+-H+ exchange in uninephrectomized animals fed a 40 vs. a 6% protein diet whereas Km was unchanged. Supplements of NaHCO3 in the 40% protein diet, to adjust for an increased rate of net acid excretion, did not prevent the increased rates of Na+-H+ exchange. However, treatment with actinomycin D (0.12 mg/kg) prevented the increased Na+-H+ activity as well as the increased renal mass and GFR noted 24 h after unilateral nephrectomy. Na+-H+ exchange rate was closely correlated with GFR (r = 0.961; P less than 0.005) and renal mass (r = .986; P less than 0.001). These observations provide evidence for modification of the luminal membrane Na+-H+ exchanger in response to changes in dietary protein content and nephron number.     

Low protein diet alters urea transport and cell structure in rat initial inner medullary collecting duct.

Low protein diets reverse the urea concentration gradient in the renal inner medulla. To investigate the mechanism(s) for this change, we studied urea transport and cell ultrastructure in initial and terminal inner medullary collecting ducts (IMCD) from rats fed 18% protein or an isocaloric, 8% protein diet for 4 wk. Serum urea, aldosterone, and albumin were significantly lower in rats fed 8% protein, but total protein and potassium were unchanged. Vasopressin stimulated passive urea permeability (Purea) threefold (P < 0.05) in initial IMCDs from rats fed 8% protein, but not from rats fed 18% protein. Luminal phloretin reversibly inhibited vasopressin-stimulated Purea. However, in terminal IMCDs from rats fed either diet, vasopressin stimulated Purea. Net transepithelial urea flux (measured with identical perfusate and bath solutions) was found only in initial IMCDs from rats fed 8% protein. Reducing the temperature reversibly inhibited it, but phloretin did not. Electron microscopy of initial IMCD principal cells from rats fed 8% protein showed expanded Golgi bodies and prominent autophagic vacuoles, and morphometric analysis demonstrated a marked increase in the surface density and boundary length of the basolateral plasma membrane. These ultrastructural changes were not observed in the terminal IMCD. Thus, 8% dietary protein causes two new urea transport processes to appear in initial but not terminal IMCDs. This is the first demonstration that "active" urea transport can be induced in a mammalian collecting duct segment.     

Non-haem iron transport in the rat proximal colon

BACKGROUND: Only 10% of dietary iron is absorbed in the duodenum which implies that 90% (approximately 9 mg day(-1)) reaches the lower small intestine and colon. Therefore the purpose of this study was to assess the iron transport capacity of the rat proximal colon and to determine whether iron absorption is regulated by changes in dietary iron content. MATERIALS AND METHODS: Rats were fed for 14 days on either iron adequate (44 mg Fe kg(-1) diet) or iron-deficient (< 0.5 mg Fe kg(-1) diet) diets. The 59Fe transport across the colonic epithelium and its subsequent appearance in the blood were measured in vivo. In separate studies the colon was excised and used to measure divalent metal transporter expression. RESULTS: Divalent metal transporter (DMT1) was expressed at the apical membrane of the surface epithelium in rat proximal colon. In animals fed an iron-deficient diet, DMT1 mRNA and protein expression were increased. This was accompanied by a significant increase in tissue 59Fe uptake. CONCLUSIONS: The proximal colon can absorb non-haem iron from the intestinal lumen. The purpose of this mechanism remains to be elucidated.     

Dietary fat and protein interact in suppressing neuropathic pain-related disorders following a partial sciatic ligation injury in rats

Chronic neuropathic sensory disorders (CNSD) of rats receiving a partial sciatic nerve ligation injury (the PSL model) are suppressed by dietary soy protein. Although previously shown to modify nociceptive behavior in acute pain models, dietary fat has never been tested for its putative analgesic properties in chronic pain states. Here we tested the role of dietary fat, protein and fat/protein interactions in the development of tactile allodynia and heat hyperalgesia in PSL-injured rats. Male Wistar rats were fed nine different diets, comprising of three proteins (soy, casein and albumin) and three fats (corn, soy and canola) for a week preceding PSL injury and for 2 weeks thereafter. Rats' responses to tactile and noxious heat stimuli were tested before surgery and 3, 7 and 14 days afterwards. Tactile and heat sensory abnormalities following PSL injury were significantly different among the nine dietary groups. Consumption of corn and soy fats suppressed the levels of tactile and heat allodynia and hyperalgesia, whereas consumption of soy and casein proteins was associated with lower levels of heat hyperalgesia but not tactile allodynia. A significant fat/protein interaction was found for the heat but not tactile stimuli. We conclude that dietary fat is a significant independent predictor of levels of neuropathic sensory disorders in rats and that this effect is accentuated by dietary protein. The mechanisms by which fat suppresses neuropathic disorders have yet to be determined.     

Genome-wide analysis of phenobarbital-inducible genes in Drosophila melanogaster

An oligoarray analysis was conducted to determine the differential expression of genes due to phenobarbital exposure in Drosophila melanogaster (w(1118) strain) third instar larvae. Seventeen genes were observed to be induced with increased expression by a statistical analysis of microarrays approach with a q < or = 0.05. At q < or = 0.12, four more genes (Cyp12d1, DmGstd4, and two genes with unknown function) were found to be up-regulated, and 11 genes with unknown function were found to be down-regulated. Fifteen of these genes, Cyp4d14, Cyp6a2, Cyp6a8, Cyp12d1, Cyp6d5, Cyp6w1, CG2065, DmGstd6, DmGstd7, Amy-p/Amy-d, Ugt86Dd, GC5724, Jheh1, Jheh2 and CG11893, were verified using quantitative real time polymerase chain reaction. Some of these genes have been shown to be over-transcribed in metabolically DDT-resistant Drosophila strains.     

Transgenic Drosophila reveals a functional in vivo receptor for the Bacillus thuringiensis toxin Cry1Ac1

The bacterium Bacillus thuringiensis synthesizes toxins (delta-endotoxins) that are highly specific for insects. Once ingested, the activated form of the toxin binds to a specific receptor(s) located on the midgut epithelial cells, inserts into the membrane causing the formation of leakage pores and eventual death of the susceptible insect larvae. Manduca sexta larvae are highly susceptible to Cry1Ac1, a toxin that is believed to bind M. sexta Aminopeptidase N, a glycoprotein located on the apical membrane. However, the binding data obtained to date only support the interaction of Cry1Ac1 with APN in vitro. To explore the in vivo role of APN, we have utilized the GAL4 enhancer trap technique to drive the expression of M. sexta APN in both midgut and mesodermal tissues of Cry1Ac1 insensitive Drosophila larvae. Transgenic Drosophila fed the toxin were now killed, demonstrating that APN can function as a receptor for Cry1Ac1 in vivo.     

Molecular analysis of the helper T cell response in murine interstitial nephritis. T cells recognizing an immunodominant epitope use multiple T cell receptor V beta genes with similarities across CDR3.

Soluble fiber consistently lowers plasma total and low density lipoprotein (LDL)-cholesterol concentrations in humans and various animal models including the hamster; however, the mechanism of this effect remains incompletely defined. We performed studies to determine the activity of dietary psyllium on hepatic 7 alpha-hydroxylase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and LDL receptor expression in the hamster. In animals fed a cholesterol-free semisynthetic diet containing 7.5% cellulose (avicel) as a fiber source, substitution of psyllium for avicel increased hepatic 7 alpha-hydroxylase activity and mRNA levels by 3-4-fold. Comparable effects on 7 alpha-hydroxylase expression were observed with 1% cholestyramine. Psyllium also increased hepatic 7 alpha-hydroxylase activity and mRNA in animals fed a diet enriched with cholesterol and triglyceride. Activation of 7 alpha-hydroxylase was associated with an increase in hepatic cholesterol synthesis that was apparently not fully compensatory since the cholesterol content of the liver declined. Although dietary psyllium did not increase hepatic LDL receptor expression in animals fed the cholesterol-free, very-low-fat diet, it did increase (or at least restore) receptor expression that had been downregulated by dietary cholesterol and triglyceride. Thus, 7.5% dietary psyllium produced effects on hepatic 7 alpha-hydroxylase and LDL metabolism that were similar to those of 1% cholestyramine. Induction of hepatic 7 alpha-hydroxylase activity by dietary psyllium may account, in large part, for the hypocholesterolemic effect of this soluble fiber.     

Dietary fat selectively alters transport properties of rat jejunum.

The influence of dietary fatty acid composition on intestinal active and passive transport function, brush border membrane composition, and morphology was examined in rats. Animals fed a semisynthetic diet high in saturated fatty acids demonstrated enhanced in vitro jejunal uptake of decanoic, dodecanoic, palmitic, stearic, and linoleic acid, as well as cholesterol and chenodeoxycholic and taurochenodeoxycholic acid, as compared with uptake in animals fed a semisynthetic diet high in polyunsaturated fatty acids but equivalent in total content of fat and other nutrients, or as compared with Purina chow. Feeding the saturated fatty acid diet was also associated with reduced jejunal uptake of a range of concentrations of glucose, enhanced ileal uptake of leucine, unchanged uptake of galactose, and lower uptake of decanol. The semisynthetic diets did not alter brush border membrane protein, sucrase or alkaline phosphatase activities, cholesterol, or total phospholipids, although the percentage of jejunal amine phospholipids was higher than in rats fed chow. The morphologic differences between the jejunum and ileum were abolished in animals fed the high polyunsaturated fatty acid diet; in rats fed the high saturated fatty acid diet, there was reduced mean ileal villus height, width, thickness, surface area, cell size, and villus density, as well as reduced mucosal surface area. The changes in jejunal transport were not correlated with the alterations in morphology, unstirred layer resistance, food intake, or body weight gain. It is proposed that small changes in the percentage of total dietary lipids composed of essential and nonessential fatty acids (without concurrent alterations in dietary total fat, carbohydrate, or protein) influence active and passive intestinal transport processes in the rat.     

Effect of different levels of protein intake on renal deterioration and nutritional state in experimental renal disease

We examined the effects of various levels of dietary protein on the course of adriamycin-induced nephropathy in rats fed with high (30%), intermediately low (10%) or strictly low (5%) protein diets for 24 weeks. In the rats fed on the 30% protein diets, there were massive proteinuria, progressive increases in serum creatinine and focal glomerular sclerosis associated with severe tubulo-interstitial changes. With the 5% dietary protein, proteinuria was decreased, the levels of serum creatinine were preserved within normal ranges and renal histological changes diminished. Weight loss and hypoproteinaemia were more marked. With intermediate protein restriction (10% protein), renal function and plasma protein were preserved but body weight did not increase normally. Aggregated human immunoglobulin G, which had been intravenously injected at weeks 12 and 24, accumulated in the glomeruli more densely in rats fed on the 30% protein diet than in those fed on the 10% or 5% protein diets. We tentatively conclude that functional and histological deterioration of focal glomerular sclerosis can be prevented by appropriate restriction of dietary protein; however, severe protein restriction does aggravate nutritional states.