Nutritional impact of excluding red meat from the Canadian diet.

The objective of the study was to examine differences in nutrient intake between consumers and non-consumers of red meat and to assess nutritional adequacy of consumers relative to Recommended Daily Allowance (RDA) in Canada. Matching estimators were used to identify differences in nutrient intake between the two groups. Statistically significant differences were observed in nutrient intake between red meat consumers and non-consumers, including lower daily intake of protein, riboflavin, niacin, vitamin D, and zinc and a higher daily intake of dietary fiber, folate, and magnesium among Canadians who did not consume red meat. Further, red meat consumers and non-consumers had nutrient intakes below RDA for dietary energy, fiber, and calcium. While individuals who did not consume red meat were at increased risk of calcium, vitamin D, energy, and potassium inadequacy, those who consumed red meat were at increased risk of dietary fiber, vitamin A, and magnesium inadequacy.

Increased renal fibrosis and expression of renal phosphatidylinositol 4-kinase-beta and phospholipase C(gamma1) proteins in piglets exposed to ochratoxin-A.

Endemic nephropathy has been linked to exposure of ochratoxin-A (OA) in grains and animal products. The underlying events surrounding this form of renal injury are not well known, partly due to the lack of a suitable animal model of the disease. Therefore, in this study, a pig model of OA-induced renal injury was established and used to examine whether elements of the phosphoinositide signalling pathway are altered in this disease. Weanling piglets were fed diets containing 0, 2, and 4 ppm OA for 6 weeks. Serum creatinine and urea and renal fibrosis were monitored biweekly using serial blood samples and renal biopsies. At termination, the protein levels of renal phosphatidylinositol 4-kinase-beta (PtdIns4Kbeta) and phospholipase C(gamma1) (PLC(gamma1)) were determined using immunoblotting and scanning densitometry. Serum creatinine was elevated by 2 weeks and renal fibrosis was elevated by 4 weeks at both levels of inclusion of OA. At the end of the experimental period, kidney size and water content were elevated, as were the protein levels of renal PtdIns4Kbeta and PLC(gamma1) in OA-exposed animals. Therefore, serial biopsies can be used to track changes in renal pathology in the OA-exposed piglet. We conclude that this is a useful model for OA-induced renal injury in which the underlying molecular events associated with this form of renal injury can be studied.

Overexpression of kidney phosphatidylinositol 4-kinasebeta and phospholipase C(gamma1) proteins in two rodent models of polycystic kidney disease.

Our studies of renal phosphoinositide levels and metabolism in the pcy mouse with polycystic kidney disease (PKD) suggest that phosphatidylinositol kinase (PtdInsK) and phospholipase C (PLC) are elevated in this renal disorder. Therefore, the steady-state levels of select isoforms of these enzymes were examined in renal cytosolic and particulate (detergent-soluble) fractions in male and female normal and CD1-pcy/pcy (pcy) mice at 60, 120 and 180 days of age, and in male and female normal and diseased (Han:SPRD-cy) rats at 28 and 70 days of age. Disease-related increases in phosphatidylinositol 4-kinasebeta (PtdIns4Kbeta) and PLC(gamma1) levels were present in both models. PtdIns4Kbeta levels were higher by as much as 233% in pcy mice and by 95% in diseased Han:SPRD-cy rats compared to normals of the same age and gender. Steady-state levels of PLC(gamma1) were as much as 74% and 35% higher in pcy mice and diseased Han:SPRD-cy rats, respectively, compared to their controls. The consistency of these alterations in two accepted models of PKD indicates the importance of the phosphoinositide signalling pathway in the evolution of this disorder, and represents a potential site for therapeutic intervention.

Dietary soy protein effects on disease and IGF-I in male and female Han:SPRD-cy rats.

BACKGROUND: Dietary soy protein compared with casein retards disease progression in a gender-specific manner in the pcy mouse. In this model of polycystic kidney disease (PKD), kidney insulin-like growth factor-I (IGF-I) levels are elevated. The present study examined the gender-specific effects of soy protein feeding on disease and IGF-I in Han:SPRD-cy rats. METHODS: Normal (+/+) and affected (cy/+) weanling male and female Han:SPRD-cy rats were given either casein- or soy protein-based diets for six weeks. Renal size, water content, cyst size and IGF-I, serum creatinine, urea and IGF-I, and creatinine clearance were determined. RESULTS: Soy protein-fed cy/+ animals had lower kidney weight, water content and cyst size, lower serum urea and creatinine, and higher creatinine clearance. In cy/+ females, dietary soy protein resulted in normalized serum creatinine and creatinine clearance. Kidney IGF-I levels (ng/kidney) were 32 to 76% higher in cy/+ compared with +/+ groups (P < 0.001). Soy protein feeding resulted in lower kidney IGF-I in cy/+ males (1123 vs. 1496 ng/kidney, P < 0.001) and cy/+ females (816 vs. 943 ng/kidney, P < 0.05). In males, soy protein feeding resulted in lower serum IGF-I concentrations in +/+ (1439 vs. 1708 ng/mL, P < 0.05) and in cy/+ (1483 vs. 2073 ng/mL, P < 0.001) animals. CONCLUSIONS: Dietary soy protein compared with casein delays the progression of disease in male and female Han:SPRD-cy rats. Overall, IGF-I was lower in +/+ animals, in females, and in animals consuming the soy protein diet, supporting a role for IGF-I in the pathogenesis of disease in the Han:SPRD-cy rat and an ameliorating role for dietary soy protein.

Creatine supplementation increases renal disease progression in Han:SPRD-cy rats.

The growing use of creatine as a potential ergogenic aid among active individuals has raised concern regarding its effects on the kidney, particularly among those individuals with compromised renal function. The object of this study is to investigate the effects of oral creatine supplementation in an accepted animal model of renal cystic disease. Han:Sprague-Dawley (SPRD)-cy rats with cystic kidney disease were administered a creatine supplement at a loading dose of 2.0 g/kg of diet for 1 week, followed by 5 weeks during which the dose was one fifth this amount, mimicking typical human consumption on a body-weight basis. Cystic kidney disease progression was assessed by measuring kidney size and fluid content and determining cyst scores. Renal function was assessed by measuring serum urea and creatinine concentrations and creatinine clearance. Creatine supplementation resulted in greater cyst growth and worsened renal function in the Han:SPRD-cy rat, evidenced by greater kidney weights (2.87 +/- 0.08 versus 2.61 +/- 0.09 g/100 g of body weight; P: = 0.0365), renal fluid contents (89.22 +/- 0.41 versus 87.38 +/- 0.48 g/100 g of kidney weight; P: = 0.0057), cyst scores (0.49 +/- 0.02 versus 0.40 +/- 0.03; P: = 0.0167) and serum urea concentrations (23.96 +/- 0.92 versus 20.65 +/- 1.06 mmol/L; P: = 0.0230), and lower creatinine clearances (0.125 +/- 0.098 versus 0.162 +/- 0.011 mL/min/100 g of body weight; P: = 0.0159). These results indicate that creatine supplements may exacerbate disease progression in an animal model of cystic renal disease. Although systematic research of the effects of creatine supplementation in humans with compromised renal function is awaited, it follows that creatine should be used with particular caution in individuals with or at risk for renal disease.

High dietary fat intake increases renal cyst disease progression in Han:SPRD-cy rats.

The effect of a high level of dietary fat on renal cyst disease was examined in the Han:SPRD-cy rat model of polycystic kidney disease. Control and diseased rats at 4 wk of age were fed either a low fat or high fat diet (5 or 20 g/100 g diet) for 6 wk. In rats with kidney disease fed the high fat rather than the low fat diet, kidneys were 17% larger, renal fluid content was 19% higher and cyst scores were 30% higher, indicating greater disease progression. In diseased rats fed the high fat diet, serum urea was 25% higher, indicating worsened renal function. Serum creatinine was 49% higher only in males. To examine whether high dietary fat worsened renal cyst disease by altering sex hormone concentrations, serum testosterone and estrogen concentrations were determined. In normal compared with diseased male rats, serum testosterone concentrations were one to three times higher. Serum testosterone concentrations were higher in normal male rats fed the high compared with the low fat diet, but were not affected by diet in diseased rats. Serum estrogen concentrations were unaffected by dietary fat levels or by disease state. Although it remains to be elucidated how dietary fat influences sex hormone concentrations in this disease, the current study demonstrates that a high dietary fat intake increases kidney disease progression in Han:SPRD-cy rats.

Safety of chronic exercise in a rat model of kidney disease.

PURPOSE: The objective of this study was to determine the effect of treadmill running on polycystic kidney disease (PKD) progression and bone mineral density in Han:SPRD-cy rats, an animal model of PKD. METHODS: Using a 2 x 2 design, normal and polycystic male rats were divided randomly into exercise and sedentary groups at 4 wk of age. The exercising group performed treadmill exercise (14 m x min(-1)) for 30 min 3 d x wk(-1) for 6 wk, whereas the control group remained sedentary. This 6-wk period represents the period of greatest cyst growth in this model. RESULTS: Both exercised and sedentary polycystic animals had significantly greater kidney weights, as well as greater concentrations of serum urea nitrogen and serum creatinine than control animals. Exercise did not alter these parameters in either normal or polycystic animals. In addition, bone mineral density and bone mineral content, determined by dual-energy x-ray absorptiometry, were not altered by exercise in these animals. Bone mineral content, however, was marginally lower in polycystic animals. CONCLUSIONS: These results support the safety of moderate exercise in PKD. Additional research in this area is needed since there may be other benefits that are derived from exercise in this population.

Dietary soy protein effects on inherited polycystic kidney disease are influenced by gender and protein level.

The effects of dietary soy protein compared to casein were examined in male and female CD1-pcy/pcy (pcy) mice with polycystic kidney disease. Animals 10 wk of age were fed purified diets containing either soy protein isolate or casein given at a level of 17.4 or 6% protein. After 13 wk on the diets, body weights and serum concentrations of albumin and protein indicated that protein nutrition was adequate on all diets. Overall, animals fed soy protein versus casein had 28% lower (P = 0.0037) relative kidney weights (g/100 g body wt), 37% lower (P = 0.0089) cyst scores (% cyst area x relative kidney weight), and 25% less (P = 0.0144) kidney water (g). Dietary protein reduction resulted in 30% lower (P = 0.0010) relative kidney weights, 25% lower (P = 0.0327) cyst scores, and 35% less (P = 0.0001) kidney water. Analysis of interactions between main effects revealed that the effects of soy protein on kidney size were significant only in females, and that effects of soy protein on cyst score were significant only in animals on the low protein diets. In addition, differences in kidney weights and cyst score due to protein reduction were significant in animals fed soy protein, but not in those fed casein as the protein source. These results show that both dietary protein source and level significantly affect polycystic kidney disease in pcy animals, with the effects of dietary soy protein being most pronounced in female animals fed the low protein diets and the effects of protein reduction being most pronounced in animals fed soy protein-based diets.

Butyrate alters activity of specific cAMP-receptor proteins in a transgenic mouse colonic cell line.

There is great interest in utilizing butyrate as a chemotherapeutic agent. To elucidate its mechanism of action, the effect of butyrate on cAMP receptor protein kinase (PKA) activity in young adult mouse colon (YAMC) cells isolated from transgenic mice bearing a temperature sensitive mutation of the SV40 large T antigen gene was investigated. Conditionally immortalized cultures were plated at the permissive temperature (33 degrees C) or growth arrested by incubation at the nonpermissive temperature (39 degrees C). In addition, cells were incubated at 33 degrees C with or without 1 mmol/L butyrate for 24 h. Butyrate treatment reduced cell proliferation by 28% and enhanced apoptosis by 350% compared with cultures not exposed to butyrate. The PKA type I/II isozyme activity ratio was lower (P < 0.05) in cells incubated with butyrate. The relative level of PKA I isozyme was higher in proliferating cells at 33 degrees C (63% of total PKA), while the relative level of PKA II was higher in nonproliferating cells undergoing apoptosis at 39 degrees C (59% of total PKA). Neither incubation conditions (33 vs. 39 degrees C) nor butyrate treatment altered total PKA activity. When YAMC cells were incubated with 8-CI-cAMP, an activator of PKA II, growth was markedly inhibited in cells at both temperatures. Consistent with in vitro data, increased PKA I isozyme levels were associated with dysregulated growth in vivo. Specifically, the relative level of PKA I isozyme was three- to fivefold higher in rat colonic tumors compared with normal nontransformed colonic mucosa. These data indicate that the biological effects of butyrate on colonocyte proliferation and apoptosis are associated with changes in PKA isozyme-dependent signal transduction, and the YAMC cell line is a relevant model to examine the molecular mechanisms by which dietary-derived factors affect relative cancer risk.

Dietary fat and fiber differentially alter intracellular second messengers during tumor development in rat colon.

The effect of fat, fiber and carcinogen on colonic epithelial intracellular second messengers 1,2-diacyl-sn-glycerol (DAG), ceramide, and the steady-state level of phospholipase C (PLC-gamma1) was determined in 160 male Sprague-Dawley rats (10 rats per group). The study was a 2 x 2 x 2 x 2 factorial design with two types of fat (corn oil or fish oil), two types of fiber (cellulose or pectin), two injected subgroups (with or without azoxymethane (AOM), and two time points (15 and 37 weeks). At the final time point (37 weeks) there were an additional 20 rats per diet in each of the carcinogen-treated groups for tumor analyses only (n = 80), for a total of 240 animals in the entire study. At each time point (15 and 37 weeks), 80 rats were killed and colonic mucosa obtained for DAG, ceramide and PLC-gamma1 assays. At the first time point (15 weeks), there was no microscopic evidence of tumors. At the final time point (37 weeks), fish oil resulted in a lower proportion of animals with adenocarcinomas relative to corn oil feeding (56.1 % versus 69.6 %, P < 0.05). There was no significant main effect of fiber on the percentage of animals with tumors. At 15 weeks post-injection, AOM injected animals fed corn oil-containing diets had a significantly (P < 0.001) higher DAG mass and steady-state levels of PLC-gamma1 compared with AOM-injected animals fed fish oil and saline injected rats on all diets. Animals fed corn oil diets also had a significantly (P < 0.01) elevated mucosal ceramide mass compared with fish oil fed animals. Moreover, rats injected with AOM had a significantly (P < 0.02) elevated colonic mucosal DAG/ceramide ratio versus saline injected animals. In contrast, dietary fiber had no effect on any of the parameters measured at 15 weeks. However, at 37 weeks post-injection, dietary fiber significantly altered DAG (P < 0.02), and PLC-gamma1 expression (P < 0.05) in the absence of an effect on tumor incidence. These data demonstrate that the ability of dietary fish oil to reduce experimental colon carcinogenesis may be mediated by changes in colonic intracellular mediators during the initial stages of tumorigenesis.

Marked enrichment of the alkenylacyl subclass of plasma ethanolamine glycerophospholipid with eicosapentaenoic acid in human subjects consuming a fish oil concentrate.

Alteration in human platelet fatty acid levels with the consumption of fish oils containing eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have been well documented, but changes in the fatty acid composition of plasma plasmalogenic phospholipid under similar circumstances have not been delineated. In the present study, subjects consumed the fish oil concentrate (MaxEPA) for 6 wk followed immediately by a 6-wk recovery period with no fish oil ingestion. Plasma total choline glycerophospholipid (GPC) and ethanolamine glycerophospholipid (GPE) subclasses isolated from blood samples obtained at 0, 3, 6, 9 and 12 wk of the experimental period were analyzed for fatty acid composition via thin-layer and gas-liquid chromatographic techniques. Consumption of fish oil for 3 or 6 wk significantly elevated the content of n-3 fatty acids while concomitantly decreasing n-6 fatty acid levels in plasma total GPC and in diacyl and alkenylacyl (plasmalogen) GPE. Alkenylacyl GPE exhibited the greatest alteration of both n-3 and n-6 fatty acid levels. Following 6 wk of supplementation with fish oil, EPA rose by 24.6 mol% in alkenylacyl GPE compared to increases of 6.7 and 7.1 mol% in diacyl GPE and total GPC, respectively. The increase in EPA (from 5.0 to 29.6 mol%) in plasma alkenylacyl GPE represents amongst the highest enrichment of EPA in any lipid yet reported in human subjects. DHA also rose by 8.0, 4.8, and 3.1 mol% in alkenylacyl GPE, diacyl GPE, and total GPC, respectively. Alkenylacyl GPE exhibited the greatest mol% decline (by 18.7 mol%) in arachidonic acid (AA, 20:4n-6) following 6 wk of fish oil supplementation. The corresponding decreases of AA in diacyl GPE and total GPC were 8.7 and 1.8 mol%, respectively. Following the 6 wk recovery period, n-3 and n-6 fatty acid levels had returned to pre-supplementation values. The marked enrichment of alkenylacyl GPE in n-3 fatty acids, especially EPA, may be of significance with respect to a unique role for this plasma phospholipid subclass in attenuating certain lipoprotein-mediated cardiovascular effects as observed with fish/fish oil consumption.

Localization of protein kinase C isozymes in rat colon.

We have demonstrated previously the presence of classical (alpha), novel (delta and epsilon), and atypical (zeta) protein kinase C (PKC) isozymes in human and rat colonic mucosa (L. A. Davidson et al., Arch. Biochem. Biophys., 312:547-553, 1994). To gain insight into the functions of individual PKC isozymes in colonic epithelium in situ, we determined the localization of the major PKC isozymes expressed in normal rat colonic epithelial cells using in situ reverse transcription (RT)-PCR and immunohistochemistry (IH). Cytokeratin, a positive biological control known to be expressed in epithelial cells, was shown by in situ RT-PCR and IH to be expressed only in epithelial cells within the colonic crypt. PKC gamma, a negative control for the colon since it is expressed only in the central nervous system, was not detectable in colon sections by either methodology. In situ RT-PCR analysis revealed that PKC alpha, delta, epsilon, and zeta mRNAs are expressed in epithelial cells along the entire colonic crypt. In addition, PKC delta and zeta mRNA are expressed in the stromal layer. All four PKC isozymes in the colonic epithelial cells were also detected by IH. However, in general, isozyme protein expression was greater at the top of the crypt axis, associated primarily with cells having acquired a differentiated phenotype. These results suggest that PKC isozyme protein expression may be localized to mature differentiated cells at the top of the colonic crypt. Therefore, PKC isozyme-dependent signal transduction may play a role in colonic epithelial cell ontogeny along the colonic crypt axis.

Diet and disease alter phosphoinositide composition and metabolism in murine polycystic kidneys.

Because diet can affect the progression of polycystic kidney disease (PKD) and because renal phosphoinositide metabolism is altered in mice with PKD, the effects of diet and disease on phosphoinositide composition and metabolism were examined in kidneys of mice with PKD. The phosphatidylinositol-phosphate (PIP) to phosphatidylinositol (PI) molar ratio was higher (0.034 +/- 0.003 vs. 0.023 +/- 0.001, P < 0.01) and the PI-bisphosphate (PIP2) to PIP molar ratio was lower (0.70 +/- 0.08 vs. 1.19 +/- 0.10, P < 0.05) in kidneys of mice with PKD [DBA/2FG-pcy (pcy)] compared with normal controls (DBA/2J). When initial incorporation (reflecting synthesis) of [3H]inositol into renal phosphoinositides in mice injected with [3H]inositol was measured, the [3H]PIP to [3H]PI ratio was higher in the diseased kidneys compared with normal kidneys (0.016 +/- 0.001 vs. 0.013 +/- 0.001, P < 0.05), whereas the [3H]PIP2 to [3H]PIP ratio was not significantly different. In a study using dietary manipulations that alter the progression of PKD in pcy mice (6 vs. 25% casein and sunflower seed oil vs. fish oil in a 2 x 2 design), animals were injected intraperitoneally with [3H]inositol 5 h before killing. In these animals, the [3H]PIP2 to [3H]PIP ratio seemed to be the best indicator of disease progression. In addition, kidney weight (as altered by diet) was positively correlated (r = 0.62, P = 0.02) with the level of the [3H]PI-3-P isomer relative to total [3H]PIP in the kidney. These results demonstrate that alterations in dietary protein level and lipid composition can modulate renal phosphoinositide signal transduction in mice with PKD.

Dietary fat and fiber alter rat colonic protein kinase C isozyme expression.

To better understand the biochemical mechanisms by which select fats and fibers modulate colonic cell proliferation, we determined the profile of protein kinase C (PKC) isozymes and cell proliferation in rat proximal and distal colonic mucosa following diet manipulation, because enhanced cell proliferation has been correlated with colon cancer incidence. Rats were assigned to one of four diets (each with 15 g fat + 6 g fiber/100 g diet) for 3 wk: fiber-free fish oil (FF), fiber-free corn oil (FC), cellulose + corn oil (CC), or pectin + corn oil (PC). Stead-state levels of colonic mucosal cytosolic and membrane PKC isozymes were determined. In vivo cell proliferation was determined by bromodeoxyuridine incorporation into DNA. In addition, viable exfoliated colonic epithelial cells were isolated from feces using Percoll-bovine serum albumin gradients. We found that 1) proximal and distal colonic mucosa possessed different steady-state levels and relative proportions of PKC isozymes; 2) PKC alpha and delta expression were significantly greater in distal membrane of the PC-fed group compared with the other dietary groups; 3) the number of exfoliated cells per 4-h fecal collection generally was proportional to the diet-induced changes in cell proliferation (PC > FC > CC > FF). These data demonstrate that dietary treatment altered colonic PKC isozyme expression, with animals fed the fiber-containing diets generally expressing higher steady-state levels of PKC alpha and delta.

Early dietary protein restriction slows disease progression and lengthens survival in mice with polycystic kidney disease.

The objective of these studies was to examine the effects of early dietary protein restriction on disease progression and survival in the DBA/2FG-pcy (pcy) mouse model of polycystic kidney disease. Male pcy mice of 70 days of age were fed either a normal protein (NP, 25% casein) or a low-protein (LP, 6% casein) diet for 105 days. At the end of the dietary treatment, kidney weight, kidney weight relative to body weight and kidney water contents were almost 50% lower, and relative renal phospholipid and triglyceride contents were almost 50% higher, in mice fed the LP diet, indicating a marked reduction in the progression of cystic disease. Morphometric analyses also revealed a lower total and percent cyst area in kidneys derived from mice on the LP compared with the NP diet. There were no significant differences in final body weight, urine volume and osmolality, GFR, proteinuria, or plasma levels of protein and urea between these two groups. In a second study, it was found that all mice fed an NP diet from 70 days of age onward had died by 310 days of age, compared with a 42% survival rate in LP-fed mice at this age. Overall, the mean lifespan for pcy mice on the LP diet was 24% longer than that for those mice on the NP diet (310 +/- 20 versus 251 +/- 16 days; P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary modulation of rat colonic cAMP-dependent protein kinase activity.

Malignant transformation of cells is associated with enhanced proliferation and alterations in cAMP-dependent protein kinase (PKA) activity. To investigate the role of PKA in normal colonic cell proliferation, PKA was characterized in rat colonic mucosa. In addition, rats were fed diets containing different fats (corn oil, fish oil) and fibers (pectin, cellulose, fiber free) to elicit varying levels of colonic cell proliferation in order to study this signaling system under normal physiologic conditions. Overall, PKA activities were higher in cytosolic compared to membrane fractions. PKA type II (PKA II) isozyme contributed 89 +/- 1% and 96 +/- 1% of total PKA activity in cytosolic and membrane fractions, respectively. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of mRNA for both the alpha and beta isoforms of the regulatory subunits of PKA II. PKA activities were 21-33% higher in distal membrane and total distal fractions in rats fed a cellulose/corn oil diet compared to animals consuming the other fiber/fat diets. These effects were seen only in the distal colon, where the number of cells per crypt column was elevated only in animals fed the cellulose/corn oil diet relative to other diets. Diet-induced mitogenic responses did not involve significant changes in the relative activity of PKA I and II isozymes. These data demonstrate that dietary effects on PKA activity in the distal colon may be related to changes in cell differentiation as indicated by the number of cells per crypt column.

Protein kinase C isoforms in human and rat colonic mucosa.

The protein kinase C (PKC) family of enzymes plays a key role in the regulation of cellular events, including cell proliferation and differentiation. Work from our laboratory has shown that the effects of dietary fat and fiber on colonic cell proliferation were positively correlated with membrane/cytosol PKC activity ratios (Chapkin et al., 1993, J. Nutr. 123, 649-655). The presence and subcellular distribution of specific PKC isoforms in rat and human colon were therefore determined in cytosolic and membrane extracts. Tissue extracts were probed with antibodies to individual PKC isoforms. PKC alpha, beta, delta, epsilon, and zeta were detected in both rat and human colonic mucosa, while PKC eta was detected in human colonic mucosa only. PKC alpha, beta, and zeta were predominantly localized in the cytosolic fraction, whereas the majority of PKC delta, epsilon, and eta were found in the membrane-associated fraction. Presence of mRNA for individual PKC isoforms was determined by reverse transcriptase PCR (RT-PCR). Using rat colonic mucosa, mRNA for PKC alpha, beta, delta, epsilon, eta, and zeta were detected by RT-PCR with identity confirmed by sequencing. The relative steady-state levels of PKC isoforms in human colon adenocarcinoma as compared with normal colonic mucosa were determined, with adenocarcinomas having higher amounts of cytosolic PKC beta, delta, epsilon, eta, and zeta. PKC isoforms were also detected in viable, exfoliated colonic cells isolated from human feces, demonstrating that this noninvasive method can be utilized to examine PKC expression in colonic cells. These results demonstrate that colonic mucosa expresses both calcium-dependent (classical) and calcium-independent (novel and atypical) PKC isoforms with distinct subcellular distributions for each. The dynamics of these PKC isoforms may have implications in the development of colon carcinogenesis.

Purified dietary n-3 polyunsaturated fatty acids alter diacylglycerol mass and molecular species composition in concanavalin A-stimulated murine splenocytes.

A low-dose, short-term dietary supplementation with highly purified (n-3) fatty acid ethyl esters was studied in mice to determine the effect on splenic cell membrane diacylglycerol mass and composition. Mice were fed diets containing either 3% safflower oil (SAF) ethyl esters, 2% SAF plus 1% eicosapentaenoic acid ethyl ester (EPA), or 2% SAF plus 1% docosahexaenoic acid ethyl ester (DHA). Following a 10-day feeding period, pathogen-free mice were sacrificed and splenic cells isolated and stimulated with concanavalin A (Con A) at 10 micrograms/ml. After 0 min (basal), 5 min, and 180 min, 1,2-diacyl, 1-O-alkyl-2-acyl, and 1-O-alkenyl-2-acyl-sn-glycerol subclasses were isolated and quantitated by HPLC. Diacylglycerol (DAG) was found to be the major diradylglycerol (DG) component in murine splenocytes. DHA-fed mice had significantly (P < 0.05) higher levels of DAG at all stimulation time points relative to EPA and SAF animals. Significant effects (P < 0.05) of diet, time, and a diet x time interaction (P < 0.05) were noted for various DAG molecular species. In general, a significantly higher (n-3) polyunsaturated fatty acid (PUFA) content in the EPA and DHA groups, and a significantly higher (n-6) PUFA content in the SAF group was noted. 18:0-22:5(n-3), 18:1-22:5(n-3) and 16:1-20:5(n-3) species were present only in EPA and DHA-DAG, confirming the incorporation of (n-3) fatty acids into splenocyte DAG. The data indicate that the molecular species composition of murine splenocyte DAG is significantly modulated by low-dose, short-term EPA and DHA feeding. In addition, substitution of SAF with DHA results in an increase in DAG mass. These alterations could potentially influence signal transduction pathways regulating lymphocyte function.

Dietary fat and fiber alter rat colonic mucosal lipid mediators and cell proliferation.

To better understand the biochemical mechanisms by which dietary fat and fiber modulate colonic cell proliferation, we determined the effect of dietary fats and fibers on rat colonic epithelial cell phospholipid mass and composition and on two metabolic products of phospholipids, prostaglandins and diacylglycerol (DAG). In a 3 x 3 factorial design, groups of 10 male Sprague-Dawley rats were fed one of nine experimental diets for 3 wk: three types of fat at 15 g/100 g (beef tallow, corn oil or fish oil) x two types of fiber (pectin or cellulose) or fiber-free as a control group. Dietary treatment did not alter phospholipid or DAG mass, although the fatty acid compositions of membrane phospholipids and DAG were altered by dietary treatment. Arachidonic acid [20:4(n-6)] and eicosapentaenoic acid [20:5(n-3)] in colonic mucosal phospholipid and DAG were associated with higher and lower indices of cell proliferation, respectively. These correlations were specific for the distal colon, which was the principle site of dietary fat effects on cell proliferation. Prostaglandin E and prostacyclin synthesis in colonic mucosa and muscle was significantly lower in fish oil-fed compared with beef tallow- and corn oil-fed animals (by 46-90%, P < 0.001), in both the proximal and distal colon. Correlations between prostaglandin production and cell proliferation, however, were significant only in the distal colon. These data raise the possibility that dietary fat and fiber may modulate intracellular events related to cell proliferation via their effects on epithelial cell phospholipid fatty acid composition, and subsequently on prostaglandin production and DAG composition.

Mass and fatty acid composition of the 3-phosphorylated phosphatidylinositol bisphosphate isomer in stimulated human platelets.

By high pressure liquid chromatography (HPLC) analysis, the occurrence of radiolabeled 3-phosphorylated phosphoinositides has been well documented in several cell systems, including agonist-stimulated platelets. The actual mass amounts and fatty acid composition of these unique lipids, however, have not been reported to date. In the present study, we report the mass and fatty acid composition of phosphatidylinositol (PI) 3,4-P2 from U46619-stimulated platelets using a thin-layer chromatographic system for the separation of PI 3,4-P2 from PI 4,5-P2. The mass of PI 3,4-P2 in the stimulated platelet was 180 +/- 9.7 pmol/1 x 10(9) platelets (mean +/- S.E., n = 4), representing 9.3% of total phosphatidylinositol bisphosphate (PIP2). Based on HPLC analysis, PI 3,4-P2 in unstimulated platelets represented < 0.5% of total PIP2 (which corresponds to < 7.0 pmol/1 x 10(9) platelets). Fatty acid analysis of this lipid revealed a composition very similar to the conventional polyphosphoinositides (stearic and arachidonic acids accounting for 44.2 and 40.4 mol %, respectively, of the fatty acids). Since PI 3,4-P2 also did not appear to be distinct from the other polyphosphoinositides, in regard to radiolabeling properties, it was concluded that this lipid is unlikely to originate from a unique precursor pool. This conclusion validates the use of HPLC analysis of radiolabeled phosphoinositides for the estimation of PI 3,4-P2 mass in agonist-stimulated platelets. The chromatographic procedure described should prove useful for the mass and fatty acid analysis of PI 3,4-P2 from other cell systems.