Turnover of carbon, nitrogen, and sulfur in bovine longissimus dorsi and psoas major muscles: Implications for isotopic authentication of meat.

Stable isotope ratio analysis of light elements (including C, N, and S) is a powerful tool for inferring the production and geographic origins of animals. The objectives of this research were to quantify experimentally the isotopic turnover of C, N, and S in bovine skeletal muscle (LM and psoas major) and to assess the implications of the turnover for meat authentication. The diets of groups (n = 10 each) of beef cattle were switched from a control diet containing barley and unlabelled urea to an experimental diet containing maize, (15)N-labeled urea, and seaweed for periods of up to 168 d preslaughter. The feeding of the experimental diet was clearly reflected by the delta(13)C, delta(15)N, and delta(34)S values of the LM and psoas major muscles, but isotopic equilibrium was not reached in either muscle for C, N, or S after 168 d of feeding the experimental diet. The slow turnover in skeletal muscle was reflected by the C and N half-lives of 151 and 157 d for LM and 134 and 145 d for psoas major, respectively, and by an S half-life of 219 d in LM. It is concluded that the turnover of light elements (C, N, and S) in bovine skeletal muscles is a slow process; therefore, skeletal muscles contain isotopic information on dietary inputs integrated over a long period of time (months to years).

Translocation of 15N indicates nitrogen recycling in the mat-forming lichen Cladonia portentosa.

Nitrogen translocation was measured in Cladonia portentosa during 2 yr growth in Scottish heathland. Translocation was predicted to occur if N is resorbed from senescent basal tissue and recycled within the thallus. (15)N was introduced into either the lower (TU thalli) or upper (TD thalli) 25 mm of 50-mm-long thalli as (15)N-NH(4) (+), (15)N-NO(3) (-) or (15)N-glycine. Labelled thalli were placed within intact lichen cushions, either upright (TU) or inverted (TD). Vertical distribution of label was quantified immediately following labelling and after 1 and 2 yr. Independently of the form of introduced label, (15)N migrated upwards in TU thalli, with new growth being a strong sink. Sink regions for (15)N during year 1 (including new growth) became sources of (15)N translocated to new growth in year 2. Upward migration into inverted bases was minimal in TD thalli, but was again marked in new growth that developed from inverted apices. Relocation of N to regions of growth could facilitate internal N recycling, a process postulated to explain the ecological success of mat-forming lichens.

Plant roots release phospholipid surfactants that modify the physical and chemical properties of soil.

• Plant root mucilages contain powerful surfactants that will alter the interaction of soil solids with water and ions, and the rates of microbial processes. • The lipid composition of maize, lupin and wheat root mucilages was analysed by thin layer chromatography and gas chromatography-mass spectrometry. A commercially available phosphatidylcholine (lecithin), chemically similar to the phospholipid surfactants identified in the mucilages, was then used to evaluate its effects on selected soil properties. • The lipids found in the mucilages were principally phosphatidylcholines, composed mainly of saturated fatty acids, in contrast to the lipids extracted from root tissues. In soil at low tension, lecithin reduced the water content at any particular tension by as much as 10 and 50% in soil and acid-washed sand, respectively. Lecithin decreased the amount of phosphate adsorption in soil and increased the phosphate concentration in solution by 10%. The surfactant also reduced net rates of ammonium consumption and nitrate production in soil. • These experiments provide the first evidence we are aware of that plant-released surfactants will significantly modify the biophysical environment of the rhizosphere.

Phenotype/genotype associations for yield and salt tolerance in a barley mapping population segregating for two dwarfing genes.

Barley traits related to salt tolerance are mapped in a population segregating for a dwarfing gene associated with salt tolerance. Twelve quantitative trait loci (QTLs) were detected for seven seedling traits in doubled haploids from the spring barley cross Derkado x B83-12/21/5 when given saline treatment in hydroponics. The location of QTLs for seedling growth stage (leaf appearance rate), stem weight prior to elongation, and tiller number are reported for the first time. In addition, four QTLs were found for the mature plant traits grain nitrogen and plot yield. In total, seven QTLs are co-located with the dwarfing genes sdw1, on chromosome 3H, and ari-e.GP, on chromosome 5H, including seedling leaf response (SGa) to gibberellic acid (GA(3)). QTLs controlling the growth of leaves (GS2) on chromosomes 2H and 3H and emergence of tillers (TN2) and grain yield were independent of the dwarfing genes. Field trials were grown in eastern Scotland and England to estimate yield and grain composition. A genetic map was used to compare the positions of QTLs for seedling traits with the location of QTLs for the mature plant traits. The results are discussed in relation to the study of barley physiology and the location of genes for dwarf habit and responses to GA.

Dietary epoxy fatty acids are absorbed in healthy women.

BACKGROUND: Epoxy fats in the diet may adversely affect human health. There are no data on the absorption of these fats in humans. METHODS: Triglycerides were synthesized containing two U-13C-labelled monoepoxy or diepoxy stearic acid molecules. Apparently healthy women consumed a standardized fatty meal (30 g fat) containing either 20 mg monoepoxy or 25 mg diepoxy fat (n = 6 and n = 7, respectively). Plasma lipid [U-13C]monoepoxy and diepoxy stearate concentrations were determined (0-24 h) by gas chromatography-mass spectrometry. RESULTS: Plasma triglycerides increased from 1.05 +/- 0.12 to 1.83 +/- 0.13 mmol L-1 (n = 6) and from 1.10 +/- 0.19 to 1.41 +/- 0.27 mmol L-1 (n = 7) (both P < 0.001). Plasma [U-13C]monoepoxy and diepoxy stearate levels increased to 0.18 +/- 0.07 micromol L-1 (n = 6) and to 0.08 +/- 0.03 micromol L-1 (n = 7), respectively. Monoepoxy triglyceride was better absorbed than diepoxy triglyceride: 17 +/- 4 vs. 8 +/- 1% of dose (determined from area under curve (plasma 13C) normalized to that of absorbed triglycerides (plasma 12C); P < 0.02 after log transformation). The absorption of monoepoxy- and diepoxy-labelled triglycerides was related to that of normal triglycerides (r = 0.80, P < 0.05 and r = 0.91, P < 0.001, respectively). CONCLUSIONS: Monoepoxy fats are better absorbed than diepoxy fats in women (17 +/- 4 vs. 8 +/- 1% of dose, P = 0.02). This difference in absorption is important when considering the relative toxicity of epoxidized material in the food chain.

Altritol synthesis by Notheia anomala.

The linear hexitol altritol has only been identified in six genera, all of them in the order Fucales of the brown algae. Five of these genera are closely related according to molecular phylogenetic and other data, while the sixth (Notheia) is an obligate epiphyte on two other altritol-containing genera with which it is symphanic. The possibility that Notheia obtains altritol from the algae on which it is epiphytic rather than by synthesizing altritol independently was investigated by supplying 13C-inorganic carbon in the light followed by mass spectrometric and nuclear magnetic resonance analysis. Notheia separated from the phorophyte Hormosira during exposure to 13C showed 13C enrichment in both altritol and mannitol, while the Hormosira only showed significant labelling of mannitol. These data show that altritol can be synthesized by Notheia, with implications for the number of gains and losses of the capacity to synthesize altritol in the evolution of the Fucales.

Development of delta(15)N stratification of NO(3)(-) in soil profiles.

New evidence, obtained using a robust method for measuring the delta(15)N of NO(3)(-)-N in soil, is consistent with denitrification being the major determinant in the vertical distribution of NO(3)(-)-delta(15)N in soil profiles. These data also suggest that varying moisture regimes result in different effects of soil NO(3)(-)-N leaching on residual whole soil delta(15)N.

Isomerization increases the postprandial oxidation of linoleic acid but not alpha-linolenic acid in men.

Human lipid intake contains various amounts of trans fatty acids. Refined vegetable and frying oils, rich in linoleic acid and/or alpha-linolenic acid, are the main dietary sources of trans-18:2 and trans-18:3 fatty acids. The aim of the present study was to compare the oxidation of linoleic acid, alpha-linolenic acid, and their major trans isomers in human volunteers. For that purpose, TG, each containing two molecules of [1-(13)C]linoleic acid, alpha-[1-(13)C]linolenic acid, [1-(13)C]-9cis,12trans-18:2, or [1-(13)C]-9cis,12cis,15trans-18:3, were synthesized. Eight healthy young men ingested labeled TG mixed with 30 g of olive oil. Total CO(2) production and (13)CO(2) excretion were determined over 48 h. The pattern of oxidation was similar for the four fatty acids, with a peak at 8 h and a return to baseline at 24 h. Cumulative oxidation over 8 h of linoleic acid, 9cis,12trans-18:2, alpha-linolenic acid, and 9cis,12cis,15trans-18:3 were, respectively, 14.0 +/- 4.1%, 24.7 +/- 6.7%, 23.6 +/- 3.3%, and 23.4 +/- 3.7% of the oral load, showing that isomerization increases the postprandial oxidation of linoleic acid but not alpha-linolenic acid in men.

Using stable isotope natural abundances (delta 15N and delta 13C) to integrate the stress responses of wild barley (Hordeum spontaneum C. Koch.) genotypes.

To integrate the complex physiological responses of plants to stress, natural abundances (delta) of the stable isotope pairs 15N/14N and 13C/12C were measured in 30 genotypes of wild barley (Hordeum spontaneum C. Koch.). These accessions, originating from ecologically diverse sites, were grown in a controlled environment and subjected to mild, short-term drought or N-starvation. Increases in total dry weight were paralleled by less negative delta 13C in shoots and, in unstressed and droughted plants, by less negative whole-plant delta 13C. Root delta 15N was correlated negatively with total dry weight, whereas shoot and whole-plant delta 15N were not correlated with dry weight. The difference in delta 15N between shoot and root varied with stress in all genotypes. Shoot-root delta 15N may be a more sensitive indicator of stress response than shoot, root or whole-plant delta 15N alone. Among the potentially most productive genotypes, the most stress-tolerant had the most negative whole-plant delta 15N, whether the stress was drought or N-starvation. In common, controlled experiments, genotypic differences in whole-plant delta 15N may reflect the extent to which N can be retained within plants when stressed.

Effects of symbiosis with Frankia and arbuscular mycorrhizal fungus on the natural abundance of 15N in four species of Casuarina.

The effect of interactions between Casuarina species, Frankia strains and AMF on nitrogen isotope fractionation within the plant were determined under conditions where changes in source nitrogen were minimized by growing plants in mineral nitrogen-deficient conditions and without added organic N. Casuarina cunninghamiana, C. equisetifolia, C. glauca, and C. junghuniana were inoculated singly with three Frankia strains or were dual inoculated with Frankia and Glomus fasciculatum. The %N and delta 15N of separated parts of plants inoculated with the three Frankia strains or with Frankia + Glomus were not significantly different within Casuarina species. However, the slow-growing C. junghuniana differed in several variables from the other three species. There was a highly significant, linear relationship between the natural logarithms of cladode N content and delta 15N of plants of the four Casuarina species when inoculated with Frankia or with Frankia + Glomus, showing that nitrogen supply and the correlated variable, plant growth rate, were major determinants of delta 15N. Provision of small quantities of (NH4)2SO4 or KNO3 increased several-fold the growth of three of the Casuarina species when inoculated with Frankia alone or with Frankia + Glomus. Within species, mycorrhizal and non-mycorrhizal plants receiving supplementary soluble phosphate were of similar dry weights at harvest. delta 15N values for cladodes of C. cunninghamiana, C. equisetifolia and C. glauca were similar, but values for the poor growing C. junghuniana were more variable and, with the exception of plants receiving KNO3, were lower than those of the other three species. Reduced growth due to suboptimal availability of N or P had a major influence on delta 15N and, in these conditions where plants could not access significant amounts of organic N, outweighed any effects on cladode delta 15N of colonization by Glomus. delta 15N values of nodules were higher than other parts of Frankia or Frankia + Glomus inoculated Casuarinas, conceivably due to retention in nodules of fixed N, with delta 15N close to zero.

Carbon and nitrogen stable isotope ratios in body tissue and mucus of feeding and fasting earthworms (Lumbricus festivus).

We used natural abundance stable isotope techniques to estimate carbon and nitrogen turnover rates in body tissue and mucus of earthworms. Isotope ratios of carbon (delta(13)C) and nitrogen (delta(15)N) were monitored simultaneously in body tissue and mucus for up to 101 days in feeding or fasting Lumbricus festivus kept in an artificial substrate. When the diet of the earthworms was switched from clover (C(3) plant, legume) to maize (C(4), non-legume), the new dietary delta(13)C signature manifested itself much more rapidly in the mucus than in the body tissue of the animals, causing a delta(13)C shift of about 4 per thousand in mucus and 1 per thousand in tissue after 13.5 days. Turnover of earthworm body tissue carbon, unlike that of mucus carbon, was described adequately by an exponential, single-pool model. Nitrogen turnover could not be assessed because the delta(15)N difference between sources was too small. Fasting for 56 days did not result in the expected whole-body (15)N or (13)C enrichment, but it caused a significant decrease in mucus and tissue C:N ratios and in the ratio (mucus C:N ratio):(tissue C:N ratio). We conclude that the separate analysis of body tissue and mucus has great potential for studying the ecophysiology, feeding ecology and role in elemental cycling of earthworms and other invertebrates.

Wild rabbit host and some parasites show trophic-level relationships for delta 13C and delta 15N: a first report.

We report the first isotopic study of an animal host-parasite system. Parasitic, intestinal nematodes, Graphidium strigosum and Passalurus ambiguus, were 15N-enriched relative to their host, the European rabbit Oryctolagus cuniculus, while parasitic cestodes, Cittataenia denticulata and Mosgovoyia pectinata, were 15N-depleted, suggesting different trophic relationships. Host embryos were more similar in their delta 13C and delta 15N values to maternal muscle than were any of the parasites. Coprophagy, the direct recycling of food by the rabbit eating its own faeces, did not lead to isotopic differences between stomach contents and faeces, suggesting that the major point for isotopic discrimination in lagomorph nitrogen metabolism is in the animal rather than in the gut. We conclude that bulk delta 13C and delta 15N can reveal valuable new information about host-parasite relationships, and these could be explored further at the biochemical level using compound-specific isotopic analyses.

AFLP variation in wild barley (Hordeum spontaneum C. Koch) with reference to salt tolerance and associated ecogeography.

Thirty-nine genotypes of Hordeum spontaneum were selected from three geographically separated areas (southwestern, northern, and southeastern) of the Fertile Crescent. The lines were subject to AFLP (amplified fragment length polymorphism) analysis from which a similarity matrix was produced. A dendrogram of the data showed, with two exceptions, that the genotypes grouped together according to area of origin. This was confirmed by principal coordinate analysis in which the first principal coordinate separated the genotypes of the southwestern area from the other two areas, which in turn could be separated by the second principal coordinate. While genotypes from the same site of origin can exhibit very similar AFLP profiles, sharp genetic differences were detected between genotypes separated by relatively short distances. Thirty of the 39 genotypes were subjected to hydroculture salt tolerance tests. These were analysed for shoot Na+ content and carbon isotope composition (δ13C) after 4 weeks of treatment (100 mol∙m−3 NaCl). Shoot Na+ content and δ13C were highly correlated. Twelve AFLP markers were found to be associated with both shoot Na+ content and shoot δ13C and were also associated with site of origin ecogeographic data, particularly longitude. The most salt tolerant genotype came from Ilam in the southeastern area and the most salt sensitive genotype originated in the southwestern area. The 12 markers were partitioned into groups that showed significant associations within groups but no significant association between groups. In a multiple regression analysis, three AFLP markers, from separate groups, accounted for more than 60% of the variation for shoot Na+ content and δ13C. The results demonstrate the effectiveness of AFLP fingerprinting in genetic studies of complex traits at the wild species and (or) population level.

Adequacy of food rations in soldiers during exercise in hot, day-time conditions assessed by doubly labelled water and energy balance methods.

The energy requirements of people doing physical work in hot climates are not clearly understood. In particular, we know little about the combined effects of heat stress and muscular work on energy requirements. During military exercises in the African bush, soldiers are supplied with standard rations, the adequacy of which is unknown. We have now assessed the adequacy of these food and water rations in 12 male Zimbabwean soldiers during 12 days of strenuous, heat-stress exercise in the field. We used two methods to measure energy expenditure: the double-labelled water method (DLW) and the energy balance method (i.e. comparing dietary energy with changes, if any, in body energy stores). Two groups were studied: one group (eight subjects) carried out field exercises; the control group consisted of four soldiers doing normal work. Mean daily energy expenditure as assessed by the DLW method was [mean (SE)] 23 (1.5) MJ x day(-1) for the field group and 14 (0.5) MJ x day(-1) for the control group (P < 0.001). By the energy balance method, daily energy expenditure was calculated to be 26 (0.7) MJ x day(-1) and 15.5 (0.4) MJ x day(-1) for the field group and control group, respectively. Body mass loss was 3 (0.1) kg [4.6 (0.3)% of body mass] for the field group, but the control group gained 1.1 (0.1) kg. Mean daily fluid intake was 11 (0.5) 1 x day(-1), suggesting that the standard ration supplied was inadequate. Body mass loss was caused by both the energy deficit and total body water loss. These results suggest strenuous work in hot, dry field conditions imposes extra energy requirements.

Wild Rabbit Host and Some Parasites Show Trophic-Level Relationships for δ(13)C and δ(15)N: A First Report.

Abstract We report the first isotopic study of an animal host-parasite system. Parasitic, intestinal nematodes, Graphidium strigosum and Passalurus ambiguus, were (15)N-enriched relative to their host, the European rabbit Oryctolagus cuniculus, while parasitic cestodes, Cittataenia denticulata and Mosgovoyia pectinata, were (15)N-depleted, suggesting different trophic relationships. Host embryos were more similar in their δ(13)C and δ(15)N values to maternal muscle than were any of the parasites. Coprophagy, the direct recycling of food by the rabbit eating its own faeces, did not lead to isotopic differences between stomach contents and faeces, suggesting that the major point for isotopic discrimination in lagomorph nitrogen metabolism is in the animal rather than in the gut. We conclude that bulk δ(13)C and δ(15)N can reveal valuable new information about host-parasite relationships, and these could be explored further at the biochemical level using compound-specific isotopic analyses.

Variations of the natural abundances of nitrogen and carbon isotopes in Triticum aestivum, with special reference to phloem and xylem exudates.

This work explored whether the natural abundances of carbon and nitrogen isotopes could be used to describe the movement of C and X within wheat plants; we also considered whether isotopic analyses of aphids or their honeydew would substitute for direct analysis of phloem exudate. The δ13 C of ears and roots (sinks) most closely matched those of the sugars + organic acids fraction (sources) in both growth stages; phloem δ13 C matched that of leaf blade sugars. Xylem exudate δ13 C matched no other putative (and measured) source in the ear-forming stage and matched that of whole roots and ears in the grain-filling stage. The δ15 N of grain and roots (sinks) resembled that of leaf amino acids (sources) in the ear-forming stage. In the gram-filling stage, ear δ15 N continued to resemble that of leaf amino acids, and δ15 N of roots most closely resembled that of whole leaves. In the grain-filling stage, phloem δ15 N fell between that of leaf blade amino acids and that of whole leaves and was 15 X-depleted relative to internal and external NO, -N. In both growth stages, xylem exudate δ15 N was less than that of soil NO3 - -N and more than that of residual soil N after mineral N extraction. The isotopic values are generally in agreement with data from other approaches, such as isotope labelling; they show NO3 - -N reduction in both shoots and roots of wheat and significant N recycling (root-shoot-phloem-root) and C movement. Aphids might serve as a substitute for isotopic analysis of phloem δ15 N. having the same value as their food source. Their excreta was 15 N-enriched relative to phloem.

Exercise and the oxidation and storage of glucose, maize-syrup solids and sucrose determined from breath 13CO2.

In order to determine which of maize syrup solids, glucose and sucrose were more readily oxidised during exercise and least readily oxidised afterwards, the rates of oxidation of three almost identical isoenergetic solutions of carbohydrates (330 ml of 18.5% w/v solutions of glucose, maize syrup solids and sucrose, 989-1050 kJ total energy) naturally enriched with 13C were examined at rest and during and after 1 h uphill walking at 75% maximum oxygen uptake (VO2max) in nine subjects [mean (SEM) VO2max, 45.4 (0.9) ml.kg-1.min-1]. Rates of production of expired 13CO2 were used to estimate rates of oxidation of each exogenous substrate. Energy expenditure and the contributions from total carbohydrate and fat oxidation were calculated from whole-body gas exchange. At rest, maize syrup solids were oxidised less than sucrose during the 1st h [glucose 2.7 (0.2) g.h-1, maize syrup solids 1.9 (0.3) g.h-1, sucrose 3.7 (0.2) g.h-1; maize syrup solids vs sucrose P < 0.01], but this difference disappeared after a further 3 h at rest [glucose 8.3 (0.5) g.h-1, maize syrup solids 7.7 (0.5) g.h-1, sucrose 8.1 (0.4) g.h-1]. During exercise, all the carbohydrates were oxidised to the same extent [glucose 23.0 (2.8) g.h-1, maize syrup solids 23.9 (3.4) g.h-1, sucrose 27.5 (2.6) g.h-1) but during 4 h of recovery after exercise, maize syrup solids were oxidised least [glucose 4.6 (0.1) g.h-1, maize syrup solids 3.7 (0.1) g.h-1, sucrose 6.4 (0.1) g.h-1; P < 0.05] suggesting that it may be stored to a greater extent. The results suggest that 18.5% glucose, maize syrup solids and sucrose solutions were equally well oxidised during exercise. During recovery from exercise maize syrup solids were oxidised less than glucose, which in turn was oxidised less than sucrose.

Energy expenditure in type 2 diabetic patients on metformin and sulphonylurea therapy.

Insulin and sulphonylurea therapies have both been reported to cause weight gain in Type 2 diabetic patients whereas metformin does not have this adverse effect. The mechanism for this difference is unclear. We have investigated in a cross-over study the effect of sulphonylurea and metformin therapy on energy expenditure and body composition in 10 Type 2 diabetic patients (7 females, 3 males) of various weights (mean body mass index 33.4 (SD 7.6 kg m-2)). Free living total energy expenditure was measured over 14 days by the doubly labelled water method adjusted for urinary glucose energy losses and resting energy expenditure by ventilated hood indirect calorimetry. Overall, total energy expenditure (12.88 +/- 4.17 vs 13.1 +/- 3.69 MJ 24 h-1) and resting metabolic rate (7.30 +/- 1.75 vs 7.23 +/- 1.74 MJ 24 h-1) were similar on metformin and sulphonylurea therapy, respectively. When adjusted for differences in fat free mass, resting metabolic rate on sulphonylurea therapy was slightly but significantly lower (mean difference -5.5 kJ 24 h-1 kg-1, 95% CI -1.2, -9.9 kJ 24 h-1 kg-1, p < 0.05). Fat free mass also increased significantly by 1.3 kg (95% CI 0.4, 2.4 kg, p < 0.05) when on sulphonylurea therapy, thus compensating for the lower resting metabolic rate per kg fat free mass to leave overall resting metabolic rate unchanged compared to metformin therapy. We also investigated the effect of adding metformin to six Type 2 diabetic patients already on insulin. This did not lead to any measurable changes in any of the components of energy expenditure.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-mediated changes in PD and glucose uptake after correction of acidosis in humans with CRF.

To test the hypothesis that acidosis contributes to the insulin resistance of chronic renal failure (CRF) and impairs the action of insulin to decrease protein degradation, eight CRF patients were studied using the combined L-[1-13C]leucine-euglycemic clamp technique before (acid) and after (NaHCO3) 4 wk treatment with NaHCO3 (pH: acid 7.29 +/- 0.01 vs. NaHCO3 7.36 +/- 0.01, P < 0.001). Protein degradation (PD) was estimated sequentially from the kinetics of a primed continuous infusion of L-[1-13C]leucine in the basal state and during a hyperinsulinemic euglycemic clamp. Insulin sensitivity was measured during the clamp. The correction of acidosis significantly increased the glucose infusion rate necessary to maintain euglycemia (acid 6.44 +/- 0.89 vs. bicarbonate 7.38 +/- 0.90 mg.kg-1.min-1, P < 0.01) and significantly decreased PD in the basal state (acid 126.4 +/- 8.1 vs. bicarbonate 100.1 +/- 6.9 mumol.kg-1.h-1, P < 0.001). Hyperinsulinemia decreased PD in both studies (acid basal 126.4 +/- 8.1 vs. clamp 96.5 +/- 7.7, P < 0.001; bicarbonate basal 100.1 +/- 6.9 vs. clamp 88.2 +/- 5.5 mumol.kg-1.h-1, P = 0.06), its effect being unaltered by acidosis, with a reduction of 24% before and 12% after the correction of acidosis. In conclusion, acidosis contributes to the insulin resistance of CRF but does not affect the action of insulin on PD.