The A0 blood group genotype modifies the jejunal glycomic binding pattern profile of piglets early associated with a simple or complex microbiota.

The intestinal epithelium glycocalyx sugar motif is an important determinant of the bacterial-host interaction and may be affected in pigs by gut microbiota and by blood group genotype. The aim was to study the effect of intestinal association with different microbiota and A0 blood group genotypes on the expressed glycomic pattern in the small intestine. Twelve caesarean-derived pigs previously associated with a simple association (SA) or complex association (CA) microbiota were selected at 26 to 37 d of age. In each subject, different jejunal loops were perfused for 8 h with enterotoxigenic K88 (ETEC), ETEC fimbriae (F4), (LAM), or a saline control. The piglets were genotyped for A0 blood group and the glycomic profile was evaluated by microscopic screening of lectin binding: peanut agglutinin (PNA), which is galactose specific; agglutinin I (UEA), which is fucose specific; lectin II (MALii), which is sialic acid specific; concavalin A, which is mannose specific; soybean agglutinin (SBA), which is -acetyl-galactosamine specific; and wheat germ agglutinin (WGA), which is -acetyl-glucosamine specific. A0 pigs had fewer UEA-positive cells, MALii-positive cells ( < 0.001), and SBA-positive cells ( < 0.10) than 00 pigs. Simple association pigs had more SBA positive cells ( < 0.01) than CA pigs. Enterotoxigenic K88-perfused intestinal loops had fewer UEA-positive cells ( < 0.01) and WGA positive cells ( < 0.001) cells and more PNA positive cells (only in SA pigs, < 0.01). No effects of introduction of F4 and LAM in the intestinal lumen were observed. The porcine A0 blood group genotype and the luminal presence of ETEC strongly affected the jejunal mucosa glycomic pattern profile whereas an early oral simple or complex microbial association had limited effects. Pig genetic background has relevance on the cross talk between intestinal epithelium glycocalyx sugar motif and ETEC and, ultimately, on the gut microbial colonization in later life.

Cholecystokinin regulates satiation independently of the abdominal vagal nerve in a pig model of total subdiaphragmatic vagotomy.

The vagal nerve and gut hormones CCK and GLP-1 play important roles in the control of food intake. However, it is not clear to what extent CCK and GLP-1 increase satiation by stimulating receptors located on abdominal vagal nerve endings or via receptors located elsewhere. This study aimed to further explore the relative contribution of the abdominal vagal nerve in mediating the satiating effects of endogenous CCK and GLP-1. Total subdiaphragmatic vagotomy or sham operation was combined with administration of CCK1 and GLP-1 receptor antagonists devazepide and exendin (9-39) in 12 pigs, applying an unbalanced Latin Square within-subject design. Furthermore, effects of vagotomy on preprandial and postprandial acetaminophen absorption, glucose, insulin, GLP-1 and CCK plasma concentrations were investigated. Ad libitum liquid meal intake (mean±SEM) was similar in sham and vagotomized pigs (4180±435 and 3760±810 g/meal). Intake increased by about 20% after blockade of CCK1 receptors, independently of the abdominal vagal nerve. Food intake did not increase after blockade of GLP-1 receptors. Blockade of CCK1 and GLP-1 receptors increased circulating CCK and GLP-1 concentrations in sham pigs only, suggesting the existence of a vagal reflex mechanism in the regulation of plasma CCK1 and GLP-1 concentrations. Vagotomy decreased acetaminophen absorption and changed glucose, insulin, CCK and GLP-1 concentrations indicating a delay in gastric emptying. Our data show that at liquid feeding, satiation is decreased effectively by pharmacological blockade of CCK1 receptors. We conclude that regulation of liquid meal intake appears to be primarily regulated by CCK1 receptors not located on abdominal vagal nerve endings.

Effects of resistant starch on behaviour, satiety-related hormones and metabolites in growing pigs.

Resistant starch (RS) has been suggested to prolong satiety in adult pigs. The present study investigated RS-induced changes in behaviour, satiety-related hormones and metabolites in catheterized growing pigs to explore possible underlying mechanisms for RS-induced satiety. In a cross-over design with two 14-day periods, 10 pigs (initial BW: 58 kg) were assigned to two treatments comprising diets containing either 35% pregelatinized starch (PS) or 34% retrograded starch (RS). Diets were isoenergetic on gross energy. Pigs were fed at 2.8× maintenance. Postprandial plasma response of satiety-related hormones and metabolites was measured at the end of each period using frequent blood sampling. Faecal and urinary energy losses were measured at the end of each period. Behaviour was scored 24 h from video recordings using scan sampling. Energy digestibility and metabolizability were ~6% lower in RS compared with PS diet (P<0.001), and metabolizable energy (ME) intake was ~3% lower in RS-fed than in PS-fed pigs (P<0.001). RS-fed pigs showed less feeder-directed (P=0.001) and drinking (P=0.10) behaviours than PS-fed pigs throughout the day. Postprandial peripheral short-chain fatty acid (SCFA) levels were higher in RS-fed than in PS-fed pigs (P<0.001). Postprandial glucose and insulin responses were lower in RS-fed than in PS-fed pigs (P<0.001). Triglyceride levels were higher in RS-fed than in PS-fed pigs (P<0.01), and non-esterified fatty acid levels did not differ between diets (P=0.90). Glucagon-like peptide-1 (GLP-1) levels were lower in RS-fed than in PS-fed pigs (P<0.001), and peptide tyrosine tyrosine (PYY) levels did not differ between diets (P=0.90). Blood serotonin levels were lower (P<0.001), whereas monoamine oxidase activity (P<0.05) and tryptophan (P<0.01) levels were higher in RS-fed than in PS-fed pigs. Despite a lower ME intake, RS seemed to prolong satiety, based on behavioural observations. Possible underlying mechanisms for RS-induced satiety include increased 24 h plasma SCFA levels, and decreased postprandial glucose and insulin responses. GLP-1 and PYY seemed not to play a role in RS-induced satiety. Low blood serotonin levels in RS-fed pigs suggested a difference in intestinal serotonin release between treatments. Increased postprandial plasma triglyceride levels corresponded with increased SCFA levels, but it is unclear whether triglycerides may have signalled satiety in RS-fed pigs.

Effects of surplus dietary L-tryptophan on stress, immunology, behavior, and nitrogen retention in endotoxemic pigs.

The possible beneficial effects of surplus dietary Trp (+5 g of Trp/kg of diet) on factors related to stress, immunology, behavior, and N retention were investigated in postweaning piglets (approximately 15 kg of BW) challenged for 10 d with intravenous bacterial lipopolysaccharide (from Escherichia coli). Two diets fed restrictively (732 kJ of NE/kg of BW(0.75)/d) were compared, 1) a basal diet (apparent ileal digestible Trp = 1.9 g/kg; the recommended amount of Trp to warrant near-optimal growth in nonendotoxemic piglets), and 2) a Trp-enriched basal diet (+5 g of free l-Trp/kg), with 8 individually housed piglets per diet. Pooled salivary cortisol, but not plasma cortisol sampled at euthanasia, showed a tendency (P = 0.07) toward reduced concentrations in the Trp group (1.1 vs. 1.4 ng/mL; pooled SE = 0.1 ng/mL). Plasma C-reactive protein was reduced (P = 0.04) in the Trp group (0.9 vs. 5.0 mg/L; pooled SE = 1.3 mg/L), but haptoglobin, IL-6, tumor necrosis factor α, and lipopolysaccharide-induced fever were similar between the 2 dietary treatments. Physical activity related to approaching a human showed a tendency (P = 0.08) toward increased latency time in the Trp group (101 vs. 60 s; pooled SE = 16 s), but the times spent standing, sitting, and lying were similar between dietary treatments. The ADFI, ADG (346 vs. 302 g/d; pooled SE = 14 g/d; P = 0.11), body N retention (11.6 vs. 11.0 g/d; pooled SE = 0.2 g/d; P = 0.18), and G:F (0.55 vs. 0.49; pooled SE = 0.03; P = 0.17) were not different between the groups fed Trp and the basal diet. In conclusion, surplus dietary Trp had limited effects on stress, immunology, behavior, and N retention in a pig model of systemic endotoxemia.

Effects of a simple or a complex starter microbiota on intestinal microbiota composition in caesarean derived piglets.

The present study was designed to develop a model in piglets that allows the investigation of the effects of postnatal association with a simple or a complex microbiota on gut health and development. Thirty piglets from 2 sows were obtained by caesarean delivery (day 0) and were equally divided over 2 treatment groups housed in separate clean, nonsterile rooms. All piglets received orally a simple microbiota consisting of Lactobacillus amylovorus, Clostridium glycolicum, and Parabacteroides spp. on days 1, 2, and 3 after birth. On day 3 and 4 the piglets received either a complex microbiota by providing them with a fecal inoculant of an adult sow [complex association (CA)] or a placebo inoculant [simple association (SA)]. Fecal microbiota composition, as determined by denaturing gradient gel electrophoresis and by pig intestinal tract chip (PITChip) analysis of 16S rRNA genes (days 3, 5, 7, 14, and 28), was less diverse in the SA group compared to the CA group. A difference in fecal microbiota composition between treatments persisted until the end of the study. It was concluded that the composition of microbiota in feces of cesarean delivery-derived piglets is influenced by bacterial association in the first days after birth. Differences in fecal microbiota composition between piglets exposed to a simple or complex inoculum at early age persisted for at least 3 wk.

Increasing weaning age of piglets from 4 to 7 weeks reduces stress, increases post-weaning feed intake but does not improve intestinal functionality.

This study tested the hypothesis that late weaning and the availability of creep feed during the suckling period compared with early weaning, improves feed intake, decreases stress and improves the integrity of the intestinal tract. In this study with 160 piglets of 16 litters, late weaning at 7 weeks of age was compared with early weaning at 4 weeks, with or without creep feeding during the suckling period, on post-weaning feed intake, plasma cortisol (as an indicator of stress) and plasma intestinal fatty acid binding protein (I-FABP; a marker for mild intestinal injury) concentrations, intestinal morphology, intestinal (macro)molecular permeability and intestinal fluid absorption as indicators of small intestinal integrity. Post-weaning feed intake was similar in piglets weaned at 4 weeks and offered creep feed or not, but higher (P < 0.001) in piglets weaned at 7 weeks with a higher (P < 0.05) intake for piglets offered creep feed compared with piglets from whom creep feed was witheld. Plasma cortisol response at the day of weaning was lower in piglets weaned at 7 weeks compared with piglets weaned at 4 weeks, and creep feed did not affect cortisol concentration. Plasma I-FABP concentration was not affected by the age of weaning and creep feeding. Intestinal (macro)molecular permeability was not affected by the age of weaning and creep feeding. Both in uninfected and enterotoxigenic Escherichia coli-infected small intestinal segments net fluid absorption was not affected by the age of weaning or creep feeding. Creep feeding, but not the age of weaning, resulted in higher villi and increased crypt depth. In conclusion, weaning at 7 weeks of age in combination with creep feeding improves post-weaning feed intake and reduces weaning stress but does not improve functional characteristics of the small intestinal mucosa.

Impact of bioactive substances on the gastrointestinal tract and performance of weaned piglets: a review.

The EU ban on in-feed antibiotics has stimulated research on weaning diets as a way of reducing post-weaning gut disorders and growth check in pigs. Many bioactive components have been investigated but only few have shown to be effective. Amongst these, organic acids (OA) have been shown to exert a bactericidal action mediated by non-dissociated OA, by lowering gastric pH, increasing gut and pancreas enzyme secretion and improving gut wall morphology. It has been postulated that they may also enhance non-specific immune responses and improve disease resistance. In contrast, relatively little attention has been paid to the impact of OA on the stomach but recent data show they can differently affect gastric histology, acid secretion and gastric emptying. Butyrate and precursors of butyric acid have received special attention and although promising results have been obtained, their effects are dependent upon the dose, treatment duration, initial age of piglets, gastrointestinal site and other factors. The amino acids (AA) like glutamine, tryptophan and arginine are supportive in improving digestion, absorption and retention of nutrients by affecting tissue anabolism, stress and (or) immunity. Glutamine, cysteine and threonine are important for maintaining mucin and permeability of intestinal barrier function. Spray-dried plasma (SDP) positively affects gut morphology, inflammation and reduces acquired specific immune responses via specific and a-specific influences of immunoglobulins and other bioactive components. Effects are more pronounced in early-weaned piglets and under poorer health conditions. Little interaction between plasma protein and antibiotics has been found, suggesting distinct modes of action and additive effects. Bovine colostrum may act more or less similarly to SDP. The composition of essential oils is highly variable, depending on environmental and climatic conditions and distillation methods. These oils differ widely in their antimicrobial activity in vitro and some components of weaning diets may decrease their activity. Results in young pigs are highly variable depending upon the product and doses used. These studies suggest that relatively high concentrations of essential oils are needed for beneficial effects to be observed and it has been assumed that these plant extracts mimic most of the effects of antibiotics active on gut physiology, microbiology and immunology. Often, bioactive substances protective to the gut also stimulate feed intake and growth performance. New insights on the effects of selected OA and AA, protein sources (especially SDP, bovine colostrum) and plant extracts with anti-bacterial activities on the gut are reported in this review.

Diurnal variation in insulin-stimulated systemic glucose and amino acid utilization in pigs fed with identical meals at 12-hour intervals.

The diurnal variation in insulin-stimulated systemic glucose and amino acid utilization was investigated in eleven pigs of approximately 40 kg. Pigs were fed isoenergetic/isoproteinic diets (366 kJ/kg BW (0.75) per meal) in two daily rations (06:00 and 18:00 h). After a 3-week habituation period, hyperinsulinemic euglycemic euaminoacidemic clamp studies (by intra-portal insulin, glucose and amino acids infusion and arterial blood sampling) were performed starting at 06:00 or 18:00 h (while skipping the meal), using a cross-over within-animal design. Basal (preclamp) plasma concentrations of insulin, glucose, lactate, individual amino acids and urea were similar in the morning compared to the evening. Insulin-stimulated ( approximately 4-fold increase over basal) systemic glucose utilization was similar (17.6+/-1.4 and 18.9+/-1.8 mg x kg (-1) x min (-1)) but amino acid utilization was 19% greater in the morning VS. the evening (2.37+/-0.21 VS. 1.99+/-0.15 mg x kg (-1) x min (-1), p<0.05), respectively. Insulin-stimulated plasma lactate concentrations remained constant in the morning (0.77+/-0.06 to 0.71+/-0.04 mmol x l (-1)) but declined in the evening (0.89+/-0.09 to 0.65+/-0.06 mmol x l (-1), p<0.05). By contrast, insulin-stimulated plasma urea concentrations declined in the morning (2.48+/-0.11 to 2.03+/-0.10 mmol x l (-1), p<0.005) but remained constant in the evening (2.18+/-0.14 to 2.12+/-0.12 mmol x l (-1)). In conclusion, pigs fed identical meals at 12-hour intervals follow a clear diurnal biorhythm in protein anabolism, with greater insulin-stimulated systemic amino acid utilization and lower plasma urea response in the morning compared to the evening.

Monitoring health by values of acute phase proteins.

A systemic acute phase reaction may develop during infection and inflammation, due to the action of peripherally liberated proinflammatory cytokines. Hepatic metabolism changes, and negative and positive acute phase proteins (APPs) can be measured in the blood: the APPs therefore represent appropriate analytes to assess health. While they are non-specific markers, their levels change with biological effects and this can be used to assess nutritional deficits and reactive processes, especially when positive and negative acute phase variables are combined in an index. Unfortunately, at present, no comprehensive, easy-to-use and cheap system is available to assess various acute phase proteins in serum or blood samples. Protein micro-array technology may satisfy this need; it will permit simultaneous analysis of numerous analytes in the same small volume sample and enable integration of information derived from systemic reactivity and nutrition with disease-specific variables. Applying such technology may help to address health problems in many countries.

Effects of supplemental L-tryptophan on serotonin, cortisol, intestinal integrity, and behavior in weanling piglets.

Stress occurs in intensive pig farming when piglets are weaned and mixed. In this study, we investigated whether this stress might be reduced with elevated dietary levels of Trp. The effects of supplemental dietary Trp (5 g/kg of feed, as-fed basis) were tested on the neuroendocrine system, intestinal integrity, behavior, and growth performance in nursery pigs, both before and after mixing. Mixing occurred 5 d after weaning and diet introduction. On d 4, 5, and 6, Trp-fed pigs vs. control pigs showed approximately a 2-fold elevation in plasma Trp concentrations (68 +/- 7 vs. 32 +/- 2 micromol/L; P < 0.001), a 38% increase in hypothalamic serotonin turnover as measured by 5-hydroxyindoleacetic acid:5-hydroxytryptamine (P < 0.001), and an 11 to 18% increase (P < 0.05) in the intestinal villus height:crypt depth. Before (d 4) and at (d 5) mixing, saliva but not plasma cortisol concentrations were reduced (P < 0.02) by approximately 2-fold in Trp-fed pigs vs. control pigs. Intestinal paracellular (horseradish peroxidase) and transcellular (fluorescein isothiocyanate) transport of macromolecules were not affected by dietary treatment, but mixing induced a 2-fold reduction (P < 0.05) in transcellular transport. Behavioral responses (lying and standing) at mixing were not affected by dietary treatment, except on d 10 after diet introduction when Trp supplementation induced more lying and less standing (P < 0.02). Average daily gain and ADFI were not different among dietary groups (P > 0.10). In conclusion, supplemental dietary Trp (5 g/kg) to piglets increased hypothalamic serotonergic activity, reduced the salivary cortisol response to mixing, improved intestinal morphology, and reduced physical activity 10 d after diet introduction. Consequently, diets containing high Trp levels improved neuroendocrine components of stress and increased gastrointestinal robustness but did not affect behavioral reactivity in nursery pigs during weaning and mixing.

Physiology, regulation and multifunctional activity of the gut wall: a rationale for multicompartmental modelling.

A rationale is given for a modelling approach to identify the mechanisms involved in the functioning and metabolic activity of tissues in the wall of the gastrointestinal tract. Maintenance and productive functions are discussed and related to the distinct compartments of the gastrointestinal tract and the metabolic costs involved. Functions identified are: tissue turnover; tissue proliferation; ion transport; nutrient transport; secretions of digestive enzymes, mucus and immunoglobulins; production of immune cells. The major nutrients involved include glucose, amino acids and volatile fatty acids. In vivo measurements of net portal fluxes of these nutrients in pigs and ruminants are evaluated to illustrate the complexity of physiology and metabolic activity of the gastrointestinal tract. Experimental evidence indicates that high, but variable and specific, nutrient costs are involved in the functioning of the gastrointestinal tract.

Acute phase reaction and acute phase proteins.

A review of the systemic acute phase reaction with major cytokines involved, and the hepatic metabolic changes, negative and positive acute phase proteins (APPs) with function and associated pathology is given. It appears that APPs represent appropriate analytes for assessment of animal health. Whereas they represent non-specific markers as biological effect reactants, they can be used for assessing nutritional deficits and reactive processes, especially when positive and negative acute phase variables are combined in an index. When such acute phase index is applied to separate healthy animals from animals with some disease, much better results are obtained than with single analytes and statistically acceptable results for culling individual animals may be reached. Unfortunately at present no cheap, comprehensive and easy to use system is available for assessing various acute phase proteins in serum or blood samples at the same time. Protein microarray or fluid phase microchip technology may satisfy this need; and permit simultaneous analysis of numerous analytes in the same small volume sample and enable integration of information derived from systemic reactivity and nutrition with disease specific variables. Applying such technology may help to solve health problems in various countries not only in animal husbandry but also in human populations.

Hyperlipidaemia is associated with increased insulin-mediated glucose metabolism, reduced fatty acid metabolism and normal blood pressure in transgenic mice overexpressing human apolipoprotein C1.

AIMS/HYPOTHESIS: Insulin resistance for glucose metabolism is associated with hyperlipidaemia and high blood pressure. In this study we investigated the effect of primary hyperlipidaemia on basal and insulin-mediated glucose and on non-esterified fatty acid (NEFA) metabolism and mean arterial pressure in hyperlipidaemic transgenic mice overexpressing apolipoprotein C1 (APOC1). Previous studies have shown that APOC1 transgenic mice develop hyperlipidaemia primarily because of an impaired hepatic uptake of very low density lipoprotein (VLDL). METHODS: Basal and hyperinsulinaemic (6 mU.kg-1.min-1), euglycaemic (7 mmol/l) clamps with 3(-)3H-glucose or 9,10(-)3H-palmitic acid infusions and in situ freeze clamped tissue collection were carried out. RESULTS: The APOC1 mice showed increased basal plasma cholesterol, triglyceride, NEFA and decreased glucose concentrations compared with wild-type mice (7.0 +/- 1.2 vs 1.6 +/- 0.1, 9.1 +/- 2.3 vs 0.6 +/- 0.1, 1.9 +/- 0.2 vs 0.9 +/- 0.1 and 7.0 +/- 1.0 vs 10.0 +/- 1.1 mmol/l, respectively, p < 0.05). Basal whole body glucose clearance was increased twofold in APOC1 mice compared with wild-type mice (18 +/- 2 vs 10 +/- 1 ml.kg-1.min-1, p < 0.05). Insulin-mediated whole body glucose uptake, glycolysis (generation of 3H2O) and glucose storage increased in APOC1 mice compared with wild-type mice (339 +/- 28 vs 200 +/- 11; 183 +/- 39 vs 128 +/- 17 and 156 +/- 44 vs 72 +/- 17 mumol.kg-1.min-1, p < 0.05, respectively), corresponding with a twofold to threefold increase in skeletal muscle glycogenesis and de novo lipogenesis from 3-(3)H-glucose in skeletal muscle and adipose tissue (p < 0.05). Basal whole body NEFA clearance was decreased threefold in APOC1 mice compared with wild-type mice (98 +/- 21 vs 314 +/- 88 ml.kg-1.min-1, p < 0.05). Insulin-mediated whole body NEFA uptake, NEFA oxidation (generation of 3H2O) and NEFA storage were lower in APOC1 mice than in wild-type mice (15 +/- 3 vs 33 +/- 6; 3 +/- 2 vs 11 +/- 4 and 12 +/- 2 vs 22 +/- 4 mumol.kg-1.min-1, p < 0.05) in the face of higher plasma NEFA concentrations (1.3 +/- 0.3 vs 0.5 +/- 0.1 mmol/l, p < 0.05), respectively. Mean arterial pressure and heart rate were similar in APOC1 vs wild-type mice (82 +/- 4 vs 85 +/- 3 mm Hg and 459 +/- 14 vs 484 +/- 11 beats.min-1). CONCLUSIONS/INTERPRETATION: 1) Hyperlipidaemic APOC1 mice show reduced NEFA and increased glucose metabolism under both basal and insulin-mediated conditions, suggesting an intrinsic defect in NEFA metabolism. Primary hyperlipidaemia alone in APOC1 mice does not lead to insulin resistance for glucose metabolism and high blood pressure.

Chronic physiologic hyperinsulinemia impairs suppression of plasma free fatty acids and increases de novo lipogenesis but does not cause dyslipidemia in conscious normal rats.

Type 2 diabetes mellitus and obesity are characterized by fasting hyperinsulinemia, insulin resistance with respect to glucose metabolism, elevated plasma free fatty acid (FFA) levels, hypertriglyceridemia, and decreased high-density lipoprotein (HDL) cholesterol. An association between hyperinsulinemia and dyslipidemia has been suggested, but the causality of the relationship remains uncertain. Therefore, we infused eight 12-week-old male catheterized conscious normal rats with insulin (1 mU/min) for 7 days while maintaining euglycemia using a modification of the glucose clamp technique. Control rats (n = 8) received vehicle infusion. Baseline FFAs were 1.07+/-0.13 mmol/L, decreased to 0.57+/-0.10 (P < .05) upon initiation of the insulin infusion, and gradually increased to 0.95+/-0.12 by day 7 (P = NS vbaseline). On day 7 after a 6-hour fast, plasma insulin, glucose, and FFA levels in control and chronically hyperinsulinemic rats were 32+/-5 versus 116+/-21 mU/L (P < .005), 122+/-4 versus 129+/-8 mg/dL (P = NS), and 1.13+/-0.18 versus 0.95+/-0.12 mmol/L (P = NS); total plasma triglyceride and cholesterol levels were 78+/-7 versus 66+/-9 mg/dL (P = NS) and 50+/-3 versus 47+/-2 mg/dL (P = NS), respectively. Very-low-density lipoprotein (VLDL) + intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and HDL2 and HDL3 subfractions of plasma triglyceride and cholesterol were similar in control and hyperinsulinemic rats. Plasma FFA correlated positively with total (r = .61, P < .005) triglycerides. On day 7 after an 8-hour fast, hyperinsulinemic-euglycemic clamps with 3-3H-glucose infusion were performed in all rats. Chronically hyperinsulinemic rats showed peripheral insulin resistance (glucose uptake, 15.8+/-0.8 v 19.3+/-1.4 mg/kg x min, P < .02) but normal suppression of hepatic glucose production (HGP) compared with control rats (4.3+/-1.0 v 5.6+/-1.4 mg/kg x min, P = NS). De novo tissue lipogenesis (3-3H-glucose incorporation into lipids) was increased in chronically hyperinsulinemic versus control rats (0.90+/-0.10 v 0.44+/-0.08 mg/kg x min, P < .005). In conclusion, chronic physiologic hyperinsulinemia (1) causes insulin resistance with regard to the suppression of plasma FFA levels and increases lipogenesis; (2) induces peripheral but not hepatic insulin resistance with respect to glucose metabolism; and (3) does not cause an elevation in VLDL-triglyceride or a reduction in HDL-cholesterol.

Neonatal de-afferentation of capsaicin-sensitive sensory nerves increases in vivo insulin sensitivity in conscious adult rats.

Sensory neuropeptides, released from the peripheral nervous system, might modulate glucose homeostasis by antagonizing insulin action. The effects of de-afferentation of functional small diameter unmyelinated C-fibres (sensory nerves) on in vivo insulin-mediated intracellular glucose metabolism were investigated by using euglycaemic insulin (6 and 18 mU/kg x min) clamps with [3-(3)H]-glucose infusion in 24 adult rats, treated neonatally with either capsaicin (CAP) (50 mg/kg) or vehicle (CON). Following the clamp, skeletal muscle groups, liver and adipose tissue were freeze-clamped. At plasma insulin levels of approximately 90 mU/l, CAP-rats showed a 21% increase in whole body glucose uptake compared with CON (24.4 +/- 1.6 vs 20.1 +/- 0.8 mg/kg min, p < 0.02), which was paralleled by a 20% increase in whole body glycolysis (12.6 +/- 0.8 vs 10.5 +/- 0.5 mg/ kg.min p < 0.05) (concentration of 3H2O in plasma). Whole body skeletal muscle glycogenesis was increased by 80% in CAP-rats (5.7 +/- 0.7 vs 3.1 +/- 0.7 mg/kg x min, p < 0.05) with increased muscle glycogen synthase activity. Whole body (muscle, liver and adipose tissue combined) de novo lipogenesis also was increased in CAP-rats compared with CON (0.69 +/- 0.10 vs 0.44 +/- 0.06 mg/kg x min, p < 0.05) (incorporation of [3-(3)H]-glucose counts into glycogen or fat). Hepatic glucose production was lower in CAP-rats compared with CON (0.6 +/- 0.6 vs 2.1 +/- 0.7 mg/kg x min, p < 0.05). Plasma glucagon, corticosterone, epinephrine and norepinephrine levels were reduced in CAP-rats: 43 +/- 2 compared with 70 +/- 6 pg/ml, 855 +/- 55 compared with 1131 +/- 138 nmol/l, 513 +/- 136 compared with 1048 +/- 164 pmol/l and 928 +/- 142 compared with 1472 +/- 331 pmol/l, respectively, p < 0.05. At plasma insulin levels of approximately 400 mU/l, CAP-rats showed no differences in peripheral and hepatic insulin action compared with CON. We conclude that the removal of endogenous sensory neuropeptides, by de-afferentation of capsaicin-sensitive sensory nerves, increases in vivo insulin sensitivity, but not responsiveness: 1) primarily through an increased sensitivity of skeletal muscle glycogen synthesis to insulin; 2) through a reduction in the levels of counter-regulatory hormones, thereby creating a milieu which favours overall in vivo insulin sensitivity with respect to glucose uptake, glucose production, glycolysis, glycogenesis and lipogenesis.

Effect of hyperinsulinemia on plasma leptin concentrations and food intake in rats.

We investigated the dose- and time-dependent effect of insulin infusion on peripheral and portal plasma leptin concentrations in normal rats. Three groups were studied: group I: euglycemic (6 mmol/l) insulin (6 mU . kg-1 . min-1) clamps for 0, 2, 4, 12, and 24 h; group II: euglycemic insulin (18 mU . kg-1 . min-1) clamp for 2 h; and group III: euglycemic insulin (3 mU . kg-1 . min-1) clamp for 7 days. In group III, food intake was quantified during days 1-7. After an overnight fast, peripheral and portal plasma leptin levels were identical (1.5 +/- 0.2 and 1.6 +/- 0.2 ng/ml). Insulin infusion (6 mU . kg-1 . min-1) for 2 h had no effect on plasma leptin levels (1.5 +/- 0.2 ng/ml). After 4 h (2.0 +/- 0.2 ng/ml), 12 h (2.2 +/- 0. 4 ng/ml), and 24 h (2.7 +/- 0.6 ng/ml; all P < 0.05) of insulin infusion, a progressive time-related increase in plasma leptin concentration was observed; portal vein leptin levels rose in parallel and were similar to peripheral levels. When insulin (18 mU . kg-1 . min-1) was infused for 2 h, plasma leptin levels increased to 3.0 +/- 0.3 ng/ml (P < 0.01). Seven days of constant insulin infusion (3 mU . kg-1 . min-1) resulted in a progressive increase in fasting plasma leptin and a parallel decrease in food intake. A mean increase in plasma leptin concentration of 1 ng/ml during the 7-day insulin infusion period was associated with a mean decrease in food intake of 2.5 g/day (multivariate ANOVA, P < 0.05). We conclude that the insulin-induced rise in peripheral and portal vein leptin levels is similar and both dose and time dependent. The inverse relationship between plasma leptin concentration and food intake during prolonged hyperinsulinemia, but not during short-term hyperinsulinemia, supports the role of leptin in long-term food consumption.

Time course of insulin action on tissue-specific intracellular glucose metabolism in normal rats.

We investigated the time course of insulin action in conscious rats exposed to constant physiological hyperinsulinemia (approximately 100 mU/l) while maintaining euglycemia (approximately 100 mg/dl) for 0, 0.5, 2, 4, 8, or 12 h. [3-3H]glucose was infused to quantitate whole body glucose disposal (rate of disappearance, Rd), glycolysis (generation of 3H2O in plasma), hepatic glucoses production (HGP), and skeletal muscle and liver glycogen synthesis ([3-3H]glucose incorporation into glycogen and time-dependent change in tissue glycogen concentration). The basal Rd, which equals HGP, was 6.0 +/- 0.3 mg.kg-1.min-1. With increased duration of hyperinsulinemia from 0 to 0.5 to 2 to 4 h, Rd increased from 6.0 +/- 0.3 to 21.0 +/- 1.1 to 24.1 +/- 1.5 to 26.6 +/- 0.6 mg.kg-1.min-1 (P < 0.05 for 2 and 4 h vs. 0.5 h). During the first 2 h the increase in Rd was explained by parallel increases in glycolysis and glycogen synthesis. From 2 to 4 h the further increase in Rd was entirely due to an increase in glycolysis without change in glycogen synthesis. From 4 to 8 to 12 h of hyperinsulinemia, Rd decreased by 19% from 26.6 +/- 0.6 to 24.1 +/- 1.1 to 21.6 +/- 1.8 mg.kg-1.min-1 (P < 0.05 for 8 h vs. 4 h and 12 h vs. 8 h). The progressive decline in Rd, in the face of constant hyperinsulinemia, occurred despite a slight increase (8-14%) in glycolysis and was completely explained by a marked decrease (64%) in muscle glycogen synthesis. In contrast, liver glycogen synthesis increased fourfold, indicating an independent regulation of muscle and liver glycogen synthesis by long-term hyperinsulinemia. In the liver, during the entire 12-h period of insulin stimulation, the contribution of the direct (from glucose) and the indirect (from C-3 fragments) pathways to net glycogen formation remained constant at 77 +/- 5 and 23 +/- 5%, respectively. HGP remained suppressed throughout the 12-h period of hyperinsulinemia.

Seven days of euglycemic hyperinsulinemia induces insulin resistance for glucose metabolism but not hypertension, elevated catecholamine levels, or increased sodium retention in conscious normal rats.

Epidemiological studies have suggested an association among chronic hyperinsulinemia, insulin resistance, and hypertension. However, the causality of this relationship remains uncertain. In this study, chronically catheterized conscious rats were made hyperinsulinemic for 7 days (approximately 90 mU/l, i.e., threefold over basal), while strict euglycemia was maintained (approximately 130 mg/dl, coefficient of variation < 10%) by using a modification of the insulin/glucose clamp technique. Control rats received vehicle infusion. Baseline mean arterial pressure and heart rate were 125 +/- 5 mmHg and 427 +/- 12 beats/min and remained unchanged during the 7-day infusion of insulin (127 +/- 7 mmHg; 401 +/- 12 beats/min) or vehicle (133 +/- 4 mmHg; 411 +/- 10 beats/min). Baseline plasma epinephrine (88 +/- 15 pg/ml), norepinephrine (205 +/- 31 pg/ml), and sodium balance (0.34 +/- 0.09 mmol) remained constant during the 7-day insulin or vehicle infusion. After 7 days of insulin or vehicle infusion, in vivo insulin action was determined in all rats using a 2-h hyperinsulinemic (1 mU/min) euglycemic clamp with [3-3H]glucose infusion to quantitate whole-body glucose uptake, glycolysis, glucose storage (total glucose uptake minus glycolysis), and hepatic glucose production. Compared with vehicle-treated rats, 7 days of sustained hyperinsulinemia resulted in a reduction (P < 0.01) in insulin-mediated glucose uptake, glucose storage, and glycolysis by 39, 62, and 26%, respectively. Hepatic glucose production was normally suppressed after 7 days of hyperinsulinemia. Neither insulin-stimulated glucose uptake nor glucose storage correlated with blood pressure or heart rate. In conclusion, 7 days of euglycemic hyperinsulinemia induces severe insulin resistance with respect to whole-body glucose metabolism but does not increase blood pressure, catecholamine levels, or sodium retention. This indicates that hyperinsulinemia-induced insulin resistance is not associated with the development of hypertension in rats who do not have a genetic predisposition for hypertension. Because hyperinsulinemia was initiated in normal rats under euglycemic conditions, additional (inherited or acquired) factors may be necessary to observe an effect of hyperinsulinemia and/or insulin resistance to increase blood pressure.

Chronic hyperinsulinemia inhibits platelet-activating factor (PAF) biosynthesis in the rat kidney.

A number of risk factors for cardiovascular disease, including hypertension, are associated with the insulin resistance syndrome. The hallmark of this syndrome is an impairment in insulin action which provokes a compensatory increase in pancreatic beta-cell insulin secretion leading to chronic hyperinsulinemia. Indirect studies show that platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine, PAF), a potent antihypertensive lipid produced by the kidney, may be decreased by hyperinsulinemia. The present study was designed to evaluate the effect of chronic hyperinsulinemia on renal PAF metabolism, arterial blood pressure and whole body insulin sensitivity. Chronic catheterized, unstressed rats were infused with saline or insulin plus glucose to create a chronic condition of sustained euglycemic (approximately 130 mg/dl) hyperinsulinemia (approximately 90 mU 1. or 3-fold over basal levels). PAF is a metabolically unstable compound being susceptible to rapid degradation to the biologically inactive lyso-PAF, a metabolite which also serves as a precursor for PAF synthesis. PAF synthesis and counter-regulatory prostaglandins may be derived from the same arachidonate precursor. The enzymes which catalyze these reactions were measured in plasma and in the subcellular fractions of the kidneys. Compared to saline-treated rats, sustained physiologic hyperinsulinemia for 7 days: (i) decreased insulin-mediated glucose disposal by 30%; (ii) caused an increased plasma PAF:acetylhydrolase, which degrades PAF to lyso-PAF, without any change in cytosolic PAF:acetylhydrolase activity; and (iii) completely inhibited microsomal lyso-PAF:acetyl CoA acetyltransferase activity which catalyzes the conversion of lyso-PAF back to bioactive PAF. The increased catabolism of PAF in plasma, combined with decreased renal PAF biosynthesis, would be expected to decrease circulating PAF levels leading to a rise in blood pressure. However, blood pressure remained unchanged. The sustained hyperinsulinemia stimulated plasma membrane CoA-independent transacylase activity, which is responsible for the mobilization of arachidonates into lyso-PAF, to form l-alkylarchidonoyl-glycerophosphocholine. The latter is the stored precursor for the synthesis of PAF and vasodilatory prostaglandins, which may have offset the effect of decreased PAF. We hypothesize that hyperinsulinemia may alter the blood pressure only if the balance between the synthesis/catabolism of PAF and vasodilatory prostaglandins is disrupted.

Oxidant-mediated repression of mitochondrial transcription in diabetic rats.

Diabetes-associated mitochondrial dysfunction is recognized, but the underlying mechanisms are unknown. Using isolated liver mitochondria from streptozotocin-induced diabetic Sprague-Dawley rats, we showed that diabetes can result in a > 95% loss in mitochondrial transcriptional capacity. Decreased transcription correlated well with both disease status, as indicated by serum lipemia and ketone levels, and with increased resistance of the mitochondrial transcription system to oxidative stress imposed by the hydrophilic AAPH [2,2'-azobis-(2-amidino-propane) hydrochloride] or the hydrophobic AMVN [2,2'-azobis-(2,4,-dimethyl-valeronitrile)]. The onset of AAPH- or AMVN-induced lipid peroxidation was also delayed; this suggests that liver mitochondrial membranes from diabetics have increased resistance to peroxyl radical-mediated lipid peroxidation. Lipid peroxidation induced endogenously was increased, however, suggesting a state of increased oxidative stress likely exists in vivo. Furthermore, changes in the rate of lipid peroxidation occurring during the propagation phase were also affected by diabetes. This implies possible changes in lipid composition or structure. Analysis indicated that the factors protecting mitochondria from lipid peroxidation differ from those involved in protecting the transcription system, and that both are independent of free radical scavenger levels. These results suggested that diabetes alters mitochondrial exposure and/or response to reactive species and provided clues to the role of oxidant stress in the development of diabetes-associated mitochondrial dysfunction.