Effect of supplemental calcium propionate on insulin action to blood glucose metabolism in adult sheep

An experiment combining a hyperinsulinemic euglycemic clamp procedure of four sequential 2-h periods and an isotope dilution method of [U-13C]glucose determined the effect of supplemental calcium propionate on blood glucose metabolism during insulin and glucose infusions in adult sheep. They were fed lucerne hay cubes and commercial concentrate with and without supplementary calcium propionate (Prop and Cont diets, respectively) in a crossover design for each 21-day period. At the preinfusion period, blood glucose turnover rate (GTR) was greater (P < 0.05) for the Prop diet than for the Cont diet. Blood GTR, endogenous glucose production rate (EGPR) and the ratio of EGPR to blood GTR were greater (P < 0.01, P < 0.05 and P < 0.05, respectively) for the Prop diet than for the Cont diet. Blood GTR and glucose infusion rate (GIR) increased (P < 0.001) and the ratio of EGPR to blood GTR was reduced (P < 0.01) with increased insulin infusion rates. The maximal GIR tended to be (P < 0.10) greater for the Prop diet than for the Cont diet but plasma insulin concentration at half maximal GIR did not differ between diets. It is suggested that in adult sheep, dietary propionate supplementation enhances insulin action on glucose metabolism, however, changes in measures of tissue responsiveness and sensitivity were not significant.     

Contribution of glucose and gluconeogenic substrates to insulin-stimulated glycogen synthesis in cultured fetal hepatocytes

The influence of medium composition on basal and insulin-stimulated glycogenesis was studied in cultured 17-day-old rat fetal hepatocytes, which contain no glycogen at the time of transplantation. Continuous-labeling ¹⁴C-glucose experiments were used to determine both glycogen content and glycogen labeling. The specific activity of glucose units in the newly formed glycogen (a) was compared to that of the medium glucose (b): the ratio a/b expresses the contribution of medium glucose to glycogen formation. In standard medium (5.5 mM glucose), this ratio averaged 0.60. Variations of glucose concentration in the medium from 1 to 40 mM were accompanied by a progressive increase in both glycogen content and the ratio a/b (up to 0.80). Supplementation of standard medium with fructose, galactose, glycerol, or lactate-pyruvate decreased the hepatocyte glucose uptake from the medium. Galactose (1 to 5 mM) or lactate-pyruvate (5 mM) enhanced the glycogen content whereas glycerol or fructose (1 to 5 mM) had no effect. The ratio a/b, not modified by glycerol or lactate-pyruvate, was decreased to 0.45 by fructose (5 mM). Galactose at concentrations as low as 1 to 2 mM brought the ratio down to 0.30, indicating that it is a superior precursor of glycogen as compared to glucose. When the hepatocytes were grown in the presence of 10 nM insulin, the glycogen content was constantly higher than in the absence of the hormone (2-fold stimulation). Also the amplitude of the glycogenic effect of insulin was similar whatever the modifications of the medium, whereas ratio a/b and glucose uptake were hardly increased by insulin. Thus several substrates can contribute to glycogen formation (especially galactose) in cultured fetal hepatocytes and the essential effect of insulin is a stimulation of the final step of the glycogenosynthetic pathway.     

Synthesis of phosphoenolpyruvate from propionate in sheep liver

1. Utilization of propionate by sheep liver mitochondria was stimulated equally by pyruvate or alpha-oxoglutarate, with formation predominantly of malate. Pyruvate increased conversion of propionate carbon into citrate, whereas alpha-oxoglutarate increased formation of phosphoenolpyruvate. The fraction of metabolized propionate converted into phosphoenolpyruvate was about 17% in the presence or absence of alpha-oxoglutarate and about 7% in the presence of pyruvate. Pyruvate consumption was inhibited by 80% by 5mm-propionate. 2. Compared with rat liver, sheep liver was characterized by very high activities of phosphoenolpyruvate carboxykinase and moderately high activities of aconitase in the mitochondria and by low activities of ;malic' enzyme, pyruvate kinase and lactate dehydrogenase in the cytosol. Activities of phosphoenolpyruvate carboxy-kinase were similar in liver cytosol from rats and sheep. Activities of malate dehydrogenase and NADP-linked isocitrate dehydrogenase in sheep liver were about half those in rat liver. 3. The phosphate-dicarboxylate antiport was active in sheep liver mitochondria, but compared with rat liver mitochondria the citrate-malate antiport showed only low activity and mitochondrial aconitase was relatively inaccessible to external citrate. The rate of swelling of mitochondria induced by phosphate in solutions of ammonium malate was inversely related to the concentration of malate. 4. The results are discussed in relation to gluconeogenesis from propionate in sheep liver. It is proposed that propionate is converted into malate by the mitochondria and the malate is converted into phosphoenolpyruvate by enzymes in the cytosol. In this way sufficient NADH would be generated in the cytosol to convert the phosphoenolpyruvate into glucose.     

Metabolism of propionate by sheep liver. Oxidation of propionate by homogenates

1. The rate and stability to aging of the metabolism of propionate by sheep-liver slices and sucrose homogenates were examined. Aging for up to 20min. at 37° in the absence of added substrate had little effect with slices, whole homogenates or homogenates without the nuclear fraction. 2. Metabolism of propionate by sucrose homogenates was confined to the mitochondrial fraction, but the mitochondrial supernatant (microsomes plus cell sap) stimulated propionate removal. 3. The rate of propionate metabolism by liver slices was higher in a high potassium phosphate–bicarbonate medium [0·88(±s.e.m. 0·16)μmole/mg. of N/hr.] than in Krebs–Ringer bicarbonate medium [0·44(±s.e.m. 0·13)μmole/mg. of N/hr.]. 4. Metabolism of propionate by sucrose homogenates freed from nuclei was dependent on the presence of oxygen, carbon dioxide and ATP. Propionate removal was stimulated 250% by Mg²⁺ ions and 670% by cytochrome c. 5. In the complete medium 2·39(±s.e.m. 0·15)μmoles of propionate were consumed/mg. of N/hr. 6. The ratio of oxygen consumption to propionate utilization was sufficient to account for the complete oxidation of half the propionate consumed. 7. The only products detected under these conditions were succinate, fumarate and malate. Propionate had no effect on the production of lactate from endogenous sources and did not itself give rise to lactate. 8. Methylmalonate did not accumulate when propionate was metabolized and was not oxidized. It was detected as an intermediate in the conversion of propionyl-CoA into succinate. The rate of this reaction sequence was adequate to account for the rate of propionate metabolism by sucrose homogenates or slices, provided that the rate of formation of propionyl-CoA was not limiting. 9. The methylmalonate pathway was predominantly a mitochondrial function. 10. The metabolism of propionate appeared to be dependent on active oxidative phosphorylation.     

Metabolism of propionate by sheep liver. Interrelations of propionate and glutamate in aged mitochondria

1. The rate of metabolism of propionate by aged sheep-liver mitochondria in the presence of oxygen + carbon dioxide (95:5) was 5.0 (+/- s.e.m. 0.8) mumoles/mg. of mitochondrial N/hr. 2. When aged in the presence of the mitochondrial supernatant the rate was increased. Mitochondria from 0.33g. of liver, when combined with the corresponding mitochondrial supernatant from 0.08g. of liver, metabolized propionate at a rate of 11.4 (+/- s.e.m. 1.2) mumoles/mg. of mitochondrial N/hr. This rate is comparable with rates previously obtained with aged nuclear-free homogenates. 3. Two factors in the mitochondrial supernatant were detected, which when combined reproduced the effect of the fresh supernatant and prevented loss of activity on aging. One of these was non-diffusible and was recovered by fractionation of the dialysed mitochondrial supernatant with ammonium sulphate. The second factor was present in an ultrafiltrate of fresh mitochondrial supernatant and in boiled mitochondrial supernatant; it was isolated and identified as l(+)-glutamate. 4. The effect of the non-diffusible factor was due to protection of the mitochondria from the aging process, whereas glutamate served both in this capacity and as a direct stimulant of propionate metabolism at low concentration.     

Regulation of glycolysis and fatty acid synthesis from glucose in sheep adipose tissue

1. The following were measured in adipose-tissue pieces, obtained from 7–9 month-old sheep, before or after the tissue pieces had been maintained in tissue culture for 24 h: the rates of synthesis from glucose of fatty acids, acylglycerol glycerol, pyruvate and lactate; the rate of glucose oxidation to CO₂; the rate of glucose oxidation via the pentose phosphate pathway; the activities of hexokinase, glucose 6-phosphate dehydrogenase, phosphofructokinase, pyruvate kinase, pyruvate dehydrogenase and ATP citrate lyase; the intra- and extra-cellular water content; the concentration of various metabolites and ATP, ADP and AMP. 2. The proportion of glucose carbon converted into the various products in sheep adipose tissue differs markedly from that observed in rat adipose tissue. 3. There was a general increase in the rate of glucose utilization by the adipose-tissue pieces after maintenance in tissue culture; largest changes were seen in the rates of glycolysis and fatty acid synthesis from glucose. These increases are paralleled by an increase in pyruvate kinase activity. There was no change in the activities of the other enzymes as measured, although the net flux through all the enzymes increased. 4. Incubation of fresh adipose-tissue pieces for 2–6h led to an increase in the affinity of pyruvate kinase for phosphoenolpyruvate. 5. The rate of pyruvate production by glycolysis was greater than the activity of pyruvate dehydrogenase of the tissue. 6. The results suggest that both pyruvate kinase and pyruvate dehydrogenase have important roles in restricting the utilization of glucose carbon for fatty acid synthesis in sheep adipose tissue.     

Metabolism of propionate by sheep liver: Pathway of propionate metabolism in aged homogenate and mitochondria

Experiments were conducted with aged nuclear-free homogenate of sheep liver and aged mitochondria in an attempt to measure both the extent of oxidation of propionate and the distribution of label from [2-¹⁴C]propionate in the products. With nuclear-free homogenate, propionate was 44% oxidized with the accumulation of succinate, fumarate, malate and some citrate. Recovery of ¹⁴C in these intermediates and respiratory carbon dioxide was only 33%, but additional label was detected in endogenous glutamate and aspartate. With washed mitochondria 30% oxidation of metabolized propionate occurred, and proportionately more citrate and malate accumulated. Recovery of ¹⁴C in dicarboxylic acids, citrate, α-oxoglutarate, glutamate, aspartate and respiratory carbon dioxide was 91%. The specific activities of the products and the distribution of label in the carbon atoms of the dicarboxylic acids were consistent with the operation solely of the methylmalonate pathway together with limited oxidation of the succinate formed by the tricarboxylic acid cycle via pyruvate. In a final experiment with mitochondria the label consumed from [2-¹⁴C]propionate was entirely recovered in the intermediates of the tricarboxylic acid cycle, glutamate, aspartate, methylmalonate and respiratory carbon dioxide.     

Kinetics of glucose metabolism in sheep

The kinetics of glucose cycling in 24 ewes bearing twins were studied 1 month before term by bolus injections of [6-3H]- and [U-14C]glucose. The function representing glucose carbon recycling was determined by deconvolution of the [3H]glucose from the [14C]glucose decay curves in plasma by using the SAAM and CONSAM programs, and a model for kinetics of glucose cycling was developed. The [3H]glucose data were fitted by four compartments, and an additional three compartments were required to explain recycling. The results show that labelled carbon was still recycling to plasma 2 days after the injection of tracer. By contrast, a similar analysis on a non-pregnant sheep, with data taken from the literature, showed that no more material was recycled after 1 day. It appears that a larger fraction (20 v. 5%) of the carbon 6 of glucose recycles in pregnant than in non-pregnant sheep. This presumably reflects the metabolism by the feto-placental unit and the increased rate of glucose metabolism during pregnancy.     

Interactions of acetate, propionate and butyrate in sheep liver mitochondria

1. Interactions in the rates of consumption of acetate, propionate and butyrate in sheep liver mitochondria were examined in the presence and absence of l-malate and alpha-oxoglutarate. 2. Acetate was not consumed in absence of ancillary substrate but utilization of acetate (7.2nmol/min per mg of protein) occurred in the presence of alpha-oxoglutarate. This consumption was abolished by propionate or butyrate but the presence of acetate did not affect consumption of propionate or butyrate. 3. Propionate consumption (10.1nmol/min per mg of protein) was unaffected by malate but was stimulated by 63% by butyrate or by 180% by alpha-oxoglutarate. 4. Butyrate consumption (3.3nmol/min per mg of protein) was stimulated by 117% by malate, by 151% by propionate and by 310% by alpha-oxoglutarate. 5. In the absence of ancillary substrates the maximum rate of total volatile fatty acid utilization (24.7nmol/min per mg of protein) occurred with a mixture of propionate and butyrate. When both propionate and butyrate were present total consumption was not affected by malate but was stimulated by 24% by alpha-oxoglutarate. With alpha-oxoglutarate present, propionate and butyrate each decreased the other's consumption by about 26%, but the total utilization was the greatest observed. 6. The inhibition of acetate consumption by propionate or butyrate is unexplained, but the remaining effects are consistent with an interaction of propionate and butyrate through oxaloacetate together with a general limitation imposed by a need for GTP to rephosphorylate AMP formed during activation of the volatile fatty acids.     

Contribution of branched-chain amino acids to uteroplacental ammonia production in sheep.

The uteroplacental tissues are a principal site of ammonia production for the conceptus. The goal of this study was to examine the effect of the composition of maternal amino acid (AA) infusate on uteroplacental ammonia production. Seven pregnant ewes (126 +/- 1. 4 days gestation) were infused through the maternal femoral vein (duration 3.5 h, rate 240 ml per hour) with three solutions of AAs. The first infusate was comparable to commercial parenteral nutrition preparations, the second infusate contained the same solution without branched-chain AAs (BCAAs), and the third infusate contained only BCAAs. Blood samples were simultaneously collected from the maternal artery, uterine vein, fetal artery, and umbilical vein to determine plasma AA concentrations and whole blood ammonia concentrations, before (control) and 2 h after (experimental) the start of infusion. Uterine and umbilical blood flows were measured using the ethanol steady-state diffusion method. Results showed that fetal arterial and venous ammonia concentrations increased significantly after infusions with all AAs or only BCAAs, but not without BCAAs. Uteroplacental ammonia production increased in response to each of the three infusates. However, this increase was much greater when the BCAAs were present in infusates. We conclude that there is a significant contribution of BCAAs to the uteroplacental ammonia production. Maternal AA infusions containing BCAAs can result in increased fetal blood ammonia concentrations.     

Contribution of the heme propionate groups to the electron transfer and electrostatic properties of myoglobin

The role of the heme propionate groups in determining the electron transfer and electrostatic properties of myoglobin have been studied by thermodynamic, kinetic, and spectroscopic studies of horse heart myoglobin in which the heme propionate groups are esterified. Spectroelectrochemical analysis has established that the E_m,7 of dimethylester heme-substituted Mb (DME-Mb) (E_m,7 = 100.2(2) mV vs. NHE (Normal Hydrogen Electrode) (25 °C) is increased  40 mV relative to that of the native protein with ΔH° = −12.9(2) kcal/mol and ΔS° = −51.0(8) cal/mol/deg (pH 7.0, μ = 0.1 M (phosphate)). The second order rate constant for reduction of DME-metMb by Fe(EDTA)²⁻ is increased  > 400-fold relative to that for reduction of native metMb to a value of 1.34(2) × 10³ M⁻¹ s⁻¹ with ΔS^‡ = −13(1) cal/mol/deg and ΔH^‡ = 9.2(3) (pH 7.0, μ = 0.1 M (phosphate)). Analysis of the pH dependences of the reduction potential and rate constant for reduction by Fe(EDTA)²⁻ demonstrates that heme propionate esterification introduces significant changes into the electrostatic interactions in myoglobin. These changes are also manifested by differences in the pH dependences of the ¹H NMR spectra of native and DME-metMb that reveal shifts in pK_a values for specific His residues as the result of heme propionate esterification. In sum, the current results establish that heme propionate esterification not only affects the electron transfer properties of myoglobin but also influences the titration behavior of specific His residues.     

Contribution to glucose tolerance of insulin-independent vs. insulin-dependent mechanisms in mice

To study the contributions of insulin-dependent vs. insulin-independent mechanisms to intravenous glucose tolerance (K(G)), 475 experiments in mice were performed. An intravenous glucose bolus was given either alone or with exogenous insulin or with substances modulating insulin secretion and sensitivity. Seven samples were taken over 50 min. Insulin [suprabasal area under the curve (DeltaAUC(ins))] ranged from 0 to 100 mU. ml(-1). 50 min. After validation against the euglycemic hyperinsulinemic clamp, the minimal model of net glucose disappearance was exploited to analyze glucose and insulin concentrations to measure the action of glucose per se independent of dynamic insulin (S(G)) and the combined effect of insulin sensitivity (S(I)) and secretion. Sensitivity analysis showed that insulin [through disposition index (DI)] contributed to glucose tolerance by 29 +/- 4% in normal conditions. In conditions of elevated hyperinsulinemia, contribution by insulin increased on average to 69%. K(G) correlated with DI but was saturated for DeltaAUC(ins) above 15 mU. ml(-1). 50 min. Insulin sensitivity related to DeltaAUC(ins) in a hyperbolic manner, whereas S(G) did not correlate with the insulin peak in the physiological range. Thus glucose tolerance in vivo is largely mediated by mechanisms unrelated to dynamic insulin and saturates with high insulin.     

Effect of propionate on the utilization of nitrogen from 15NH4Cl for urea synthesis in hepatocytes isolated from sheep liver

1. The effect of ornithine (2.0 mM) and propionate (5.0 mM) on the utilization of N from 15NH4Cl (5.0 mM) for urea synthesis in hepatocytes isolated from sheep liver was investigated. 2. The capacity of sheep hepatocytes to utilize [15N]ammonia in the absence of the other exogenous substrates was very low and amounted 132 +/- 37.3 mumol/hr per 1 g dry wt. 3. Ornithine failed to affect the total [15N]ammonia uptake and total urea synthesis, but at the same time it markedly increased the utilization of [15N]ammonia for ureagenesis and diminished the rate of urea synthesis from endogenous sources. 4. Propionate markedly increased total [15N]ammonia utilization and total urea formation; this increase resulted from the rise of ammonia utilization for urea synthesis and it was similar in the presence or absence of ornithine. 5. The capacity of sheep liver cells to utilize ammonia in the presence of propionate (in the presence or absence of ornithine) amounted to 256 mumol/hr per 1 g dry wt, thus being similar to the values in vivo. 6. It is concluded that in sheep hepatocytes both ornithine and propionate stimulate the utilization of ammonia for urea synthesis and these effects take place independently and occur by different mechanisms.     

Ontogeny of the insulin response to glucose in fetal and adult sheep

The purpose of the present experiments was to examine in sheep whether the fetal insulin response to glucose was present by day 110 (d110) of pregnancy and whether the magnitude of the fetal insulin response changed between d110 and d145 (term). We also compared the responses observed in fetuses to those of adult nonpregnant sheep. Basal concentrations of glucose measured in plasma collected from the fetal femoral artery rose progressively between d110 and d145 of gestation, but did not attain the plasma glucose concentrations measured in adult sheep. Peak glucose concentrations in fetuses were achieved 10 min following the bolus injection of glucose (0.8 g/kg estimated fetal body weight) into the fetal femoral vein, and peak values increased with gestational age. Significantly higher peak glucose concentrations were achieved in adult sheep. The concentration of insulin rose rapidly in fetuses at d110, and a similar time course of insulin release in plasma was seen at all gestational ages. The peak plasma insulin concentrations were achieved at 20 min and were significantly greater in older (d140-145) than younger (d125-130) fetuses (p less than 0.05). Peak insulin values in fetuses were much less than in adult sheep. In adult sheep glucose and insulin concentrations remained elevated at 120 min following the injection of glucose, whereas in the fetus the concentration of insulin had returned to preinjection values by 60 min. The insulin/glucose ratio did not change in fetal lambs over the last one third of gestation and was not different from the adult sheep.(ABSTRACT TRUNCATED AT 250 WORDS)