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Maturation in liver mitochondria of Ruthenium Red-sensitive calcium-ion-transport activity and the influence of glucagon administration in vivo and in utero.

ABSTRACT: The maturation of Ca(2+) transport in mitochondria isolated from rat liver was examined, from 5 days before birth. The mitochondria used were isolated from liver homogenates by centrifugation at 22000g-min. Ca(2+) transport by mitochondria isolated from foetal liver is energy-dependent and Ruthenium Red-sensitive. The transmembrane pH gradient in these mitochondria is higher by about 7mV and the membrane potential lower by about 20mV than in adult mitochondria. The inclusion of 2mm-P(i) in the incubation medium enhances the protonmotive force by approx. 30mV. The rate of Ca(2+) influx in foetal mitochondria measured in buffered KCl plus succinate is low until about 2-3h after birth, when it increases to about 60% of adult values; approx. 24h later it has reached near-adult values. Higher rates of Ca(2+) influx are observed in the presence of 2mm-P(i); 3-5 days before birth the rates are about one-third of adult values and decline slightly as birth approaches. By 2-3h post partum they have reached adult values. The inclusion of 12.5mum-MgATP with the P(i) stimulates further the initial rate of Ca(2+) influx in foetal mitochondria. The rates observed are constant over the prenatal period examined and are 50-60% of those observed in adult mitochondria. Mitochondria isolated from foetal livers 4-5 days before birth retain the accumulated Ca(2+) for about 50min in the presence of 2mm-P(i). In the period 2 days before birth to birth, this ability is largely lost, but by 2-3h after birth Ca(2+) retention is similar to that of adult mitochondria. The presence of 12.5mum-MgATP progressively enhances the Ca(2+) retention time as development proceeds until 2-3h after birth, when it becomes less sensitive to added MgATP. Glucagon administration to older foetuses in utero enhances both the rate of mitochondrial Ca(2+) influx assayed in the presence of 2mm-P(i) and the time for which mitochondria retain accumulated Ca(2+) in the presence of 12.5mum-MgATP and 2mm-P(i). Its administration to neonatal animals leads to an increase in mitochondrial Ca(2+) retention similar to that seen in adult mitochondria. The data provide evidence that the Ruthenium Red-sensitive Ca(2+) transporter is potentially as active in foetal mitochondria 5 days before birth as it is in adult mitochondria. They also show that foetal mitochondria have an ability to retain accumulated Ca(2+) reminiscent of mitochondria from tumour cells and from hormone-challenged rat liver.

SUBMITTER: Prpic V

PROVIDER: S-EPMC1162984 | biostudies-other | 1981 Apr

REPOSITORIES: biostudies-other

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Project description:1. The subcellular distribution and maturation of Ruthenium Red-insensitive Ca(2+) transport activity were determined in livers of rats ranging in age from 3 days pre-term to 10 weeks of adult life and compared with those of glucose 6-phosphatase, 5'-nucleotidase and Ruthenium Red-sensitive Ca(2+) transport. Initial rates of Ruthenium Red-insensitive Ca(2+) transport were highest in those fractions enriched in glucose 6-phosphatase, i.e. the microsomal fraction; this fraction was devoid of Ruthenium Red-sensitive Ca(2+) transport activity. Although the heaviest fraction (nuclear) contained significant amounts of 5'-nucleotidase activity it was devoid of Ruthenium Red-insensitive Ca(2+) transport activity. 2. Foetal rat liver contain minimal amounts of Ruthenium Red-insensitive Ca(2+) transport activity, glucose 6-phosphatase and 5'-nucleotidase activities. These begin to be expressed concomitantly soon after birth; Ruthenium Red-insensitive Ca(2+) transport is maximal by 3 to 4 days and remains so for up to at least 10 weeks of adult life. Glucose 6-phosphatase also reaches a peak at 3-4 days, but then rapidly decreases to approach adult values. Maximal activity of 5'-nucleotidase in the microsomal and nuclear fractions is seen about 4-6 days after birth; this enzyme activity remains increased for up to about 10 days and then falls, but not as rapidly as glucose 6-phosphatase. It is tentatively suggested that the bulk of the Ruthenium Red-insensitive Ca(2+) transport is attributable to the system derived from the endoplasmic reticulum. 3. Administration of glucagon to adult rats enhances by 2-3-fold the initial rate of Ruthenium Red-insensitive Ca(2+) transport in the intermediate but not the microsomal fraction. The hormone-induced effect is fully suppressed by co-administration of puromycin, is dose-dependent with half-maximal response at approx. 1mug of glucagon/100g body wt. and time-dependent exhibiting a half-maximal response about 1h after administration of the hormone. 4. Ruthenium Red-insensitive Ca(2+) transport in the post-mitochondrial fraction of foetal liver also responds to the administration in situ of glucagon. The response, which also is prevented by co-administration of puromycin, is maximal in those foetuses nearing term. The suggestion is made that these effects of the hormone on Ruthenium Red-insensitive Ca(2+) transport are an integral part of the physiological network in the liver cell.

Project description:1. Seven fractions sedimenting at between 3000 and 120000g-min were prepared from a rat liver homogenate by differential centrifugation in buffered iso-osmotic sucrose. The following measurements were carried out on each of these fractions: Ruthenium Red-sensitive Ca(2+) transport in the absence and in the presence of P(i) as well as in the presence of N-ethylmaleimide to prevent P(i) cycling, succinate-supported respiration in the absence and in the presence of ADP, the DeltaE and -59 DeltapH components of the protonmotive force, cytochrome oxidase, uncoupler-stimulated adenosine triphosphatase, alpha-glycerophosphate dehydrogenase, P(i) content and the effect on the ;resting' rate of respiration of repeated additions of a fixed Ca(2+) concentration. 2. Ca(2+) transport either in the presence or in the absence of added P(i) and in the presence of N-ethylmaleimide exhibits significantly higher rates in the fraction sedimenting at 8000g-min. By contrast, respiration in the presence or in the absence of added ADP and the values for DeltaE and -59 DeltapH were similar in those fractions sedimenting between 4000 and 20000g-min, indicating that the driving force for Ca(2+) transport was similar in each of these fractions. 3. Experiments designed to determine the capacity of the individual fractions for Ca(2+), as measured by the effect of repeated additions of Ca(2+) on the resting rate of respiration, showed that fraction 2, i.e. that sedimenting at 8000g-min, also exhibited the greatest tolerance towards the uncoupling action of the ion. 4. Of the three enzyme activity profiles, only that of alpha-glycerophosphate dehydrogenase was similar to that of Ca(2+) transport. Because previous workers have assigned this enzyme to loci in the inner peripheral membrane [Werner & Neupert (1972) Eur. J. Biochem.25, 379-396], it is concluded that the Ruthenium Red-sensitive Ca(2+)- transport system also is located in this domain of the inner membrane. The relation of these findings to the mechanisms of mitochondrial Ca(2+) transport and the biogenesis of mitochondria is discussed.

Project description:The objective of the current study was to evaluate the effects of postruminal casein infusion and exogenous glucagon-like peptide 2 (GLP-2) administration on transcriptomic pathways and functions of the jejunal mucosa in cattle using next-generation RNA sequencing. Twenty-four Holstein steers [250 ± 23 kg body weight (BW)] received a continuous abomasal infusion of 3.94 g raw corn starch/kg of BW combined with either 0 or 1.30 g casein/kg of BW for 7 d. Steers received subcutaneous injections in two equal portions daily of excipient (0.5% bovine serum albumin) or 100 µg GLP-2/kg of BW per day. At the end of the 7-d treatment period, steers were slaughtered for collection of the jejunal mucosa. Total RNA was extracted from jejunal mucosal tissue, strand-specific cDNA libraries were prepared, and RNA sequencing was conducted to generate 150-bp paired-end reads at a depth of 20 M reads in each direction. Differentially expressed genes (DEG), KEGG pathway enrichment, and gene ontology enrichment between treatments were determined based on the FDR-corrected P-value. Postruminal casein infusion downregulated (padj < 0.05) 7 genes of the jejunal mucosa and did not result in any enriched KEGG pathways or gene ontologies. Exogenous GLP-2 administration upregulated (padj < 0.05) 667 DEGs, 26 KEGG pathways, 198 biological processes, 56 cellular components, and 60 molecular functions. Exogenous GLP-2 downregulated (padj < 0.05) 1101 DEGs, 14 KEGG pathways, 270 biological processes, 105 cellular components, and 46 molecular functions. Enrichment analyses revealed that exogenous GLP-2 administration affected several pathways involving mucosal growth, nutrient digestion, absorption, and metabolism, and intestinal barrier function. Results of the current study identified pathways affected by exogenous GLP-2 administration that could have implications for improved animal growth and feed efficiency in cattle. This research generated novel targets for future research to elucidate mechanisms of GLP-2-mediated responses in the jejunal mucosa.

Project description:We previously demonstrated that postruminal casein infusion and exogenous glucagon-like peptide 2 (GLP-2) administration independently stimulated growth and carbohydrase activity of the pancreas and jejunal mucosa in cattle. The objective of the current study was to profile the jejunal mucosal transcriptome of cattle using next-generation RNA sequencing in response to postruminal casein infusion and exogenous GLP-2. Twenty-four Holstein steers [250 ± 23.1 kg body weight (BW)] received a continuous abomasal infusion of 3.94 g raw corn starch/kg of BW combined with either 0 or 1.30 g casein/kg of BW for 7 d. Steers received subcutaneous injections at 0800 and 2000 h to provide either 0 or 100 μg GLP-2/kg of BW per day. At the end of the 7-d treatment period, steers were slaughtered for collection of the jejunal mucosa. Total RNA was extracted from jejunal mucosal tissue, strand-specific cDNA libraries were prepared, and RNA sequencing was conducted to generate 150-bp paired-end reads at a depth of 40 M reads per sample. Differentially expressed genes (DEG), KEGG pathway enrichment, and gene ontology enrichment were determined based on the FDR-corrected P-value (padj). Exogenous GLP-2 administration upregulated (padj < 0.05) 667 genes and downregulated 1,101 genes of the jejunal mucosa. Sphingolipid metabolism, bile secretion, adherens junction, and galactose metabolism were among the top KEGG pathways enriched with upregulated DEG (padj < 0.05) in response to exogenous GLP-2 administration. The top gene ontologies enriched with upregulated DEG (padj < 0.05) in response to exogenous GLP-2 administration included nutrient metabolic processes, brush border and bicellular tight junction assembly, and enzyme and transporter activities. Exogenous GLP-2 administration increased or tended to increase (padj < 0.10) brush border carbohydrase (MGAM, LCT, TREH), hexose transporter (SLC5A1, SLC2A2), and associated transcription factor (HNF1, GATA4, KAT2B) mRNA expression of the jejunal mucosa. Gene ontologies and KEGG pathways that were downregulated (padj < 0.05) in response to exogenous GLP-2 were related to genetic information processing. Postruminal casein infusion downregulated (padj < 0.05) 7 jejunal mucosal genes that collectively did not result in enriched KEGG pathways or gene ontologies. This study highlights some of the transcriptional mechanisms associated with increased growth, starch assimilation capacity, and barrier function of the jejunal mucosa in response to exogenous GLP-2 administration.

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Maturation in liver mitochondria of Ruthenium Red-sensitive calcium-ion-transport activity and the influence of glucagon administration in vivo and in utero.

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