Conjugated linoleic acid and betaine affect lipolysis in pig adipose tissue explants

Consumers’ demand of leaner meat products is a challenge. Although betaine and conjugated linoleic acid (CLA) have the potential to decrease porcine adipose tissue, their mode of action is poorly understood. The aim of the study was to determine the lipolytic effect of betaine and CLA in the adipose tissue of Iberian pigs. Adipose tissue explants from five pigs (38 kg BW) were prepared from dorsal subcutaneous adipose tissue samples and cultivated for 2 h (acute experiments) or 72 h (chronic experiments). Treatments included 100 µM linoleic acid (control), 100 µM trans-10, cis-12 CLA, 100 µM linoleic acid + 1 mM betaine and 100 µM trans-10, cis-12 CLA + 1 mM betaine (CLABET). To examine the ability of betaine or CLA to inhibit insulin’s suppression of isoproterenol-stimulated lipolysis, test medium was amended with 1 µM isoproterenol ±10 nM insulin. Media glycerol was measured at the end of the incubations. Acute lipolysis (2 h) was increased by CLA and CLABET (85% to 121%; P < 0.05) under basal conditions. When lipolysis was stimulated with isoproterenol (1090%), acute exposure to betaine tended to increase (13%; P = 0.071), while CLA and CLABET increased (14% to 18%; P < 0.05) isoproterenol-stimulated lipolysis compared with control. When insulin was added to isoproterenol-stimulated explants, lipolytic rate was decreased by 50% (P < 0.001). However, supplementation of betaine to the insulin + isoproterenol-containing medium tended to increase (P = 0.07), while CLABET increased (45%; P < 0.05) lipolysis, partly counteracting insulin inhibition. When culture was extended for 72 h, CLA decreased lipolysis under basal conditions (18%; P < 0.05) with no effect of betaine and CLABET (P > 0.10). When lipolysis was stimulated by isoproterenol (125% increase in rate compared with basal), CLA and CLABET decreased glycerol release (27%; P < 0.001) compared with control (isoproterenol alone). When insulin was added to isoproterenol-stimulated explants, isoproterenol stimulation of lipolysis was completely blunted and neither betaine nor CLA altered the inhibitory effect of insulin on lipolysis. Isoproterenol, and especially isoproterenol + insulin, stimulated leptin secretion compared with basal conditions (68% and 464%, respectively; P < 0.001), with no effect of CLA or betaine (P > 0.10). CLA decreased leptin release (25%; P < 0.001) when insulin was present in the media, partially inhibiting insulin stimulation of leptin release. In conclusion, betaine and CLA produced a biphasic response regarding lipolysis so that glycerol release was increased in acute conditions, while CLA decreased glycerol release and betaine had no effect in chronic conditions. Furthermore, CLA and CLABET indirectly increased lipolysis by reducing insulin-mediated inhibition of lipolysis during acute conditions.     

The impact of aneuploidy on cellular homeostasis

Genomic instability is a common feature of tumours that has a wide range of disruptive effects on cellular homeostasis. In this review we briefly discuss how instability comes about, then focus on the impact of gain or loss of DNA (aneuploidy) on oxidative stress. We discuss several mechanisms that lead from aneuploidy to the production of reactive oxygen species, including the effects on protein complex stoichiometry, endoplasmic reticulum stress and metabolic disruption. Each of these are involved in positive feedback loops that amplify relatively minor genetic changes into major cellular disruption or cell death, depending on the capacity of the cell to induce antioxidants or processes such as mitophagy that can moderate the disruption. Finally we examine the direct effects of reactive oxygen species on mitosis and how oxidative stress can compromise centrosome number, cytoskeletal integrity and signalling processes that are vital for mitotic fidelity.     

Inhibition of oxidative metabolism by nitric oxide restricts EMCV replication selectively in pancreatic beta-cells

Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1β, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for β-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in β-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a β-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.     

Characterization of the new β-glucuronidase from <Emphasis Type="Italic">Streptococcus equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Recently, a new gene encoding β-glucuronidase from Streptococcus equi subsp. zooepidemicus (SEZ) was identified and expressed in Escherichia coli. In this paper, the characterization of the enzyme is described. Specific enzyme activity was 120,000 U/mg purified protein at 37°C and pH = 7.0. The temperature and pH value, at which the enzyme has the highest specific activity, were determined and were found to be approximately 52°C and 5.6, respectively. The mutant strain SEZ glcHis was designed for the efficient isolation of β-glucuronidase from S. equi subsp. zooepidemicus. It was observed that the specific activity of β-glucuronidase in the cytoplasmic extract of a mutated strain was about 45% lower than in the cytoplasmic extract of a wild-type strain. The specific activity of purified β-glucuronidase from SEZ glcHis was four times as low as β-glucuronidase purified from E. coli. Comparing the specific activity of purified streptococcal β-glucuronidase from E. coli with E. coli β-glucuronidase (the enzyme with the highest specific activity was supplied by Sigma), the former is 1.8 higher than the latter.     

Effect of fluorocitrate on cerebral oxidation of lactate and glucose in freely moving rats

Glucose is the primary carbon source to enter the adult brain for catabolic and anabolic reactions. Some studies suggest that astrocytes may metabolize glucose to lactate; the latter serving as a preferential substrate for neurons, especially during neuronal activation. The current study utilizes the aconitase inhibitor fluorocitrate to differentially inhibit oxidative metabolism in glial cells in vivo. Oxidative metabolism of 14C-lactate and 14C-glucose was monitored in vivo using microdialysis and quantitating 14CO2 in the microdialysis eluate following pulse labeling of the interstitial glucose or lactate pool. After establishing a baseline oxidation rate, fluorocitrate was added to the perfusate. Neither lactate nor glucose oxidation was affected by 5 micromol/L fluorocitrate. However, 20 and 100 micromol/L fluorocitrate reduced lactate oxidation by 55 +/- 20% and 68 +/- 12%, respectively (p < 0.05 for both). Twenty and 100 micromol/L fluorocitrate reduced 14C-glucose oxidation by 50 +/- 14% (p < 0.05) and 24 +/- 19% (ns), respectively. Addition of non-radioactive lactate to (14)C-glucose plus fluorocitrate decreased 14C-glucose oxidation by an additional 29% and 38%, respectively. These results indicate that astrocytes oxidize about 50% of the interstitial lactate and about 35% of the glucose. By subtraction, neurons metabolize a maximum of 50% of the interstitial lactate and 65% of the interstitial glucose.     

Characterization of the Saccharomyces cerevisiae galactose mutarotase/UDP-galactose 4-epimerase protein, Gal10p

Saccharomyces cerevisiae and some related yeasts are unusual in that two of the enzyme activities (galactose mutarotase and UDP-galactose 4-epimerase) required for the Leloir pathway of d-galactose catabolism are contained within a single protein-Gal10p. The recently solved structure of the protein shows that the two domains are separate and have similar folds to the separate enzymes from other species. The biochemical properties of Gal10p have been investigated using recombinant protein expressed in, and purified from, Escherichia coli. Protein-protein crosslinking confirmed that Gal10p is a dimer in solution and this state is unaffected by the presence of substrates. The steady-state kinetic parameters of the epimerase reaction are similar to those of the human enzyme, and are not affected by simultaneous activity at the mutarotase active site. The mutarotase active site has a strong preference for galactose over glucose, and is not affected by simultaneous epimerase activity. This absence of reciprocal kinetic effects between the active sites suggests that they act independently and do not influence or regulate each other.