Search

Aims/hypothesis: Targeting regulators of adipose tissue lipoprotein lipase could enhance adipose lipid clearance, prevent ectopic lipid accumulation and consequently ameliorate insulin resistance and type 2 diabetes. Angiopoietin-like 8 (ANGPTL8) is an insulin-regulated lipoprotein lipase inhibitor strongly expressed in murine adipose tissue. However, Angptl8 knockout mice do not have improved insulin resistance. We hypothesised that pharmacological inhibition, using a second-generation antisense oligonucleotide (ASO) against Angptl8 in adult high-fat-fed rodents, would prevent ectopic lipid accumulation and insulin resistance by promoting adipose lipid uptake.Methods: ANGPTL8 expression was assessed by quantitative PCR in omental adipose tissue of bariatric surgery patients. High-fat-fed Sprague Dawley rats and C57BL/6 mice were treated with ASO against Angptl8 and insulin sensitivity was assessed by hyperinsulinaemic-euglycaemic clamps in rats and glucose tolerance tests in mice. Factors mediating lipid-induced hepatic insulin resistance were assessed, including lipid content, protein kinase Cε (PKCε) activation and insulin-stimulated Akt phosphorylation. Rat adipose lipid uptake was assessed by mixed meal tolerance tests. Murine energy balance was assessed by indirect calorimetry.Results: Omental fat ANGPTL8 mRNA expression is higher in obese individuals with fatty liver and insulin resistance compared with BMI-matched insulin-sensitive individuals. Angptl8 ASO prevented hepatic steatosis, PKCε activation and hepatic insulin resistance in high-fat-fed rats. Postprandial triacylglycerol uptake in white adipose tissue was increased in Angptl8 ASO-treated rats. Angptl8 ASO protected high-fat-fed mice from glucose intolerance. Although there was no change in net energy balance, Angptl8 ASO increased fat mass in high-fat-fed mice.Conclusions/interpretation: Disinhibition of adipose tissue lipoprotein lipase is a novel therapeutic modality to enhance adipose lipid uptake and treat non-alcoholic fatty liver disease and insulin resistance. In line with this, adipose ANGPTL8 is a candidate therapeutic target for these conditions. [ABSTRACT FROM AUTHOR]

The modeling of long bone surfaces during linear growth is a key developmental process, but its regulation is poorly understood. We report here that parathyroid hormone-related peptide ( PTHr P) expressed in the fibrous layer of the periosteum ( PO) drives the osteoclastic ( OC) resorption that models the metaphyseal-diaphyseal junction ( MDJ) in the proximal tibia and fibula during linear growth. PTHr P was conditionally deleted (c KO) in the PO via Scleraxis gene targeting ( Scx- Cre). In the lateral tibia, c KO of PTHr P led to a failure of modeling, such that the normal concave MDJ was replaced by a mound-like deformity. This was accompanied by a failure to induce receptor activator of NF-kB ligand ( RANKL) and a 75% reduction in OC number ( P ≤ 0.001) on the cortical surface. The MDJ also displayed a curious threefold increase in endocortical osteoblast mineral apposition rate ( P ≤ 0.001) and a thickened cortex, suggesting some form of coupling of endocortical bone formation to events on the PO surface. Because it fuses distally, the fibula is modeled only proximally and does so at an extraordinary rate, with an anteromedial cortex in CD-1 mice that was so moth-eaten that a clear PO surface could not be identified. The c KO fibula displayed a remarkable phenotype, with a misshapen club-like metaphysis and an enlargement in the 3 D size of the entire bone, manifest as a 40-45% increase in the PO circumference at the MDJ ( P ≤ 0.001) as well as the mid-diaphysis ( P ≤ 0.001). These tibial and fibular phenotypes were reproduced in a Scx- Cre-driven RANKL c KO mouse. We conclude that PTHr P in the fibrous PO mediates the modeling of the MDJ of long bones during linear growth, and that in a highly susceptible system such as the fibula this surface modeling defines the size and shape of the entire bone. [ABSTRACT FROM AUTHOR]

In Gram-positive bacteria and other prokaryotes containing succinate:menaquinone reductases, it has previously been shown that the succinate oxidase and succinate:menaquinone reductase activities are lost when the transmembrane electrochemical proton potential, Δp, is abolished by the rupture of the bacteria or by the addition of a protonophore. It has been proposed that the endergonic reduction of menaquinone by succinate is driven by the electrochemical proton potential. Opposite sides of the cytoplasmic membrane were envisaged to be separately involved in the binding of protons upon the reduction of menaquinone and their release upon succinate oxidation, with the two reactions linked by the transfer of two electrons through the enzyme. However, it has previously been argued that the observed Δp dependence is not associated specifically with the succinate:menaquinone reductase. Definitive insight into the mechanism of catalysis of this reaction requires a corresponding functional characterization of an isolated, membrane-bound succinate:menaquinone reductase from a Gram-positive bacterium. Here, we describe the purification, reconstitution into proteoliposomes, and functional characterization of the diheme-containing succinate:menaquinone reductase from the Gram-positive bacterium Bacillus licheniformis and, with the help of the design, synthesis, and characterization of quinones with finely tuned oxidation/reduction potentials, provide unequivocal evidence for Δp-dependent catalysis of succinate oxidation by quinone as well as for Δp generation upon catalysis of fumarate reduction by quinol. [ABSTRACT FROM AUTHOR]

Objective: Spermatogonia are male germ line progenitors of sperm cells. In bovine, rodents and primates, undifferentiated spermatogonia and spermatogonial stem cells (SSCs) express the surface marker THY1. Since cells can be enriched from mixed populations by positive or negative selection on physical or functional characteristics such as cell surface markers, present study was aimed to compare the efficiency of two different enrichment methods: Magnetic activated cell sorting (MACS) and differential plating (DP) in pre-pubertal goat testes cell population. To comparison of two enrichment methods efficiency, real time PCR analysis, protein expression, and transplantation into recipient mice data were analyzed. For control group, the intact testis cells population were used. Materials and Methods: After 3 times enzymatic digestion of pre-pubertal goat testicular tissues, cells were cultured for 16 h in laminin-coated flasks and DP carried out. For MACS method, cell suspension were sorted using THY1 surface marker. Results: Real time PCR analysis revealed that expression of THY1, PLZF, VASA, BCL6B, UCHL1 as SCCs characteristic genes in THY1 positive cells were significantly higher than in the DP isolation cells and unselected total cells. Transplantation of THY1positive, DP isolated and intact population of testis cells (control) into the mice recipient seminiferous tubules revealed that THY1 positive cells had higher capacity for colony formation were compared to DP isolated and intact cells. Conclusion: The results indicated that isolation of undifferentiated testis cells using MACS method can be more efficient than differential plating [ABSTRACT FROM AUTHOR]