While there is substantial evidence that brown adipose tissue (BAT) is insulin-responsive, it remains unclear whether BAT becomes insulin resistant with high fat diet (HFD) feeding. Here, we examined ...the hypothesis that the sn-1,2-diacylglycerol (DAG)/Protein Kinase C epsilon (PKCε)/insulin receptor kinase (IRK)T1150 phosphorylation pathway may trigger HFD-induced BAT insulin resistance. To address this hypothesis we first studied two groups of male Sprague Dawley rats fed either a regular chow diet (RC) or a HFD. HFD-fed rats manifested BAT insulin resistance as reflected by reduced insulin-stimulated phosphorylation of IRKT1158 (p=0.03), AKTT308 (p=0.003), PDHS232 (p=0.0002), and PDHS300 (p=0.0004) compared to RC rats during a hyperinsulinemic-euglycemic clamp (HEC). These reductions in insulin signaling occured at the level of the IRK and were associated with increased sn-1,2 diacylglycerols (p=0.03) and increased PKCε translocation (p<0.004). These defects in insulin signaling were also associated with a 35% (p<0.009) reduction in insulin-stimulated BAT mitochondrial pyruvate dehydrogenase/citrate synthase flux (VPDH/VCS) as assessed by LC-MS/MS and GC-MS measurements of C4C5 glutamate (m+2)/pyruvate (m+3) during a HEC where 13C6glucose was the infusate. In order to assess the role of PKCε-induced IRKT1150 phosphorylation in this process we next studied the effects of HFD feeding in: 1) wild type mice fed a regular chow diet (WT), 2) wild type mice fed a HFD (WT-HFD) and 3) IRKT1150A knockin mice fed a HFD (IRKT1150A-HFD) and found that IRKT1150A-HFD mice have preserved levels of insulin-stimulated AKTS473 phosphorylation compared to WT-HFD mice (p=0.04).
Conclusion: These results demonstrate a critical role of the sn-1,2-DAG-PKCε-IRKT1150 phosphorylation pathway in mediating HFD-induced insulin resistance and mitochondrial dysfunction in BAT.
Disclosure
R.C.Gaspar: None. B.T.Hubbard: None. I.Sakuma: None. T.E.Lamoia: None. D.Zhang: None. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc.
AGPAT2 (1-acyl-sn-glycerol-3-phosphate acyltransferase 2) converts lysophosphatidic acid (LPA) into phosphatidic acid (PA) and mutations of the AGPAT2 gene are responsible for the most common form of ...congenital generalized lipodystrophy (CGL) which leads to diabetes and steatohepatitis. The underlying mechanism by which AGPAT2 deficiency leads to these abnormalities has not been elucidated. We explored this question using an antisense oligonucleotide (ASO) to knock down expression of AGPAT2 in liver and white adipose tissue (WAT) of adult male SD rats. AGPAT2 ASO treatment induced lipodystrophic features in which adipose and hepatic inflammation were associated with increased LPA content in WAT (Control ASO=0.4 ± 0.1 nmol/g tissue; AGPAT2 ASO=0.8 ± 0.4, P<0.05) and liver (Control ASO=2.7 ± 0.4 nmol/g tissue; AGPAT2 ASO=4.2 ± 0.9, P<0.01) whereas PA content was unchanged. We hypothesized that increased LPA content may lead to inflammation in these tissues. We tested if either a controlled-release mitochondrial protonophore (CRMP) or an ASO against glycerol-3-phosphate acyltransferase, mitochondrial (GPAM) would reduce LPA content and inflammation in these tissues in AGPAT2 ASO treated rats. CRMP treatment (20 mg/kg-day) primarily prevented LPA accumulation in WAT (Control ASO=0.4 ± 0.1 nmol/g tissue; AGPAT2 ASO=0.9 ± 0.1; AGPAT2 ASO+CRMP=0.6 ± 0.1) (AGPAT2 ASO vs AGPAT2+CRMP P<0.05) and prevented WAT inflammation. In contrast GPAM ASO treatment primarily prevented hepatic LPA accumulation (Control ASO=3.6 ± 0.4 nmol/g tissue; AGPAT2 ASO=4.8 ± 0.2; AGPAT2+GPAM ASO=2.7 ± 0.4) (AGPAT2 ASO vs AGPAT2+GPAM ASO P<0.01) and prevented hepatic inflammation.
Conclusion: These results demonstrate a key role of LPA acid in mediating inflammation in liver and WAT in AGPAT2 deficient animals and identify novel therapeutic targets to treat CGL in patients with AGPAT2 deficiency.
Disclosure
I.Sakuma: None. R.C.Gaspar: None. P.Luukkonen: None. M.Kahn: None. S.Murray: Employee; Ionis Pharmaceuticals. V.Samuel: None. K.Petersen: Advisory Panel; Novo Nordisk, Research Support; Merck Sharp & Dohme Corp., Gilead Sciences, Inc. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc.
Recently, our group demonstrated that 3-week high fat diet feeding (HFD) impairs insulin signaling in the renal cortex at the level of the insulin receptor kinase (IRK). Specifically, IRKY1162 and ...AKTS473 phosphorylation were reduced by ~50% during hyperinsulinemic-euglycemic clamp (HEC); however, it is unknown how renal cortical insulin resistance (IR) may affect gluconeogenic (GNG) and oxidative metabolism. To address this, here we measure renal cortical GNG fluxes in vivo using Q-Flux. In regular chow (RC) fed mice, hyperinsulinemia significantly suppressed mitochondrial pyruvate carboxylase (PC), as PC flux relative to α-ketoglutarate dehydrogenase flux (VPC/VOGDH) was 1.25 under basal conditions, but decreased by ~50%, to 0.57 during HEC. Similarly, PEPCK flux relative OGDH flux (VPEPCK/VOGDH) was reduced by ~60% during HEC. In contrast, insulin failed to suppress both PC and PEPCK fluxes with HFD. Using a 13C6glucose tracer, we also find insulin-stimulated mitochondrial pyruvate oxidation to be impaired by ~30% with HFD. Mechanistically, we previously found HFD led to a 2-fold increase in plasma membrane (PM) sn-1,2-diacylglycerol (DAG) and a 4-fold increase in Protein Kinase Cε (PKCε) translocation, suggesting renal cortical IR may be mediated by an sn-1,2-DAG-PKCε-IRK axis. We further interrogated this possibility here by assessing inhibitory phosphorylation at IRKT1150, the key target of PKCε, finding a 42% increase with HFD. In addition, IRKT1150A knockin mice, whose IRK cannot be phosphorylated by PKCε, were protected from HFD-induced signaling and pyruvate oxidation defects.
Conclusion: HFD inhibits renal cortical insulin signaling at the level of the IRK, which is associated with perturbed mitochondrial GNG and oxidative metabolism. IRKT1150A mice are protected from HFD-induced defects, indicating a PM sn-1,2-DAG-PKCε-pIRKT1150 mechanism for lipid-induced renal cortical IR.
Disclosure
B.T.Hubbard: None. R.C.Gaspar: None. D.Zhang: None. M.Kahn: None. A.Nasiri: Other Relationship; Medtronic. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc.
The striatum, the primary input nucleus of the basal ganglia, is crucially involved in motor and cognitive function and receives significant glutamate input from the cortex and thalamus. Increasing ...evidence suggests fundamental differences between these afferents, yet direct comparisons have been lacking. We describe a slice preparation that allows for direct comparison of the pharmacology and biophysics of these two pathways. Visualization of slices from animals previously injected with BDA into the parafascicular nucleus revealed the presence of axons of thalamic origin in the slice. These axons were especially well-preserved after traversing the reticular nucleus, the location chosen for stimulation of thalamostriatal afferents. Initial characterization of the two pathways revealed both non-NMDA and NMDA receptor-mediated currents at synapses from both afferents and convergence of the afferents in 51% of striatal efferent neurons. Annihilation of action potentials was not observed in collision experiments, nor was current spread from the site of stimulation to striatum found. Differences in short-term plasticity suggest that the probability of release differs for the two inputs. The present work thus provides a novel rat brain slice preparation in which the effects of selective stimulation of cortical versus thalamic afferents to striatum can be studied in the same preparation.
Carbon nanotubes (CNTs) and nanofibers (CNFs) were synthesized on clinker and silica fume particles in order to create a low cost cementitious nanostructured material. The synthesis was carried out ...by an in situ chemical vapor deposition (CVD) process using converter dust, an industrial byproduct, as iron precursor. The use of these materials reduces the cost, with the objective of application in large-scale nanostructured cement production. The resulting products were analyzed by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and thermogravimetric analysis (TGA) and were found to be polydisperse in size and to have defective microstructure. Some enhancement in the mechanical behavior of cement mortars was observed due to the addition of these nano-size materials. The contribution of these CNTs/CNFs to the mechanical strength of mortar specimens is similar to that of high quality CNTs incorporated in mortars by physical mixture.
SignificanceMetformin is the most commonly prescribed drug for the treatment of type 2 diabetes mellitus, yet the mechanism by which it lowers plasma glucose concentrations has remained elusive. Most ...studies to date have attributed metformin's glucose-lowering effects to inhibition of complex I activity. Contrary to this hypothesis, we show that inhibition of complex I activity in vitro and in vivo does not reduce plasma glucose concentrations or inhibit hepatic gluconeogenesis. We go on to show that metformin, and the related guanides/biguanides, phenformin and galegine, inhibit complex IV activity at clinically relevant concentrations, which, in turn, results in inhibition of glycerol-3-phosphate dehydrogenase activity, increased cytosolic redox, and selective inhibition of glycerol-derived hepatic gluconeogenesis both in vitro and in vivo.
Food packaging trends have been changing in the last years, mainly due to consumer concerns about plastic pollution and agrifood waste. Recent advances in the development of edible films and coatings ...are discussed. These systems can be much more than packaging, with additional functions such as antioxidant, antimicrobial, and nutritional properties, among others. Challenges in industrial processes and/or in some films’ properties are addressed from the authors’ point of view. The use of agrifood residues in these packaging systems is included as a promising strategy that promotes sustainability and circular economy. The authors consider that more research and actions are needed to achieve the best packaging material for each food product with reduced production costs.
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•The food packaging sector has changed from conventional to sustainable systems.•Edible films and coatings appear as a new trend with advantages and challenges.•Use of food residues is promising in the packaging sector.•More research/actions are needed to have packaging based on residues in the market.
The LHCb experiment at the LHC accelerator at CERN collects collisions of particle bunches at 40 MHz. After a first level of hardware trigger with an output rate of 1 MHz, the physically interesting ...collisions are selected by running dedicated trigger algorithms in the High Level Trigger (HLT) computing farm. This farm consists of up to roughly 25000 CPU cores in roughly 1750 physical nodes each equipped with up to 4 TB local storage space. This work describes the LHCb online system with an emphasis on the developments implemented during the current long shutdown (LS1). We will elaborate the architecture to treble the available CPU power of the HLT farm and the technicalities to determine and verify precise calibration and alignment constants which are fed to the HLT event selection procedure. We will describe how the constants are fed into a two stage HLT event selection facility using extensively the local disk buffering capabilities on the worker nodes. With the installed disk buffers, the CPU resources can be used during periods of up to ten days without beams. These periods in the past accounted to more than 70% of the total time.
•Male, but not female, rats expressed fear-conditioned analgesia.•Expression of other fear-related behaviours was higher in males than females.•Fear-related increases in periaqueductal grey (PAG) ...levels of OEA and GABA in male rats, but not females.•Evidence for sex differences in expression of endocannabinoid-related receptors in the PAG.
The endocannabinoid system within the periaqueductal grey (PAG) has been implicated in fear-conditioned analgesia (FCA), the profound suppression of pain upon re-exposure to a context previously paired with an aversive stimulus. Since the endocannabinoid and nociceptive systems exhibit sexual dimorphism, the aim of the present study was to assess possible sex differences in the expression of FCA, fear in the presence of nociceptive tone, and associated sex-dependent alterations in the endocannabinoid system within the PAG. Male and female Sprague-Dawley rats received footshock (10 × 1s; 0.4 mA; every 60 s) or no-footshock in a conditioning arena and 23.5 h later received intraplantar injection of formalin (2.5%) under brief isoflourane anaesthetic into the right hind paw. Nociceptive and fear-related behaviours were assessed 30 min later. Levels of endocannabinoids, N-acylethanolamines and neurotransmitters in the PAG were assessed by LC–MS/MS and expression of endocannabinoid system-related proteins by Western immunoblotting. Male, but not female, rats exhibited robust FCA and greater expression of fear-related behaviours than females. Fear-conditioned formalin-treated males, but not females, had higher levels of N-oleoylethanolamine (OEA) and γ-aminobutyric acid (GABA) in the PAG, compared with non-fear-conditioned controls. There was no effect of fear conditioning on the levels of FAAH or CB1 receptor expression (CB1R) in the PAG of male or female formalin-treated rats. Non-fear-conditioned females had higher levels of CB1R and PPARγ expression than non-fear-conditioned male counterparts. In summary, our results provide evidence of sexual dimorphism in the expression of FCA and fear-related behaviours, and associated alterations in components of the endocannabinoid system and GABA within the PAG.
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