Insulin resistance may be linked to incomplete fatty acid b-oxidation and the subsequent increase in acylcarnitine species in different tissues including skeletal muscle. It is not known if acylcarnitines participate in muscle insulin resistance or simply reflect dysregulated metabolism. The aims of this study were to determine whether acylcarnitines can elicit muscle insulin resistance and to better understand the link between incomplete muscle fatty acid b-oxidation, oxidative stress, inflammation, and insulin-resistance development. Differentiated C2C12, primary mouse, and human myotubes were treated with acylcarnitines (C4:0, C14:0, C16:0) or with palmitate with or without carnitine acyltransferase inhibition by mildronate. Treatment with C4:0, C14:0, and C16:0 acylcarnitines resulted in 20-30% decrease in insulin response at the level of Akt phosphorylation and/or glucose uptake. Mildronate reversed palmitateinduced insulin resistance concomitant with an ∼25% decrease in short-chain acylcarnitine and acetylcarnitine secretion. Although proinflammatory cytokines were not affected under these conditions, oxidative stress was increased by 2-3 times by short-or long-chain acylcarnitines. Acylcarnitine-induced oxidative stress and insulin resistance were reversed by treatment with antioxidants. Results are consistent with the conclusion that incomplete muscle fatty acid b-oxidation causes acylcarnitine accumulation and associated oxidative stress, raising the possibility that these metabolites play a role in muscle insulin resistance.-Aguer, C., McCoin, C. S., Knotts, T. A., Thrush, A. B., Ono-Moore, K., McPherson, R., Dent, R., Hwang, D. H., Adams, S. H., Harper, M.-E. Acylcarnitines: potential implications for skeletal muscle insulin resistance. FASEB J. 29, 336-345 (2015). www.fasebj.org

Perturbations in metabolic pathways can cause substantial increases in plasma and tissue concentrations of long-chain acylcarnitines (LCACs). For example, the levels of LCACs and other acylcarnitines rise in the blood and muscle during exercise, as changes in tissue pools of acylcoenzyme A reflect accelerated fuel flux that is incompletely coupled to mitochondrial energy demand and capacity of the tricarboxylic acid cycle. This natural ebb and flow of acylcarnitine generation and accumulation contrasts with that of inherited fatty acid oxidation disorders (FAODs), cardiac ischaemia or type 2 diabetes mellitus. These conditions are characterized by very high (FAODs, ischaemia) or modestly increased (type 2 diabetes mellitus) tissue and blood levels of LCACs. Although specific plasma LCAC concentrations and chain-lengths are widely used as diagnostic markers of FAODs, research into the potential effects of excessive LCAC accumulation or the roles of acylcarnitines as physiological modulators of cell metabolism is lacking. Nevertheless, a growing body of evidence has highlighted possible effects of LCACs on disparate aspects of pathophysiology, such as cardiac ischaemia outcomes, insulin sensitivity and inflammation. This Review, therefore, aims to provide a theoretical framework for the potential consequences of tissue build-up of LCACs among persons with metabolic disorders.

Incomplete β-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete β-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed d,l isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant l-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. l-C14 carnitine (5–25 μM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, l-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, l-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.

In this study, we explored the roles of microRNAs in adipocyte differentiation and metabolism. We first knocked down Argonaute2 (Ago2), a key enzyme in the processing of micro-RNAs (miRNAs), to investigate a potential role for miRNAs in adipocyte differentiation and/or metabolism. Although we did not observe dramatic differences in adipogenesis between Ago2 knock-down and control 3T3-L1 cells, incorporation of [(14)C]glucose or acetate into triacylglycerol, and steady-state levels of triacyglycerol were all reduced, suggesting a role for miRNAs in adipocyte metabolism. To study roles of specific miRNAs in adipocyte biology, we screened for miRNAs that are differentially expressed between preadipocytes and adipocytes for the 3T3-L1 and ST2 cell lines. Distinct subsets of miRNAs decline or increase during adipocyte conversion, whereas most miRNAs are not regulated. One locus encoding two miRNAs, 378/378*, contained within the intron of PGC-1beta is highly induced during adipogenesis. When overexpressed in ST2 mesenchymal precursor cells, miRNA378/378* increases the size of lipid droplets and incorporation of [(14)C]acetate into triacylglycerol. Although protein and mRNA expression levels of C/EBPalpha, C/EBPbeta, C/EBPdelta, and PPARgamma1 are unchanged, microarray and quantitative RT-PCR analyses indicate that a set of lipogenic genes are upregulated, perhaps due to increased expression of PPARgamma2. Knock-down of miRNA378 and/or miRNA378* decreases accumulation of triacylglycerol. Interestingly, we made the unexpected finding that miRNA378/378* specifically increases transcriptional activity of C/EBPalpha and C/EBPbeta on adipocyte gene promoters.

plays an important role in the maintenance of white adipose tissue (WAT) architecture and function, and proper ECM remodeling is critical to support WAT malleability to accomodate changes in energy storage needs. Obesity and adipocyte hypertrophy place a strain on the ECM remodeling machinery, which may promote disordered ECM and altered tissue integrity and could promote proinflammatory and cell stress signals. To explore these questions, new methods were developed to quantify omental and subcutaneous WAT tensile strength and WAT collagen content by three-dimensional confocal imaging, using collagen VI knockout mice as a methods validation tool. These methods, combined with comprehensive measurement of WAT ECM proteolytic enzymes, transcript, and blood analyte analyses, were used to identify unique pathophenotypes of metabolic syndrome and type 2 diabetes mellitus in obese women, using multivariate statistical modeling and univariate comparisons with weightmatched healthy obese individuals. In addition to the expected differences in inflammation and glycemic control, approximately 20 ECMrelated factors, including omental tensile strength, collagen, and enzyme transcripts, helped discriminate metabolically compromised obesity. This is consistent with the hypothesis that WAT ECM physiology is intimately linked to metabolic health in obese humans, and the studies provide new tools to explore this relationship. matrix metalloproteinase; bariatric surgery; adipose inflammation; type 2 diabetes mellitus; extracellular matrix WHITE ADIPOSE TISSUE (WAT) is the primary site of energy storage in humans and by its nature must be malleable to respond to changes in energy balance through expansion and contraction. Under healthy conditions, changes in WAT architecture and the extracellular matrix (ECM) are coordinated with alterations in adipocyte size to accommodate fat storage needs. In contrast, sustained excessive energy intake with concomitant development of hypertrophic adipocytes creates a strain on ECM adaptation with pathophysiological consequences such as inflammation and insulin resistance.Adipose transcript abundances of several ECM-related genes were increased in obese compared with nonobese human subjects, including cell adhesion molecules and ECM receptor interaction genes, whereas weight loss after gastric bypass surgery led to decreased expression of these genes (18). Collagen VI is enriched in adipose tissue, and visceral WAT mass is related to increased expression of collagen 6␣3 in humans and in genetically obese mice (21, 38). Some studies have indicated that, at least in severe obesity, WAT fibrosis is more prevalent compared with nonobese individuals and that fibrosis decreases with weight loss (13,14), and this is believed to play a role in obesity-associated inflammation. Genetically obese ob/ob mice deficient in collagen VI (and thus lower fibrosis) fed a high-fat diet have improved glucose tolerance, insulin signaling, and triglyceride disposal with greater adipocyte size along with reduced WAT inf...

On our initial discovery that prion protein (PrP)-derived peptides were capable of capturing the pathogenic prion protein (PrP Sc ), we have been interested in how these peptides interact with PrP Sc . After screening peptides from the entire human PrP sequence, we found two peptides (PrP 19 -30 and PrP100-111) capable of binding full-length PrP Sc in plasma, a medium containing a complex mixture of other proteins including a vast excess of the normal prion protein (PrP C ). The limit of detection for captured PrP Sc was calculated to be 8 amol from a Ϸ10 5 -fold dilution of 10% (wt/vol) human variant Creutzfeldt-Jakob disease brain homogenate, with >3,800-fold binding specificity to PrP Sc over PrP C . Through extensive analyses, we show that positively charged amino acids play an important, but not exclusive, role in the interaction between the peptides and PrP Sc . Neither hydrophobic nor polar interactions appear to correlate with binding activity. The peptide-PrP Sc interaction was not sequence-specific, but amino acid composition affected binding. Binding occurs through a conformational domain that is only present in PrP Sc , is species-independent, and is not affected by proteinase K digestion. These and other findings suggest a mechanism by which cationic domains of PrP C may play a role in the recruitment of PrP C to PrP Sc .plasma ͉ Creutzfeldt-Jakob disease ͉ detection ͉ cationic interaction ͉ diagnostic

Hepatic mitochondrial adaptations to physical activity may be regulated by biogenesis- and mitophagy-associated pathways in a sex-dependent manner. Here, we tested if mice with targeted deficiencies in liver-specific peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α; LPGC1α ) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3)-mediated mitophagy (BNIP3 ) would have reduced physical activity-induced adaptations in respiratory capacity, H O emission and mitophagy compared to wild-type (WT) controls and if these effects were impacted by sex. Male and female WT, LPGC1α and BNIP3 C57BL6/J mice were divided into groups that remained sedentary or had access to daily physical activity via voluntary wheel running (VWR) (n = 6-10/group) for 4 weeks. Mice had ad libitum access to low-fat diet and water. VWR reduced basal mitochondrial respiration, increased mitochondrial coupling and altered ubiquitin-mediated mitophagy in a sex-specific manner in WT mice. Female mice of all genotypes displayed higher electron transport system content, displayed increased ADP-stimulated respiration, produced less mitochondrially derived reactive oxygen species, exhibited reduced mitophagic flux, and were less responsive to VWR compared to males. Males responded more robustly to VWR-induced changes in hepatic mitochondrial function resulting in a match to adaptations found in females. Deficiencies in PGC1α and BNIP3 alone did not largely alter mitochondrial adaptations to VWR. However, VWR restored sex-dependent abnormalities in mitophagic flux in LPGC1α . Finally, BNIP3 mice had elevated mitochondrial content and increased mitochondrial respiration putatively through repressed mitophagic flux. In conclusion, hepatic mitochondrial adaptations to physical activity are more dependent on sex than PGC1α and BNIP3.

The impact of sexual dimorphism and mitophagy on hepatic mitochondrial adaptations during the treatment of steatosis with physical activity are largely unknown. Here, we tested if deficiencies in liver-specific peroxisome proliferative activated-receptor-γ coactivator-1α (PGC-1α), a transcriptional coactivator of biogenesis, and BCL-2/ADENOVIRUS EIB 19-kDa interacting protein (BNIP3), a mitophagy regulator, would impact hepatic mitochondrial adaptations (respiratory capacity, H2O2 production, mitophagy) to a high-fat diet (HFD) and HFD plus physical activity via voluntary wheel running (VWR) in both sexes. Male and female wild-type (WT), liver-specific PGC-1α heterozygote (LPGC-1α), and BNIP3 null mice were thermoneutral housed (29–31°C) and divided into three groups: sedentary-low-fat diet (LFD), 16 wk of (HFD), or 16 wk of HFD with VWR for the final 8 wk (HFD + VWR) ( n = 5–7/sex/group). HFD did not impair mitochondrial respiratory capacity or coupling in any group; however, HFD + VWR significantly increased maximal respiratory capacity only in WT and PGC-1α females. Males required VWR to elicit mitochondrial adaptations that were inherently present in sedentary females including greater mitochondrial coupling control and reduced H2O2 production. Females had overall reduced markers of mitophagy, steatosis, and liver damage. Steatosis and markers of liver injury were present in sedentary male mice on the HFD and were effectively reduced with VWR despite no resolution of steatosis. Overall, reductions in PGC-1α and loss of BNIP3 only modestly impacted mitochondrial adaptations to HFD and HFD + VWR with the biggest effect seen in BNIP3 females. In conclusion, hepatic mitochondrial adaptations to HFD and treatment of HFD-induced steatosis with VWR are more dependent on sex than PGC-1α or BNIP3.