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1.  Mitochondrial Substrate Availability and Its Role in Lipid-Induced Insulin Resistance and Proinflammatory Signaling in Skeletal Muscle 
Diabetes  2013;62(10):3426-3436.
The relationship between glucose and lipid metabolism has been of significant interest in understanding the pathogenesis of obesity-induced insulin resistance. To gain insight into this metabolic paradigm, we explored the potential interplay between cellular glucose flux and lipid-induced metabolic dysfunction within skeletal muscle. Here, we show that palmitate (PA)-induced insulin resistance and proinflammation in muscle cells, which is associated with reduced mitochondrial integrity and oxidative capacity, can be attenuated under conditions of glucose withdrawal or glycolytic inhibition using 2-deoxyglucose (2DG). Importantly, these glucopenic-driven improvements coincide with the preservation of mitochondrial function and are dependent on PA oxidation, which becomes markedly enhanced in the absence of glucose. Intriguingly, despite its ability to upregulate mitochondrial PA oxidation, glucose withdrawal did not attenuate PA-induced increases in total intramyocellular diacylglycerol and ceramide. Furthermore, consistent with our findings in cultured muscle cells, we also report enhanced insulin sensitivity and reduced proinflammatory tone in soleus muscle from obese Zucker rats fed a 2DG-supplemented diet. Notably, this improved metabolic status after 2DG dietary intervention is associated with markedly reduced plasma free fatty acids. Collectively, our data highlight the key role that mitochondrial substrate availability plays in lipid-induced metabolic dysregulation both in vitro and in vivo.
PMCID: PMC3781443  PMID: 23733201
2.  Metabolic Signatures of Cultured Human Adipocytes from Metabolically Healthy versus Unhealthy Obese Individuals 
PLoS ONE  2014;9(4):e93148.
Background and Aims
Among obese subjects, metabolically healthy and unhealthy obesity (MHO/MUHO) can be differentiated: the latter is characterized by whole-body insulin resistance, hepatic steatosis, and subclinical inflammation. Aim of this study was, to identify adipocyte-specific metabolic signatures and functional biomarkers for MHO versus MUHO.
10 insulin-resistant (IR) vs. 10 insulin-sensitive (IS) non-diabetic morbidly obese (BMI >40 kg/m2) Caucasians were matched for gender, age, BMI, and percentage of body fat. From subcutaneous fat biopsies, primary preadipocytes were isolated and differentiated to adipocytes in vitro. About 280 metabolites were investigated by a targeted metabolomic approach intracellularly, extracellularly, and in plasma.
Among others, aspartate was reduced intracellularly to one third (p = 0.0039) in IR adipocytes, pointing to a relative depletion of citric acid cycle metabolites or reduced aspartate uptake in MUHO. Other amino acids, already known to correlate with diabetes and/or obesity, were identified to differ between MUHO's and MHO's adipocytes, namely glutamine, histidine, and spermidine. Most species of phosphatidylcholines (PCs) were lower in MUHO's extracellular milieu, though simultaneously elevated intracellularly, e.g., PC aa C32∶3, pointing to increased PC synthesis and/or reduced PC release. Furthermore, altered arachidonic acid (AA) metabolism was found: 15(S)-HETE (15-hydroxy-eicosatetraenoic acid; 0 vs. 120pM; p = 0.0014), AA (1.5-fold; p = 0.0055) and docosahexaenoic acid (DHA, C22∶6; 2-fold; p = 0.0033) were higher in MUHO. This emphasizes a direct contribution of adipocytes to local adipose tissue inflammation. Elevated DHA, as an inhibitor of prostaglandin synthesis, might be a hint for counter-regulatory mechanisms in MUHO.
We identified adipocyte-inherent metabolic alterations discriminating between MHO and MUHO.
PMCID: PMC3973696  PMID: 24695116
3.  Cinnamon Extract Improves Insulin Sensitivity in the Brain and Lowers Liver Fat in Mouse Models of Obesity 
PLoS ONE  2014;9(3):e92358.
Treatment of diabetic subjects with cinnamon demonstrated an improvement in blood glucose concentrations and insulin sensitivity but the underlying mechanisms remained unclear. This work intends to elucidate the impact of cinnamon effects on the brain by using isolated astrocytes, and an obese and diabetic mouse model.
Cinnamon components (eugenol, cinnamaldehyde) were added to astrocytes and liver cells to measure insulin signaling and glycogen synthesis. Ob/ob mice were supplemented with extract from cinnamomum zeylanicum for 6 weeks and cortical brain activity, locomotion and energy expenditure were evaluated. Insulin action was determined in brain and liver tissues.
Treatment of primary astrocytes with eugenol promoted glycogen synthesis, whereas the effect of cinnamaldehyde was attenuated. In terms of brain function in vivo, cinnamon extract improved insulin sensitivity and brain activity in ob/ob mice, and the insulin-stimulated locomotor activity was improved. In addition, fasting blood glucose levels and glucose tolerance were greatly improved in ob/ob mice due to cinnamon extracts, while insulin secretion was unaltered. This corresponded with lower triglyceride and increased liver glycogen content and improved insulin action in liver tissues. In vitro, Fao cells exposed to cinnamon exhibited no change in insulin action.
Together, cinnamon extract improved insulin action in the brain as well as brain activity and locomotion. This specific effect may represent an important central feature of cinnamon in improving insulin action in the brain, and mediates metabolic alterations in the periphery to decrease liver fat and improve glucose homeostasis.
PMCID: PMC3958529  PMID: 24643026
4.  Identification of the Amino Acids 300–600 of IRS-2 as 14-3-3 Binding Region with the Importance of IGF-1/Insulin-Regulated Phosphorylation of Ser-573 
PLoS ONE  2012;7(8):e43296.
Phosphorylation of insulin receptor substrate (IRS)-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique for IRS-2 while the other residues are conserved in IRS-1 and IRS-2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS-2 in transfected HEK293 cells and this binding was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS-2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus and refeeding. Using different IRS-2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300–600. The 24 identified residues on IRS-2 included several 14-3-3 binding candidates in the region 300–600. Single alanine mutants of these candidates led to the identification of serine 573 as 14-3-3 binding site. A phospho-site specific antibody was generated to further characterize serine 573. IGF-1-dependent phosphorylation of serine 573 was reduced by inhibition of PI 3-kinase and Akt/PKB. A negative role of this phosphorylation site was implicated by the alanine mutant of serine 573 which led to enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. To conclude, our data suggest a physiologically relevant role for IGF-1/insulin-dependent 14-3-3 binding to IRS-2 involving serine 573.
PMCID: PMC3422239  PMID: 22912850
5.  Genetic Ablation of cGMP-Dependent Protein Kinase Type I Causes Liver Inflammation and Fasting Hyperglycemia 
Diabetes  2011;60(5):1566-1576.
The nitric oxide/cGMP/cGMP-dependent protein kinase type I (cGKI) signaling pathway regulates cell functions that play a pivotal role in the pathogenesis of type 2 diabetes. However, the impact of a dysfunction of this pathway for glucose metabolism in vivo is unknown.
The expression of cGKI in tissues relevant to insulin action was analyzed by immunohistochemistry. The metabolic consequences of a genetic deletion of cGKI were studied in mice that express cGKI selectively in smooth muscle but not in other cell types (cGKI-SM mice).
In wild-type mice, cGKI protein was detected in hepatic stellate cells, but not in hepatocytes, skeletal muscle, fat cells, or pancreatic β-cells. Compared with control animals, cGKI-SM mice had higher energy expenditure in the light phase associated with lower body weight and fat mass and increased insulin sensitivity. Mutant mice also showed higher fasting glucose levels, whereas insulin levels and intraperitoneal glucose tolerance test results were similar to those in control animals. Interleukin (IL)-6 signaling was strongly activated in the liver of cGKI-SM mice as demonstrated by increased levels of IL-6, phospho-signal transducer and activator of transcription 3 (Tyr 705), suppressor of cytokine signaling-3, and serum amyloid A2. Insulin-stimulated tyrosine phosphorylation of the insulin receptor in the liver was impaired in cGKI-SM mice. The fraction of Mac-2–positive macrophages in the liver was significantly higher in cGKI-SM mice than in control mice. In contrast with cGKI-SM mice, conditional knockout mice lacking cGKI only in the nervous system were normal with respect to body weight, energy expenditure, fasting glucose, IL-6, and insulin action in the liver.
Genetic deletion of cGKI in non-neuronal cells results in a complex metabolic phenotype, including liver inflammation and fasting hyperglycemia. Loss of cGKI in hepatic stellate cells may affect liver metabolism via a paracrine mechanism that involves enhanced macrophage infiltration and IL-6 signaling.
PMCID: PMC3292332  PMID: 21464444
6.  Fra-2 mediates oxygen-sensitive induction of transforming growth factor β in cardiac fibroblasts 
Cardiovascular Research  2010;87(4):647-655.
In the ischaemia-reperfused heart, transforming growth factor β (TGFβ) proteins trigger the differentiation of cardiac fibroblasts (CFs) contributing to fibrosis. Reoxygenation of the heart, in addition to being a trigger for reperfusion injury, induces tissue remodelling by hyperoxia-sensitive signalling processes involving TGFβ. Here, we sought to characterize the molecular mechanisms responsible for the O2-sensitive transcriptional induction of TGFβ in murine CF and to test the significance of such findings in the infarcted myocardium in vivo using laser capture microdissection.
Methods and results
All three isoforms of TGFβ were induced in the CF-rich peri-infarct tissue as well as in CF exposed to hyperoxic challenge. Reporter studies demonstrated that TGFβ transcription is hyperoxia inducible. Deletion of any one or both of the activating protein-1 (AP-1) binding sites in the TGFβ reporter construct resulted in loss of O2 sensitivity, demonstrating that AP-1 confers O2 sensitivity to TGFβ transcription. Fos-related AP-1 transcription factor (Fra-2) and Ask-1 (apoptosis signal-regulating kinase-1) were identified as key mediators of AP-1-dependent O2-sensitive TGFβ transcription. Knockdown of Fra-2 significantly blunted O2-induced expression of TGFβ1 as well as TGFβ3 in CF. Knockdown of Ask-1 blunted hyperoxia-induced Fra-2 gene expression and nuclear localization in CF. Collectively, these observations point towards a central role of Ask-1 and Fra-2 in O2-inducible AP-1 activation and induction of TGFβ.
Taken together with the observation that Fra-2-regulated genes are implicated in fibrosis, identification of Fra-2 as an O2-sensitive transcriptional regulator of inducible TGFβ expression positions Fra-2 as an important player in reoxygenation-induced fibrosis.
PMCID: PMC2920807  PMID: 20427335
Oxygen; Reperfusion; TGF
7.  Insulin Sensitivity Is Reflected by Characteristic Metabolic Fingerprints - A Fourier Transform Mass Spectrometric Non-Targeted Metabolomics Approach 
PLoS ONE  2010;5(10):e13317.
A decline in body insulin sensitivity in apparently healthy individuals indicates a high risk to develop type 2 diabetes. Investigating the metabolic fingerprints of individuals with different whole body insulin sensitivity according to the formula of Matsuda, et al. (ISIMatsuda) by a non-targeted metabolomics approach we aimed a) to figure out an unsuspicious and altered metabolic pattern, b) to estimate a threshold related to these changes based on the ISI, and c) to identify the metabolic pathways responsible for the discrimination of the two patterns.
Methodology and Principal Findings
By applying infusion ion cyclotron resonance Fourier transform mass spectrometry, we analyzed plasma of 46 non-diabetic subjects exhibiting high to low insulin sensitivities. The orthogonal partial least square model revealed a cluster of 28 individuals with alterations in their metabolic fingerprints associated with a decline in insulin sensitivity. This group could be separated from 18 subjects with an unsuspicious metabolite pattern. The orthogonal signal correction score scatter plot suggests a threshold of an ISIMatsuda of 15 for the discrimination of these two groups. Of note, a potential subgroup represented by eight individuals (ISIMatsuda value between 8.5 and 15) was identified in different models. This subgroup may indicate a metabolic transition state, since it is already located within the cluster of individuals with declined insulin sensitivity but the metabolic fingerprints still show some similarities with unaffected individuals (ISI >15). Moreover, the highest number of metabolite intensity differences between unsuspicious and altered metabolic fingerprints was detected in lipid metabolic pathways (arachidonic acid metabolism, metabolism of essential fatty acids and biosynthesis of unsaturated fatty acids), steroid hormone biosyntheses and bile acid metabolism, based on data evaluation using the metabolic annotation interface MassTRIX.
Our results suggest that altered metabolite patterns that reflect changes in insulin sensitivity respectively the ISIMatsuda are dominated by lipid-related pathways. Furthermore, a metabolic transition state reflected by heterogeneous metabolite fingerprints may precede severe alterations of metabolism. Our findings offer future prospects for novel insights in the pathogenesis of the pre-diabetic phase.
PMCID: PMC2955523  PMID: 20976215
8.  Lipidomics Analysis Reveals Efficient Storage of Hepatic Triacylglycerides Enriched in Unsaturated Fatty Acids after One Bout of Exercise in Mice 
PLoS ONE  2010;5(10):e13318.
Endurance exercise induces lipolysis, increases circulating concentrations of free fatty acids (FFA) and the uptake and oxidation of fatty acids in the working muscle. Less is known about the regulation of lipid metabolism in the liver during and post-exercise.
Methodology/Principal Findings
We performed an ultra fast liquid chromatography-mass spectrometry (UFLC-MS) based lipidomics analysis of liver tissue samples obtained from C57Bl/6J mice immediately after a 60 min treadmill run of moderate intensity, and after 3 h of recovery. The PLS-DA scores plot for 115 quantified lipid molecular species revealed a clear separation of the hepatic lipid profile of sedentary from recovering mice, but not from mice immediately after running. 21 lipid species were considered to be most responsible for the difference in the hepatic lipid profiles, including 17 triacylglycerides (TG), one lysophosphatidylcholine (LPC) and three phosphatidylcholines (PC). TG species were found to be more abundant in the recovery phase, while PC species were decreased. The degree of accumulation of individual TG species correlated well with the amount of theoretical energy stored whereas no increase was found for TG species containing only saturated or one monounsaturated fatty acid. Total liver TG content as assayed by an enzymatic method was increased to 163% in the recovery phase, while it was significantly decreased in skeletal muscle by the exercise bout and remained less in the recovery phase. Results from fasted and refed mice indicate that fasting-induced lipolysis was associated with a pronounced accumulation of hepatic TG, which is reversed by refeeding for 5 h. Thus food intake per se did not elevate hepatic TG.
These data indicate that high availability of FFA induced by endurance exercise or fasting resulted in a transient hepatic TG accumulation, while muscle TG content was decreased during exercise presumably due to increased muscle fatty acid oxidation.
PMCID: PMC2954156  PMID: 20967198
9.  Individual Stearoyl-CoA Desaturase 1 Expression Modulates Endoplasmic Reticulum Stress and Inflammation in Human Myotubes and Is Associated With Skeletal Muscle Lipid Storage and Insulin Sensitivity In Vivo 
Diabetes  2009;58(8):1757-1765.
Increased plasma levels of free fatty acids occur in obesity and type 2 diabetes and contribute to the development of insulin resistance. Saturated fatty acids (SFAs) such as palmitate especially have lipotoxic effects leading to endoplasmatic reticulum (ER) stress, inflammation, and insulin resistance. Stearoyl-CoA desaturase 1 (SCD1) plays a key role in preventing lipotoxic effects, as it converts SFAs to less harmful monounsaturated fatty acids. Here, we tested the hypothesis that individual differences in the regulation of SCD1 expression by palmitate exist and influence insulin sensitivity and the cellular response to palmitate.
Palmitate-induced gene expression was studied in primary human myotubes of 39 metabolically characterized individuals, as well as in an SCD1-overexpressing cell culture model.
SCD1 mRNA expression and inducibility by palmitate in cultured myotubes showed a broad interindividual variation, presumably due to inheritable characteristics of the donors. Overexpression of SCD1 prevented the inflammatory and ER stress response to palmitate exposure. In primary human myotubes, high SCD1 inducibility was associated with a low inflammatory (interleukin [IL]-6, IL-8, and chemokine [CXC motif] ligand 3 [CXCL3]) and ER stress (CCAAT/enhancer binding protein [C/EBP] homologous protein, activating transcription factor 3 [ATF3], and X-box binding protein 1 [XBP1]) response to palmitate exposure. Finally, palmitate-stimulated SCD1 mRNA expression, positively correlated with intramyocellular lipid (IMCL) content of the donors, was measured by 1H-magnetic resonance spectroscopy. After adjustment for IMCL, SCD1 expression and inducibility were positively correlated with insulin sensitivity.
We hypothesize that myocellular SCD1 inducibility by palmitate is an individual characteristic that modulates lipid storage, palmitate-induced inflammation, ER stress, and insulin resistance. This may describe individuals with increased capability of innoxious free fatty acid handling and benign triglyceride storage.
PMCID: PMC2712792  PMID: 19478146
10.  Medium Chain Acylcarnitines Dominate the Metabolite Pattern in Humans under Moderate Intensity Exercise and Support Lipid Oxidation 
PLoS ONE  2010;5(7):e11519.
Exercise is an extreme physiological challenge for skeletal muscle energy metabolism and has notable health benefits. We aimed to identify and characterize metabolites, which are components of the regulatory network mediating the beneficial metabolic adaptation to exercise.
Methodology and Principal Findings
First, we investigated plasma from healthy human subjects who completed two independent running studies under moderate, predominantly aerobic conditions. Samples obtained prior to and immediately after running and then 3 and 24 h into the recovery phase were analyzed by a non-targeted (NT-) metabolomics approach applying liquid chromatography-qTOF-mass spectrometry. Under these conditions medium and long chain acylcarnitines were found to be the most discriminant plasma biomarkers of moderately intense exercise. Immediately after a 60 min (at 93% VIAT) or a 120 min run (at 70% VIAT) a pronounced, transient increase dominated by octanoyl-, decanoyl-, and dodecanoyl-carnitine was observed. The release of acylcarnitines as intermediates of partial β-oxidation was verified in skeletal muscle cell culture experiments by probing 13C-palmitate metabolism. Further investigations in primary human myotubes and mouse muscle tissue revealed that octanoyl-, decanoyl-, and dodecanoyl-carnitine were able to support the oxidation of palmitate, proving more effective than L-carnitine.
Medium chain acylcarnitines were identified and characterized by a functional metabolomics approach as the dominating biomarkers during a moderately intense exercise bout possessing the power to support fat oxidation. This physiological production and efflux of acylcarnitines might exert beneficial biological functions in muscle tissue.
PMCID: PMC2902514  PMID: 20634953

Results 1-10 (10)