A hypothesis for insulin resistance in primary human adipocytes involving MRTF-A and suppression of PPARγ.

Obesity is the main risk factor behind insulin resistance and type 2 diabetes. Still, the mechanism behind adipocyte dysfunction is not yet resolved. Recently, we reported that rapid actin remodeling correlates with adipose cell size changes after short-term overfeeding. Therefore, we hypothesized that the actin-driven myocardin-related transcription factor (MRTF-A) contributes to impaired mature adipocyte function. Primary human adipocytes were subjected to adenoviral overexpression of MRTF-A or MRTF-B, followed by Western blot analysis and tracer glucose uptake assay. Further, we assessed cell size distribution, insulin response, MRTF-A localization, actin organization and degree of polymerization in adipocytes isolated from Ob/Ob mice. Overexpression of MRTF-A, but not MRTF-B, markedly suppressed PPARγ expression. Further, MRTF-A expression resulted in decreased IRS-1 level, shifted phosphorylation of Akt (pS473/pT308), IRS-1 (pS302) and AS160 (pT642), and lowered insulin-stimulated glucose uptake. Hypertrophic adipocytes from Ob/Ob mice displayed an increased proportion of polymerized actin, and increased nuclear translocation of MRTF-A compared with control (Ob/+). Similar with human adipocytes overexpressing MRTF-A, adipocytes isolated from Ob/Ob mice had reduced expression of IRS-1 and PPARγ, as well as impaired insulin response. Together, these data demonstrate that MRTF-A negatively influences insulin sensitivity and the expression of key targets in fully mature human adipocytes. This suggests that MRTF-A is poised to exert a transcriptional response in hypertrophic adipocytes, contributing to adipocyte dysfunction and insulin resistance.

Early accentuated muscle hypertrophy is strongly associated with myonuclear accretion.

The current study explored whether the marked hypertrophic response noted with a short-term unilateral concurrent exercise paradigm was associated with more prominent changes in myonuclei accretion, ribosome biogenesis, and capillarization compared with resistance exercise alone (RE). Ten men (age 25 ± 4 yr) performed aerobic and resistance exercise (AE + RE) for one leg while the other leg did RE. Muscle biopsies were obtained before and after 5 wk of training and subjected to fiber-type specific immunohistochemical analysis, and quantification of total RNA content and mRNA/rRNA transcript abundance. Type II fiber cross-sectional area (CSA) increased with both AE + RE (22%) and RE (16%), while type I fiber CSA increased mainly with AE + RE (16%). The change score tended to differ between legs for type I CSA (P = 0.099), and the increase in smallest fiber diameter was greater in AE + RE than RE (P = 0.029). The number of nuclei per fiber increased after AE + RE in both fiber types, and this increase was greater (P = 0.027) than after RE. A strong correlation was observed between changes in number of nuclei per fiber and fiber CSA in both fiber types, for both AE + RE and RE (r > 0.8, P < 0.004). RNA content increased after AE + RE (24%, P = 0.019), but the change-scores did not differ across legs. The capillary variables generally increased in both fiber types, with no difference across legs. In conclusion, the accentuated hypertrophic response to AE + RE was associated with more pronounced myonuclear accretion, which was strongly correlated with the degree of fiber hypertrophy. This suggests that myonuclear accretion could play a role in facilitating muscle hypertrophy also during very short training periods.

Skeletal muscle signaling responses to resistance exercise of the elbow extensors are not compromised by a preceding bout of aerobic exercise.

The current study examined the effects of a preceding bout of aerobic exercise (AE) on subsequent molecular signaling to resistance exercise (RE) of the elbow extensors. Eleven men performed unilateral elbow-extensor AE (~45 min at 70% peak workload) followed by unilateral RE (4 × 7 maximal repetitions) for both arms. Thus, one arm performed AE+RE interspersed with 15 min recovery, whereas the other arm conducted RE alone. Muscle biopsies were taken from the triceps brachii of each arm immediately before (PRE) and 15 min (POST1) and 3 h (POST2) after RE. Molecular markers involved in translation initiation, protein breakdown, mechanosignaling, and ribosome biogenesis were analyzed. Peak power during RE was reduced by 24% (±19%) when preceded by AE (P < 0.05). Increases in PGC1a and MuRF1 expression were greater from PRE to POST2 in AE+RE compared with RE (18- vs. 3.5- and 4- vs. 2-fold, respectively, interaction, P < 0.05). Myostatin mRNA decreased in both arms (P < 0.05). Phosphorylation of AMPK (Thr172) increased (2.5-fold), and 4E-BP1 (Thr37/46) decreased (2.0-fold), after AE (interactions, P < 0.05). p70 S6K, yes-associated protein, and c-Jun NH2-terminal kinase phosphorylation were unaltered, whereas focal adhesion kinase decreased ~1.5-fold, and ß1-integrin increased ~1.3- to 1.5-fold, (time effect, P < 0.05). Abundance of 45S pre-ribosomal (r)RNA (internally transcribed spacer, ITS) decreased (~30%) after AE (interaction, P < 0.05), whereas CMYC mRNA was greater in AE+RE compared with RE (12-fold, P < 0.05). POLR1B abundance increased after both AE+RE and RE. All together, our results suggest that a single bout of AE leads to an immediate decrease in signaling for translation initiation and ribosome biogenesis. Yet, this did not translate into altered RE-induced signaling during the 3-h postexercise recovery period.

Serotonin (5-HT) and 5-HT2A receptor agonists suppress lipolysis in primary rat adipose cells.

Serotonin (5-HT) is a biogenic monoamine that functions both as a neurotransmitter and a circulating hormone. Recently, the metabolic effects of 5-HT have gained interest and peripheral 5-HT has been proposed to influence lipid metabolism in various ways. Here, we investigated the metabolic effects of 5-HT in isolated, primary rat adipose cells. Incubation with 5-HT suppressed ß-adrenergically stimulated glycerol release and decreased phosphorylation of protein kinase A (PKA)-dependent substrates, hormone sensitive lipase (Ser563) and perilipin (Ser522). The inhibitory effect of 5-HT on lipolysis enhanced the anti-lipolytic effect of insulin, but sustained in the presence of phosphodiesterase inhibitors, OPC3911 and isobuthylmethylxanthine (IBMX). The relative expression of 5-HT1A, -2B and -4 receptor class family were significantly higher in adipose tissue compared to adipose cells, whereas 5-HT1D, -2A and -7 were highly expressed in isolated adipose cells. Similar to 5-HT, 5-HT2 receptor agonists reduced lipolysis while 5-HT1 receptor agonists rather decreased non-stimulated and insulin-stimulated glucose uptake. Together, these data provide evidence of a direct effect of 5-HT on adipose cells, where 5-HT suppresses lipolysis and glucose uptake, which could contribute to altered systemic lipid- and glucose metabolism.

Improvements in Maximal Oxygen Uptake After Sprint-Interval Training Coincide with Increases in Central Hemodynamic Factors.

INTRODUCTION: Sprint-interval training has been shown to improve maximal oxygen uptake, in part through peripheral muscle adaptations that increase oxygen utilization. In contrast, the adaptations of central hemodynamic factors in this context remain unexplored. PURPOSE: The aim of the current study was to explore the effects of sprint-interval training on maximal oxygen uptake and central hemodynamic factors. METHODS: Healthy men and women (n = 29; mean age, 27 ± 5 yr; height, 175 ± 8 cm; body mass, 72.5 ± 12.0 kg) performed 6 wk of sprint-interval training consisting of three weekly sessions of 10-min low-intensity cycling interspersed with 3 × 30-s all-out sprints. Maximal oxygen uptake, total blood volume, and maximal cardiac output were measured before and after the intervention. RESULTS: Maximal oxygen uptake increased by 10.3% (P < 0.001). Simultaneously, plasma volume, blood volume, total hemoglobin mass, and cardiac output increased by 8.1% (276 ± 234 mL; P < 0.001), 6.8% (382 ± 325 mL; P < 0.001), 5.7% (42 ± 41 g; P < 0.001), and 8.5% (1.0 ± 0.9 L·min-1; P < 0.001), respectively. Increased total hemoglobin mass along with measures of body surface area had a significant impact on the improvements in maximal oxygen uptake. CONCLUSIONS: Six weeks of sprint-interval training results in significant increases in hemoglobin mass, blood volume, and cardiac output. Because these changes were associated with marked improvements in maximal oxygen uptake, we conclude that central hemodynamic adaptations contribute to the improvement in maximal oxygen uptake during sprint-interval training.

Analysis of the effect of mobile phone base station antenna loading on localized SAR and its consequences for measurements.

In this work, the effect of antenna element loading on the localized specific absorption rate (SAR) has been analyzed for base station antennas. The analysis was conducted in order to determine whether localized SAR measurements of large multi-element base station antennas can be conducted using standardized procedures and commercially available equipment. More specifically, it was investigated if the antenna shifting measurement procedure, specified in the European base station exposure assessment standard EN 50383, will produce accurate localized SAR results for base station antennas larger than the specified measurement phantom. The obtained results show that SAR accuracy is affected by the presence of lossy material within distances of one wavelength from the tested antennas as a consequence of coupling and redistribution of transmitted power among the antenna elements. It was also found that the existing standardized phantom is not optimal for SAR measurements of large base station antennas. A new methodology is instead proposed based on a larger, box-shaped, whole-body phantom.

Acute endurance exercise stimulates circulating levels of mitochondrial-derived peptides in humans.

Mitochondrial-derived peptides (MDPs) humanin (HN) and mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) are involved in cell survival, suppression of apoptosis, and metabolism. Circulating levels of MDPs are altered in chronic diseases such as diabetes type 2 and chronic kidney disease. Whether acute resistance (RE) or endurance (EE) exercise modulates circulating levels of HN and MOTS-c in humans is unknown. Following familiarization, subjects were randomized to EE (n = 10, 45 min cycling at 70% of estimated V̇O2max), RE (n = 10, 4 sets × 7RM, leg press and knee extension), or control (CON, n = 10). Skeletal muscle biopsies and blood samples were collected before and at 30 min and 3 h following exercise. Plasma concentration of HN and MOTS-c, skeletal muscle MOTS-c as well as gene expression of exercise-related genes were analyzed. Acute EE and RE promoted changes in skeletal muscle gene expression typically seen in response to each exercise modality (c-Myc, 45S pre-rRNA, PGC-1α-total, and PGC-1α-ex1b). At rest, circulating levels of HN were positively correlated to MOTS-c levels and age. Plasma levels of MDPs were not correlated to fitness outcomes [V̇O2max, leg strength, or muscle mitochondrial (mt) DNA copy number]. Circulating levels of HN were significantly elevated by acute EE but not RE. MOTS-C levels showed a trend to increase after EE. These results indicate that plasma MDP levels are not related to fitness status but that acute EE increases circulating levels of MDPs, in particular HN.NEW & NOTEWORTHY In this manuscript, we report for the first time, to our knowledge, the response of circulating levels of mitochondrial-derived peptides humanin and MOTS-c to acute resistance and endurance exercise. Our data support that acute endurance exercise stimulates MDP levels in plasma, whereas acute resistance exercise does not.

Adipose cell size changes are associated with a drastic actin remodeling.

Adipose tissue plays a major role in regulating whole-body insulin sensitivity and energy metabolism. To accommodate surplus energy, the tissue rapidly expands by increasing adipose cell size (hypertrophy) and cell number (hyperplasia). Previous studies have shown that enlarged, hypertrophic adipocytes are less responsive to insulin, and that adipocyte size could serve as a predictor for the development of type 2 diabetes. In the present study, we demonstrate that changes in adipocyte size correlate with a drastic remodeling of the actin cytoskeleton. Expansion of primary adipocytes following 2 weeks of high-fat diet (HFD)-feeding in C57BL6/J mice was associated with a drastic increase in filamentous (F)-actin as assessed by fluorescence microscopy, increased Rho-kinase activity, and changed expression of actin-regulating proteins, favoring actin polymerization. At the same time, increased cell size was associated with impaired insulin response, while the interaction between the cytoskeletal scaffolding protein IQGAP1 and insulin receptor substrate (IRS)-1 remained intact. Reversed feeding from HFD to chow restored cell size, insulin response, expression of actin-regulatory proteins and decreased the amount of F-actin filaments. Together, we report a drastic cytoskeletal remodeling during adipocyte expansion, a process which could contribute to deteriorating adipocyte function.

EHD2 regulates adipocyte function and is enriched at cell surface-associated lipid droplets in primary human adipocytes.

Adipocytes play a central role in energy balance, and dysfunctional adipose tissue severely affects systemic energy homeostasis. The ATPase EH domain-containing 2 (EHD2) has previously been shown to regulate caveolae, plasma membrane-specific domains that are involved in lipid uptake and signal transduction. Here, we investigated the role of EHD2 in adipocyte function. We demonstrate that EHD2 protein expression is highly up-regulated at the onset of triglyceride accumulation during adipocyte differentiation. Small interfering RNA-mediated EHD2 silencing affected the differentiation process and impaired insulin sensitivity, lipid storage capacity, and lipolysis. Fluorescence imaging revealed localization of EHD2 to caveolae, close to cell surface-associated lipid droplets in primary human adipocytes. These lipid droplets stained positive for glycerol transporter aquaporin 7 and phosphorylated perilipin-1 following adrenergic stimulation. Further, EHD2 overexpression in human adipocytes increased the lipolytic signaling and suppressed the activity of transcription factor PPARγ. Overall, these data suggest that EHD2 plays a key role for adipocyte function.

Insulin induces Thr484 phosphorylation and stabilization of SIK2 in adipocytes.

AIMS/HYPOTHESIS: Salt-inducible kinase 2 (SIK2) is downregulated in adipose tissue from obese or insulin-resistant individuals and inhibition of SIK isoforms results in reduced glucose uptake and insulin signalling in adipocytes. However, the regulation of SIK2 itself in response to insulin in adipocytes has not been studied in detail. The aim of our work was to investigate effects of insulin on various aspects of SIK2 function in adipocytes. METHODS: Primary adipocytes were isolated from human subcutaneous and rat epididymal adipose tissue. Insulin-induced phosphorylation of SIK2 and HDAC4 was analyzed using phosphospecific antibodies and changes in the catalytic activity of SIK2 with in vitro kinase assay. SIK2 protein levels were analyzed in primary adipocytes treated with the proteasome inhibitor MG132. RESULTS: We have identified a novel regulatory pathway of SIK2 in adipocytes, which involves insulin-induced phosphorylation at Thr484. This phosphorylation is impaired in individuals with a reduced insulin action. Insulin stimulation does not affect SIK2 catalytic activity or cellular activity towards HDAC4, but is associated with increased SIK2 protein levels in adipocytes. CONCLUSION/INTERPRETATION: Our data suggest that downregulation of SIK2 in the adipose tissue of insulin-resistant individuals can partially be caused by impaired insulin signalling, which might result in defects in SIK2 expression and function.

Intact glucose uptake despite deteriorating signaling in adipocytes with high-fat feeding.

To capture immediate cellular changes during diet-induced expansion of adipocyte cell volume and number, we characterized mature adipocytes during a short-term high-fat diet (HFD) intervention. Male C57BL6/J mice were fed chow diet, and then switched to HFD for 2, 4, 6 or 14 days. Systemic glucose clearance was assessed by glucose tolerance test. Adipose tissue was dissected for RNA-seq and cell size distribution analysis using coulter counting. Insulin response in isolated adipocytes was monitored by glucose uptake assay and Western blotting, and confocal microscopy was used to assess autophagic activity. Switching to HFD was accompanied by an immediate adipocyte size expansion and onset of systemic insulin resistance already after two days, followed by recruitment of new adipocytes. Despite an initially increased non-stimulated and preserved insulin-stimulated glucose uptake, we observed a decreased phosphorylation of insulin receptor substrate-1 (IRS-1) and protein kinase B (PKB). After 14 days of HFD, both the insulin-stimulated phosphorylation of Akt substrate of 160 kDa (AS160) and glucose uptake was blunted. RNA-seq analysis of adipose tissue revealed transient changes in gene expression at day four, including highly significant upregulation of Trp53inp, previously demonstrated to be involved in autophagy. We confirmed increased autophagy, measured as an increased density of LC3-positive puncta and decreased p62 expression after 14 days of HFD. In conclusion, HFD rapidly induced systemic insulin resistance, whereas insulin-stimulated glucose uptake remained intact throughout 6 days of HFD feeding. We also identified autophagy as an early cellular process that potentially influences adipocyte function upon switching to HFD.

Rosiglitazone drives cavin-2/SDPR expression in adipocytes in a CEBPα-dependent manner.

Caveolae are abundant adipocyte surface domains involved in insulin signaling, membrane trafficking and lipid homeostasis. Transcriptional control mechanisms for caveolins and cavins, the building blocks of caveolae, are thus arguably important for adipocyte biology and studies in this area may give insight into insulin resistance and diabetes. Here we addressed the hypothesis that one of the less characterized caveolar components, cavin-2 (SDPR), is controlled by CCAAT/Enhancer Binding Protein (CEBPα) and Peroxisome Proliferator-Activated Receptor Gamma (PPARG). Using human mRNA expression data we found that SDPR correlated with PPARG in several tissues. This was also observed during differentiation of 3T3-L1 fibroblasts into adipocytes. Treatment of 3T3-L1-derived adipocytes with the PPARγ-activator Rosiglitazone increased SDPR and CEBPα expression at both the mRNA and protein levels. Silencing of CEBPα antagonized these effects. Further, adenoviral expression of PPARγ/CEBPα or Rosiglitazone-treatment increased SDPR expression in primary rat adipocytes. The myocardin family coactivator MKL1 was recently shown to regulate SDPR expression in human coronary artery smooth muscle cells. However, we found that actin depolymerization, known to inhibit MKL1 and MKL2, was without effect on SDPR mRNA levels in adipocytes, even though overexpression of MKL1 and MKL2 had the capacity to increase caveolins and cavins and to repress PPARγ/CEBPα. Altogether, this work demonstrates that CEBPα expression and PPARγ-activity promote SDPR transcription and further supports the emerging notion that PPARγ/CEBPα and MKL1/MKL2 are antagonistic in adipocytes.

Parameter estimation of human nerve C-fibers using matched filtering and multiple hypothesis tracking.

We describe how multiple-target tracking may be used to estimate conduction velocity changes and recovery constants of human nerve C-fibers. These parameters discriminate different types of C-fibers and pursuing this may promote new insights into differential properties of nerve fiber membranes. Action potentials (APs) were recorded from C-fibers in the peroneal nerve of awake human subjects. The APs were detected by a matched filter constituting a maximum-likelihood constant false-alarm rate detector. Using the multiple-hypothesis tracking method and Kalman filtering, the detected APs (targets) in each trace (scan) were associated to individual nerve fibers (tracks) by their typical conduction latencies in response to electrical stimulation. The measurements were one-dimensional (range only) and the APs were spaced in time with intersecting trajectories. In general, the AP amplitude of each C-fiber differed for different fibers. Amplitude estimation was therefore incorporated into the tracking algorithm to improve the performance. The target trajectory was modeled as an exponential decay with three unknowns. These parameters were estimated iteratively by applying the simplex method on the parameters that enter nonlinearly and the least squares method on the parameters that enter linearly.

Improved emission uniformity from a liquid-jet laser-plasma extreme-ultraviolet source.

Many modern compact soft-x-ray and extreme-ultraviolet (EUV) imaging systems operate with small fields of view and therefore benefit from the use of small high-brightness sources. Such systems include water-window microscopes and EUV lithography tools. We show that the photon losses in such systems can be minimized while uniformity of object-plane illumination is maintained by controlled scanning of the source. The improved collection efficiency is demonstrated both theoretically and experimentally for a scanned laser-plasma source compared with static sources. A prospective aerial image microscope and a liquid-xenon-jet laser-plasma source are offered as examples of modern imaging tools that may benefit from such scanning of the source.