Excess membrane cholesterol is an early contributing reversible aspect of skeletal muscle insulin resistance in C57BL/6NJ mice fed a Western-style high-fat diet

Skeletal muscle insulin resistance manifests shortly after high-fat feeding, yet mechanisms are not known. Here we set out to determine whether excess skeletal muscle membrane cholesterol and cytoskeletal derangement known to compromise glucose transporter (GLUT)4 regulation occurs early after high-fat feeding. We fed 6-wk-old male C57BL/6NJ mice either a low-fat (LF, 10% kcal) or a high-fat (HF, 45% kcal) diet for 1 wk. This HF feeding challenge was associated with an increase, albeit slight, in body mass, glucose intolerance, and hyperinsulinemia. Liver analyses did not reveal signs of hepatic insulin resistance; however, skeletal muscle immunoblots of triad-enriched regions containing transverse tubule membrane showed a marked loss of stimulated GLUT4 recruitment. An increase in cholesterol was also found in these fractions from HF-fed mice. These derangements were associated with a marked loss of cortical filamentous actin (F-actin) that is essential for GLUT4 regulation and known to be compromised by increases in membrane cholesterol. Both the withdrawal of the HF diet and two subcutaneous injections of the cholesterol-lowering agent methyl-β-cyclodextrin at 3 and 6 days during the 1-wk HF feeding intervention completely mitigated cholesterol accumulation, cortical F-actin loss, and GLUT4 dysregulation. Moreover, these beneficial membrane/cytoskeletal changes occurred concomitant with a full restoration of metabolic responses. These results identify skeletal muscle membrane cholesterol accumulation as an early, reversible, feature of insulin resistance and suggest cortical F-actin loss as an early derangement of skeletal muscle insulin resistance

A Noisy Paracrine Signal Determines the Cellular NF-ΚB Response to Lipopolysaccharide

Nearly identical cells can exhibit substantially different responses to the same stimulus. We monitored the nuclear localization dynamics of nuclear factor KB (NF-KB) in single cells stimulated with tumor necrosis factor–α (TNF-α) and lipopolysaccharide (LPS). Cells stimulated with TNF-α have quantitative differences in NF-KB nuclear localization, whereas LPS-stimulated cells can be clustered into transient or persistent responders, representing two qualitatively different groups based on the NF-KB response. These distinct behaviors can be linked to a secondary paracrine signal secreted at low concentrations, such that not all cells undergo a second round of NF-KB activation. From our single-cell data, we built a computational model that captures cell variability, as well as population behaviors. Our findings show that mammalian cells can create "noisy" environments to produce diversified responses to stimuli

Blocking paracrine signaling by TNF across all concentrations and preparations.

<p>The average time course for N number of active cells is plotted for cells stimulated in the absence (blue trace, top value of n) and presence (orange trace, bottom value) of sTNFRII, which competes to bind TNF. Concentrations are indicated at left, and the preparation at top.</p

The potency window for each of the LPS preparations.

<p>The fraction of active cells is plotted as a function of concentration for values spanning nine orders of magnitude, as shown.</p

Comparison of single-cell NF-κB activation dynamics for three different LPS preparations.

<p>Intensity represents the relative nuclear localization of p65-dsRed fusion protein, calculated as mean nuclear intensity divided by initial mean cytoplasmic intensity. The concentration for all three preparations was 0.5 µg/mL. The black line shows the average time course for all cells; the light blue traces are ten randomly selected individual cells. The number of active cells (N), as well as the maximum peak amplitude (Peak Amp), and time elapsed until the maximum amplitude is reached (Time to Peak) are also shown. The maximum intensity is indicated by a dot and the two dashed lines indicate how Peak Amp and Time to Peak are determined. The duration of the first peak (Peak Width) is also shown. This value is determined by drawing a horizontal line at the intensity that is halfway between the minimum and maximum peak value. The region above the line and shaded in green denotes the time during which the p65-dsRed nuclear intensity is more than half of the maximum p65-dsRed nuclear intensity. Below each plot, corresponding representative microscope images are shown for the first 200 minutes after stimulation, as labeled.</p

The effect of blocking TNF on the NF-κB activation time courses as a function of concentration and preparation.

<p>The cosine vector distances between the average time courses in the presence and absence of sTNFRII are shown. Almost no cells were activated by UP LPS at low concentrations; hence the dashed light blue line is included for completeness but is not statistically defensible.</p

Activation dynamics for each of the LPS preparations and several concentrations and summary statistical comparisons.

<p>(A) As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053222#pone-0053222-g001" target="_blank">Figure 1</a>, the average and ten randomly selected traces of active cells are shown, as well as Time to Peak, Peak Amp and N. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053222#pone-0053222-g001" target="_blank">Figure 1</a> legend for more details. Concentrations are indicated at left, and the preparation at top. The very lowest and highest concentrations are not shown here (but appear in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053222#pone-0053222-g004" target="_blank">Figure 4</a>), because virtually no cells were found to be active in the first case, and the traces are essentially identical to their nearest neighbor in the second. (B) Time-to-peak values for each LPS preparation are shown with standard deviations, across each concentration. LPS preparations are indicated with different colors as labeled in D. (C) Peak amplitude values for each LPS preparation are shown with standard deviations across each concentration. (D) The correspondence between time-to-peak and peak amplitude is shown for each LPS preparation, including all concentrations.</p

An early, reversible cholesterolgenic etiology of diet-induced insulin resistance

Objective: A buildup of skeletal muscle plasma membrane (PM) cholesterol content in mice occurs within 1 week of a Western-style high-fat diet and causes insulin resistance. The mechanism driving this cholesterol accumulation and insulin resistance is not known. Promising cell data implicate that the hexosamine biosynthesis pathway (HBP) triggers a cholesterolgenic response via increasing the transcriptional activity of Sp1. In this study we aimed to determine whether increased HBP/Sp1 activity represented a preventable cause of insulin resistance. Methods: C57BL/6NJ mice were fed either a low-fat (LF, 10% kcal) or high-fat (HF, 45% kcal) diet for 1 week. During this 1-week diet the mice were treated daily with either saline or mithramycin-A (MTM), a specific Sp1/DNA-binding inhibitor. A series of metabolic and tissue analyses were then performed on these mice, as well as on mice with targeted skeletal muscle overexpression of the rate-limiting HBP enzyme glutamine-fructose-6-phosphate-amidotransferase (GFAT) that were maintained on a regular chow diet. Results: Saline-treated mice fed this HF diet for 1 week did not have an increase in adiposity, lean mass, or body mass while displaying early insulin resistance. Consistent with an HBP/Sp1 cholesterolgenic response, Sp1 displayed increased O-GlcNAcylation and binding to the HMGCR promoter that increased HMGCR expression in skeletal muscle from saline-treated HF-fed mice. Skeletal muscle from these saline-treated HF-fed mice also showed a resultant elevation of PM cholesterol with an accompanying loss of cortical filamentous actin (F-actin) that is essential for insulin-stimulated glucose transport. Treating these mice daily with MTM during the 1-week HF diet fully prevented the diet-induced Sp1 cholesterolgenic response, loss of cortical F-actin, and development of insulin resistance. Similarly, increases in HMGCR expression and cholesterol were measured in muscle from GFAT transgenic mice compared to age- and weight-match wildtype littermate control mice. In the GFAT Tg mice we found that these increases were alleviated by MTM. Conclusions: These data identify increased HBP/Sp1 activity as an early mechanism of diet-induced insulin resistance. Therapies targeting this mechanism may decelerate T2D development