Hepatic Pdk4 knockdown moderately improves glucose tolerance in IrsLDKO mice.

<p>A, Gene knockdown efficiency was analyzed by real-time PCR in IrsLDKO livers transduced with shRNA adenoviruses against GFP (shGFP), Pdk2 (shPdk2), or Pdk4 (shPdk4). B, Glucose tolerance tests were performed in shRNA adenoviruses infected IrsLDKO mice. C, Area under curve analysis (AUC) was performed for the above glucose tolerance test data. Data are presented as means ± SEM, n = 4–5. *, <i>P</i><0.05 relative to corresponding controls.</p

Ablation of Pdks improves glucose tolerance in IrsLDKO mice.

<p>A, Glucose tolerance tests (GTT) were performed in age-matched control and knockout mice (n = 8–12). B and C, Expression of gluconeogenic genes <i>Pck1</i> and <i>G6pc</i> was analyzed in the liver of overnight fasted control and knockout mice (n = 3). Data are presented as means ± SEM. *, <i>P</i><0.05 relative to corresponding controls.</p

Knockout of the <i>Pdk</i> genes in wild-type and IrsLDKO mice.

<p>A, Control wild-type and IrsLDKO mice (n = 3) were fasted overnight for 16 hours and half of them were fed for 4 hours immediately after the fasting. <i>Pdks</i> gene expression in the liver was analyzed by real-time PCR and data were normalized to an internal control gene — Ppia. B, Western blot analysis of liver lysates from control and knockout mice. C, Body weight measurements in control and knockout mice (n = 6–20). D, Serum triglycerides (TG) were measured in overnight fasted control and knockout mice (n = 6–8). E, Liver TG analysis in control and knockout mice (n = 6–8). Pdk2KO, Pdk2 knockout; Pdk4KO, Pdk4 knockout; IrsLDKO, Irs1/2 liver-specific double knockout. Data are presented as means ± SEM. *, <i>P</i><0.05 relative to corresponding controls.</p

Deletion of the <i>Pdk4</i> gene improves hyperglycemia in IrsLDKO mice.

<p>A, Blood glucose was measured in overnight fasted control and knockout mice. B, Blood glucose was measured in <i>ad libitum</i> fed control and knockout mice. Data are presented as means ± SEM, n = 8–23. *, <i>P</i><0.05 relative to corresponding controls.</p

Pdk2 or Pdk4 knockdown has no significant effect on insulin tolerance in IrsLDKO mice.

<p>A, Insulin tolerance tests were performed on IrsLDKO mice (n = 4–5) that were injected with shGFP, shPdk2, and shPdk4 adenoviruses. B, Area under curve was analyzed for the above ITT data. C–F, Akt phosphorylation was analyzed in the liver and skeletal muscle of mice injected with shGFP, shPdk2, and shPdk4 adenoviruses. Western blot signals were also quantified using the Quantity One software. Data are presented as means ± SEM. *, <i>P</i><0.05 relative to corresponding controls.</p

Insulin signaling analysis in the control and knockout mice.

<p>A and B, Animals were stimulated with 5 units of human insulin (saline as a vehicle control) for 3 min before liver and skeletal muscle samples were collected for Akt and Erk phosphorylation analyses. Western blot signals were quantified using the Quantity One software (Bio-Rad). Data are presented as means ± SEM. *, <i>P</i><0.05 relative to corresponding controls.</p

Inactivation of Pdks improves insulin sensitivity in IrsLDKO mice.

<p>A, Insulin tolerance tests (ITT) were performed in age-matched control and knockout mice (n = 8–20). B, Fasting plasma insulin was analyzed in age-matched control and knockout mice (n = 5–9). C, HOMA-IR (homeostatic model assessment-insulin resistance) was analyzed using fasting glucose and insulin data. Data are presented as means ± SEM. *, <i>P</i><0.05 relative to corresponding controls.</p

Establishing a Proteomics-Based Monocyte Assay To Assess Differential Innate Immune Activation Responses

Innate immune cells are complex systems that can be simultaneously activated in a variety of ways. Common methods currently used to estimate the response of innate immune cells to stimuli are usually biased toward a single mode of activation. The aim of this study was to assess the possibility of designing an assay based on unbiased proteome analysis that would be capable of predicting the complex response of the innate immune system to various challenges. Monocytes were used as representative cells of the innate immune system. The underlying hypothesis was that their proteome response to different activating molecules would reflect the immunogenicity of these molecules. To identify the main modes of response, we treated the human monocytic THP-1 cell line with nine different stimuli. Differentiation and activation were determined to be the two major modes of monocyte response, with PMA causing the strongest differentiation and Pam3CSK4 causing the strongest proinflammatory activation. The established assay was applied to characterize the monocyte response to epidermal growth factor peptide containing isoaspartate, which induced differentiation but not proinflammatory activation. Because of its versatility, robustness, and specificity, this new assay is likely to find a niche among the more established immunological methods

Proteomics Reveals a Role for Attachment in Monocyte Differentiation into Efficient Proinflammatory Macrophages

Monocytes are blood-borne cells of the innate immune system. They can be differentiated and activated into proinflammatory macrophages that might be employed in tumor immune therapy. Monocyte exposure to lipopolysaccharide (LPS) is a standard method to induce a pro­inflammatory macrophage state, with the resultant population comprising both adherent and nonadherent cells. In the current study, we aimed to identify the differences in proteomes of these monocyte subpopulations, which addresses a more general question about the role of attachment in monocyte differentiation. Label-free proteomics of a model of human monocytes (THP-1 cell line) revealed that the cells remaining in suspension upon LPS treatment were activated by cytokines and primed for rapid responsiveness to pathogens. In terms of proteome change, the adhesion process was orthogonal to activation. Adherent cells exhibited signs of differentiation and enhanced innate immune responsivity, being closer to macrophages. These findings indicate that adherent, LPS-treated cells would be more appropriate for use in tumor therapeutic applications

Proteomics Reveals a Role for Attachment in Monocyte Differentiation into Efficient Proinflammatory Macrophages

Monocytes are blood-borne cells of the innate immune system. They can be differentiated and activated into proinflammatory macrophages that might be employed in tumor immune therapy. Monocyte exposure to lipopolysaccharide (LPS) is a standard method to induce a pro­inflammatory macrophage state, with the resultant population comprising both adherent and nonadherent cells. In the current study, we aimed to identify the differences in proteomes of these monocyte subpopulations, which addresses a more general question about the role of attachment in monocyte differentiation. Label-free proteomics of a model of human monocytes (THP-1 cell line) revealed that the cells remaining in suspension upon LPS treatment were activated by cytokines and primed for rapid responsiveness to pathogens. In terms of proteome change, the adhesion process was orthogonal to activation. Adherent cells exhibited signs of differentiation and enhanced innate immune responsivity, being closer to macrophages. These findings indicate that adherent, LPS-treated cells would be more appropriate for use in tumor therapeutic applications