Alteration of JNK-1 Signaling in Skeletal Muscle Fails to Affect Glucose Homeostasis and Obesity-Associated Insulin Resistance in Mice

<div><p>Obesity and associated metabolic disturbances, such as increased circulating fatty acids cause prolonged low grade activation of inflammatory signaling pathways in liver, skeletal muscle, adipose tissue and even in the CNS. Activation of inflammatory pathways in turn impairs insulin signaling, ultimately leading to obesity-associated type 2 diabetes mellitus. Conventional JNK-1 knock out mice are protected from high fat diet-induced insulin resistance, characterizing JNK-1-inhibition as a potential approach to improve glucose metabolism in obese patients. However, the cell type-specific role of elevated JNK-1 signaling as present during the course of obesity has not been fully elucidated yet. To investigate the functional contribution of altered JNK-1 activation in skeletal muscle, we have generated a ROSA26 insertion mouse strain allowing for Cre-activatable expression of a JNK-1 constitutive active construct (JNK^C). To examine the consequence of skeletal muscle-restricted JNK-1 overactivation in the development of insulin resistance and glucose metabolism, JNK^C mice were crossed to Mck-Cre mice yielding JNK^SM-C mice. However, despite increased muscle-specific JNK activation, energy homeostasis and glucose metabolism in JNK^SM-C mice remained largely unaltered compared to controls. In line with these findings, obese mice with skeletal muscle specific disruption of JNK-1, did not affect energy and glucose homeostasis. These experiments indicate that JNK-1 activation in skeletal muscle does not account for the major effects on diet-induced, JNK-1-mediated deterioration of insulin action and points towards a so far underappreciated role of JNK-1 in other tissues than skeletal muscle during the development of obesity-associated insulin resistance.</p> </div

Unaltered glucose metabolism and insulin sensitivity in JNK^SM-C mice.

<p>(A) Glucose tolerance tests of control (white bar) and JNK^SM-C (grey bar) mice when feeding a NCD were performed at 11 weeks of age (n = 10–13). (B) Insulin tolerance tests of control (white bar) and JNK^SM-C (grey bar) mice upon NCD feeding were performed at 12 weeks of age (n = 13–15). (C) Insulin levels from sera isolated at week 17 from mice with the indicated genotypes upon NCD feeding determined by ELISA (n = 8–10). (D) Representative insulin-induced AKT phosphorylation of muscle and liver lysates isolated from control and JNK^SM-C mice when feeding NCD using Western Blot analysis with the indicated antibodies. Values are means ± SEM.</p

Muscle specific JNK overactivation.

<p>(A) Western Blot analysis (IB) of protein lysates isolated from muscles of WT mice at 20 weeks of age when exposed to NCD and HFD using P-JNK and JNK antibodies. (B) JNK-kinase assay of protein lysates isolated from muscles of WT mice at 20 weeks of age when exposed to NCD and HFD. Calnexin antibodies were used for input control. (C) Quantitation of radioactively labeled cJun peptide in skeletal muscle of NCD fed WT and HFD WT mice. (D) Targeting strategy for the Cre-activatable JNK^C construct into the ubiquitously expressed ROSA26 locus. The CAG modified STOP-EGFP-ROSA targeting vector was described elsewhere. In the unique AscI site, a fusion cDNA between the mutant MKK7D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054247#pone.0054247-Wang1" target="_blank">[36]</a> and JNK-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054247#pone.0054247-Zheng1" target="_blank">[37]</a> was inserted to generate the final ROSA JNK^C targeting vector. Homologous recombination between the homology arms and the genomic ROSA26 locus generated the targeted allele which was identified by Southern Blot analysis of EcoRI-digested clonal DNA using the ROSA probe <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054247#pone.0054247-Mao1" target="_blank">[38]</a> resulting in the 16 kb WT band besides the 6 kb targeted band as shown in (E). Such positive C57/BL6-derived ES cell clones were used to generate the ROSA JNK^C mouse strain. (F) Determination of relative JNK-1 mRNA expression in skeletal muscle of WT and JNK^SM-C mice by quantitative realtime PCR using Hprt1, Gusb and Tfrc as housekeeping genes (n = 8). (G) Western Blot analysis of protein lysates isolated from skeletal muscle of WT and JNK^SM-C mice using JNK-1 and AKT antibodies. (H) Expression of the JNK^C fusion protein only in skeletal muscle of JNK^SM-C mice indicated by Western blot analysis. (I) Functionality of the JNK^C construct in skeletal muscle of JNK^SM-C mice was revealed by increased phosphorylation of cJun in JNK kinase assay (KA) experiments. (J) Quantitation of radioactively labeled cJun peptide in skeletal muscle of NCD fed WT, JNK^SM-C and HFD WT mice. Values are means ± SEM. ***, p≤0.001.</p

Unaltered glucose metabolism and insulin sensitivity in JNK-1^SM-KO under normal and obese conditions.

<p>(A) Glucose tolerance tests of control NCD fed (open circles) and HFD fed (open squares) mice and JNK-1^SM-KO mice fed a NCD (black circles) or a HFD (black squares) were performed at 11 weeks of age (n = 9–49). (B) Insulin tolerance tests of control NCD fed (open circles) and HFD fed (open squares) mice and JNK-1^SM-KO fed a NCD (black circles) or a HFD (black squares) were performed at 12 weeks of age (n = 5–30). (C) Insulin levels from sera isolated at week 17 from mice with the indicated genotypes upon NCD or HFD feeding determined by ELISA (n = 5–9). (D) Representative insulin-induced AKT phosphorylation of muscle and liver lysates isolated from control and JNK-1^SM-KO mice when feeding NCD and HFD using Western Blot analysis with the indicated antibodies. Values are means ± SEM. **, p≤0.01; ***, p≤0.001.</p

Unaltered body composition and energy homeostasis in JNK-1^SM-KO mice under normal and obese conditions.

<p>(A) The average bodyweight of control NCD fed (open circles) and HFD fed (open squares) mice was compared with JNK-1^SM-KO mice fed a NCD (black circles) or a HFD (black squares) from 3 to 17 weeks of age (n = 15–45). (B) Body length of control (white bar) and JNK-1^SM-KO (black bar) mice upon NCD and HFD feeding. (C) Body composition of control (white bar) and JNK-1^SM-KO (black bar) mice when exposed to NCD or HFD were determined by using a Brucker minispec in week 17 (n = 11–24). (D) Weight of epigonadal fat pads from NCD and HFD fed control (white bar) and JNK-1^SM-KO (black bar) mice in week 17 (n = 13–40). (E) Serum leptin levels of control (white bar) and JNK-1^SM-KO (black bar) mice upon NCD and HFD feeding at the age of 17 weeks (n = 8–18). (F) Daily food intake of control (white bar) and JNK-1^SM-KO (black bar) mice upon HFD feeding at the age of 14 weeks (n = 5–27). (G) Energy expenditure revealed by the daily and nightly volume of O₂ consumption and CO₂ release of control (white bar) and JNK-1^SM-KO (black bar) mice upon HFD feeding (n = 8). (H) Respiratory exchange rate (RER) of control (white bar) and JNK-1^SM-KO (black bar) mice upon HFD feeding (n = 8). Values are means ± SEM. **, p≤0.01; ***, p≤0.001.</p

<i>CaMKIIa-Cre</i>-mediated inactivation of the mouse <i>Phb2</i> gene in forebrain neurons.

<p>(A) <i>In-situ</i> hybridization of <i>Phb2</i> mRNA in the hippocampus of 8-week-old <i>Phb2^NKO</i> and <i>Phb2^fl/fl</i> control mice. Scale bar: 500 µm. (B) Immunoblot analysis of tissue lysates generated from the indicated brain regions of <i>Phb2^NKO</i> (KO) and <i>Phb2^fl/fl</i> (WT) control mice of different age using PHB1- and PHB2-specific antibodies. Ponceau S (PoS) staining was used to monitor equal gel loading. Cortex (CO), striatum (ST), hippocampus (HC), cerebellum (CB). (C) Representative photographs of 20-week-old <i>Phb2^NKO</i> mice of the indicated genotypes showing lordokyphosis (left panel) and excessive pathological grooming (right panel). White arrows indicate regions of self-inflicted open skin lesions. (D) Body weight analysis of <i>Phb2^NKO</i> and <i>Phb2^fl/fl</i> control animals. n = 20. ***<i>P</i><0.001. Error bars indicate SEM. (E) Kaplan-Meier survival plot of <i>Phb2^NKO</i> (n = 30) and control animals (<i>Phb2^fl/fl</i> (n = 59), <i>Phb2^HET</i> (n = 19)). <i>P</i><0.0001.</p

Defective mitochondrial morphogenesis and ultrastructure in <i>Phb2</i>-deficient neurons <i>in vivo</i>.

<p>(A) Transmission electron microscopy analysis of the mitochondrial ultrastructure in DG neurons of 6-week-old <i>Phb2^NKO</i> and <i>Phb2^fl/fl</i> control mice. The enlargements show the double membrane of the mitochondrion and the emergence of one crista. Scale bar: 400 nm. (B) Fragmentation and perinuclear clustering of PHB2-deficient neuronal mitochondria. Primary hippocampal neurons isolated from E18.5 <i>Phb2^fl/fl</i> embryos were infected with lentiviruses expressing mitochondrially targeted EGFP and Cre recombinase (NLS-Cre) as indicated. Fixed samples were immunostained with antibodies directed against GFP and neuronal <i>β</i>III-tubulin followed by DAPI staining. a′, b′ are magnifications of the boxed insets shown in a, b. Scale bars: 10 µm. (C) Quantification of mitochondrial morphology in PHB2-deficient and control primary hippocampal neurons. Cells were infected with lentiviruses expressing Cre recombinase when indicated and processed as described in (B). Cells containing tubular (white bars) or fragmented mitochondria (red bars) were classified. >200 cells were scored in three independent experiments. ***<i>P</i><0.001. Error bars indicate SEM. (D) Quantification of mitochondria per neurites in PHB2-deficient primary hippocampal neurons. <i>Phb2^fl/fl</i> neurons were infected with lentiviruses expressing Cre recombinase when indicated and processed as described in (B). >30 cells were scored in three independent experiments. **<i>P</i><0.01. Error bars indicate SEM. (E) Immunoblot analysis of hippocampal tissue lysates from <i>Phb2^NKO</i> (KO) and <i>Phb2^fl/fl</i> (WT) control mice of the indicated age. Lysates were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Antibodies directed against VDAC and the 70 kDa subunit of complex II were used to monitor equal gel loading. b/e: long/short OPA1 isoforms.</p

Interleukin-6 Signaling in Liver-Parenchymal Cells Suppresses Hepatic Inflammation and Improves Systemic Insulin Action

The contribution of interleukin (IL)-6 signaling in obesity-induced inflammation remains controversial. To specifically define the role of hepatic IL-6 signaling in insulin action and resistance, we have generated mice with hepatocyte-specific IL-6 receptor (IL-6R) a deficiency (IL-6RaL-KO mice). These animals showed no alterations in body weight and fat content but exhibited a reduction in insulin sensitivity and glucose tolerance. Impaired glucose metabolism originated from attenuated insulin-stimulated glucose transport in skeletal muscle and fat. Surprisingly, hepatic IL-6Ra-disruption caused an exaggerated inflammatory response during euglycemic hyperinsulinemic clamp analysis, as revealed by increased expression of IL-6, TNF-a, and IL-10, as well as enhanced activation of inflammatory signaling such as phosphorylation of IkBa. Neutralization of TNF-a or ablation of Kupffer cells restored glucose tolerance in IL-6RaL-KO mice. Thus, our results reveal an unexpected role for hepatic IL-6 signaling to limit hepatic inflammation and to protect from local and systemic insulin resistance