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. 2017 Aug;16(4):847-858.
doi: 10.1111/acel.12613. Epub 2017 May 29.

Sustained NFκB Inhibition Improves Insulin Sensitivity but Is Detrimental to Muscle Health

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Free PMC article

Sustained NFκB Inhibition Improves Insulin Sensitivity but Is Detrimental to Muscle Health

Ning Zhang et al. Aging Cell. .
Free PMC article

Abstract

Older adults universally suffer from sarcopenia and approximately 60-70% are diabetic or prediabetic. Nonetheless, the mechanisms underlying these aging-related metabolic disorders are unknown. NFκB has been implicated in the pathogenesis of several aging-related pathologies including sarcopenia and type 2 diabetes and has been proposed as a target against them. NFκB also is thought to mediate muscle wasting seen with disuse, denervation, and some systemic diseases (e.g., cancer, sepsis). We tested the hypothesis that lifelong inhibition of the classical NFκB pathway would protect against aging-related sarcopenia and insulin resistance. Aged mice with muscle-specific overexpression of a super-repressor IκBα mutant (MISR) were protected from insulin resistance. However, MISR mice were not protected from sarcopenia; to the contrary, these mice had decreases in muscle mass and strength compared to wild-type mice. In MISR mice, NFκB suppression also led to an increase in proteasome activity and alterations in several genes and pathways involved in muscle growth and atrophy (e.g., myostatin). We conclude that the mechanism behind aging-induced sarcopenia is NFκB independent and differs from muscle wasting due to pathologic conditions. Our findings also indicate that, while suppressing NFκB improves insulin sensitivity in aged mice, this transcription factor is important for normal muscle mass maintenance and its sustained inhibition is detrimental to muscle function.

Keywords: NFκB; aging; insulin resistance; sarcopenia; skeletal muscle.

Figures

Figure 1
Figure 1
Improved insulin action in MISR mice. Glucose infusion rate (GIR) required to maintain euglycemia and M value in 3‐ and 18‐month‐old male mice. (A) GIR during the clamp. (B) Mean GIR during the last 30 min of the clamp. (C) M value. = 4–5 per group. *< 0.05 by two‐way ANOVA followed by Tukey's post hoc test. All data are means ± SE. (D) Akt Ser473 phosphorylation assessed by Western blotting in quadriceps muscle collected at the end of the insulin clamp. Representative blots are shown for each group.
Figure 2
Figure 2
Ceramide and acylcarnitine content in muscles from MISR mice. (A) Ceramide and sphingolipid content in quadriceps muscle.. n = 3–6 per group. (B) mRNA levels of sptlc1 (SPT) in WT (white) and MISR (black) mice. SPT catalyzes the rate‐limiting step in the de novo synthesis of sphingolipids. For B, = 4–6 per group. (C) Acylcarnitine content in quadriceps. = 3–6 per group. (D) mRNA levels of the carnitine biosynthetic enzyme aldh9a1 (TMABADH). = 5–6 per group. (E) Carnitine muscle content. (F) Muscle content of the precursor lysine. For panels E and F, = 3–6 per group. †Genotype effect < 0.05 by two‐way ANOVA; §Age effect < 0.05 by two‐way ANOVA; *< 0.05 by Tukey's post hoc test. All data are means ± SE.
Figure 3
Figure 3
NFκB suppression leads to reduced muscle mass and function. (A) Whole‐body mass and (B) total lean mass in male wild‐type (WT) and MISR mice (cohort 1; = 9–20 per group). Wet weight (normalized to total weight) of (C) gastrocnemius, (D) quadriceps, (E) tibialis anterior (TA), and (F) soleus muscles; = 5 per group. (G) Grip strength; = 9–20 per group. (H) Force generation in electrically stimulated hindlimb muscles in WT (white) and MISR (black) mice; = 5–9 per group (I) Sciatic nerve conduction velocity; = 5–6 per group. All mice were male. *< 0.05 by two‐way ANOVA followed by Tukey's post hoc test. All data are means ± SE.
Figure 4
Figure 4
Genes and pathways regulated by aging and NFκB suppression. (A) Number of differentially expressed genes (DEGs) in WT vs. MISR (< 0.05). Gene lists in these groups are provided in Table S1 (Supporting information) . (B) Gene Ontology and IPA analyses of pathways/gene sets differentially regulated by aging and NFκB suppression (p‐values and gene lists for each corresponding functions are provided in Table S2). (C–K) mRNA levels of mstn, igfbp5, fgfbp1, myod1, map3k14 (NIK), tbkbp1, traf2, traf3, and c‐met in quadriceps muscle from WT (white) and MISR (black) mice. = 5–6 per group. Data analyzed by two‐way ANOVA; *< 0.05 by Tukey's post hoc test. All data are means ± SE.
Figure 5
Figure 5
NFκB suppression promotes muscle cell differentiation. C2C12 myoblasts were transduced with Ad‐IκBα‐SR or Ad‐GFP, as described in the Methods. (A) Mstn mRNA level was measured by real‐time RTPCR. NIK (B) and TRAF2 (C) protein content was measured by capillary electrophoresis. *< 0.05 from two‐way ANOVA followed by Tukey's post hoc test. Data are means ± SE from three independent experiments performed with an = 2–6 per condition per experiment. (D) C2C12 myoblasts were transduced with Ad‐IκBα‐SR or Ad‐GFP. Fast myosin (SKM) heavy‐chain protein was measured by Western blotting during progressing differentiation stages. *< 0.05 Ad‐IκBα‐SR vs. Ad‐GFP and §< 0.05 Ad‐IκBα‐SR vs no‐virus from two‐way ANOVA followed by Tukey's post hoc test. Data are means ± SE from one experiment done with an = 4 per condition, and the experiment was repeated two more times with similar results.
Figure 6
Figure 6
Elevated proteasome activity in MISR mice. (A) 20S and (B) 26S proteasome activity in quadriceps muscle from WT and MISR male mice. = 4 per group. (C) Total proteasomal protein content represented by the α4 subunit of the 20S proteasome core in quadriceps muscle from WT (white) and MISR (black) mice. = 5 per group. (D) Psme4 (PA200), (E) fbxo32 (atrogin), and (F) trim63 (MuRF1) mRNA levels in quadriceps. = 5–6 per group. Data analyzed by two‐way ANOVA; *< 0.05 by Tukey's post hoc test. All data are means ± SE.

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