Atypical expression of circadian clock genes in denervated mouse skeletal muscle

<div><p>The central circadian clock in the suprachiasmatic nucleus of the hypothalamus synchronizes peripheral clocks through neural and humoral signals in most mammalian tissues. Here, we analyzed the effects of unilateral sciatic denervation on the expression of circadian clock- and clock-controlled genes in the gastrocnemius muscles of mice twice per day on days 0, 3, 7, 9, 11 and 14 after denervation and six times on each of days 7 and 28 after denervation to assess the regulation mechanism of the circadian clock in skeletal muscle. Sciatic denervation did not affect systemic circadian rhythms since core body temperature (Day 7), corticosterone secretion (Days 7 and 28), and hepatic clock gene expression remained intact (Days 7 and 28). Expression levels of most circadian clock-related genes such as <i>Arntl, Per1, Rora, Nr1d1</i> and <i>Dbp</i> were reduced in accordance with the extent of muscle atrophy, although circadian <i>Per2</i> expression was significantly augmented (Day 28). Cosinor analysis revealed that the circadian expression of <i>Arntl</i> (Days 7 and 28) and <i>Dbp</i> (Day 28) was phase advanced in denervated muscle. The mRNA expression of <i>Clock</i> was significantly increased in denervated muscle on Day 3 when the severe atrophy was absent, and it was not affected by atrophic progression for 28 days. Sciatic denervation did not affect the expression of these genes in the contralateral muscle (Days 7 and 28), suggesting that humoral changes were not involved in denervation-induced muscle clock disruption. We then analyzed genome-wide gene expression using microarrays to determine the effects of disrupting the molecular clock in muscle on circadian rhythms at Day 7. Among 478 circadian genes, 313 lost rhythmicity in the denervated muscles. These denervation-sensitive genes included the lipid metabolism-related genes, <i>Nrip1, Bbs1, Ptgis, Acot1, Scd2, Hpgd, Insig1, Dhcr24, Ldlr</i> and <i>Mboat1</i>. Our findings revealed that sciatic denervation disrupts the circadian expression of clock and clock-controlled genes either directly or indirectly via muscle atrophy in the gastrocnemius muscles of mice in a gene-specific manner.</p></div

Pharmacokinetic parameters of flavonoids after oral administration of 8-PN and naringenin (50 mg/kg body weight) in a single dose in mice.

<p>C_max: maximum concentration in plasma; AUC: area under the plasma concentration–time curve; Tmax: time to maximum plasma concentration; T_1/2: half-life of flavonoid in the elimination phase.</p><p>Each flavonoid (50 mg/kg bw) was administered to mice once by stomach intubation. Plasma concentration was analyzed by HPLC–UV. Data are the mean ± S.E (n = 4). Asterisks indicate significant differences between two groups (C_max: <i>p</i> = 0.026; AUC: <i>p</i> = 0.0039, Student’s <i>t-</i>test).</p

8-PN can prevent disuse muscle atrophy by enhancing Akt phosphorylation.

<p>(a) Muscle atrophy induced by denervation. The weight of the GM was measured after denervation for the indicated period. Open bar: sham leg (left); closed bar: denervated leg (right). Data are the mean ± S.E (n = 4). Asterisks indicate significant differences between sham and the denervated leg (Student’s <i>t</i>-test, <i>p</i><0.007). (b) Effect of dietary intake of 8-PN or naringenin on muscle atrophy. Mice consumed each flavonoid-mixed diet for 18 days, and denervation was then carried out. After 4 (black bar) or 6 (white bar) days, the level of atrophy in the GM was calculated as the ratio of the weight of denervated muscle to the weight of sham muscle in each mouse. Data are the mean ± S.E (n = 4). C: control-diet group, 8-PN: 8-PN-containing diet group. Asterisks indicate significant differences to the control diet, which was analyzed by the Tukey multiple comparison test with one-way ANOVA (day 4: <i>p</i> = 0.0034; day 6: <i>p</i> = 0.041). (c) Phosphorylation of Akt and atrogin-1 in the GM (which was collected on the 6th day after denervation) was detected by western blotting (upper) and the density of each image analyzed (bottom). The black bar and white bar in left graph denote phosphorylated Akt and total Akt, respectively. Data are the mean ± S.E (n = 4). C: control-diet group, 8-PN: 8-PN-containing diet group. *Significant differences to the control diet-denervation group (<i>p</i>0.05). #Significant differences to the control diet-sham group.</p

Accumulation of 8-PN in the GM. (a)–(e): HPLC chromatograms for quantitative analyses of 8-PN or naringenin in the GM.

<p>Chromatograms from mice fed an 8-PN-containing diet (a) and control diet (b). Chromatograms from mice fed a naringenin-containing diet (c) and control diet (d). (a) and (b) were obtained by the analytical condition for 8-PN. (c) and (d) were obtained by the analytical condition for naringenin. These analyses were undertaken by HPLC with electrochemical detection. (e) Contents of these flavonoids in the GM as determined by HPLC analysis. Data are the mean ± S.E (n = 4). Different letters indicate significant differences analyzed by the Tukey multiple comparison test with two-way ANOVA (<i>p</i> = 0.00043).</p

Preventive effect of <i>Humulus lupulus</i> on disuse muscle atrophy.

<p>Mice consumed a <i>Humulus lupulus</i>-mixed diet for 14 days, after which denervation was carried out. After 4 days, the weight of the GM was measured. The level of atrophy was calculated to be the ratio of the weight of denervated muscle to the weight of sham muscle in each mouse. Data are the mean ± S.E (n = 3). Asterisks indicate significant differences analyzed by the Student’s <i>t</i>-test (P = 0.0046).</p

Conversion of xanthohumol to 8-prenylnaringenin (8-PN).

<p>Conversion of xanthohumol to 8-prenylnaringenin (8-PN).</p

Cellular uptake of 8-PN in mouse C2C12 myotubes.

<p>Differentiated C2C12 cells seeded on 60-mm dishes were used. (a) 8-PN and (b) naringenin (10 µM) were administered to the cells for the indicated time. Cell homogenates were prepared and then each flavonoid was extracted. Amounts of flavonoids were determined by HPLC with UV detection. Data are the mean ± S.E (n = 3).</p

Protein and water content in the GM.

<p>Data are the mean ± S.E. There was no significant difference upon analyses by the Tukey multiple comparison test with one-way ANOVA (<i>p</i><0.05).</p

Accumulation of 8-PN and naringenin in the GM and plasma after their dietary intake for 22 days.

<p>Blood was collected from mice fed flavonoid (5.6 mmol flavonoid/kg diet) for 22 days, and then total body reflux was carried out. The GM was collected after the reflux. Plasma was prepared from the blood as described in the Materials and Methods section. Values of the total 8-PN and total naringenin indicate the sum of aglycone and conjugated metabolites obtained by the HPLC analysis with deconjugation treatment. Amount of flavonoids was determined by HPLC analyses as described in the Materials and Methods section. Data are the mean ± S.E. Different letters indicate significant differences upon analyses by the Tukey multiple comparison test with two-way ANOVA (<i>p</i> = 1.79×10^–9).</p