Rengaraj Anantharaj1, Jadegoud Yaligar1, Giang Thi Thu Le1, Venkatesh Gopalan1, Sanjay Kumar Verma1, Kavita Kaur1, Kasthuri Thirumurugan2, Johan G Eriksson2,3, Brian Kennedy4, and S Sendhil Velan1,2
1Laboratory of Molecular Imaging, Singapore Bioimaging Consortium, A*STAR, Singapore, Singapore, 2Singapore Institute for Clinical Sciences, A*STAR, Singapore, Singapore, 3Department of Obstetrics & Gynecology, National University of Singapore, Singapore, Singapore, 4Center for Healthy Aging, National University of Singapore, Singapore, Singapore
Synopsis
Aging associated loss of
muscle mass leads to metabolic diseases
and compromised quality of life. Increase in
intramyocellular lipid (IMCL) and reduced skeletal muscle mass are associated
with insulin resistance and diabetes. Rapamycin
increases muscle mass by inhibiting the mTOR signalling pathway. In this study, we investigated IMCL
metabolism and muscle mass in response to rapamycin intervention in an
aging rodent model. We observed significant reduction in IMCL along with increase in muscle mass indicating
improved muscle metabolism with rapamycin intervention. Muscle differentiation
gene, MyoD was
upregulated and myostatin which is negative regulator of
muscle growth factor was down regulated.
Introduction
Aging is associated with
loss of skeletal muscle mass or Sarcopenia.
Muscle tissue is critical for glucose disposal and loss of muscle mass
may contribute to the development and progression of type 2 diabetes through compromised
glucose disposal and with accumulation of intramyocellular lipid (IMCL)1. Interventions increasing muscle
mass and reducing IMCL are of great clinical interest both in relation to aging
and diabetes. Rapamycin increases muscle mass and extends the life span of mice
by inhibiting the mechanistic target of rapamycin (mTOR) signalling pathway2.Animals and Methods
Twelve months old, male Wister rats (n=10) maintained on chow diet were randomized into group 1
(vehicle, n=5) and group 2 (Rapamycin, n = 5). Rapamycin at a concentration of 20 mg/ml, in
ethanol was resuspended in vehicle (0.25% PEG, 0.25% Tween-80) at a final concentration
of 4 mg/ml, and administered (8 mg/kg BW,
route i.p) on alternative days for 2
weeks. MR imaging/spectroscopy was performed using a
9.4T Bruker BioSpec (Bruker,
Germany, ParaVision 6.0.1). A 72-mm transmit /receive volume coil and a phase
array receive-only coil was utilized for all the experiments. Anatomical imaging was performed by high
resolution gradient-echo 3D T1 weighted imaging with TR of 6.2 ms; TE of 2.46
ms; FOV with 78 ×78 mm2; matrix size 256 × 256; 30 slices with 1 mm
thickness. 3D multi-echo (6 echoes) GRE sequence was utilized for fat-water
imaging with repetition time TR 11 ms, TE’s (1.28, 1.47, 1.66, 1.85, 2.04 and 2.23
ms); flip angle 40, field of view, 78 × 78 mm2; matrix
size 256 × 256; 30 slices with 1 mm thickness. A water suppressed localized spectrum from tibialis anterior muscle was obtained using PRESS sequence with TR /TE 4000/17.08 ms, voxel volume 3.5 × 3.5 × 3.5 mm3, averages 256. A
water unsurpassed spectrum was collected from the same voxel. Muscle volumes were
estimated by manual segmentation of high resolution images using ITK-SNAP3. IMCL was quantified by processing the MRS data using
LC-model4. After the terminal MR imaging, oral glucose tolerance test was performed
on all animals. Relative mRNA expression of MyoD and Myostatin
levels were estimated RT-PCR.. Haematoxylin (H) and eosin (E) staining was
performed on 5µm tissue sections of tibialis anterior muscle tissue. Results were compared
between vehicle and rapamycin groups and statistical
significance was
calculated by welch’s t test. Statistical
significance (P < 0.05) was calculated between
two groups.Results and Discussion
Figure 1 shows body weight in the vehicle and
rapamycin treated groups. The rapamycin
group showed significant reduction in body weight after 2 weeks of intervention
(P < 0.01). Figure 2A
shows increased (P
< 0.01) postprandial
glucose concentrations in the rapamycin group indicating impaired
glucose-stimulated insulin release. Earlier studies have also shown that
rapamycin negatively impacts glucose stimulated insulin release by pancreatic
beta cells in C57BL/6 mice5. Plasma triglycerides (Figure 2B) were significantly
(P < 0.01) reduced in the rapamycin group compared to the vehicle treated
group. Figure 3 shows the representative T1
weighted coronal images from skeletal muscle of vehicle (A) and rapamycin treated
animals. Figure 3B shows the segmented
muscle volumes for both groups. The muscle volume increased significantly (P
< 0.01) in the rapamycin treated group. Rapamycin, inhibits the mTOR
signalling pathway and improved/increases the skeletal muscle volumes6. Figure
4A shows the skeletal muscle IMCL measured from tibialis anterior compartment for both vehicle and
rapamycin treated groups. The IMCL is significantly (P <
0.05) reduced
in rapamycin treated animals compared to vehicle group. Reduction in IMCL and
increase in muscle mass with rapamycin indicates the enhanced lipid utilization
due to expansion of muscle mass. Figure 4B shows the IMCL levels before
intervention (day 0) and after two weeks of intervention. Increase in muscle volume and muscle fibres in
the rapamycin group is further confirmed by H & E stained sections (Figure
5A). Figure 5B shows the mRNA analysis of muscle markers
MyoD (P < 0.05) and myostatin (P < 0.05). Muscle differentiation marker
MyoD is increased in the rapamycin group supporting improved muscle differentiation7. In aging, myostatin is known to inhibit muscle differentiation8. In the
rapamycin treated group myostatin levels were significantly reduced promoting
muscle differentiation. Conclusions
Rapamycin
treatment significantly increased muscle mass along with reduction in IMCL,
body weight and plasma triglycerides. Increased postprandial glucose in
rapamycin treated group is due to impaired glucose-stimulated insulin release9. Increased myogenic differentiation marker
MyoD in the rapamycin group is positively associated with functional effects of
the skeletal muscle in aged rats. Reduction
of myostatin
is in agreement with increased muscle volumes in the rapamycin treated group. Further studies are required to evaluate effect of rapamycin on glucose-insulin metabolism. Acknowledgements
No acknowledgement found.References
- Cleasby
ME et al Insulin resistance and sarcopenia: mechanistic links between common
co-morbidities J Endocrinol. 2016 May;229(2): R67-81.
- Kennedy
BK et al Rapamycin reverses elevated mTORC1 signaling in lamin A/C-deficient
mice, rescues cardiac and skeletal muscle function, and extends survival Sci
Transl Med. 2012 Jul 25;4(144):144ra103.
- Paul A.
Yushkevich, Joseph Piven, Heather Cody Hazlett, Rachel Gimpel Smith, Sean Ho,
James C. Gee, and Guido Gerig. User-guided 3D active contour segmentation of
anatomical structures: Significantly improved efficiency and reliability.
Neuroimage 2006 Jul 1;31(3):1116-28.
- Provencher
SW. Automatic quantitation of localized in vivo 1H spectra with LCModel. NMR
Biomed. 2001; 14:260-264.
- Yang
SB, Lee HY, Young DM, Tien AC, Rowson-Baldwin A, Shu YY, Jan YN, Jan LY (2012)
Rapamycin induces glucose intolerance in mice by reducing islet mass, insulin
content, and insulin sensitivity. J. Mol. Med. (Berl) 90, 575–585.
- Mee-Sup Yoon, mTOR as a Key Regulator in Maintaining
Skeletal Muscle Mass Front Physiol. 2017; 8: 788.
- Jeff Ishibashi et al, MyoD induces myogenic
differentiation through cooperation of its NH2- and COOH-terminal
regions J Cell Biol. 2005 Nov 7; 171(3): 471–482.
- Langley
B et al, Myostatin inhibits myoblast differentiation by down-regulating MyoD
expression J Biol Chem. 2002 Dec 20;277(51):49831-40.
- Lamming
DW et al, Young and old genetically heterogeneous HET3 mice on a rapamycin diet
are glucose intolerant but insulin sensitive Aging Cell. 2013 Aug;12(4):712-8.