Background: Obesity-related metabolic disorders are among the leading causes of morbidity and mortality in the developed world. Structural and functional changes in skeletal muscles and the microvasculature are critically involved in the mediation of obesity-related insulin resistance. A shift from expression of slow to fast type myosin heavy chain isoforms in slow twitch muscles has been shown to contribute to reduced insulin sensitivity in obesity, while in fast-twitch weight-bearing muscles no fibre type shifting was observed. Furthermore, in obesity, accumulation of lipid droplets in skeletal muscle fibres can also contribute to insulin resistance. However, it is not yet clear how intramyocellular lipid accumulation and fibre type changes are associated. Moreover, in advanced obesity with insulin resistance, reduced capillary network density in skeletal muscles and impaired capillary recruitment has been demonstrated. On the other hand, in the early stage of obesity with insulin resistance, an increased functional vascular response to insulin has been described. However, it remains unclear whether such functional vascular response is associated with morphological alterations in the capillary network, or if the switch of fibre types toward fast type isoforms also occurs in fast-twitch, non-weight-bearing muscles in the early stages of obesity with insulin resistance.
Methods: The study was carried out using eighteen 54-week-old C57BL/6JOlaHsd mice, divided into two study groups of nine mice each: the high fat diet-induced obese and the standard diet-treated lean groups. Insulin resistance status was assessed by the oral glucose tolerance test and fasting glucose measurements. We determined the capillary network characteristics using 3D analysis of 100 µm thick transverse sections of gluteus maximus muscle, and employed immunofluorescent techniques to mark the capillary endothelium and the basement membrane of capillaries and muscle fibres, which were then captured with a confocal microscope and analysed with the Ellipse software. We used the indirect immunohistochemical method to determine the myosin heavy chain isoform expression in muscle fibres. Antibodies against myosin heavy chain isoforms type 1, 2a, 2x/d and 2b were used to determine the expression of individual myosin heavy chain isoforms in successive 10 µm thick sections of gluteus maximus, gastrocnemius, plantaris and soleus muscles. We performed analysis of intramyocellular lipid content using 10 µm thick sections of gastrocnemius, plantaris and soleus muscles stained with Sudan Black B, and calculated the lipid content index as 100 times the ratio of the cross-sectional area of muscle fibre occupied by lipid droplets to the cross-sectional area of muscle fibre. The deformations of skeletal muscle thick tissue sections in horizontal plane were determined by comparison of gluteus maximus muscle fibre diameters in 10 µm thick native tissue sections to 100 µm thick fixed and immunofluorescently labelled tissue sections, respectively.
Results: Compared to the standard diet-treated lean mice, mice fed on a high fat diet had a significantly increased body mass (p = 0.0001) and basal glycaemia, and decreased glucose tolerance (p < 0.05). The obese mice also showed denser capillary network of the gluteus maximus muscle compared to lean mice. Compared to the lean mice, capillary length per muscle fibre length, and capillary length per muscle fibre surface area were significantly larger around small muscle fibres (< 40 µm) (p < 0.05) in the obese mice, while there were no significant differences around large fibres in both groups. Other capillary characterization indices such as tortuosity, anisotropy and fibre diameter did not significantly differ between the study groups. In the slow-twitch soleus and fast-twitch non-weight-bearing gluteus maximus muscles, we noted a shift towards fast type myosin heavy chain isoform expression in the obese mice (p < 0.05), while in the weight-bearing fast-twitch gastrocnemius and intermediate plantaris muscles there were no significant differences in myosin heavy chain expression between the two study groups. Moreover, in obese mice muscle fibre size and intramyocellular lipid content were significantly increased in type 2a and 2x/d muscle fibres (p<0.05), with greater prominence in the fast- and intermediate-twitch than slow-twitch skeletal muscles. During the preparation of thick transverse sections of skeletal muscle for confocal microscopy, we demonstrated significant dilation of the sections in horizontal direction (p < 0.001) and shrinkage in the axial direction (p < 0.001). In addition, we noted a positive correlation between the magnitude of horizontal dilation and the magnitude of shrinkage in the axial direction (r = 0.493, p < 0.01), the latter being more pronounced in transversely than obliquely cut tissue sections.
Conclusions: We found a selective increase in capillarisation around small muscle fibres of the more oxidative fibre types in our obese insulin-resistant mice, which could be an early compensatory mechanism ameliorating obesity-related insulin resistance. Our findings further suggest that in obesity with insulin resistance, both slow- and fast-twitch muscles exhibit the tendency for a shift toward fast type myosin heavy chain isoforms, and that increased weight bearing may condition the resistance of fast-twitch muscles to fibre type shifting. Our results also reaffirm that in obesity, intramyocellular lipid accumulation is specific for both skeletal muscle and fibre types, with greater prominence in fast-twitch muscles and muscle fibre types 1, 2a and 2x/d. Finally, our study also provide evidence on horizontal dilation and axial shrinkage of thick transverse sections of skeletal muscle. The magnitude of the former was partially dependent on the latter, suggesting that even though axial shrinkage can be corrected by calibration, histological protocols should be optimised to minimize the axial collapse that could cause horizontal dilation.
|
