Quantitative Ultrastructural Morphometry and Gene Expression of mTOR-Related Mitochondriogenesis within Glioblastoma Cells

In glioblastoma (GBM) cells, an impairment of mitochondrial activity along with autophagy suppression occurs. Autophagy suppression in GBM promotes stemness, invasion, and poor prognosis. The autophagy deficit seems to be due, at least in part, to an abnormal up-regulation of the mammalian target of rapamycin (mTOR), which may be counteracted by pharmacological mTORC1 inhibition. Since autophagy activation is tightly bound to increased mitochondriogenesis, a defect in the synthesis of novel mitochondria is expected to occur in GBM cells. In an effort to measure a baseline deficit in mitochondria and promote mitochondriogenesis, the present study used two different GBM cell lines, both featuring mTOR hyperactivity. mTORC1 inhibition increases the expression of genes and proteins related to autophagy, mitophagy, and mitochondriogenesis. Autophagy activation was counted by RT-PCR of autophagy genes, LC3- immune-fluorescent puncta and immune-gold, as well as specific mitophagy-dependent BNIP3 stoichiometric increase in situ, within mitochondria. The activation of autophagy-related molecules and organelles after rapamycin exposure occurs concomitantly with progression of autophagosomes towards lysosomes. Remarkably, mitochondrial biogenesis and plasticity (increased mitochondrial number, integrity, and density as well as decreased mitochondrial area) was long- lasting for weeks following rapamycin withdrawal.

Influence of endurance exercise training and sex on intramyocellular lipid and mitochondrial ultrastructure, substrate use, and mitochondrial enzyme activity

Impaired mitochondrial function and structure and intramyocellular lipid (IMCL) accumulation have been associated with obesity and Type 2 diabetes. We examined whether endurance exercise training and sex influenced IMCL and mitochondrial morphology using electron microscopy, whole-body substrate use, and mitochondrial enzyme activity. Untrained men ( n = 5) and women ( n = 7) were tested before and after 7 wk of endurance exercise training. Testing included 90 min of cycle ergometry at 60% V̇o2 peak with preexercise muscle biopsies analyzed for IMCL and mitochondrial size/area using electron microscopy and short-chain β-hydroxyacyl-CoA dehydrogenase (SCHAD) and citrate synthase (CS) enzyme activity. Training increased the mean lipid area density ( P = 0.090), the number of IMCL droplets ( P = 0.055), the number of IMCL droplets in contact with mitochondria ( P = 0.010), the total mitochondrial area ( P < 0.001), and the size of individual mitochondrial fragments ( P = 0.006). Women had higher mean lipid area density ( P = 0.030) and number of IMCL droplets ( P = 0.002) before and after training, but higher individual IMCL area only before training ( P = 0.013), compared with men. Women oxidized more fat ( P = 0.027) and less carbohydrate ( P = 0.032) throughout the study. Training increased V̇o2 peak ( P < 0.001), %fat oxidation ( P = 0.018), SCHAD activity ( P = 0.003), and CS activity ( P = 0.042). In summary, endurance exercise training increased IMCL area density due to an increase in the number of lipid droplets, whereas the increase in total mitochondrial area was due to an increase in the size of individual mitochondrial fragments. In addition, women have higher IMCL content compared with men due mainly to a greater number of individual droplets. Finally, endurance exercise training increased the proportion of IMCL in physical contact with mitochondria.

Ultrastructural changes in skeletal muscle after fatiguing exercise

Thoroughbred horses were exercised to fatigue at 40, 85, and 100% of their maximal oxygen consumption (VO2max) on a treadmill and completed a 1,600-m gallop on a track to identify the effect of exercise of various durations and intensities on the ultrastructure of mitochondria and sarcoplasmic reticulum (SR) from the middle gluteal muscle. The percentage of the total area occupied by mitochondria and SR increased in electron micrographs of muscle samples collected at the termination of exercise and at 30 and 60 min of recovery compared with those collected before exercise. Mitochondrial area increased 3- to 4-fold and SR area approximately 1.6-fold after exercise at the intensities greater than 40% of the VO2max. Smaller increases occurred in response to exercise at 40% of the VO2max. Areas were not different from rest in samples collected after 60 min of recovery. The reversal of ultrastructural alterations paralleled the trend toward normalization of muscle temperature, muscle pH, and the concentrations of selected muscle metabolites.

Morphometric analysis of reserve substances and ultrastructural changes during caulogenic determination and loss of competence of Eastern White pine (Pinus strobus) cotyledons in vitro

Lipid, starch, and protein reserves were monitored cytochemically and, along with certain ultrastructural changes, were quantified morphometrically during adventitious shoot determination or loss of competence in cotyledons from cultured Pinus strobus zygotic embryos. Initial expiants were rich in lipid, which declined during culture. There was more lipid retention in explants on cytokinin medium by day 5 than on basal medium. However, by day 7, five- and six-celled clusters showed greater lipid utilization than most other cells on cytokinin, which may have indicated a shift towards shoot determination in these cells. Except for an initially greater retention on cytokinin, a similar pattern for storage protein degradation was observed in both treatments. Starch levels increased during culture, but did not differ between treatments during the first 7 days. Vacuolation increased during culture and was greater on basal medium than on cytokinin. Relative nuclear size also increased, but was greater on cytokinin. Relative mitochondrial area increased during culture, except in noncluster cells on basal medium. While few differences were noted between cell types within each treatment, distinct differences in nuclear size, vacuolation, and lipid content existed between cells on cytokinin and basal medium. These coincided with the timing of caulogenic determination in cytokinin-treated expiants. The loss of lipid reserves, as well as ultrastructural changes associated with the maturation of cells on basal medium, was associated with the loss of competence.