Effects of Aerobic Exercise in Hepatic Lipid Droplet-Mitochondria interaction in Non-alcoholic Fatty Liver Disease

Background

Lipid droplets (LD) are composed of excess lipids which can be derived from diet (increasing lipids in the circulation and storage in tissues) or from the recycling of lipoprotein remnants and lipolysis (dependent on energy status). Storing lipids in LD prevents their toxicity and maintains lipid metabolism homeostasis by maintaining energy storage, for example [1]. 

Many disease conditions are marked by lipid overload which results in the storage of lipids in unusual or unprepared tissues. Examples include metabolic syndrome and non-alcoholic fatty liver disease (NAFLD), which are driven by obesity and its consequences [2]. According to the National Institute of Health (NIH) it is estimated that 24% of adults in the United States have NAFLD [3].These numbers are alarming due to the possibility of progression to non-alcoholic steatohepatitis (NASH) without health promotion and prevention measures. 

Exercise has a systemic beneficial role in reducing the risk of developing obesity and obesity-related diseases, such as metabolic syndrome, hypertension, type 2 diabetes mellitus (T2DM) and liver disease [4]. The aim of this study was to investigate the effects of exercise on modifying the LD-mitochondria interaction in hepatocytes and its association with the severity of liver disease.

 

Key findings

Exercise improves HFD-induced obesity parameters that correlate with NAFLD

Using an aerobic exercise treadmill, the authors demonstrated that obese mice submitted to 4 weeks of exercise improved glucose metabolism (lower fasting glycemia and decreased intolerance), lipid metabolism (lower cholesterol and triglycerides) and muscle metabolism (higher grip strength, speed and fiber hypertrophia) as well as liver function (lower AST and ALT levels) compared to sedentary mice. Exercised lean mice showed lower weight gain and a better grip strength and speed. As expected, the liver of obese mice showed steatotic characteristics, such as higher weight and number of lipid droplets. In addition, mitochondrial content was reduced and PLIN5 expression increased in the liver of obese mice. 

Exercise impacts the lipid droplet (LD) characteristics by reducing LD-mitochondria interaction in obese mice

The authors observed that exercised obese mice had a higher number of small LD (<140 μm²). This also occurred in white adipose tissue which presented a lower area size of adipocytes without a mass difference. Mitochondrial dysfunction induced by obesity was confirmed by a lower number and density of mitochondria and exercise did not alter these parameters. The authors also demonstrated that obese mice showed more LD-mitochondria interaction and that exercise could reverse this. The observed LD-mitochondria interaction was characterized by a larger size of LD which positively correlated with ALT levels as well as with glucose intolerance. 

Figure 1: Lipid droplet (LD) and mitochondria interaction occurs in a different pattern in obese mice and exercise can modulate this interaction. HFD-induced obese and sedentary mice seem to have a smaller number of mitochondria and these mitochondria seem to be interacting with LD. When exposed to aerobic exercise, this interaction seems to decrease and the number of mitochondria increases, which may be related to an improvement in hepatic steatosis in these mice.

 

Exercise improves the efficiency of peridroplet mitochondia 

The authors showed a similar profile of peridroplet (PDM) and cytosolic (CM) mitochondria in both sedentary and exercised obese mice. However, the authors observed that PDM in exercised obese mice were more elongated than the PDM from sedentary obese mice. Also, PDM of obese exercised mice had lower complex I expression, but a higher basal and ATP-linked oxygen consumption ratio (OCR) compared to PDM and CM from sedentary obese mice.

Exercise improves MCD-induced fibrosis and reduces LD-mitochondria interaction in mice

The authors induced non-alcoholic steatohepatitis (NASH) through the methionine-choline deficient diet (MCD) and investigated the effect of this progressive condition on liver metabolism. The authors demonstrated that MCD-induced fibrosis was accompanied by a higher number and size of LD as well as LD-mitochondria interaction. Mitochondrial dysfunction was also observed in this model and exercise could revert some of the phenotypes, such as fibrosis and mitochondrial function, without changing PLIN5 expression. Also, the authors showed a negative correlation between fibrosis and LD-mitochondria interaction. This negative correlation was somewhat unexpected due to the possible positive role of the reduction of LD-mitochondria interaction in the HFD-induced obese mice.

 

Why I think this preprint is important 

As obesity is a condition that causes liver disorders with a significant impact on public health costs, it is important to better understand how these disorders develop and how we can either prevent or treat them. This study focuses on the interaction between mitochondria and lipid droplets in the context of obesity and liver disease. By distinguishing the roles of cytosolic mitochondria and those associated with lipid droplets, this study demonstrates how exercise can counterbalance the presence of these different populations of mitochondria and drive a better health status. 

 

Questions and Suggestions for the Authors

1. Given that HOMA-IR is calculated by fasting insulin (µUI/mL) x fasting glucose (mmol/L*)/ 22,5 [5], the authors could consider the values of fasting to calculate the HOMA-IR as presented in figure 1J. 
2. A small correction: the authors should include reference size bars on all microscopy images such as on figures 2G, 3A, 3G, 4A, 5B, 5F, 6E, 6K and 7A.
3. Considering the genetic characteristics of the C57BL/6J mouse strain, such as the mutation on the nicotinamide nucleotide transhydrogenase (Nnt) gene [6] which is involved in mitochondrial antioxidant defense and its susceptibility to diet-induced obesity [7], how do the authors interpret the results in light of the genetic background of this animal model? Also, the authors could consider describing the limitations of the cold stress (occasioned by the temperature of 20-23ºC) and the use of anesthesia for euthanasia.
4. How did the authors measure the Non-alcoholic fatty liver disease score (NAS score)? The authors could consider using the scale and histological features described in literature [8], thus describing the calculations used to assess this score. Also, in figure 2D it seems like the values of the “CD+Ex” and “HFD+Ex” groups are lacking.
5. NAFLD and NASH are both driven by fat accumulation and this drives subsequent inflammation and cell death in NASH [2]. Thus, how can the authors interpret and characterize the results of HFD- from MCD-treated animals considering an inflammatory and apoptotic background?
6. As lipophagy and lipolysis could be triggered and diminish the LD [1], how can a role for PLIN5 be attributed without assessing lipophagy and/or lipolysis targets? The authors could consider discussing these pathways related to PLIN5 activity. 
7. The LD are synthesized and secreted in the endoplasmic reticulum (ER) [1]. HFD-induced obesity and MCD-induced NASH exhibit characteristic ER stress and unfolded protein response (UPR) activation. This UPR activation acts as a molecular link between hepatic disturbances and others dysfunctions [9]. Given that, could the authors discuss the UPR pathway and the LD in these models?
8. The authors could revise the legends of figures 4 and 6  including the description of the statistical significances. 

 

References

[1] Jarc, E. & Petan, T. Lipid Droplets and the Management of Cellular Stress. Yale J Biol Med. 2019 Sep 20;92(3):435-452;

[2] Mashek, D.G. Hepatic lipid droplets: A balancing act between energy storage and metabolic dysfunction in NAFLD. Mol Metab. 2021 Aug;50:101115;

[3]<https://www.niddk.nih.gov/health-information/liver-disease/nafld-nash/definition-facts> Accessed in 06/25/2023;

[4] Thyfault, J.P. & Bergouignan, A. Exercise and metabolic health: beyond skeletal muscle. Diabetologia. 2020 Aug;63(8):1464-1474;

[5] Gayoso-Diz, P. et al. Insulin resistance (HOMA-IR) cut-off values and the metabolic syndrome in a general adult population: effect of gender and age: EPIRCE cross-sectional study. BMC Endocr Disord. 2013 Oct; 13(47):1-10;

[6] Enríquez, J.A. Mind your mouse strain. Nat Metab. 2019 Jan;1(1):5-7;

[7] <https://www.jax.org/strain/000664> Accessed in 06/25/2023;

[8] Lucas, C. et al. A systematic review of the present and future of non-alcoholic fatty liver disease. Clin Exp Hepatol. 2018 Sep;4(3):165-174;

[9] Zhang, X.Q., et al. Role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease. World J Gastroenterol. 2014 Feb 21;20(7):1768-76.

Tags: exercise, lipid droplet, mitochondria, nafld

doi: https://doi.org/10.1242/prelights.35897

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