1. Young Investigators’ session
Mitochondrial function and insulin resistance in humans with obesity, diabetes mellitus and non-alcoholic fatty liver disease (NAFLD) C. Koliaki (Greece)

2. Chrysi Koliaki, MD PhD Greece
Hepatic mitochondrial function in humans with insulin resistance and NAFLD 15th MGSD Meeting, Young Investigators’ session
Chrysi Koliaki, MD PhD
Greece
Athens,

3. No financial relationships to disclose
Conflict of interest
No financial relationships to disclose

4. Research fellowship

5. Postdoctoral research

6. Background (I)
Mitochondrial function and its association with insulin resistance in different tissues, is a hot topic of metabolic research worldwide.
Liver is a key organ involved in energy homeostasis and pathogenesis of type 2 diabetes mellitus (T2DM) and NAFLD.
NAFLD comprises benign steatosis (non-alcoholic fatty liver, NAFL), steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma, and associates with both hepatic and peripheral insulin resistance.
Roden M. Nat. Clin. Pract. Endocrinol. Metab. 2006;2:335–348

7. Background (II) Mitochondria are dynamic intracellular organelles
Major orchestrator of cellular energy metabolism
β-oxidation
tricarboxylic acid cycle (TCA)
adenosine triphosphate (ATP) synthesis through oxidative phosphorylation (OXPHOS)
reactive oxygen species (ROS) formation and detoxification
Murphy MP. Biochem. J. 2009;417:1–13

8. Background (III)
In skeletal muscle, insulin resistance may coexist with lower mitochondrial density, reduced OXPHOS gene expression and ATP synthesis.
Whether similar mitochondrial alterations are also present in livers of insulin resistant humans remains unclear.
While there was some evidence for impaired hepatic mitochondrial function in T2DM and NASH, other investigators reported an increased hepatic mitochondrial function in obese humans with NAFLD.
Prior to our study, no data on simultaneous direct measurement of mitochondrial content and capacity were available in livers of humans at different stages of insulin resistance and NAFLD.
Mootha VK et al. Nat. Genet. 2003;34:267–273; Morino K et al. J. Clin. Invest. 2005;115:3587–3593; Szendroedi J et al. PLoS Med. 2007;4:e154; Szendroedi J et al. Hepatology 2009;50:1079–1086; Perez-Carreras M et al. Hepatology 2003;38:999–1007; Sunny NE et al. Cell Metab. 2011;14:804–810

9. Our study Registered clinical trial, NCT01477957
Ex vivo high-resolution respirometry (HRR) to quantify hepatic mitochondrial respiration
Combined with measures of mitochondrial content
Intra-operative liver samples from obese humans
without NAFL (OBE NAFL-), n=18
with NAFL (OBE NAFL+), n=16
with NASH (OBE NASH), n=7
lean humans without NAFLD (CON), n=12

10. Methods
Metabolic characterization (euglycemic-hyperinsulinemic clamps with [6,6-2H2]glucose)
Intra-operative liver biopsies ( mg tissue)
Liver histology (steatosis, NAFLD score)
Ex vivo high resolution respirometry in liver tissue & isolated mitochondria (Oroboros oxygraphs, Innsbruck)
Citrate synthase activity (CSA) and mtDNA for mitochondrial content
TBARS (thiobarbituric acid reactive substances) for lipid peroxidation
H2O2 production for oxidative stress
Gene expression analyses

11. Study groups

12. Obese groups without NASH exhibit upregulated hepatic mitochondrial respiration
Koliaki et al. Cell Metab. 2015;21:739–746

13. Obese with NASH have more mitochondria but increased proton leakage
Koliaki et al. Cell Metab. 2015;21:739–746

14. Obese with NAFL and NASH exhibit impaired mitochondrial biogenesis
Koliaki et al. Cell Metab. 2015;21:739–746

15. Only NASH patients present with hepatic oxidative DNA damage
Koliaki et al. Cell Metab. 2015;21:739–746

16. Hepatic mitochondrial adaptation in NAFLD
Obese humans with NAFL presented with elevated hepatic mitochondrial O2 fluxes, which associate with increased hepatocellular lipid content. They further featured increased hepatic lipid peroxidation, impaired mitochondrial biogenesis and reduced ETC complex expression in the face of unchanged hepatic insulin sensitivity. These abnormalities were exacerbated in patients with NASH, who further exhibited profound hepatic insulin resistance, an increased number of inefficient leaking mitochondria, and excess hepatic oxidative stress in parallel with reduced anti-oxidant defense capacity, leading to oxidative DNA damage and activation of hepatic proinflammatory pathways.
Taken together, we propose that increased lipid availability in the liver of obese humans with steatosis stimulates hepatic mitochondrial capacity and thereby serves to protect against NAFLD progression. However, augmented respiration may not be bioenergetically efficient due to leaking mitochondria
and thereby promote excessive hepatic oxidative stress, challenging hepatocellular anti-oxidant defense mechanisms. Once these mechanisms fail, mitochondrial functionality decreases and hepatic insulin resistance, NAFLD progression to NASH, and systemic inflammation develop. In conclusion, these data suggest an adaptation of hepatic mitochondria in obese humans without NASH. This ‘‘hepatic mitochondrial flexibility’’ associated with early stages of human obesity could serve as future target for the prevention and treatment of NAFLD.

17. Hypothesis of hepatic mitochondrial flexibility
Non-alcoholic fatty liver (NAFL) adapts to increased lipid availability by upregulating its mitochondrial oxidative capacity, as illustrated by increased burning activity (fire/flames). This overoxidizing state overwhelms mitochondria (“sweating”) and promotes accumulation of hydrogen peroxides (H2O2), which are scavenged by catalase activity. In advanced non-alcoholic fatty liver disease (NAFLD) such as steatohepatitis (NASH), mitochondria are already “exhausted” and oxidative capacity is low (no fire). This associates with a larger number of hypofunctional leaking mitochondria, hepatic insulin resistance and excess oxidative stress in parallel with reduced anti-oxidant capacity. On pp. xxx-xxx of this issue, Koliaki et al. show that obese humans with NAFL feature elevated but bioenergetically inefficient mitochondrial respiration rates. This adaptation (“metabolic flexibility”) is abolished in obese humans with NASH through mechanisms related to inflammation and oxidative stress.
Idea of the cartoon inspired by the album of Eagles “Hell freezes over„ (released in 1994) and “The Road to Hell„ by Chris Rea (released in 1989).
Concept by M. Roden, artwork by V. Koliaki

18. Tissue-specific alterations of mitochondrial content and function in states of insulin resistance
In addition to the original research described above, we published two review articles (Annu Rev Nutr 2016, Moll Cell Endocrinol 2013) and one commentary (Mol Metab 2014), regarding the controversial association between mitochondrial function and insulin sensitivity in obesity, T2DM and NAFLD. The scope of our review work was to provide a consolidated update of the basic features of mitochondrial function and their implications for metabolic diseases, and to critically review data reporting tissue-specific mitochondrial alterations in obesity, T2DM and NAFLD, within the conceptual framework of a global multiparametric approach of mitochondrial function.

19. Major publications
Koliaki C et al. Adaptation of hepatic mitochondrial function in humans with non-alcoholic fatty liver is lost in steatohepatitis. Cell Metab 2015; 21:
Koliaki C, Roden M. Alterations of Mitochondrial Function and Insulin Sensitivity in Human Obesity and Diabetes Mellitus. Annu Rev Nutr 2016; 36:337-67
Koliaki C, Roden M. Do mitochondria care about insulin resistance? Mol Metab 2014; 3:351-3
Koliaki C, Roden M. Hepatic energy metabolism in human diabetes mellitus, obesity and non-alcoholic fatty liver disease. Mol Cell Endocrinol 2013; 379:35-42

20. Significance of data
The pathophysiologic concept of “hepatic mitochondrial flexibility” at early stages of human obesity-associated insulin resistance, could serve as a relevant future target for the prevention and treatment of NAFLD and other metabolic diseases.

21. Future perspectives
We aim to expand our data on mitochondrial function and insulin resistance by recruiting additional patients from Obesity Outpatient Clinics in Greece, undergoing bariatric surgery, liver biopsies and metabolic phenotyping, in order to further corroborate our concept of hepatic mitochondrial adaptation to insulin resistance and NAFLD.

22. Acknowledgements Prof. Michael Roden for guidance and support
Study volunteers
KT, ME, KR for technical assistance
National Foundation of State Scholarships of Greece
EFSD, Albert Renold Research Fellowship
Ministry of Science and Research of the State of North Rhine-Westphalia (MIWF NRW)
German Federal Ministry of Health (BMG)

23. Thank you!