**4. Discussion**

Mutations of the mitochondrial DNA (mtDNA) involve different organs. These genetic changes can affect the translation of mtDNA-encoded proteins, including respiratory chain complexes [15,16]. The m.3243A>G mutation has been described to be of strong diabetogenic nature [17]. Elevated FFAs are discussed to be a major cause of insulin resistance in skeletal muscle and liver [18].

It is generally accepted, that saturated FAs like palmitate induce insulin resistance, whereas the monounsaturated ones like oleate increase insulin sensitivity in diabetic patients and healthy individuals [4]. It has been shown, that oleate, but not palmitate, increased the expression of genes related to the FAO pathway in a sirtuin (Sirt)1-peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α dependent manner. This, in turn, led to an increase in complete FA oxidation in mice skeletal muscle [19]. There are other reports about the preventive effect of oleate on saturated-fatty-acid-induced endoplasmic reticulum (ER) stress, inflammation, and insulin resistance through adenosine monophosphate-activated protein kinase (AMPK). In the above-mentioned study, oleate, in contrast to palmitate, did not increase the levels of ER stress markers, which is involved in the link between lipid-induced inflammation and insulin resistance [20].

In the present study, taking palmitate as standard substrate for FAO measurements and oleate as a comparison, the data showed generally similar ß-oxidation rates in m.3243A>G patients and controls. Both, patients and controls showed decreased parameters in this pathway at 40 ◦C compared to 37 ◦C. ATP-R was reduced to about 35–45% at 40 ◦C compared to that at 37 ◦C for both, patients and controls.

Using glucose as substrate for the Mito stress test, all key parameters of mitochondrial respiration were significantly lower in patients than in controls.

The long-chain FFAs are poorly soluble in aqueous solutions. Moreover, in vitro exposure to high levels of FFAs might lead to lipotoxicity and cellular dysfunction [21,22]. The FAs used in the present study were conjugated with BSA in all experiments (ratio FFA: BSA = 6:1).

The treatment of fibroblasts from healthy controls with palmitic acid for 16 h prior to OCR measurement has been reported to significantly increase the MR and SRC for about 20% and 45%, accordingly [23]. However, oleic acid displayed no effect on OCR in the above-mentioned study. In the present study, the extra addition of FAs oleate and palmitate affected the OCR key parameters in Mito stress test differently. While oleate generally showed positive effects on OXPHOS values, palmitate had an impaired them. Considering the values obtained in presence of FAs excess, oleate seemed to show a slightly positive effect on respiratory factors in patients. After addition of oleate, ATP-R was about 30% higher in controls compared to the values with palmitate. In contrast, the negative effect of palmitate excess was significant in patients. MR and ATP-R were reduced about 17% and 30% in patients after addition of palmitate, accordingly.

The different effects in myoblasts in presence of oleate and palmitate could be explained as follows: before performance of FAO measurements, the cells are cultured for 16 h in a nutrient restricted medium (Section 2.2.2). Thus, the cells are forced to take the FAs as the only present substrate to survive. On the other hand, by Mito stress test, the cells have sufficient nutrients and the added FAs (oleate and palmitate) count as excessive substrates. FAs are the major energy source for skeletal muscle. However, the balance between energy demand, uptake, and β-oxidation of FAs should be regulated. An imbalance between fatty acid uptake and β-oxidation might lead to an insulin resistance [24]. The elevated lipid levels, which exceed the cell's capacity to store or utilize FAs, can, as well, lead to a lipotoxic response to activate stress pathways and apoptosis. For ß-oxidation in mitochondria, both, saturated and unsaturated long-chain FAs are used as substrate. However, only long-chain saturated fatty acyl CoAs serve as substrates for de novo ceramide synthesis, which is involved in initiation of apoptosis [25].

A reduction in palmitic acid, but not in oleic acid oxidation, has been reported in myotubes of patients with diabetes type II compared to controls [26]. It has been shown that monounsaturated fatty acids, such as oleic acid, are metabolized and then accumulated in the form of low-toxic triacylglycerol (TAG). However, a large amount of palmitate inhibits the TAG synthesis at the DAG stage, which leads

to the accumulation of DAG in the cell [27,28]. Based on these findings, a substitution of palmitate and other saturated or unsaturated FFAs has been recommended in patients with diabetes type II to reduce the accumulation of DAG and TAG to not promote insulin resistance [26].

The data confirmed the reported negative effect of palmitate on respiratory function of cells. This negative effect was more pronounced in m.3243A>G patients, which led to approximately 30% reduction of ATP production. The controls seemed to benefit slightly more than patients from the positive effect of oleate clearing the excess of saturated FAs via increasing the FA Oxidation. This counteracts with inflammation and insulin resistance in skeletal muscle [19]. Nevertheless, this positive effect should be noticed for keeping the balance of the body weight in over nutrition or pathological states, such as mitochondrial disorders.

An induced mitochondrial reactive oxygen species (ROS) production, mitochondrial dysfunction, and insulin resistance in skeletal muscle cells have been reported upon supplementation of palmitate, but not of oleate. Oleate should even have a protective benefit against palmitate-induced insulin resistance and might enhance the mitochondrial function, protecting against apoptosis, and increasing insulin sensitivity [29]. The analysis of potential apoptosis and ROS production might be evaluated in upcoming projects.
