How Glucagon-Like Peptide-1 Receptor Activators May Affect Energy Production in Skeletal Muscle

Searches were conducted to identify any relevant studies using the following databases: MEDLINE, Scopus, CINAHL and clinicaltrials.gov. Databases were searched from inception to 22^nd of February 2024. Because of the difficulty in translation, only studies in English were included. In addition, these searches were supplemented with internet searches (e.g., Google Scholar) and further screening of reference lists of accepted papers.

Inclusion criteria were as follows: (1) either randomized controlled trials, randomized crossover trials, cluster randomized control trials and basic science experimental trials; (2) studies using rodent animal model, in vitro models or human samples within the context of obesity with or without diabetes; (3) studies that investigated the use of GLP‐1 RA therapy with or without the combination of GIP as a pharmacological intervention with a comparison model of either no GLP‐1 RA therapy, use of a placebo or usual care; (4) primary outcome was skeletal muscle mitochondrial function either in the form of mitochondrial mass (measured by citrate synthase activity, polymerase chain reaction [PCR], western blotting or transmission electron microscopy [TEM]), oxidative capacity/mitochondrial function (by respirometry, western blotting or PCR), mitochondrial dynamics (by western blotting, PCR or TEM), mitochondrial biogenesis (by western blotting or PCR) and mitophagy (by western blotting or PCR); (5) full‐text published in English.

Exclusion criteria were as follows: (1) non‐English papers; (2) not skeletal muscle focused; (3) not mitochondrial related; (4) review articles.

Two independent reviewers conducted the study selection (VO, EW), based on the inclusion and exclusion criteria. The initial screening excluded duplicate papers and irrelevant papers. The remaining studies were reviewed with full texts screened against the inclusion/exclusion criteria. Any conflicts were resolved via discussion. The Cochrane flow diagram displays the selection process [35].

Data were extracted by VO using a standardized form and cross‐checked by EW. The following data were extracted: general study information, type of model used, cell line, GLP‐1 RA drug tested and comparator intervention, length of time of the intervention and outcome data. Studies were assessed for quality, eligibility and bias by one investigator (VO) using the Cochrane Risk of Bias Tool across five domains.

The studies included in this review involved different methodologies and animal models. A meta‐analysis was not suitable because of the wide differences between outcome measures recorded and techniques used to assess. A qualitative review was performed.

This review encompassed a total of eight studies; the study selection flow diagram can be found in Figure 1. Only one study used semaglutide [36], three used liraglutide [37, 38, 39], two used exenatide [40, 41] and two used Exendin‐4 [42, 43]. No studies using GLP‐1 RA GIP were found. All studies included were preclinical trials; no human studies were found. Of these, two trials were conducted using rat models, spontaneously diabetic torii rats (SDT‐rats) [37] or Sprague Dawley rats [40]; five trials used mice, including the C57BL/6J mouse line [36, 39, 42], KKAy mice (a cross breed between diabetic KK mice and lethal yellow Ay mice that carry a mutation of the agouti gene) and C57BL/6J mice were used by [38] and C57bL/6JOlaHSD mice (a subline of mice carrying a deletion of the alpha synuclein locus gene) were used by [41]. One in vitro study investigated GLP‐1 RA therapy using the C2C12 cell line [43]. In terms of our primary outcome, three studies reported the effect of GLP‐1 RA on mitochondrial density, area or morphology [36, 38, 40]; one study examined mitochondrial content, focusing on citrate synthase activity [37]; oxygen consumption rate was explored in two studies following different GLP‐1 RA therapy [41, 43]; two studies explored effects of GLP‐1 RA on mitochondrial biogenesis [37, 39] and two on metabolism [42, 43]. Study characteristics details can be found in Table 1.

Summaries of the studies' risk of bias are provided in Figure 2. Only one trial was rated as low risk of bias, with the remaining eight seen as either high risk or some concerns.

A summary of the results generated by this review can be found in Figure 3. Details can be found in the subsections below.

Ji and colleagues treated KKAy mice with 250 μg/kg/day of liraglutide compared with a saline control. They investigated mitochondrial area and density using transmission electron microscopy (mitochondrial area/100 μm² and mitochondrial number/10 μm² were determined by analysing 10 images taken at 20 000× magnification using image J). They reported a significant increase in mitochondrial area (+113%) and number (+441%) in the liraglutide treated animals compared with the control group [38]. The authors also reported improvements in morphology; The KKAy mouse model (a rodent model of T2DM) presented with disarranged, swollen mitochondria clusters which visually improved following the 6‐week liraglutide treatment. Ren and colleagues treated C57BL/6 mice with semaglutide or a placebo following a high‐fat diet, which resulted in weight gain compared with a group on a normal diet. They used TEM to investigate effects of semaglutide upon mitochondrial number and area. They reported the high fat diet reduced mitochondrial number and area determined by TEM, which was then reversed following semaglutide treatment together with improvements in morphology, that is, reductions in swelling [36]. They also investigated the effect of the drug upon sarcoplasmic reticulum area, but found no difference [36]. Wu and colleagues investigated the effect of exenatide treatment in mice with T2DM upon mitochondria morphology [40]. Using TEM, they found T2DM caused severe disruption to mitochondria morphology, which included swelling and disordered cristae. This was largely reversed after treatment with exenatide leading to the conclusion that the drug was able to reduce mitochondrial damage induced by T2DM.

The effect of liraglutide on mitochondrial mass and complex abundance was investigated in SDT fatty rats (an obese T2DM model) by Yamada and colleagues [37]. They found that increasing doses of liraglutide over 6 weeks increased protein expression of Cox5B (subunit of Complex V) when compared with the control SDT fatty rats [37]. There was also a strong trend for an increase in citrate synthase activity (SDT untreated vs. SDT + liraglutide p = 0.05) in the soleus muscle, but no effect was seen in the EDL. CS activity is commonly used as a marker of mitochondrial mass and COX5B was used by the authors to determine mitochondrial preservation.

Mitochondrial respiration was explored in three studies following different GLP‐1 RA therapies. The first study utilized a Seahorse XF24 Extracellular Flux Analyzer to assess oxygen consumption rates (OCR) in C2C12 cells (cells were incubated ± oleate and palmitate acid to mimic obesity) following incubation with 20‐nM Exendin‐4/day for 2 days [43]. Exendin‐4 treatment increased basal and maximal respiration, increased uncoupling efficiency and proton leak compared with untreated cells. The authors commented that this suggested Exendin‐4 promoted an increase in fatty acid oxidation. They also reported an upregulation of UCP1 mRNA after 24 h of 20 nM of Exendin‐4 in differentiated C2C12 cells, which interestingly was not replicated in the animal arm of the study [43]. No effect was seen of exendin treatment on UPC2 or UPC3 mRNA expression. Gao and colleagues noted an increased protein and RNA expression of UCP3 in the EGLP‐1 analogue group when compared with a control and a group treated with Exendin‐4 [42]. Mitochondrial uncoupling proteins are a family of transmembrane proteins localized in the inner mitochondrial membrane that are involved in the transport of protons across the mitochondrial membrane and thereby inducing mitochondrial uncoupling [44]. They are not direct measures of respiratory function but can provide information on thermogenesis and energy expenditure.

Ex vivo mitochondrial respiration using a high‐resolution respirometry via oxygraphy‐2k system (OROBOROS) in C57BL/6Jola HSD mouse models was performed by Jansen and colleagues. High‐fat diet (HFD‐fed) mice were administered 10 μg/kg/day exenatide for 8 weeks. The protocols used specifically probed LEAK and OXPHOS states and electron transfer system capacity [41]. Overall, the authors concluded that mitochondrial respiration was not influenced by exenatide treatment. They did however report that the flux control efficiency showed a significant decrease as well as the maximum complex I activity in HFD‐fed mice supplemented with Exenatide. However, the majority of the LEAK respiration (O2 compensating for the lack of ATP synthesis), OXPHOS respiration (the NADH pathway) and electron transfer system (ETS) respiration were not significantly different when compared with the HFD.

C57BL/6J mice given 0.1 mg/kg per day liraglutide for 4 weeks showed an upregulation of PGC‐1α protein expression by 85% in mice on a normal diet and by 204% in those on a high‐fat‐high‐sucrose (HFHS) diet. This effect was independent of effects upon AMPK and SIRT‐1, key proteins within the canonical pathway for increased PGC‐1α expression [39]. However, Yamada and colleagues found no significance in PGC‐1α protein expression following liraglutide in the soleus muscle between the control SD mice and the SDT mice treated with liraglutide, but there was a significantly higher expression of PGC‐1α in the extensor digitorum longus (EDL) muscle [37]. Choung and colleagues [43] reported an increase in PCG‐1α protein expression following incubation of C2C12 cells with 20‐nM Exendin‐4 for 12 h.

Overall, the studies analysed showed a beneficial effect of GLP‐1 RA therapy upon mitochondria morphology. This included increased size and density [38] [36], [40] increased absolute numbers/content [36, 37, 38], reduction in signs of mitochondrial swelling [40] and complex abundance [37]. Such improvements are vital for efficient mitochondrial function. An increase in mitochondrial size and density improves skeletal muscle oxidative capacity, which has been linked to increasing levels of resting metabolic rate (BMR) [60] supporting an overall increase in BMR. This increase in BMR could help support people who are prescribed GLP‐1 RA with their overall weight loss effect due to the natural decrease in BMR as people lose weight [61]. Ren and colleagues noticed increased mitochondrial content following 12 weeks of semaglutide treatment [36]—increased mitochondria content is correlated to improvements in skeletal muscle function and health [62]. Similarly, Ji and colleagues also saw increases in mitochondria number/content and area along with improvements within the myofibrils structure following liraglutide treatment (clearer cross striation and less atrophic myofibril compared with the mice with diabetes) [38]. These improvements show potential for liraglutide to protect and repair damaged mitochondria through decreasing oxidative stress, which is generated by dysfunctional mitochondria [63]. Exenatide was also shown to improve mitochondrial morphology after 8 weeks in rats with T2DM, with a smaller number of swollen mitochondria found when compared with the control T2DM rat without exenatide treatment [40]. This decrease allows for better ATP production and utilization within the muscle, which can support in the maintenance of skeletal muscle structure and integrity [64].

Mitochondria content improvements were noted by increased CS and upregulated Cox5B in the soleus muscle of SDT rats following liraglutide treatment for 8 weeks [37]. This study demonstrates the possible role of liraglutide in upregulation of fatty acid oxidation by preserving mitochondria content in type I muscle fibres [65]. This preservation can reduce the risk of mitochondrial dysfunction and cell death, helping to maintain the skeletal muscle integrity in patients.

It is important that researchers combine measures of mitochondrial content and function, as one does not always follow the other (i.e., increased content does not always lead to increased function), and careful consideration should be made to the different types of mitochondria function beyond ATP synthesis.

There is no clear overall effect of GLP‐1 RA on mitochondrial respiration. Choung and colleagues used the Seahorse XF24 extracellular Flux analyser to explore the effects of Exendin‐4 on C2C12 cells, which were given palmitate to induce obesity‐like properties [43]. OCR, basal respiration rate, and proton leak where all seen to increase following the Exendin‐4 treatment; there was also an increase in UCP1 expression, which can correlate with the increased energy metabolism [66]. A similar upregulation of UCP2 or UCP3 though was not reported [43], whereas Gao and colleagues did report upregulation of UCP2 and UCP3 following EGLP‐1 analogue treatment when compared with Exendin‐4 treatment in C2C12 cells [42]. UCP2 regulation has been noted to support ATP/ADP ratio, whereas UCP3 is involved in regulation of ROS and the handling of fatty acids [67]. These changes following GLP‐1 therapy may protect the mitochondria from overaccumulation of fatty acids, which may play a part in reducing skeletal muscle insulin resistance [68].

High‐resolution respirometry was used to understand the effects of exenatide on mitochondrial respiration in HFD‐fed mice [41]. There was deemed no significant overall effect as there was no difference reported in LEAK, OXPHOS or ETS respiration in the treatment mice compared with the control HFD mice. They did however report a decrease in both flux control efficiency and maximum complex I activity [41], which have been noted to improve glucose homeostasis [69]. This is the only known study to date that has investigated mitochondrial respiration using high‐resolution respirometry in skeletal muscle following GLP‐1 RA therapy. It is, therefore, still unclear if there is any benefit of GLP‐1 RA's on mitochondrial respiration health.

Two papers focused on the expression of PGC‐1α [37, 39]. Yamanda and colleagues noted an upregulation of PGC‐1α expression in SDT rats EDL muscle following liraglutide treatment, but not in the soleus muscle [37], which is interesting given the predominance of type I fibres within the soleus muscle [70]. Protein expression of PGC‐1α was also shown to be upregulated in HFHS mice given liraglutide over 4 weeks by Zhou and colleagues; however, clear indication of which muscle was used to find this expression was not given. This upregulation was not mediated through the AMPK‐SIRT‐1 cell signalling pathway, although it shows signs of supporting mitochondria capacity through other pathways, which need further investigation [39]. The authors also reported an increase in PGC‐1α protein expression in the control + liraglutide compared with the control group alone [39]. Zhou and colleagues did not report on the relationship between PGC‐1α and mitochondrial content present, which would have been beneficial in showing a clearer understanding of the effects of GLP‐1 RA. PGC‐1α is a critical regulator in mitochondria biogenesis, and its expression is often used as an indicator of the activation of this process. In addition to this, it supports quality control mechanisms within mitochondria through fission, mitophagy and fusion [71]. Therefore, determining the effects of GLP‐1 therapy on PGC‐1α are a vital component to help us understand the effects this class of drugs might be having upon on key aspects of mitochondria health [71].

The biggest limitation to the current available data is the lack of human studies that have been performed to investigate skeletal muscle respiratory function following GLP‐1 RA–based therapies. This is a major gap within the current research that is vital in understanding the effects of GLP‐1 RA–based therapies on skeletal muscle. The effect of weight loss drugs in general upon skeletal muscle is an emerging area of research clearly at an early stage, which likely accounts for the absence of human data. Future studies should include assessments of respiratory function, which can be made by magnetic resonance spectroscopy (MRS) to avoid the necessity for muscle biopsies in this population. This need for human research is also emphasized by the poor translation of drug trials from animal to human models [72]. In addition, different weight management therapies may influence mitochondria in different ways; a meta‐analysis on the mitochondrial effects comparing caloric restriction (CR) and bariatric surgery found a reduction of complex IV in CR group but not in the bariatric group [73]. There is no clear understanding on whether the effects of GLP‐1 RA on mitochondria are caused by the drug or due to weight‐independent mechanisms, which will need further evaluation. Furthermore, this review only found data on the effect of single GLP‐1 RA's on mitochondrial respiration. As multiple novel pharmacotherapies combine GLP‐1 with other enteropancreatic hormones with diverse actions as dual or triple agonists, it is important to understand over the next years the effect of the different treatment combinations with GLP‐1 on mitochondria.

There is a lack of studies looking at the impact of exercise alongside GLP‐1 RA administration on skeletal muscle, which is vital because of the key role exercise plays in weight management as well as the role mitochondria plays in exercise. A study by Yates and colleagues found a decrease in physical activity following GLP‐1 RA therapy despite the overall decline in weight loss [74], yet there is a positive effect on physical function [54]. Reasoning for this is conflicting relation is limited and warrants further attention towards the involvement of exercise during administration. It is also important to understand what effects persist when individuals stop using GLP‐1 RAs. For example, it is not known how long improvements in physical function or mitochondrial content will persist after stopping GLP‐1 RAs and whether potential changes in these parameters will be linked to weight regain after stopping GLP‐1 RAs.

Because of differences in the drug therapy, dosages, outcome measures, duration of application and model used, a meta‐analysis could not be completed and so an overall effect cannot be determined based on current data available. Only one trial was considered as low risk of bias, as majority of papers did not blind the outcome reporter to the groups or failed to report their randomization/blinding procedure if applicable. None of the studies included have reported direct number statistics in their results, leaving interpretation difficult due to the only values given being through bar graphs or their own interpretation of the results. A standardization of dosage levels could help to untangle the relative effect as the studies assessed here did not present a rationale for their dosage levels or their prior optimization. Furthermore, how the dosages used within preclinical trials were selected or how they relate to those administered to humans was not discussed in any study we evaluated. This is a limitation as it puts into question the degree of translation in the effects seen in these animal models and what might be expected to be seen in humans.