Investigation of molecular mechanisms regulating palmitate-induced metabolic inflexibility in a cell-based skeletal muscle model: physiological and pharmacological interventions.

Chien, Hung-Che (2020) Investigation of molecular mechanisms regulating palmitate-induced metabolic inflexibility in a cell-based skeletal muscle model: physiological and pharmacological interventions. PhD thesis, University of Nottingham.

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Present evidence indicates that increased systemic circulating fatty acids following chronic

high-fat dietary intake leads to skeletal muscle (SKM) metabolic inflexibility, and this is

associated with an impairment of the mitochondrial pyruvate dehydrogenase complex (PDC)

controlled carbohydrate (CHO) oxidation. However, the molecular mechanisms behind the

reduction in the PDC controlled CHO oxidation during chronic high-fat dietary intake are

poorly understood. Nevertheless, previously published evidence advocated that transcription

factors such as PPARα, PPARδ, and FOXO1 might contribute to the PDK4 mediated PDC


This thesis aimed, therefore, (1) to elucidate molecular and metabolic evidence supporting

this contention in a metabolic inflexible C2C12 SKM cell model, and (2) to determine whether

activators of CHO oxidation, such as exercise (delivered as electrical pulse stimulation - EPS),

dichloroacetate (DCA) or R-α-lipoic acid (RALA) could rescue CHO oxidation in the presence

of palmitate by altering the same molecular and metabolic events identified at (1).


In chapter 3, we established what concentration of palmitate could be tolerated by our in vitro

SKM cell model (500 M) without jeopardising the viability of the muscle cells. It was found

in our SKM cell-based model that palmitate supplementation replicated well-defined markers

of in vivo impaired cellular CHO oxidation: i.e. decreased glucose uptake, lower acetylcarnitine

accumulation, lower pyruvate mediated mitochondrial ATP production rate, increased media

lactate concentration and decreased PDC activity. It was also found that palmitate significantly

increased the gene and protein expression of PDK4, PPARα, PPARδ, and FOXO1.

In chapter 4, it was found that the palmitate-induced reduction in PDC activity could be

reversed in tandem with a reduction in the PDK4 protein expression by siRNA PPARδ and

FOXO1 gene silencing. It appeared, however, that although PPARα gene silencing restored the

palmitate-induced reduction in PDC activity, this occurred independently from any changes in

the PDK4 protein expression. siRNA PPARα, PPARδ, and FOXO1 gene silencing rescued the

palmitate-induced reduction in the cellular glucose uptake. In contrast, only PPARδ and

FOXO1 gene silencing reversed the palmitate-induced changes in media lactate and

acetylcarnitine concentrations. The latter findings, which are in line with the contention these

transcription factors are directly targeting PDC, also provide further evidence that the effects

of PPAR on PDC activity occur independently of the involvement of PDK4 protein.

Additionally, siRNA silencing of PPARα and FOXO1 restored the palmitate-induced

reductions in the mitochondrial maximal ATP production rates (MAPR). In contrast, PPARδ

silencing did not, surprisingly, rescue the palmitate-induced decline in MAPR. It was also

found that the siRNA PPARδ gene silencing decreased FOXO1 protein expression. In contrast,

the siRNA gene silencing of FOXO1 did not change the PPARδ protein expression, which

collectively suggests that FOXO1 may be a PPARδ downstream target, thereby modulating

PDK4 protein expression.

In chapter 5, it was shown that administration of RALA or DCA rescued the palmitate-induced

inhibition of PDC activity and the increase in media lactate concentration. However, only

RALA corrected the deleterious effects of palmitate on cell glucose uptake and MAPR. At the

molecular level, DCA, but not RALA, reduced PDK4 protein expression in the presence of

palmitate. We also found that DCA and RALA reversed the palmitate-induced upregulation of

PPARα and PPARδ and total-FOXO1 protein expression.

In chapter 6, it was demonstrated that the treatment of palmitate-supplemented cells with

electrical pulse stimulation (EPS) rescued the palmitate-induced dysregulation of glucose

uptake, PDC activity, and its flux, and MAPR. Furthermore, these events were accompanied

by a restoration/lowering of the protein expression levels of PPARδ and its downstream target

FOXO1, but not those of PPARα and the p-FOXO1/t-FOXO1 ratio normalised to that of

nuclear to cytoplasmic ratio. These molecular findings indicate benefits of sustained muscle

contraction in maintaining CHO flux in the presence of palmitate.


This thesis provides persuasive evidence that the mechanism by which palmitate downregulates

the PDC-mediated reduction in CHO oxidation in muscle occurs according to the following

series of events:

Palmitate PPAR FOXO1 PDK4 PDC CHO oxidation.

However, the action of PPAR seems not to involve the upregulation of the PDK4 protein.

This thesis also provides evidence that DCA, RALA, and EPS can rescue the palmitate-induced

reduction in CHO oxidation. Collectively, these findings are of clinical relevance as they open

the path for potential therapy to the lifestyle-induced metabolic inflexibility and insulin


Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Constantin-Teodosiu, Tim
Greenhaff, Paul
Keywords: Molecular mechanisms, Metabolic inflexibility, Skeletal muscle metabolic inflexibility, SKM
Subjects: R Medicine > RC Internal medicine > RC 321 Neuroscience. Biological psychiatry. Neuropsychiatry
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 61197
Depositing User: Chien, Hung
Date Deposited: 31 Dec 2020 04:40
Last Modified: 31 Dec 2020 04:40

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