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.
Abstract
Background
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
inhibition.
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).
Results
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.
Conclusions
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
resistance.
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