*2.3. Excess CPT1A Is Associated with Serine and Glycine Metabolism and Glutathione Homeostasis Metabolites*

To understand the type of stress imposed in the CPT1A OE cells, we performed global, non-targeted metabolomics in the four cells lines: non-targeting (NT), KD, EV, and OE cells. In parallel to the RNAseq analysis, we focused on the metabolic pathway differences between OE and KD cells after controlling for their respective control lines (EV and NT respectively). Analysis of the top 25 significant pathways revealed that lipid metabolism, glycine and serine metabolism, and glutathione homeostasis were the most significantly changed pathways (Figure 3A and Figure S3). Glycolysis was also significantly different across groups, with the OE cells showing less glycolysis compared to KD cells (Figure S3).

Considering the RNAseq data, we next analyzed the metabolites corresponding to the serine/glycine metabolism. The serine, glycine and one carbon metabolism pathway are a metabolic network upregulated in tumors and of high clinical relevance [30,31]. Except glucose, individual metabolites of the serine/glycine pathway were not changed significantly in OE versus KD (Figure 3B). However, the OE cells showed more intracellular glucose being shunted towards de novo serine biosynthesis compared to control EV cells (Figure S3A). In fact, less glucose seemed to be used for glycolysis in the OE cells. These results correlated with significantly increased expression of key serine/glycine pathway genes: D-3-phosphoglycerate dehydrogenase (PHGDH), phosphohydroxythreonine aminotransferase (PSAT1), and Serine hydroxymethyltransferase (SHMT2) (Figures 2E and 3B). SHMT2 is a mitochondrial enzyme that converts serine (3 carbons) into glycine (2 carbons), transferring one carbon to tetrahydrofolate (mitochondrial folate cycle). The increased dimethylglycine and cystathionine levels in OE cells compared to KD cells supports a higher folate cycle activity in these cells (Figure 3B,C).

**Figure 2.** RNAseq analysis of CPT1A OE cells shows increased endoplasmic reticulum (ER) stress response, serine metabolism, and less androgen receptor (AR) signaling. (**A**) Schematic of the RNAseq analysis paradigm with the CPT1A-KD and OE cells. (**B**) Gene set enrichment analysis was performed on the fold change of the comparison. Normalized Enrichment Scores (NES) and False discovery rate (FDR) adjusted *p*-values are shown for select pathways, as well as the rank of those genes in fold change ranking, are plotted. (**C**) Heatmap of the leading-edge genes for select pathways. (**D**–**F**) Gene expression graphs of significant genes associated with lipid catabolism (**D**), serine biosynthesis (**E**) and ER stress (**F**) in OE versus KD comparison after adjustment to their respective control cell lines. Adj *p* value < 0.001.

**Figure 3.** Excess CPT1A is associated with serine and glycine metabolism pathway and glutathione homeostasis metabolites. (**A**) Metabolite set enrichment analysis of OE metabolites versus KD cell metabolites after normalization to their own controls. The *p*-value is defined by the color scale, and the enrichment by the size of the circle. (**B**) Relative abundance of metabolites associated with the serine and glycine biosynthesis via the SHMT2 gene in the mitochondria. A scheme of the pathway is shown under the graphs, highlighting the sequence of events from glucose to synthesis of serine, glycine and the one carbon metabolism compound dimethylglycine. The genes significantly altered in OE cells from the RNAseq analysis involved in the pathway are shown in red. (**C**) Relative abundance of metabolites associated glutathione (GSH) homeostasis. The S-glutathionyl-L-Cys metabolite is associated with ineffective handling of oxidative stress. A scheme to the pathway is shown below. \* *p* < 0.05, \*\* *p* < 0.01. (**D**) Gene expression analysis of OE versus KD cells for CTH (*<sup>p</sup>* <sup>=</sup> 6.29 <sup>×</sup> <sup>10</sup><sup>−</sup>13), CHAC1 (*<sup>p</sup>* <sup>=</sup> 3.6 <sup>×</sup> <sup>10</sup><sup>−</sup>12) and GSTO2 (*p* = 0.003) genes (adj *p*-value).

Metabolites involved in Glutathione (GSH) homeostasis were also significantly changed in the OE cells compared to the KD cells (Figure 3C), suggesting better antioxidant defense. Particularly, cystathionine and cysteine were significantly increased in OE versus KD cells, and between OE cells and controls, suggesting increased cysteine synthesis and availability to generate glutathione. These increases are associated with increased expression of CTH, a cystathionine gamma lyase that breaks down cystathionine to generate cysteine [32], Figure 3D. Unexpectedly, we also observed increased generation of two breakdown products of glutathione via the ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1) enzyme. These enzymes are gamma-glutamyl cyclotransferases, which are induced by ER stress and have specific activity towards glutathione [33]. CHAC1 has been shown to break glutathione into 5-oxoproline and cysteinyl-glycine (Cys-Gly), promoting the depletion of glutathione and stress-induced apoptosis in cysteine deprived cancer cells [34]. However, we did not observe a depletion of glutathione in OE cells with higher CHAC1 expression (Figure 3C), suggesting that metabolite recycling mechanisms or compensatory increased production of glutathione may exist in OE cells [35]. Another indication that OE may have more antioxidant defense than KD cells comes from the increased ratio of ascorbate to dehydroascorbate, which is likely maintained by the dehydroascorbate activity of Glutathione S-Transferase Omega 2 (GSTO2) [36], which is significantly upregulated in the OE cells and can recycle the glutathione to a reduced state, Figure 3C,D.
