Colorectal cancer (CRC) cells shift metabolism toward aerobic glycolysis and away from using oxidative substrates such as butyrate. Pyruvate kinase M1/2 (PKM) is an enzyme that catalyzes the last step in glycolysis, which converts phosphoenolpyruvate to pyruvate. M1 and M2 are alternatively spliced isoforms of the Pkm gene. The PKM1 isoform promotes oxidative metabolism, whereas PKM2 enhances aerobic glycolysis. We hypothesize that the PKM isoforms are involved in the shift away from butyrate oxidation towards glycolysis in CRC cells. Here, we find that PKM2 is increased and PKM1 is decreased in human colorectal carcinomas as compared to non-cancerous tissue. To test whether PKM1/2 alter colonocyte metabolism, we created a knockdown of PKM2 and PKM1 in CRC cells to analyze how butyrate oxidation and glycolysis would be impacted. We report that butyrate oxidation in CRC cells is regulated by PKM1 levels, not PKM2. Decreased butyrate oxidation observed through knockdown of PKM1 and PKM2 is rescued through re-addition of PKM1. Diminished PKM1 lowered mitochondrial basal respiration and decreased mitochondrial spare capacity. We demonstrate that PKM1 suppresses glycolysis and inhibits hypoxia-inducible factor-1 alpha. These data suggest that reduced PKM1 is, in part, responsible for increased glycolysis and diminished butyrate oxidation in CRC cells.

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Increased glucose utilization and glycolysis is an important component in colorectal cancer. Inflammation is a known promoter of cancer development and progression. It has been reported that increased production of pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNFα) promote glycolysis. Previously, in colorectal cancer cells, we have demonstrated that IL-1β increased glycolysis, while suppressing oxidative metabolism. However, the mechanism underlying how this pro-inflammatory cytokine increased glycolysis was unclear. We hypothesized that IL-1β promoted glycolysis by increasing AKT activation given the metabolic role of this protein in metabolism. AKT is downstream of the interleukin-1 receptor, which is the major receptor IL-1β signals through to elicit a cellular response. Here, we show that upregulation of glycolysis by IL-1β is mediated through the AKT and more specifically the downstream transcription factor hypoxia inducible factor-1 alpha (HIF-1α). Knockout of HIF-1α completely abolished the elevated glycolysis induced by IL-1β. In addition, IL-1β had profound effects on mitochondrial function through reducing basal respiration, spare respiratory capacity, and maximal respiration. These data suggest a mechanism by which IL-1β through AKT activation and HIF-1α, regulates glycolysis in colorectal cancer cells. This work was supported by USDA NIFA (2019-67017-29261). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Objectives Dietary fiber has been proposed to protect against colorectal cancer. Butyrate, a fiber metabolite that is produced by bacteria in the colon, is known to inhibit cell proliferation and promote cell differentiation, while also inducing apoptotic cell death in colorectal cancer cells at physiologically relevant concentrations. Unlike the majority of cells in the human body that prefer utilizing glucose, non-cancerous colonocytes use butyrate as their primary energy source. However, colorectal cancer cells shift away from utilizing butyrate towards glucose (the Warburg effect). A decrease in butyrate utilization by the colonocyte has been reported in ulcerative colitis (UC) and colorectal cancer (CRC). In both of these diseases, the protein called Pyruvate Kinase Isoform M2 (PKM2) is a factor that has been found to be elevated in colonocytes and is known to catalyze a key step in glycolysis. We hypothesize that upregulation of PKM2 in ulcerative colitis and colorectal cancer results in diminished butyrate oxidation, and increased glucose utilization in colonocytes. Methods Mitochondrial function, substrate utilization will be analyzed in several colorectal cell lines, isolated colonocytes, or colonocytes grown in 3-D culture where PKM2 is knocked down, knocked-out, or overexpressed. An in vivo mouse model of colitis will be used to study the impact of PKM2 in the injury and repair process. Results Knockdown of PKM2 in cancerous colonocytes was associated with reduced proliferation and increased apoptosis. Butyrate oxidation was also increased in PKM2 knockdown cells. PKM2 regulated mitochondrial function and impacted the expression of uncoupling proteins (UCPs). Elevated PKM2 in primary colonocytes was associated with diminished butyrate utilization. Finally, conditional knockout of PKM2 in the colon resulted inhibited DSS-induced colitis. Conclusions These results show an important role for PKM2 in promoting ulcerative colitis and colorectal cancer through shifting colonocyte metabolism away from butyrate utilization. Funding Sources University of Tennessee - Start-up Funds.

Colorectal cancer cell (CRC) increases the utilization of glucose and decreases the oxidation of butyrate, which is a primary energy source for non‐cancerous colonocytes. Butyrate is a short‐chain fatty acid that is a bacterial‐derived fiber fermentation product. It takes years for the colorectal adenoma to manifest as it progresses toward invasive carcinoma. Within this timeframe, the adenoma must acquire key genomic and metabolic modifications that promote tumor growth and progression. Understanding the metabolic shift mechanisms away from butyrate oxidation to glycolysis in the CRC is significant. The overexpression of the pyruvate kinase isoform M2 (PKM2) is observed in biopsies of colorectal cancer patients, which is a hallmark of cancer. Pyruvate kinase is a metabolic enzyme that catalyzes the last step in glycolysis, which converts phosphoenolpyruvate to pyruvate generating ATP. PKM1 is generally expressed in adult differentiated tissues, such as normal non‐cancerous colonic tissue, where it promotes oxidative metabolism. In contrast, PKM2 is expressed in proliferating cells, including colorectal cancer cells, where it enhances aerobic glycolysis. However, the function of PKM2 in colorectal cancer tumorigenesis remains controversial. Our preliminary studies suggest that PKM2 drives colorectal metabolism away from butyrate and toward glucose utilization. A stable knockdown of PKM2 in colorectal cancer cells increased the oxidation of butyrate. The major hypothesis for this project is that through enhancing glucose utilization and suppressing butyrate, utilization PKM2 promotes colorectal tumor progression. Here, we demonstrate that, when expressed rather than PKM2, the PKM1 increases butyrate oxidation in cancerous colonocytes. Support or Funding Information USDA R011770176