The role of progesterone in regulating glucose metabolism in the uterine epithelium

Early embryonic loss in cattle occurs in 40-60% of pregnancies. The uterus and developing embryo need glucose. The embryonic demand for glucose increases by the blastocyst stage. How glucose is regulated to match the changing needs of the embryo is unclear. Glycogen is composed of thousands of glucose molecules and is present in the uterus of many species. Our lab has shown that the glycogen content of the bovine uterine epithelium was low on day 11 of the cycle when progesterone is high. Therefore, our objectives were to 1) make an immortalized bovine uterine epithelial (BUTE) bovine uterine fibroblast (BFIB) cell lines to investigate the hormonal control of glycogen metabolism in the uterine epithelium, 2) to elucidate the role of progesterone on glycogen breakdown in BUTE cells, 3) determine if acid α-glucosidase (GAA) is present in the endometrium and 4) determine how progesterone regulates expression of glucose metabolizing enzymes. Fresh endometrial biopsies were collected from a Holstein dairy cow, digested into a single cell suspension, and plated to allow the formation of individual colonies. Cells were immortalized with large T-antigen, and colonies with cobblestone or spindle-like morphologies were isolated, expanded, and confirmed to be epithelial (BUTE) or fibroblasts (BFIB). To determine if progesterone was glycogenolytic, BUTE cells were treated with IGF-1 (50 µg/ml to stimulate glycogenesis) and treated with progesterone for 48 hours. Progesterone decreased glycogen levels in BUTE cells by 99%. RU486, which antagonizes the nuclear progesterone receptor (nPR), did not block progesterone's effect. BUTE cells expressed all five membrane progesterone receptors (mPRs; α, β, γ, δ, and ε), and a mPR agonist (Org OD 02-0) reduced glycogen levels in BUTE cells by 94%. In vivo, immunohistochemistry showed that the bovine uterine epithelium expressed high levels of mPRα. These results suggest that progesterone acts through mPRs to stimulate glycogenolysis in BUTE cells. Neither progesterone nor Org OD 02-0 changed intracellular cAMP concentrations. In agreement, the adenylyl cyclase activator forskolin increased cAMP concentration but did not decrease glycogen levels. We found that progesterone increased phospho-AMPK levels by 18% at 24 hours compared to the control. Supporting these results in vivo, phospho-AMPK levels in the uterine epithelium were high in the bovine uterine epithelium during the luteal phase when glycogen levels were low. However, the AMPK activator (A-769662) did not reduce glycogen in BUTE cells. Dorsomorphin, an AMPK inhibitor, did not block the effect of progesterone on glycogen breakdown. Meanwhile, BUTE cells treated with D942, which increases intracellular AMP concentrations, showed a 92% decrease in glycogen levels compared to the control group. AMP allosterically activates glycogen phosphorylase, which would directly decrease glycogen levels. In human Ishikawa cells that express mPRs but not nPRs, progesterone and Org OD 02-0 also decreased glycogen levels. Glycogen can also be broken down in lysosomes by acid α-glucosidase (GAA). GAA was expressed in the luminal epithelium and BUTE cells. However, progesterone did not alter GAA protein levels or activity. To identify other pathways regulated by progesterone, BUTE cells were treated with progesterone for 24 hours, and RNAseq was performed. BUTE cells treated with progesterone had 1,623 genes upregulated and 1,449 genes downregulated. GO analysis found several over-represented pathways linked to metabolism. Interestingly, 3 of 7 genes in the pentose phosphate pathway were altered. To determine if the pentose phosphate pathway was upregulated, BUTE cells will be treated with progesterone to measure NADPH. Progesterone increased the level of NADPH in BUTE cells. In conclusion, our study has significant implications for understanding the role of progesterone in glucose and glycogen metabolism. We generated cell lines to model the bovine endometrium and showed that progesterone acts through the mPR in BUTE cells to stimulate glycogenolysis by glycogen phosphorylase. However, GAA is present in the cow endometrium but is not regulated by progesterone. The breakdown of glycogen could be crucial in providing glucose to endometrial tissue for glucose metabolism through the pentose phosphate pathway to increase NADPH within the cell.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Animal Sciences

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Berg, Malia Danielle

Contributors dc:contributor

• Dean, Matthew
• Miller, David
• Nowak, Romana
• Wheeler, Matthew

• Uterus
• Cow
• Glycogen
• Glucose Metabolism
• Membrane Progesterone Receptors
• Pentose Phosphate Pathway

Statement dc:rights

• Copyright 2024 Malia Berg

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/125599

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F125599