Investigation of the control of glucose metabolism in sertoli cells in culture

Cross, Michael (1985). Investigation of the control of glucose metabolism in sertoli cells in culture PhD Thesis, School of Biomedical Sciences, The University of Queensland. doi:10.14264/uql.2015.28

       
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Author Cross, Michael
Thesis Title Investigation of the control of glucose metabolism in sertoli cells in culture
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
DOI 10.14264/uql.2015.28
Publication date 1985
Thesis type PhD Thesis
Supervisor Unknown
Total pages 221
Language eng
Subjects 060104 Cell Metabolism
Formatted abstract
The following experiments are an investigation into various aspects of glucose metabolism by Sertoli cells in culture. Initially, and in most experiments, glucose metabolism was measured as an index of Sertoli cell function to determine how endocrine and physical parameters, specifically the polypeptide hormones FSH, LH and insulin; testosterone (T); Ca2+ and Ca-calmodulin (Ca.CM) and incubation temperature, influenced Sertoli cell function. The mechanism of FSH action on Sertoli cell metabolism was of particular interest and activities of glycolytic enzymes, the involvement of cAMP and Ca.CM were investigated for this reason. Interwoven with these considerations was the role of aerobic glycolysis, as an integrated cell function of importance to normal Sertoli cell and testicular function.

Glucose is metabolised by cells through several pathways. In these experiments total glucose uptake and metabolism, glycolysis, CO2 (oxidative metabolism) and protein (acid precipitable material) were measured and compared to ABP production by the cells, also used as an index of Sertoli cell function.

Overall glucose uptake and conversion of glucose to lactate and protein is specifically stimulated by FSH. Both LH and T had only slight and variable effects on these parameters 'while hCG had no effect on glucose uptake, suggesting the action of LH resulted from minor FSH contamination, which varied from preparation to preparation.

In combination with FSH, both LH and T had little or no additional effect on FSH-stimulated metabolism. The secretion of ABP was also not affected when LH was added with FSH, suggesting that FSH does not stimulate LH receptors in a manner analogous to FSH stimulation of LH receptors in the ovary. With FSH and T together ABP secretion was increased relative to FSH alone, but since glucose uptake and glycolysis were not markedly altered, the indication is that FSH and T have separate, but overlapping domains of influence on Sertoli cell function, and that FSH is the major determinant of increased glucose metabolism.

Glucose uptake and glycolysis were stimulated in a graded fashion by levels of FSH ranging from 1ng/ml to 1µg/ml. Stimulation occurred within 2-4 hours whereas glucose incorporation into protein was not affected until after 16 hours. Increased secretion of ABP was usually not observed until 24 hours after hormonal additions. The differences in times of stimulation suggest that glycolysis may be controlled by cytoplasmic factors which are readily organised, while incorporation into protein and ABP production requires stimulation of the genome.

Glucose metabolism was stimulated by cAMP and isoprenaline, a β-adrenergic agent, which in other tissues exerts its action via cAMP. Phenylephrine, an α-adrenergic agent was ineffective. The results suggest that cAMP mediated phosphorylation is important for FSH action. On the other hand Ca2+ and Ca.CM were not involved in FSH action to stimulate glucose metabolism. This suggests that there is no interaction between Ca2+-ion and cAMP in FSH action on the Sertoli cell. Calcium-ion inhibited protein synthesis while FSH stimulated, suggesting two opposite, independent controls on protein synthesis in the Sertoli cell.

Glucose metabolism was stimulated by Ca2+ and was reduced by inhibition of Ca.CM by trifluoperazine. This result suggests the presence of two independent controls of glucose metabolism in the Sertoli cell. The system controlled by Ca.CM may regulate basal metabolism in cells not stimulated by FSH, and may change in response to agents other than FSH.

The Sertoli cells converted about 5 times more glucose to lactate than to CO2 suggesting the cell is aerobically glycolytic. The proportion of glucose converted to lactate (aerobic glycolysis) was significantly increased by FSH, but other factors appear to be also involved. Oxidation of glucose by the cells was inhibited by FSH on the same time scale that glycolysis was increased, further indicating that aerobic glycolysis is an important function of Sertoli cells. The significance of aerobic glycolysis in Sertoli cell function is discussed.

Possible means by which FSH stimulates glucose metabolism and glycolysis were investigated. Although not fully documented in this thesis, glucose transport using OMG was measured, but no conclusions were reached. Activities of HK, PFK and PK considered rate-limiting in glycolysis and LDH were measured following hormone stimulation. The activity of LDH was consistently increased relative to unstimulated cells, the activity of PFK was occasionally increased but was usually unaffected and activities of HK and PK were unchanged by FSH treatment. Increased LDH activity was specific and sensitive to FSH addition at 10ng/ml, but showed no graded response with FSH concentrations up to 1µg/ml. LDH activity was first increased at 8 hours after FSH addition, which was much later than the earliest increase in glucose uptake and glycolysis. It was concluded that total enzyme activity did not reflect the flux of glucose through glycolysis, and that LDH, while not acting as a rate-limiting enzyme may be important in maintaining aerobic glycolysis in Sertoli cells, stimulated by FSH.

When cells were incubated at 37°C for 72 hours glucose metabolism was changed when compared to cells at 32°C. Glycolysis fell more rapidly at 37°C and aerobic glycolysis was reduced within 24 hours. Basal glucose metabolism was reduced at 37°C after 24 hours but remained unchanged at 32°C. FSH-stimulated metabolism was not changed by incubation at the elevated temperature. The results again suggest that there are two independent controls of glucose metabolism and activity of the Ca.CM system, mentioned previously, is reduced by the higher incubation temperature.

In conclusion FSH stimulates glucose metabolism, represented by glucose uptake, lactate accumulation and incorporation into protein, while LH and T has little affect either alone or in combination with FSH. Aerobic glycolysis appears to be a normal function of Sertoli cells which respond to FSH by increasing lactate production and decreasing CO2 production. Both FSH and Ca2+ ion stimulate glucose metabolism, but the mechanisms of action are independent of each other. Two controls of glucose metabolism are indicated by these experiments, one which responds to FSH and is not affected by elevated incubation temperature (37°C) and the other which is controlled by Ca.CM and is reduced by incubation at 37°C.
Keyword Glucose -- Metabolism
Additional Notes Other Title: Glucose metabolism in cultured sertoli cells.

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
 
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Created: Mon, 05 Jan 2015, 14:54:02 EST by Mary-Anne Marrington on behalf of Scholarly Communication and Digitisation Service