OBJECTIVE: The close relationship between obesity and type 2 diabetes (T2D) highlights the fact that most diabetes patients are overweight or obese. We propose that elevated glucose and free fatty acid levels in those patients cause beta-cell dysfunction. Chronic exposure to excess nutrients (glucose and free fatty acid) leads to glucolipotoxicity, characterized by basal insulin hypersecretion, a left-shift in the glucose dose-dependent insulin secretion curve, and blunted glucose-stimulated insulin secretion. One of the suggested reasons for this defect is elevated intracellular lipid. In this study, our objective was to investigate whether reducing beta-cell lipid levels can reverse basal insulin hypersecretion. METHODS: INS-1 (823/13) cells were cultured in 4 or 11 mM glucose media. Elevated glucose and KCl doses were added to cells in the insulin secretion experiments. In the KCl-induced insulin secretion experiment, cells were treated with a combination of 12 mM glucose and 250 μM diazoxide, then assigned to different test concentrations with elevated KCl doses. Insulin release and content were measured by the insulin ultra-sensitive homogenous time-resolved fluorescence (HTRF) kit (Cisbio). Following that, we monitored intracellular Ca2+ activity of KCl-induced insulin secretion on a fluorescence spectrophotometer F-2000 (Hitachi). Additionally, we acutely added Adipo C (20 µM) or fatty acid-free BSA to cells to reduce the lipids levels in the ß-cells. We also stained with Nile Red (Sigma) to examine the intrinsic lipid droplets in those cells. RESULTS: ß-cells cultured in excess nutrients (11 mM glucose) exhibited a left shift in the glucose dose-dependent response curve. The hypersecretion at low glucose could be blocked by the KATP channel activator, diazoxide, indicating that Ca2+ influx drives the increase in secretion at glucose concentrations normally considered basal. Here we extend this left shift to include KCl-induced insulin secretion, supporting a role for Ca2+ in the observed hypersensitivity. KCl-induced Ca2+ influx was also left-shifted. Interestingly, we found acute exposure to Adipo C or fatty acid-free BSA reversed the basal hypersecretion in cells cultured in excess nutrients. CONCLUSION: The work presented in this study provided supporting evidence that ß-cells cultured in excess nutrients were hypersensitive to glucose while extending these studies to KCl-induced insulin release. The excess nutrient-induced left shift in both glucose and KCl-stimulated insulin secretion was mediated by increased Ca2+. Thus, we postulate that excess nutrient exposure increases ß-cell plasma membrane lipids that alter Ca2+ handling to allow increased Ca2+ influx at inappropriate low glucose concentrations. Our results demonstrated that cells acutely exposed to the putative long-chain acyl-CoA synthetase inhibitor Adipo C or fatty acid-free BSA reversed basal insulin hypersecretion and supports a role for lipids mediating the adverse effect. T2D patients with obesity have a similar physiologically elevated fasting blood glucose and lipid. Thus, our findings suggest lowering lipid levels in ß-cells may have therapeutic potential in treating hyperinsulinemia leading to T2D.

Background: Tissue non-specific alkaline phosphatase (TNAP) is an enzyme which functions within the body to catalyze the hydrolysis of pyrophosphate to phosphate, and is a well-known mediator of bone mineralization. It has also been identified as a positive mediator of intracellular lipid accumulation (ICLA) in both murine and human preadipocytes as well as in the hepatocellular cell line HepG2. However, the mechanism through which TNAP functions to control ICLA is not known. Both osteoblasts and adipocytes are both of mesenchymal origin and thus may share conserved mechanisms through which TNAP functions. Within bone, TNAP converts pyrophosphate (which inhibits mineralization) to phosphate. This phosphate is essential to the mineralization process through binding to hydroxyapatite crystals, and it also activates the transcription of genes whose products function in osteoblast differentiation, including NRF2. This thesis therefore aimed to determine the role of both pyrophosphate and TNAP-generated phosphate in ICLA. In addition, it is possible that TNAP may interact with other proteins, as it is known that TNAP is able to dephosphorylate proteins such as tau. This thesis therefore aimed to determine whether TNAP binds to other proteins in the context of ICLA. Lipids are not only stored within hepatocytes and adipocytes, but are also found in cells of the adrenal cortex, and TNAP is known to be expressed within such cells. Therefore, this thesis also aimed to determine whether TNAP is involved in the accumulation of cholesterol esters within lipid droplets in the adrenal cortex. Methods: To determine the effect of high intracellular pyrophosphate levels on ICLA, 3T3-L1 cells (a preadipocyte cell line) were cultured in the presence and absence of probenecid, an inhibitor of the pyrophosphate transporter ANK, and induced to accumulate lipids. Lipid accumulation was monitored through Oil red O staining. The effect of probenecid treatment on TNAP activity and intracellular pyrophosphate levels was also analysed. To determine whether TNAP functions in ICLA by producing phosphate for gene induction, 3T3-L1 cells were stimulated to undergo ICLA in the presence and absence of the TNAP inhibitor levamisole, which in turn blocks ICLA. Levamisole treated cells were also incubated with phosphate to see if this would overcome the inhibitory effect of levamisole on ICLA. The ability of phosphate to induce gene expression of NRF2 was determined through real-time PCR. In addition, an NRF2 expressing plasmid was transfected into cells treated with the TNAP inhibitor levamisole to determine if this would also overcome the block on ICLA caused by TNAP inhibition. In silico analysis identified TRAF2 as a potential binder of TNAP. The expression of TRAF2 during ICLA was determined through real time PCR, and the effect of overexpression of TRAF2 on intracellular lipid accumulation was determined through the transfection of a TRAF2 expressing plasmid in cells induced to undergo ICLA. To determine whether TNAP modulates lipid accumulation in cells of the adrenal cortex, the Y1 murine adrenocortical cell line was cultured in the presence and absence of TNAP inhibitor levamisole, and ICLA measured by Oil Red O staining. The location of TNAP within Y1 cells was identified by histochemical staining. Results: Cells treated with probenecid showed increased pyrophosphate levels (expressed as a % of levels observed at baseline) when compared to untreated controls (155.5 ± 15.1 % vs 51.1 ± 18.9 %; p=0.001) after 24 hours of culture. Increased pyrophosphate levels resulted in ICLA within 3T3-L1 cells surpassing levels seen in untreated controls (507.4 ± 30.4 % vs 337.6 ± 16.17 %; p=0.004). This increase in pyrophosphate was coupled to an increase in TNAP activity within the initial 24 hours (291.5 ± 72.8 % vs baseline of 100%; p=0.038) compared to that seen in control experiments (103.43 ± 24.3 % vs baseline of 100%; p=0.848). Cells treated with levamisole showed minimal ICLA and when exogenous phosphate was added, lipid levels were reconstituted to levels similar to that seen in cells induced to accumulate lipids in the absence of levamisole (284.01 ± 62.52% vs 275.86 ± 35.52%; p= 0.83). In the presence of levamisole plus exogenous phosphate, NRF2 expression was upregulated within 1 hour of treatment to levels greater than that seen in the absence of phosphate but presence of levamisole (216.64 ± 19.24% vs 98.28 ± 3.79%; p=0.004). Expression of NRF2 (through transfection with an NRF2 expression plasmid) in cells deficient in TNAP activity (via levamisole treatment), and induced to accumulate lipids, was not able to completely reconstitute ICLA when compared to cells not treated with levamisole (193.72 ± 16.51 vs 326.46 ± 47.64; p = 0.019), but ICLA was still greater than that observed at baseline. In silico analysis predicted that TNAP would bind to TRAF2, yet neither band shift assays nor immune co-precipitation showed evidence of this. However, TRAF2 mRNA was down regulated within 3T3-L1 cells during adipogenesis, reaching levels of 15.27 ± 10.27% (p= 0.014) of baseline (levels prior to induction of intracellular lipid accumulation) by day 4 of lipid accumulation. Overexpression of TRAF2 during adipogenesis markedly reduced intracellular lipid accumulation (147.88 ± 11.28% vs 326.46 ± 47.64%; p=0.028 (after 8 days of culture)). In Y1 cells TNAP activity is upregulated during ICLA, reaching 233 ± 37.56% (p=0.019 vs. baseline) of baseline levels within the initial 24 hours. Inhibition of TNAP activity through levamisole treatment resulted in a decrease in ICLA when compared to cells not treated with levamisole. Histochemical analysis showed that TNAP activity was localised to the lipid droplet. Discussion and Conclusions: Within 3T3-L1 cells TNAP mediates intracellular lipid accumulation through the generation of phosphate. The phosphate is able to increase the expression of NRF2, however it is likely that NRF2 is not the only gene whose expression is regulated by TNAP-generated phosphate. It was found that TNAP and TRAF2 do not bind to each other in the context of ICLA; however TRAF2 is a negative mediator of ICLA through a TNAP-independent mechanism. Functional TNAP is necessary for the accumulation of cholesterol esters within the Y1 cell line, suggesting that TNAP is essential for lipid accumulation in cell types that store lipids in intracellular membrane-bound droplets in the form of triglycerides or cholesterol esters. / GR2018