1. Daniel R. Lathen, Jeffery S. Tessem
β-cell Adaptation to Elevated Palmitate Concentrations (Hyperlipidemia)
Daniel R. Lathen, Jeffery S. Tessem
Nutrition, Dietetics and Food Science Department, College of Life Sciences, Brigham Young University, Provo, Utah, 84602
BYU
Abstract
Palmitate Initially Enhances, But Ultimately Hinders, β-cell Proliferation
Palmitate Alters Expression of Nkx6.1 A. B. C.
It is estimated that over 370 million people worldwide suffer from diabetes, and this number is increasing rapidly. Normally, β-cells in the pancreas secrete insulin, which is necessary for proper glucose absorption and storage. Both Type 1 and Type 2 diabetes are characterized by decreased functional β-cell mass and insulin production, and increased circulating glucose and fatty acid levels. Diabetics’ pancreata maintain small amounts of functional β-cells, but these surviving cells are damaged and destroyed over time due to the harmful effects of hyperglycemia and hyperlipidemia. These β-cells must adapt to survive. Hence, diabetes is itself a selective process. We have mimicked and analyzed this selection process in vitro by culturing β-cells under conditions of increasingly elevated palmitate concentration applied step-wise, creating distinct cell lines acclimated to various levels of palmitate concentration with corresponding control lines. Through analysis of these lines, we aimed to simulate the gradual progression of hyperlipidemia seen in diabetic patients, and thus determine mechanisms and effects of β-cell adaptation to these conditions. Respiratory control ratios (RCR) suggest that β-cell lines adapted to higher palmitate concentrations do not adversely affect mitochondrial functionality, and Oroboros (O2K) analysis in fact implies increased cellular respiration in these cell lines. Further, DCF analysis reveals decreased free radical formation in treated cells. Importantly, cell counts indicated that proliferation increased when cells were treated with low (0.15 mM) concentrations of sodium palmitate, but decreased at 0.3 mM concentrations and higher. As measured with RT-PCR, expression of transcription factor Nkx6.1, a gene key to β-cell function and survival, follows a similar pattern. Expression of transcription factors Nr4a1 and Nr4a3, conversely, is generally down-regulated for all concentrations of palmitate. These data imply that β-cell proliferation is boosted by low-level exposure to palmitate but inhibited by excessive exposure. Respiratory functionality is not adversely affected by palmitate treatment and may in fact be improved, thus the observed change in proliferation rates may potentially be modulated by expression of key transcription factors such as Nkx6.1, Nr4a1, and Nr4a3. These results represent a significant step towards understanding the causes and effects of the transition that occurs between low and high levels of palmitate adaptation, ultimately promoting investigation into methods by which endogenous β-cells could increase cellular function, survival, and proliferation under hyperlipidemic conditions.
Figure 2: Palmitate Enhances β-cell Proliferation at Low Concentrations, But Inhibits Proliferation at Higher Concentrations A) Cells raised in 0.15 mM palmitate show significantly enhanced proliferation by 96 hours. B) Cells raised in 0.3 mM palmitate show significantly reduced proliferation by 72 hours. C) Cells raised in 0.5 mM palmitate show significantly reduced proliferation by 96 hours. It is significant that proliferation continues successfully under these higher concentrations, albeit at a reduced rate compared to controls. * = P < .05, ** = P < .01, *** = P < n ≥ 4. Bars show SEMs.
Figure 5: Palmitate Raises Expression of Nkx6.1 at Low Concentrations But Reduces Expression at Higher Concentrations We have shown previously that expression of the transcription factor Nkx6.1, which lies upstream in the regulation pathway of the Nr4a genes, directly correlates with expression of Nr4a1 and Nr4a3. β-cells cultured in 0.15 mM palmitate showed increased, though statistically insignificant (P = .260), expression of Nkx6.1, compared to decreases expression at the 0.3 and 0.5 levels, in keeping with patterns of proliferation demonstrated in Figure 2. P0.3 = .006, P0.5 = n ≥ 7. Bars show SEMs.
Palmitate Promotes Healthy Cellular Respiration A. B.
Conclusions
We have shown that adaptation to low concentrations of palmitate leads to enhanced proliferation of β-cells. Further, β-cells are capable of sufficient adaptation to increasingly higher levels of palmitate, as is theoretically present in diabetics, to permit continued, albeit reduced, proliferation.
The mechanisms responsible for the transformation in relative proliferation rates that occurs between the 0.15 mM and 0.3 mM levels require further characterization before they can be fully understood. Based on the data presented here, it is evident that changes in mitochondrial populations or function are present but are not deciding factors, since enhanced cellular respiration in treated cells is not consistent with observed patterns of proliferation. Observed alterations in expression of the key transcription factors Nkx6.1, Nr4a1, and Nr4a3 are consistent with our previous findings that up-regulation of these genes is sufficient and necessary to drive proliferation in β-cells. It is likely that changes in the expression of these genes are responsible for the variation in proliferation observed between the different levels of palmitate exposure. However, because these expression variations were generally insignificant, while cell count variations exhibited clear statistical differences, it must be assumed that other, hitherto untested factors must also play a role.
Further experimentation is necessary to establish the significance of the above-mentioned variations in transcription factor expression, assuming that said variation does in fact exist, as well as to further characterize the adaptations that occur that permit enhanced proliferation of the 0.15 mM cell line but inhibited proliferation of the 0.3 mM cell line. Tests of apoptotic rates and Glucose Stimulated Insulin Secretion (GSIS), for instance, will further elucidate the observed phenomena and potentially establish more effective methods to treat diabetes. C.
Experimental Design
GSIS Mechanism in b-cells
INS β-cells were cultured under conditions of increasingly high concentrations of palmitate, in sync with corresponding controls, (Fig. 1). At the 0.15 mM level, for instance, treatment media sufficient for one tissue culture dish was created by mixing 60 μL of 25 mM palmitic acid dissolve in pure ethanol with 10 mL of media enhanced with 2% BSA protein carrier. The corresponding control was formulated by mixing 60 μL pure ethanol into 10 mL of the same BSA-enhanced media to eliminate all known sources of variability other than the palmitate itself.
Each cell line was given time to adapt to its new conditions before any assays were performed.
Figure 3: Palmitate Promotes Cellular Respiration A) No decreases in function of the TCA cycle or the ETC were observed when palmitate-treated cell lines were tested with Oroboros assay (O2K); in fact, oxygen flux was significantly up-regulated in several instances. GM = glutamate + malate, D = ADP, S = succinate, F = FCCP. n = 3
B) Representative DCF assay reveals significantly higher fluorescence in the palmitate line, signifying reduced free radical formation from cellular respiration. n = 12.
C) Respiratory Control Ratio reveals no significant difference in mitochondrial function between treatments and controls. n = 3. * = P < .05, ** = P < .01, *** = P < .001.
n = 3. Bars show SEMs.
Palmitate Causes Decreased Expression of Nr4a1 and Nr4a3
Figure 1
Figure 4: Palmitate Causes Decreased Expression of Nr4a1 and Nr4a3 β-cells cultured in all levels of palmitate tested exhibited reduced expression of both genes, as measured with RT-PCR, although only the 0.3 level is statistically significant. (For Nr4a1: P0.15 = .215, P0.3 = .022, P0.5 = .089; for Nr4a3: P0.15 = .084, P0.3 = .008, P0.5 = .071). n ≥ 7. Bars show SEMs.
Acknowledgements
Brigham Young University Office of Research & Creative Activities (ORCA) Grant.
Jeremy Reid and Jordan Tingey for contributions to preliminary experiments.
Dr. Jeffery Tessem for unwavering assistance and guidance.