An Investigation of the Effects of Fatty Acid Type on Pancreatic β-Cell

FunctionAnne-Marie Reilly

Supervisor: Dr. Rosaleen DeveryX185

Project Aims and Objectives• Demonstrate that the long chain saturated fatty acid; palmitate (C16:0)

is cytotoxic to pancreatic β-cells.• β -ells exposed to palmitate for 24hrs-48hrs (LipoAcute) can recover

when palmitate is removed (LipoRecovered).• W–n3 Polyunstaturated Fatty Acids (PUFAs) such as (EPA) (C20:5) can

aid in this recovery.• Study the effect of insulin content and GSIS on the LipoAcute and

LipoRecovered model.• Show that lipotoxicity effects histone modifying Enzymes(HAT Activity).

Experimental Model- DesignBased on Malmgren et al. “Co-Ordinate Changes in Histone Modifications, mRNA Levels and Metabolite Profiles in Clonal INS-1832/13 β-Cells Accompany Functional Adaptions to Lipotoxicity”

LipoAcute

Control

LipoRecovered

0.5 mM Palmitate RPMI 1640

0.5 mM Palmitate

48 h

48 h 4 d Recovery

Figure 1. Experimental Model: 1.1E7 β-Cells were cultured for 2-3 d to reach ~ 50% ConfluencyLipoAcute: Cells were cultured for 6 d after seeding in RPMI 1640 medium, then exposed to 0.5 mM Palmitate for 48 hLipoRecovered: Cells were exposed to 0.5 mM Palmitate for 48 h 2 d, palmitate was then removed and cells were allowed to recover for 4 d in RPMI-1640.

Experimental Methods

1. Cell Line: Human Pancreatic β-Cells,1.1E7 Cells, Source: Sigma Aldrich, Recognised by the

ECACC, Depositor: Peter. R. Flatt

2. Cell Viability - Acid Phosphatase Assay

3. Insulin Content and Secretion - Mercodia Insulin ELISA Kit

4. Histone Acetyltransferase (HAT) Activity – Enzo Life Science HAT Colorimetric Activity Kit

Preparation of Palmitic Acid [0.5mM ]and BSA [10%] Solution

Ratio 6:1Palmitate : BSA

10% BSA (1mL Stock)10g / 100mL

100g / L1M BSA = 66000 Da.

100g/66000 = 1.5mM in 1 mL BSA

1 M Palmitic Acid = 256.43g/L

1mM = 0.256 g/L10mM = 0.0256 g/10mL

Dilute 10mM Palmitic Acid in 1mL Ethanol and top up with 9mL PB

S = 10mM SolutionPrecipitation occurs

Heat at 70oC until the solution becomes homogenous

Carry out a 1 in 10 Dilution of 10mM Palmitic Acid = 1mM

Take 9mL of a 70oC 1mM Palmitic Acid (0.9mM) solution and add it

to 1mL BSA (0.15mM)0.9mM Palmitate : 0.15mM BSA

Ratio 6: 1

Conjugate the Palmitic Acid and BSA solution at 37oC

1% BSA0.1% Ethanol

Palmitate (0.5 -1.0 mM) has a cytotoxic effect on 1.1E7 β-Cells

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 10

1000

2000

3000

4000

5000

6000

7000

*

[Palmitate] (mM)

Cell

Num

ber

Change in Cell Density ~70%

Figure 2. 48 h Exposure to 0-1mM Palmitate on Pancreatic 1.1E7 β-CellsSeeded 1000 Cells, 6 d in culture with a doubling time of 20 h, day of Acid Phosphatase Test ~ 6000 Cells in control wells.

30 h treatment with [EPA] < 50 mM had negligible effect on b-Cell Proliferation

0 5 10 15 20 25 30 35 40 45 500

1000

2000

3000

4000

5000

6000

[EPA] (mM)

Cell

Num

ber

Figure 3. Effect of EPA on Cell Viability on 1.1E7 Pancreatic β-Cells at Varying Concentrations (0-50 mM)Seeded at a density of 1x103 Cells in a 96-well plate, 3 d in culture on the third day EPA was added for 30 h. After which it was removed and cell viability was analysed.

4.5-27mM EPA has a Proliferative Effect on b-Cells36-45 mM EPA has a Cytotoxic Effect on b-Cells

Figure 4. Effect of EPA on Cell Viability on 1.1E7 Pancreatic β-Cells at Varying Concentrations (0-45 mM)Seeded 1000 cells in a 96-well plate, kept 2 d in culture. Day 3 EPA was added (0-45 mM). After 24 h the plate was analysed by Acid Phosphatase Assay.

0 4.5 9 13.5 18 22.5 27 31.5 36 40.5 450

20406080

100120140160

[EPA ] (mM)

% C

ell P

rolif

erati

on 60 % Increase in Cell Proliferation between [EPA ] 4.5-27 (mM)

EPA may have a Cytoprotective Effect on LipoAcute Cells

0 5 10 15 20 25 30 35 40 45 500

20

40

60

80

100

120

[EPA] (mM)

% C

ell P

rolif

erati

on

25% Increase in Cell Proliferation

Figure 5. Effect of EPA (0-50 mM) on LipoAcute Cells (0.5 mM Palmitate)Seeded at a density of 1x103 , 3 d in culture on the third day 0.5 mM Palmitate was added along with 0-50 mM EPA. After 30 h cell viability was assessed.

Initial LipoAcute

&LipoRecovered Model

Control LipoAcute LipoRecovered0

5000

10000

15000

20000

25000

30000

35000

Cell

Num

ber

1.1E7 B-cells kept in RPMI Medium for 6 d.

LipoAcute

Control

LipoRecovered

0.5 mM Palmitate RPMI 1640

0.5 mM Palmitate

48 h

48 h 4 d Recovery

Control - Standard Media 6 d

LipoAcute - 4 d standard media 48hr palmitate

treatment

LipoRecovered – 48 h palmitate treatment, 4 d

recoveryFigure 6. First Plate: Seeded 3x104 Cells d 1

Control LipoAcute LipoRecovered0

1000

2000

3000

4000

5000

6000

7000

Cell

Num

ber

Figure 7. Second Plate: Seeded 660 Cells on 1 d. 1.1E7 Cells double every 23 h

~ 30% Decrease in Cell Viability

Control LipoAcute LipoRecovered0

2000

4000

6000

8000

10000

12000

14000

16000

18000

Cell

Num

ber

Figure 8. Third Plate: Seeded 2x103 Cells/Well 1 d. 1.1E7 Cells double every 23 h, 48 h 0.5mM palmitate, 3 d recovery.

~55% Decrease in Cell Viability

Where to go from here????Future Experiments Determine the lipotoxic time frame of 1.1E7 b-cells (24 h,48 h???)

Can b-cells recover after undergoing lipotoxicity?

Phenol Red promotes cell growth – Use RPMI 1640 Phenol Red Free Media when treating cells.

LipoAcute and LipoRecovered Insulin content/secretion and HAT activity.

1.1E7 b-Cells Recover after undergoing Lipotoxic Conditions for 24 h

Control β-Cells 5 d

Palmitate 5 d LipoAcute 5 d RPMI, Palmitate

LipoRecovered Palmitate, 3 d

Recovery

LipoRecovered Palmitate, 3 d

Recovery + Fresh RPMI-1640 every

day

0

1000

2000

3000

4000

5000

6000

7000

24 Hr Palmitate Exposure

48 Hr Palmitate Exposure

Cell

Num

ber.

Figure 9. 24 h vs 48 h Palmitate Exposure Across LipoAcute and LipoRecovered Models

Glucose Stimulated Insulin Secretion(GSIS)

Standard Curve for Insulin ELISA

0 5 10 15 20 250

0.5

1

1.5

2

2.5

f(x) = 0.107706075517468 x + 0.0228479223503456R² = 0.995486322226772

[Insulin ] mU/L

Aver

age

Abso

rban

ce fo

r Ins

ulin

450

nm

HRPTMBH2O2

Figure 10.

LipoRecovered Cells have a higher Insulin Content than untreated 1.1E7 b-Cells

Figure 11. Control: 8 d in culture, Palmitate: 5 d exposure to 0.5mM palmitate, EPA: 5 d exposure to 5mM EPA, LipoAcute: 6 d in RPMI-1640 standard, 24 h 0.5mM palmitate, LipoR. RPMI-1640 Standard: 24 h 0.5mM palmitate, 5 d recovery, LipoR. RPMI-1640 Phenol Free: 24 h 0.5mM palmitate, 5 d recovery, LipoR. RPMI-1640 Phenol Free + 5mM EPA: 24 h 0.5mM palmitate, 5 d recovery.

0

2

4

6

8

10

12

Insu

lin/S

ampl

e m

U/L

Chronic 0.5mM Palmitate and 5 mM EPA compromises GSIS

-50

-40

-30

-20

-10

0

10

20

% C

hang

e

Figure 12. % Change in Basal Insulin Secretion 2.8mM Vs. Stimulated Secretion 16.7mM

55% Decrease in Insulin Secretion

Histone Acetyltransferase Activity(HAT)

HAT Activity- Kinetic Study Ranging from 30-240 min

Ctrl. R

PMI-1640 Standard

Ctrl. R

PMI-1640 Phenol R

ed Free

Palmita

te EPA

LipoAcu

te

LipoReco

vered RPMI-1640 Standard

LipoReco

vered RPMI-1640 Phenol F

ree

LipoReco

vered + EPA NE-1

-0.5

0

0.5

1

1.5

2

30 Min60 Min90 Min120 Min240 Min

Abso

rban

ce 4

40nm

Figure 13.

HAT Activity - 60min

Ctrl. RPMI-1640 Standard

Palmitate LipoAcute LipoRecovered RPMI-1640

Standard

NE0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6Ab

sorb

ance

440

nm

~3 Fold Decrease

Figure 14.

Revised LipoAcute and LipoRecovered Model

LipoAcute

Control

LipoRecovered

> 0.5 mM Palmitate RPMI 1640- Phenol Free

> 0.5 mM Palmitate

24 h

24 h 3 d Recovery

5 d in Culture

1 d Before Treatment

Figure 15. Experimental Model: 1.1E7 β-Cells treat at ~ 50% Confluency in RPMI-1640-Phenol Red Free, FBS FreeLipoAcute: Grow for 5 d after seeding in RPMI 1640 medium, then treat with >0.5 mM Palmitate for 24 hLipoRecovered: Expose to >0.5 mM Palmitate for 24 h, remove palmitate was then remove palmitate and allow to recover for 3 d in RPMI-1640.

Conclusion and Future ExperimentsConclusion0.5mM Palmitate depletes 1.1E7 cells4.5-27mM EPA has a proliferative effect on

1.1E7 cells1.1E7 cells can recover from 24 h acute

palmitate exposureLipoRecovered cells capable of producing

insulin to the capacity of control cells (Content).Chronic exposure of LCSSFA and PUFA results in

loss of Insulin Secretion where as acute exposure mildly disrupts GSIS. Liporecovered cells can recover insulin secretion function

HAT activity is decreased 8 fold in chronic palmitate and acute cells whilst recovered cells have HAT activity that is comparable to control cells.

Future workLipoAcute and LipoRecovered Model requires

revision• Use lower concentration of palmitate >0.5mM for

1.1E7 cells• Final concentration of BSA = 0.5 % • EPA – Conjugate with BSA• Determine a concentration of EPA which aids

LipoRecovered cells and prevents acute effects of palmitate on b-cells

HDAC AssayDetermine Viability of treated cells by FACs

analysis: Staining for FFAR1Carry out Western Blot analysis for FFAR1

Thank You!

Questions?