Novel mechanism of chronic exposure of oleic acid-induced...

42
JPET #105759 1 Novel mechanism of chronic exposure of oleic acid-induced insulin release impairment in rat pancreatic β-cells Takanori Kudo, Jie Wu, Yoshiji Ogawa, Sechiko Suga, Noriyuki Hasegawa, Toshihiro Suda, Hiroki Mizukami, Soroku Yagihashi, and Makoto Wakui Third Department of Internal Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562 (T.K., Y.O., N.H., T.S.) Division of Neurology, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, Phoenix AZ 85013, USA (J.W.) Department of Physiology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan (S.S., M.W.) Department of Pathology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan (H.M., S.Y.) JPET Fast Forward. Published on June 6, 2006 as DOI:10.1124/jpet.106.105759 Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics. This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759 at ASPET Journals on February 26, 2020 jpet.aspetjournals.org Downloaded from

Transcript of Novel mechanism of chronic exposure of oleic acid-induced...

Page 1: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

1

Novel mechanism of chronic exposure of oleic acid-induced insulin

release impairment in rat pancreatic β-cells

Takanori Kudo, Jie Wu, Yoshiji Ogawa, Sechiko Suga, Noriyuki Hasegawa, Toshihiro

Suda, Hiroki Mizukami, Soroku Yagihashi, and Makoto Wakui

Third Department of Internal Medicine, Hirosaki University School of Medicine, 5

Zaifu-cho, Hirosaki 036-8562 (T.K., Y.O., N.H., T.S.)

Division of Neurology, Barrow Neurological Institute, St. Joseph’s Hospital and

Medical Center, Phoenix AZ 85013, USA (J.W.)

Department of Physiology, Hirosaki University School of Medicine, 5 Zaifu-cho,

Hirosaki 036-8562, Japan (S.S., M.W.)

Department of Pathology, Hirosaki University School of Medicine, 5 Zaifu-cho,

Hirosaki 036-8562, Japan (H.M., S.Y.)

JPET Fast Forward. Published on June 6, 2006 as DOI:10.1124/jpet.106.105759

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 2: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

2

Running title: Chronic oleic acid alters ATP sensitivity of KATP channel.

Please send all correspondence to: Jie Wu, M.D., Ph.D.

Director, Neural Physiology Laboratory

Neurology Research

Barrow Neurological Institute

St. Joseph’s Hospital and Medical Center

Phoenix, AZ 85013-4496

Tel: (602)-406-6376

Fax: (602)-406-7172

e-mail: [email protected]

Number of text pages: 37

Number of tables: 0

Number of figures: 6

Number of references: 40

Number of words in Abstract: 249

Number of words in Introduction: 607

Number of words in Discussion: 1,466

Nonstandard Abbreviations: OA, oleic acid; KATP channel, ATP-sensitive potassium

channel

Section Assignment: Endocrine and Diabetes

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 3: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

3

Abstract

A sustained, high circulating level of free fatty acids (FFAs) is an important risk factor

for the development of insulin resistance, islet β-cell dysfunction and pathogenesis of

type 2 diabetes. Here, we report a novel mechanism of chronic exposure of oleic acid

(OA)-induced rat insulin release impairment. Following a four-day exposure to OA (0.1

mM), there was no significant difference in basal insulin release when comparing

OA-treated and untreated islets in the presence of 2.8 mM glucose, while glucose (16.7

mM)-stimulated insulin release increased 2-fold in control, but not in OA-treated, islets.

Perforated patch-clamp recordings showed that untreated β-cells exhibited a resting

potential of –62.1 ± 0.9 mV and were electrically silent, whereas OA-treated β-cells

showed more positive resting potentials and spontaneous action potential firing.

Cell-attached single channel recordings revealed spontaneous opening of ATP-sensitive

potassium (KATP) channels in control, but not in OA-treated, β-cells. Inside-out excised

patch recordings showed similar activity in both OA-treated and untreated β-cells in the

absence of ATP on the inside of the cellular membrane, while in the presence of ATP,

KATP channel activity was significantly reduced in OA-treated β-cells. Electron

microscopy demonstrated that chronic exposure to OA resulted in the accumulation of

triglycerides in β-cell cytoplasm and reduced both the number of insulin-containing

granules and insulin content. Collectively, chronic exposure to OA closed KATP

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 4: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

4

channels by increasing the sensitivity of KATP channels to ATP, which in turn led to the

continuous excitation of β-cells, depletion of insulin storage and impairment of

glucose-stimulated insulin release.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 5: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

5

Introduction

Diabetes mellitus is a collection of diseases characterized by high levels of blood

glucose resulting from defects in insulin production, insulin action, or both. Type 2

diabetes may account for more than 90% of all diagnosed cases of diabetes. Onset

usually begins as insulin resistance develops—a disorder in which cells do not use

insulin properly and/or a gradual loss arises of the ability of pancreatic β-cells to

produce insulin as the need for insulin increases due to elevated circulating glucose

levels.

Patients afflicted with type 2 diabetes often are associated with having an elevated

body weight. A sustained, high circulating level of free fatty acids (FFAs) is thought to

cause an accumulation of triglycerides in tissues (Zhou et al., 1996; Man et al., 1997;

Shimabukuro et al., 1997), insulin resistance (Ferrannini et al., 1983; Lillioja et al.,

1985; Lee 1988) and islet β-cell dysfunction (Lee et al., 1994; Zhou and Grill, 1994;

Zhou et al., 1996; Bollheimer et al., 1998; Roche et al., 2000). Although it is still not

well-understood, several possible mechanisms have been postulated to interpret

FFA-induced insulin resistance. For instance, inhibition of both insulin-activated

carbohydrate oxidation (Bonadonna et al., 1989; Felley et al., 1989; Bevilacqua et al.,

1990) and glucose uptake appear to be involved in FFA-induced insulin resistance

(Boden et al., 1994). In addition, inhibition of glycogen synthesis by FFAs may also

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 6: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

6

compete with insulin action, resulting in an increase of blood glucose concentration

(Boden et al., 1994). Also, FFAs may inhibit insulin synthesis (Lee et al., 1994;

Furukawa et al., 1999) and the glucose-fatty acid cycle may be responsible for the

impairment of insulin biosynthesis (Lee et al., 1994; Furukawa et al., 1999).

Furthermore, FFA-induced inhibition of insulin secretion may be mediated by a

decrease in glucose effectiveness on β-cells. Glucose-stimulated insulin secretion from

islet β-cells is triggered by an elevation of intracellular Ca2+ concentration ([Ca2+]i)

(Wollheim and Sharp, 1981), which occurs via the following steps (Ashcroft and

Ashcroft, 1992): 1) levels of intracellular ATP increase in the cell; 2) KATP channel

activity is inhibited by ATP, and 3) the opening of the Ca2+ entry pathway (via L-type

Ca2+ channel activation) leads to a subsequent increase in [Ca2+]i. Finally, with respect

to the effects of FFAs on the membrane properties of islet β-cells in vitro, acute

application of FFAs has been shown to induce activation of KATP channels and

hyperpolarization of β-cells (Larsson et al., 1996; Branstrom et al., 1997; Gribble et al.,

1998; Gravena et al., 2002; Branstrom et al., 2004), which then makes it more difficult

for β-cells to respond to glucose stimulation. Chronic exposure of β-cells to FFAs has

been shown to increase basal insulin release but decrease glucose-stimulated insulin

secretion (Elks, 1993; Zhou and Grill, 1994; Prentki and Corkey, 1996; Bjorklund et al.,

1997; Hosokawa et al., 1997; Bollheimer et al., 1998; McGarry and Dobbins, 1999;

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 7: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

7

Zhang et al., 2005). However, the chronic effects of FFAs on β-cell membrane

properties, especially on KATP channel function, have not been studied.

The aim of the present investigation was, therefore, to examine the effects of

chronic exposure to OA on insulin release, β-cell electrophysiological properties, KATP

channel activity, insulin-containing granules and insulin content using multiple

experimental approaches. The presented results show that after exposure to OA for four

days, rat islet β-cells were excited rather than silent, and that an increased sensitivity of

KATP channels to ATP underlay the OA-induced β-cell excitation. The elucidation of

this novel mechanism—that chronic exposure to OA altered the sensitivity of KATP

channels to ATP—provides new insights into more fully understanding the cellular

mechanisms of insulin resistance associated with type 2 diabetes patients.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 8: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

8

Materials and Methods

This study was carried out in accordance with the Guidelines for Animal

Experimentation, Hirosaki University, Japan.

Islet isolation and OA loading. Adult, male Sprague-Dawley rats were used.

Islets were isolated from the pancreas by following the collagenase digestion method

(Suga et al., 1997) with some modifications. Briefly, rats were anesthetized with

pentobarbital (Dainabot, IL, USA) at 30 mg/kg body weight, and collagenase (type S,

Nitta Zelatin, Osaka, Japan), which was dissolved in Hank’s balanced salt solution

(HBSS, Sigma Chemical Co., St Louis, MO, USA) at 1 mg/mL, was injected into the

pancreatic duct through the bile duct. The distended pancreas was removed and

incubated for digestion at 37°C for 18 min. The digested pancreas was then washed with

HBSS, which included 2% bovine serum albumin (BSA), and filtered through stainless

mesh, and finally the islets were separated by the histopaque (specific gravity 1.077;

Sigma Chemical Co., St Louis, MO, USA) gradient method. Islets were incubated for 2

days with 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, NY, USA)

with 10% bovine serum, 10000 U/mL penicillin and 10 mg/mL streptomycin. Islets

were then separated into two groups. Islets in one group were transferred to culture

medium of the same composition as described above and incubated for 3 days. Islets in

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 9: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

9

the other group were transferred to culture medium which contained 0.1 mM oleic acid

(Nacalai Tesque, Inc., Kyoto, Japan) and were also incubated for 3 days.

Isolation of islet β-cells. Following incubation for 3 days in culture medium with and

without OA, islets were further digested by disperse (1000 U/mL, Godo Shusei, Tokyo,

Japan) in order to be separated into single cells as previously described (Suga et al.,

1997). Separated cells that had been previously exposed to OA were then incubated for

an additional 24 h in the presence of 0.1 mM OA and used for electrophysiological

experiments. During the isolated cell preparations, the percentages of α-cells (8%),

β-cells (87%) and δ-cells (4%) were determined by immunohistochemical staining as

previously described (Suga et al., 2003).

For identification of β-cells in electrophysiological experiments, excitatory

responses to 16.7 mM glucose or 0.5 mM tolbutamide (Sigma Chemical Co., St Louis,

MO, USA ), tested at the end of experiments, were confirmed. Furthermore, the values

of percentages of cells showing the same results were used for judging whether β-cells

were included in those islets.

Histology of islet β-cells. Two dishes of islets, one incubated for 4 days with and the

other without OA, were prepared. For electron microscopy, a pellet of pancreatic islets

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 10: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

10

from each group was fixed in ice-cold 2.5% glutaraldehyde in 0.05 M cacodylate buffer

(pH 7.4) overnight at 4°C and postfixed with 1% osmium tetroxide (Sigma Chemical

Co., St Louis, MO, USA) for 1 h at room temperature. After dehydration through an

ascending series of ethanol, the samples were embedded in epon. Ultrathin sections

from epon-embedded blocks were stained with uranyl acetate and lead citrate. They

were examined by a JEOL TEM-2000EX electron microscope (Nihon Densi, Tokyo,

Japan).

Measurement of total protein and insulin in islets. In order to measure the amount of

total protein in islets, two groups of islets, one incubated with and the other without OA

for 4 days, were prepared. In each dish, 200 islets were plated at the start of culture, and

the number of islets in the dish after 3 days of incubation was not different. The 200

islets were homogenized in 100 µL cold TE buffer (50 mM Tris, 1 mM EDTA, pH 7.4)

with 1 mM phenylmethyl-sulfonyl fluoride and 0.2 mM N-ethylmaleimide (Sigma

Chemical Co., St Louis, MO, USA). The protein concentration in each sample was

measured by a Bio-Rad protein assay (Bio-Rad, CA, USA). For measurement of the

amount of insulin in islets, islets were prepared in a similar way as described for

measurement of total protein. 20 islets from each group (i.e., one with and the other

without OA treatment) were hand-picked and homogenized in 100 µL cold acid ethanol.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 11: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

11

The homogenate was stored at 4°C overnight and then centrifuged at 2500 r.p.m for 30

min. The supernatant was collected and stored at –20°C until insulin assay experiments.

Samples were diluted 1:5000 in normal saline which contained 2% bovine serum

albumin. Insulin concentration was measured using a Rat Insulin ELISA (Mercodia,

Uppsala, Sweden).

Measurement of insulin secretion. In each experiment, 20 islets were placed on 8.0

mm polyethylene terephthalate membrane (FALCON, Cell Culture Insert, NJ, USA),

and then the membrane with islets was placed in HBSS which contained 2% BSA and

2.8 mM glucose for 1 h before measurement of insulin secretion. The medium was then

discarded and replaced with fresh medium having the same composition. During

exposure of the islets to the medium for 15 min, the medium was collected every 1 min

for insulin measurement (basal secretion). Islets were then exposed to a medium which

contained 16.7 mM glucose for 15 min and insulin secretion was also measured as

recently described. Then, the medium was replaced with one which contained the

original concentration of glucose (2.8 mM) and insulin secretion was again measured as

recently described. Each sample was then stored at –20°C until insulin assay

experiments were carried out as described above.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 12: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

12

Patch-clamp recordings. Isolated cells were kept in a 35-mm Petri dish and the dish

was placed on the stage of an inverted microscope (IMT-2, Olympus, Tokyo, Japan). A

patch-clamp amplifier (EPC-7, HEKA Electronik, Lambrecht, Germany) was used to

measure both membrane potentials and currents. The amphotericin B (Sigma Chemical

Co., St Louis, MO, USA)-perforation method was used to measure the membrane

potential of the cell. The resistance of the electrode, when filled with pipette solution,

ranged from 2 to 4 MΩ. During whole-cell current recordings, the membrane

capacitance of single cells ranged from 8 to 14 pF. Only experiments with series

resistances below 30 MΩ were accepted for data analysis. In the whole-cell

configuration, current-voltage relationships were obtained. In such experiments, the cell

membrane was held at -90 mV, and 10 mV-increasing step pulses having duration of

200 ms were applied. To record single channel currents, the cell-attached or inside-out

configuration was used. In the cell-attached configuration, the pipette potential with

reference to the bath solution potential was kept at 0 mV. In the inside-out configuration,

the potential difference through the membrane was kept at –60 mV, inside negative. The

open-time probability (Po) was calculated according to the equation Po = 1-Pc1/N, where

Pc is the total closed time probability and N is the total number of channels present. The

Pos of KATP channels are shown as a function of the concentration of adenosine

5’-triphosphate (ATP, Sigma Chemical Co., St Louis, MO, USA). The theoretical

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 13: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

13

curves for ATP inhibition of KATP channel activity were obtained by using the Hill

equation (P/P(o)) = 1/1 + ([ATP]/ki)h, where P is the open-time probability with ATP

present at each concentration, P(o) is the open-time probability without ATP present,

[ATP] is the concentration of ATP, ki is the ATP concentration at which inhibition of

KATP channel activity is half-maximal, and h is the Hill coefficient. The mean values of

ki and h were used for obtaining the theoretical curves, which were taken from several

series of experiments in which various concentrations of ATP were examined in the

same patch. All electrophysiological experiments were carried out at room temperature

(22 ± 1° C).

Solutions. The extracellular solution for electrical recordings contained 135 mM NaCl,

5.6 mM KCl, 1.2 mM MgCl2, 1 mM CaCl2, 2.8 mM glucose and 10 mM HEPES. The

pH of the solution was 7.3. In experiments where the extracellular concentration of

glucose was elevated to 16.7 mM, the osmolarity of the control solution was adjusted by

adding sucrose. To record the membrane potential of the cell, the pipette solution

contained 135 mM KCl, 1.2 mM MgCl2, 2.8 mM glucose, 0.5 mM ethylene

glycol-bis(b-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), 10 mM

N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES) and 240 µg/mL

amphotericin B (Sigma Chemical Co., St Louis, MO, USA). The pH of this solution

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 14: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

14

was 7.2. To record KATP channel activity in the cell-attached or inside-out configuration,

the ionic composition of the pipette solution was the same as that for membrane

potential recordings, but amphotericin B was omitted. For inside-out single channel

recordings, immediately following establishment of the inside-out configuration, the

bath solution was changed to a solution whose ionic composition was the same as the

pipette solution. During electrophysiological studies, cells in the experimental bath were

continuously exposed to a stream of extracellular solution throughout the experiments.

Statistics and data analysis. Data are expressed as mean ± S.E.M from several

experiments, and statistical significance was evaluated by using the two-tailed paired or

unpaired Student’s t-test. ANOVA analysis was also used. A value of p less than 0.05

was considered to be significant.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 15: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

15

RESULTS

Chronic exposure to OA induced impaired glucose-stimulated insulin secretion

from rat islets.

Initial experiments were performed to test insulin secretion after exposing β−cells to

OA (0.1 mM) for four days. Figure 1A shows the rates of insulin secretion from rat

islets. The rates of insulin secretion in a solution which contained 2.8 mM glucose were

19.4 ± 4.3 pg/islet/min (n = 6) in control (untreated) islets and 19.0 ± 2.4 pg/islet/min (n

= 6) in OA-treated islets (Figure 1B, left columns). There was no significant difference

when comparing basal insulin release between OA-treated and untreated islets (p >

0.05). When the glucose concentration was elevated to 16.7 mM (glucose stimulation),

control islets showed an elevation of insulin secretion (Fig. 1A, open symbols), while

OA-treated islets exhibited no clear elevation of insulin secretion due to high glucose

stimulation (Fig. 1A, black symbols). The average rates of insulin secretion during high

glucose stimulation were 39.2 ± 5.8 pg/islet/min (n = 6) in control islets and 24.6 ± 3.6

pg/islet/min (n = 6) in OA-treated islets (Fig. 1B, middle columns). The difference

when comparing high glucose-stimulated insulin secretion between control and

OA-treated islets is significant.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 16: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

16

Chronic exposure to OA affected isolated islet β-cell membrane potentials and

excitability.

In order to explore the possible cellular mechanisms of glucose-stimulated insulin

secretion impairment by chronic exposure to OA, we characterized the

electrophysiological properties of single β-cells isolated from rat islets treated and

untreated with OA. Under basal conditions (2.8 mM glucose in the external solution),

β-cells from control islets were mostly electrically silent (Fig. 2A), but β-cells from

OA-treated islets showed spontaneous action potential firing (Fig. 2B). The resting

membrane potentials of control β-cells at day 0 and day 4 were –62.1 ± 0.9 mV (n = 32)

and –64.3 ± 1.2 mV (n = 19), respectively, while in β-cells treated with OA at day 1, 2

and 4, it was –45.7 ± 3.1 mV (p > 0.05 vs. control cells at day 0, n = 14), –41.7 ± 3.3

mV (p > 0.05 vs. control cells at day 0, n = 10) and –36.4 ± 1.6 mV (p < 0.01 vs. control

cells at day 4, n = 18), respectively (Fig. 2C). These results suggest a time-dependent

depolarization of β-cells during chronic exposure to OA. An increase in the glucose

concentration to 16.7 mM depolarized membrane potential and elicited action potential

firing in control β-cells (Fig. 2A), but the glucose-stimulated effect was weaker in

OA-treated β-cells (Fig. 2B). These results indicate that unlike the previously observed

acute effects of FFAs, which resulted in β-cell hyperpolarization (Gribble et al., 1998),

chronic exposure to OA depolarized and excited rat β-cells.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 17: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

17

Current-voltage relationship curves of β-cells treated and untreated with OA.

In order to explore the ionic mechanism of depolarization of islet β-cells due to OA

treatment, β-cell membrane conductance was measured by obtaining current-voltage

relationships. As demonstrated in Fig. 3, the current-voltage relationship curves were

obtained by a series of step pulses from -90 to -30 mV at 10-mV increments, and the

results showed that the whole-cell current density (pA/pF) was clearly larger in control

β-cells compared to OA-treated β-cells (Fig. 3B), but the current density at around -90

mV was not changed, suggesting that a decrease in K+ conductance was likely

responsible for depolarization of OA-treated β-cells.

Effects of chronic exposure to OA on KATP channel activity in the cell-attached

recording configuration.

Why does chronic exposure to OA induce β-cell excitation? One possible explanation

is that chronic exposure to OA may close β-cell KATP channels, which would result in

β-cell depolarization and consequently excitation. To test this hypothesis, KATP channel

activity in both control and OA-treated β-cells was monitored using the cell-attached

single channel patch recording configuration. In control β-cells, spontaneous, inward

single channel currents were recorded at the pipette potential of 0 mV under basal

conditions (2.8 mM glucose in the bath; Fig. 4Aa, before tolbutamide application). The

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 18: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

18

value of the open-time probability (Po) of the channel activity was 0.12 ± 0.2 (n = 12).

Application of the KATP channel blocker tolbutamide (0.5 mM) to the bath solution

completely inhibited channel activity (Fig. 4Ab, n = 5), suggesting that spontaneous

KATP channel activity was recorded. On the other hand, in β-cells isolated from islets

exposed to OA for four days, spontaneous KATP channel activity was rarely observed

(Fig. 4Ba), but application of the KATP channel opener diazoxide (0.1 mM) dramatically

opened KATP channels (Fig. 4Bb). These results clearly demonstrate that chronic

exposure to OA closed β-cell KATP channels.

Effects of chronic exposure to OA on the sensitivity of KATP channels to ATP in the

inside-out recording configuration.

In order to further examine the mechanisms by which chronic exposure to OA closed

β-cell KATP channels, the inside-out excised patch recording was employed.

Interestingly, in the absence of ATP on the inside of the cellular membrane, the values

of Po of KATP channel activity were 0.19 ± 0.02 (n = 8) in untreated β-cells (Figs. 5A, C)

and 0.20 ± 0.04 (p > 0.05, n=6) in OA-treated β-cells (Fig. 5B, C). However, when 10

µM ATP was applied to the inside of the membrane, KATP channel activity was

obviously reduced in OA-treated cells (Fig. 5B, C). In the presence of 10 µM ATP,

from six patches using OA-treated β-cells, the value of Po of KATP channel activity was

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 19: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

19

0.02 ± 0.01, while from eight patches using control β-cells, the value of Po of KATP

channel activity was 0.12 ± 0.01. When comparing OA-treated and untreated β-cells in

the presence of 10 µM ATP, the values of Po are significantly different (p < 0.01, Fig.

5C, right columns). As shown in Fig. 5D, the sensitivity of KATP channels to different

concentrations of ATP was compared between OA-treated and untreated β-cells. Based

on estimations from the Hill equation, the values of ki and the Hill coefficient for ATP

inhibition of KATP channels were 12.5 ± 1.0 µM and 0.88 ± 0.04 in control β-cells (n =

6), but were 1.0 ± 0.4 µM (n = 5) and 0.92 ± 0.05 in OA-treated β-cells (n = 5). The ki

value was significantly lower in OA-treated β-cells (p < 0.01) but the values of the Hill

coefficient were not significantly different when comparing OA-treated and untreated

β-cells. These results indicate that chronic exposure to OA increased the sensitivity of

KATP channels to cytosolic ATP, which in turn resulted in the closure of KATP channels

when glucose was present at low concentrations (< 5.5 mM).

Effects of chronic exposure to OA on histological features and insulin content of

islet β-cells.

Data presented thus far demonstrate that in contrast to the acute effects of FFAs,

which lead to the opening of β-cell KATP channels, chronic exposure to OA closed β-cell

KATP channels. It is well known that the closure of β-cell KATP channels excites β-cells

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 20: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

20

and triggers insulin release. To understand why chronic OA treatment excited β-cells,

but impaired glucose-stimulated insulin release (see Fig. 1), we compared histological

alterations of insulin-containing secretory granules and insulin content between

OA-treated and untreated islet β-cells using electron microscopy. As shown in Fig. 6,

insulin-containing secretory granules and Golgi apparata were well-developed in control

β-cells (A); however, in OA-treated β-cells, electron-dense inhomogeneous lipid

droplets were often deposited in the cytoplasm (B, dashed circle) and the total numbers

of insulin-containing granules were lower compared to control β-cells (A, B, arrows).

Furthermore, biochemical analysis demonstrated that total protein content was 44.9 ±

4.3 µg/islet in control islets (Fig. 6C, open column) and 48.7 ± 7.8 µg/islet in

OA-treated islets (Fig. 6C, black column, p > 0.05), suggesting that there was no

significant difference in total protein content when comparing OA-treated and untreated

islets. However, insulin content in OA-treated islets was significantly reduced compared

to control islets (Fig. 6D). The average insulin content was 19.5 ± 1.0 ng/islet in control

islets (n = 8) and 13.4 ± 0.7 ng/islet in OA-treated islets (p < 0.01, n = 8). These results

suggest that chronic exposure of islets to OA continuously excited β-cells, which

gradually depleted insulin content and led to an impairment of glucose-stimulated

insulin release.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 21: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

21

Discussion

The major and important discovery in the present study is that chronic exposure to

OA modulated rat pancreatic β-cell function in a different manner compared to acute

exposure to FFAs. In contrast to the acute effects resulting from exposure to FFAs,

which lead to the opening of β-cell KATP channels and consequently the silencing of

β-cells due to decreased sensitivity of KATP channels to ATP, chronic exposure (four

days) to OA closed β-cell KATP channels and excited β-cells by increasing the

sensitivity of KATP channels to ATP. After sustained β-cell excitation, both the total

numbers of intracellular insulin-containing granules and insulin content were decreased,

which caused an impairment of glucose-stimulated insulin release. Therefore, although

both acute and chronic exposure to FFAs results in a similar impairment of

glucose-induced insulin release, the underlying mechanism is different. Considering that

the pathogenesis of type 2 diabetes in patients having an elevated body weight is a

gradual process, this novel mechanism, which interprets how a sustained, high

circulating level of FFAs induces insulin release impairment and pancreatic β-cell

dysfunction, appears to closely resemble clinical conditions, and these new findings

help improve our understanding of the cellular mechanisms underlying insulin

resistance associated with type 2 diabetes.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 22: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

22

Chronic exposure to OA caused electrical excitation of islet β-cells when glucose

was present at low concentrations.

We found that chronic exposure to OA resulted in β-cell spontaneous action potential

firing even when glucose was present at low concentrations (Fig. 2B). Normally,

isolated islet β-cells are silent when the glucose concentration is below 5.5 mM (Fig.

2A) because in these conditions the membrane potential is below –50 mV. This resting

membrane potential is both established and maintained in part by KATP channel activity

(Suga et al., 2003), which is enough to prevent spontaneous action potential

development in low glucose (< 5.5 mM) conditions (Ashcroft and Rorsman, 1989).

When the glucose concentration is elevated, ATP production increases, which then

leads to the closure of KATP channels, β-cell depolarization and action potential firing.

Action potentials in islet β-cells allow Ca2+ entry through L-type Ca2+ channels

(Ashcroft and Rorsman, 1989), causing elevation of levels of [Ca2+]i, which triggers

exocytosis of insulin granules (Wollheim and Sharp, 1981). Therefore, it is clear that

the alteration of electrical properties of rat islet β-cells by chronic OA treatment plays

an important role in the development of OA-induced β-cell dysfunction and insulin

release impairment.

Accumulating lines of evidence indicate that acute or chronic exposure to FFAs

affects β-cell function and insulin release, hence FFAs are thought to be a critical risk

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 23: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

23

factor in the development of insulin resistance associated with type 2 diabetes (Elks,

1993; Zhou and Grill, 1994; Larsson et al., 1996; Prentki and Corkey, 1996; Bjorklund

et al., 1997; Hosokawa et al., 1997; Bollheimer et al., 1998; Gribble et al., 1998;

McGarry and Dobbins, 1999; Gravena et al., 2002; Branstrom et al., 2004; Zhang et al.,

2005). Using both native pancreatic β-cells and heterologously expressed Kir6.2/SUR1

KATP channels, acute exposure of a recorded cell to FFAs strongly opened KATP

channels and hyperpolarized or silenced the tested cell under patch-clamp recording

configuration (Larsson et al., 1996; Gribble et al., 1998; Gravena et al., 2002;

Branstrom et al., 2004), and the underlying mechanisms of the FFA-induced KATP

channel opening included a reduction in the sensitivity of KATP channels to ATP

(Gribble et al., 1998). In experiments studying the effects of chronic exposure to FFAs,

it has been reported that a 1-2-day exposure to FFAs increased basal insulin release but

decreased glucose-stimulated insulin secretion (Elks, 1993; Zhou and Grill, 1994;

Prentki and Corkey, 1996; Bjorklund et al., 1997; Hosokawa et al., 1997; Bollheimer et

al., 1998; McGarry and Dobbins, 1999; Zhang et al., 2005). However, the membrane

properties of rat pancreatic β–cells and the electrophysiological alterations, especially

the functional changes of KATP channel activity, after chronic exposure to FFAs have

not been studied. The present investigation provides this missing evidence and proposes

that after chronic exposure (four days) to OA, an increased sensitivity of KATP channels

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 24: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

24

to ATP may serve as an important mechanism, at least in part, to underlie chronic FFA

treatment-induced insulin release impairment.

Chronic exposure to OA increased the sensitivity of KATP channels to ATP.

As demonstrated in Fig. 5, KATP channel activity in the absence of ATP on the inside

of the cellular membrane was similar when OA-treated and untreated islet β-cells were

compared using inside-out patch recordings, suggesting that the basic function of KATP

channels was not altered by OA treatment. However, using the cell-attached

configuration, KATP channel activity in OA-treated β-cells mostly disappeared (Fig. 4).

This finding indicates that in OA-treated β-cells, KATP channels are functionally closed

by some intracellular mechanism. Since KATP channel activity in islet β-cells assists in

both establishing and maintaining the resting membrane potential (Ashcroft and

Rorsman, 1989), functional inhibition of KATP channel activity results in membrane

depolarization, which is responsible for the development of Ca2+-dependent action

potentials. Therefore, the functional closure of KATP channels under basal glucose

conditions (< 5.5 mM) explains why OA-treated β-cells showed spontaneous action

potential firing (Fig. 2).

KATP channel activity in islet β-cells is basically regulated by levels of intracellular

ATP and/or the ratio of ATP/ADP (Detimary et al., 1988). However, the sensitivity of

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 25: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

25

KATP channels to ATP is also known to be modulated by other intracellular molecules.

For instance, intracellular Ca2+ was shown to increase the sensitivity of KATP channels

to ATP in rat islet β-cells (Nakano et al., 2002). Mg2+ has also been reported to inhibit

KATP channel activity by reducing intracellular concentrations of free ATP (Findley,

1987) and/or by increasing the affinity of KATP channels to ATP (Ashcroft and Kakei,

1989). Furthermore, the membrane phospholipid phosphatidylinositol-4,5-bis-phosphate

(PIP2) reduced the sensitivity of KATP channels to ATP (Baukrowitz et al., 1998; Huang

et al., 1998; Shyng and Nichols, 1998) by the mechanism of its minus charges

interfering with the efficacy of ATP binding to KATP channels (Fan and Makielski,

1997). In the present study, the ki value for ATP inhibition of KATP channel activity in

OA-treated β-cells was significantly lower when compared to untreated β-cells (Fig. 5),

indicating that chronic exposure of islet β-cells to OA increased the affinity of KATP

channels to ATP. At the present time, the precise mechanisms of how chronic exposure

to OA induced a change in the affinity of KATP channels to ATP are not clear. As

mentioned before, elevation of [Ca2+]i following chronic exposure to OA may be

involved in increasing the sensitivity of KATP channels to ATP (Nakano et al., 2002).

Chronic exposure to OA decreased both total numbers of insulin-containing

granules and insulin content.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 26: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

26

As illustrated in Fig. 6, total numbers of insulin-containing granules and insulin

content in islets were reduced following four-day exposure to OA, which is consistent

with previous findings (Zhou and Grill, 1994; Furukawa et al., 1999). However, the

rates of insulin secretion from both OA-treated and untreated islets in 2.8 mM glucose

were similar (Fig. 1), which is not consistent with previous studies that reported that

chronic exposure to FFAs elevated basal insulin release (Zhou and Grill, 1994; Zhou et

al., 1996; Bollheimer et al., 1998; McGarry and Dobbins, 1999; Zhang et al., 2005).

These different results can be explained by the present findings that β-cells isolated

from OA-treated islets were electrically excited even in low glucose (2.8 mM)

conditions (Fig. 2), and that over-activity of β-cells following long-term (four days)

exposure to OA seems to gradually deplete insulin storage. Under these conditions,

excitation of β-cells was sustained by a closure of KATP channels, and consequently

insulin was continuously secreted at a maximal, saturated level, but this saturated level

was obviously lower. This interpretation is supported by the following lines of

evidence: (1) in chronic OA-treated β-cells, both total numbers of insulin-containing

secretion granules and insulin content were clearly lower compared to untreated cells

(Fig. 6); (2) in chronic OA-treated β-cells, glucose (16.7 mM)-stimulated insulin release

was clearly impaired (Fig. 1); (3) the exposure time of β-cells to FFAs was longer in the

present study (four days) compared to previous studies (one or two days), and (4)

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 27: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

27

FFA-induced hypersensitivity of islet β-cells to glucose has already been demonstrated

(Hosokawa et al., 1997; Liu et al., 1998).

Taken together, the present study provides a novel mechanism to interpret, at least in

part, chronic FFA-induced insulin release impairment. Unlike acute exposure to FFAs,

which results in the reduced sensitivity of KATP channels to ATP, chronic exposure to

OA increased the sensitivity of KATP channels to ATP, which led to the closure of KATP

channels when glucose was present at low concentrations (< 5.5 mM); therefore, β-cells

were continuously depolarized and excited, which in turn triggered long-term insulin

release under basal conditions, and ultimately led to gradual depletion of insulin storage

and impairment of glucose-stimulated insulin release.

Acknowledgements: Authors thank Kevin Ellsworth for his assistance in preparing the

manuscript.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 28: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

28

References

Ashcroft FM and Ashcroft SJH (1992) Mechanism of insulin secretion. In Insulin:

Molecular Biology to Pathology (Ashcroft FM and Ashcroft SJH eds) pp 97-150,

Oxford, IRL.

Ashcroft FM and Kakei M (1989) ATP-sensitive K+ channels in rat pancreatic β-cells:

modulation by ATP and Mg2+ ions. J Physiol 416:349-367.

Ashcroft FM and Rorsman P (1989) Electrophysiology of the pancreatic β-cell. Pro

Biophys Molec Biol 54:87-143.

Baukrowitz T, Schulte U, Oliver D, Herlitze S, Krauter T, Tucker SJ, Ruppersberg JP,

and Fakler B (1998) PIP2 and PIP as determinants for ATP inhibition of KATP

channels. Science 282:1141-1144.

Bevilacqua S, Buzzigoli G, Bonadonna R, Brandi S, Oleggini M, Boni C, Geloni M,

and Ferrannini E (1990) Operation of Randle's cycle in patients with NIDDM.

Diabetes 39:383-389.

Bjorklund A, Yaney G, McGarry JD, and Weir G (1997) Fatty acids and beta-cell

function. Diabetologia 40:B21-B26.

Boden G, Chen X, Ruiz J, White JV, and Rossetti L (1994) Mechanism of fatty

acid-induced inhibition of glucose uptake. J Clin Invest 93:2438-2446.

Bollheimer LC, Skelly RH, Chester MW, McGarry JD, and Rhodes CJ (1998) Chronic

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 29: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

29

exposure to free fatty acid reduces pancreatic B-cell insulin content by increasing

basal insulin secretion that is not compensated for by a corresponding increase in

proinsulin biosynthesis translation. J Clin Invest 101:1094-1101.

Bonadonna RC, Zych K, Boni C, Ferrannini E, and DeFronzo RA (1989) Time

dependence of the interaction between lipid and glucose in humans. Am J Physiol

57:E49-E56.

Branstrom R, Aspinwall CA, Valimaki S, Ostensson CG, Tibell A, Eckhard M,

Brandhorst H, Corkey BE, Berggren PO, and Larsson O (2004) Long-chain CoA

esters activate human pancreatic beta-cell KATP channels: potential role in Type 2

diabetes. Diabetologia 47:277-283.

Branstrom R, Corkey BE, Berggren PO, and Larsson O (1997) Evidence for a unique

long chain acyl-CoA ester binding site on the ATP-regulated potassium channel in

mouse pancreatic beta cells. J Biol Chem 272:17390-17394.

Detimary P, Gilon P, and Henquin JC (1988) Interplay between cytoplasmic Ca2+ and

the ATP/ADP ratio: a feedback control mechanism in mouse pancreatic islets.

Biochem J 15:269-274.

Elks ML (1993) Chronic perifusion of rat islets with palmitate suppresses

glucose-stimulated insulin release. Endocrinology 133:208-214.

Fan Z and Makielski JC (1997) Anion phospholipids activate ATP-sensitive potassium

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 30: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

30

channels. J Biol Chem 272:5388-5395.

Felley CP, Felly EM, van Melle GD, Frascarolo P, Jequier E, and Felber JP (1989)

Impairment of glucose disposal by infusion of triglycerides in humans: role of

glycemia. Am J Physiol 256:E747-E757.

Ferrannini E, Barrett EJ, Bevilacqua S, and De Fronzo RA (1983) Effect of fatty acids

on glucose production and utilization in man. J Clin Invest 72:1737-1747.

Findley I (1987) The effects of magnesium upon adenosine triphosphate-sensitive

potassium channels in rat insulin-secreting cell line. J Physiol 391:611-629.

Furukawa H, Carroll RJ, Swift HH, and Steiner DF (1999) Long-term elevation of free

fatty acids leads to delayed processing of proinsulin and prohormone convertases 2

and 3 in the pancreatic beta-cell line MIN6. Diabetes 48:1395-1401.

Gravena C, Mathias PC, and Ashcroft SJ (2002) Acute effects of fatty acids on insulin

secretion from rat and human islets of Langerhans. J Endocrinol 173:73-80.

Gribble FM, Proks P, Corkey BE, and Ashcroft FM (1998) Mechanism of cloned

ATP-sensitive potassium channel activation by oleoyl-CoA. J Biol Chem

273:26383-26387.

Hosokawa H, Corkey BE, and Leahy JL (1997) Beta-cell hypersensitivity to glucose

following 24-h exposure of rat islets to fatty acids. Diabetologia 40:392-397.

Huang CL, Feng S, and Hilgemann DW (1998) Direct activation of inward rectifier

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 31: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

31

potassium channels by PIP2 and its stabilization by Gβγ. Nature 391:803-806.

Larsson O, Deeney JT, Branstrom R, Berggrent PO, and Corkey BE (1996) Activation

of the ATP-sensitive K+ channel by long chain acyl-CoA. J Biol Chem

271:10623-10626.

Lee HU, Lee HK, Koh CS, and Min HK (1988) Artificial induction of intravascular

lypolysis by lipid-heparin infusions leads to insulin resistance in man. Diabetologia

31:285-290.

Lee Y, Hirose H, Ohneda M, Johnson JH, McGarry JD, and Unger RH (1994) B-cell

lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese

rats: impairment in adipocyte-B-cell relationships. Proc Natl Acad Sci USA

91:10878-10882.

Lillioja S, Bogardus C, Mott D, Kennedy LA, Knowler WC, and Howard BV (1985)

Relationship between insulin-mediated glucose disposal and lipid metabolism in

man. J Clin Invest 75:1106-1115.

Liu YQ, Tornheim K, and Leahy JL (1998) Fatty acid-induced beta cell hypersensitivity

to glucose. Increased phosphofructokinase activity and lowered

glucose-6-phosphate content. J Clin Invest 101:1870-1875.

Man ZW, Zhu M, and Shima K (1997) Impaired beta-cell function and deposition of fat

droplets in the pancreas as a consequence of hypertriglyceridemia in OLET rat, a

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 32: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

32

model of spontaneous NIDDM. Diabetes 46:1718-1724.

McGarry JD and Dobbins RL (1999) Fatty acids, lipotoxicity and insulin secretion.

Diabetologia 42:128-138.

Nakano K, Suga S, Takeo T, Ogawa Y, Suda T, Kanno T, and Wakui M (2002)

Intracellular Ca2+ modulation of ATP-sensitive K+ channel activity in

acetylcholine-induced activation of rat pancreatic β-cells. Endocrinology

143:569-576.

Prentki M and Corkey BE (1996) Are the beta-cell signaling molecules malonyl-CoA

and cystolic long-chain acyl-CoA implicated in multiple tissue defects of obesity

and NIDDM? Diabetes 45:273-283.

Roche E, Maestre I, Martin F, Fuentes E, Casero J, Reig JA, and Soria B (2000)

Nutrient toxicity in pancreatic beta-cell dysfunction. J Physiol Biochem 56:119-128.

Shimabukuro M, Koyama K, Chen G, Wang MY, Trieu F, Lee Y, Newgard CB, and

Unger RH (1997) Direct antidiabetic effect of leptin through triglyceride depletion

of tissues. Proc Natl Acad Sci USA 94:4637-4641.

Shyng SL and Nichols CG (1998) Membrane phospholipid control of nucleotide

sensitivity of KATP channels. Science 282:1138-1141.

Suga S, Kanno T, Nakano K, Takeo T, Dobashi Y, and Wakui M (1997) GLP-I (7-36)

amide augments Ba2+ current through L-type Ca2+ channel of rat pancreatic β-cell in

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 33: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

33

a cAMP-dependent manner. Diabetes 46:1755-1760.

Suga S, Nakano K, Takeo K, Osanai T, Ogawa Y, Yagihashi S, Kanno T, and Wakui M

(2003) Masked excitatory action of noradrenaline on rat islet β-cells via activation

of phospholipase C. Eur J Physiol 447:337-344.

Wollheim CB and Sharp GWG (1981) Regulation of insulin release by calcium. Physiol

Rev 61:914-973.

Zhang Y, Xiao M, Niu G, and Tan H (2005) Mechanisms of oleic acid deterioration in

insulin secretion: role in the pathogenesis of type 2 diabetes. Life Sci 77:2071-2081.

Zhou YP and Grill VE (1994) Long-term exposure of rat pancreatic islets to fatty acids

inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty

acid cycle. J Clin Invest 93:870-876.

Zhou YP, Ling ZC, and Grill VE (1996) Inhibitory effects of fatty acids on

glucose-regulated B-cell function: association with increased islet triglyceride stores

and altered effect of fatty acid oxidation on glucose metabolism. Metabolism

45:981-986.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 34: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

34

Figure legends

Fig. 1. Insulin secretion from islets treated and untreated with OA.

A, time course of insulin secretion from untreated (open circles) and OA-treated (filled

circles) islets. The glucose concentration was elevated from 2.8 mM to 16.7 mM. The

mean values from 6 experiments in each are shown. Vertical bars represent S.E.M. B,

the average rates of insulin secretion 30 min before, during and after stimulation with

glucose from 6 experiments each. *p < 0.05, **p < 0.01.

Fig. 2. Membrane potential recordings of islet β-cells.

A, untreated β-cell. The extracellular glucose concentration was 2.8 mM first, and then

elevated to 16.7 mM. B, OA-treated β-cell. Spontaneous action potential firing was seen

when the solution contained 2.8 mM glucose. Representative tracings from 12 and 20

experiments are shown in A and B, respectively. C, the values of β-cell resting

potentials were compared following different exposure times to OA. Each column

represents the average from ten to thirty-two cells, and the vertical bars represent ± S.E.

**p < 0.01.

Fig. 3. Current-voltage relationships in β-cells treated and untreated with OA.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 35: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

35

A, results from control (untreated) β-cells. Voltage pulses were applied to cells in the

presence of 1 µM nifedipine. Values are the means from 7 experiments. B, results from

OA-treated β-cells. Values are the means from 7 experiments.

Fig. 4. Single channel recordings of KATP channel activity in the cell-attached

recording configuration.

Aa, untreated β-cell. The pipette potential was 0 mV. Ab, tolbutamide (0.5 mM) was

applied to the bath solution. Ba, OA-treated β-cell. Bb, diazoxide (0.1 mM) was applied

to the bath solution. Representative traces from 5-12 experiments are shown. Dotted

lines indicate the level of channel closure.

Fig. 5. Sensitivity of KATP channels to ATP examined in the inside-out recording

configuration.

A, recordings from the membrane of an untreated β-cell. The potential difference

through the patch membrane was held at –60 mV, inside negative. The ATP

concentration in the bath was elevated from zero to 10 µM. B, recordings from the

membrane of an OA-treated β-cell. Dotted lines indicate the level of channel closure (A,

B). C, open-time probabilities (Po) of KATP channels with and without ATP examined in

membranes from untreated (open columns) and OA-treated (filled columns) β-cells.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 36: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

JPET #105759

36

Mean values from 6 experiments with no ATP and 8 experiments with 10 µM ATP are

shown. Vertical bars represent S.E.M. D, concentration-response relationships for ATP

inhibition of KATP channel activity for untreated (open circles) and OA-treated (filled

circles) β-cells. KATP channel activity at each concentration of ATP is expressed relative

to that without ATP. Mean values from 5 experiments each are shown. Solid lines were

drawn based on the Hill equation. **p < 0.01.

Fig. 6. Electron micrographs of rat islet β-cells and amounts of total protein and

insulin in islets.

A, untreated β-cell. Insulin-containing secretory granules and Golgi apparata are

well-developed. B, OA-treated β-cell. Electron-dense inhomogeneous lipid droplets

were deposited in the cytoplasm. Total numbers of insulin granules were lower

compared to control cells. C, amount of total protein per islet, measured in untreated

and OA-treated islets. Mean values from 7 experiments each are shown. Vertical bars

represent S.E.M. The two values are not significantly different. D, amount of insulin per

islet, measured from untreated and OA-treated islets. Mean values from 8 experiments

each are shown. **p < 0.01.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 37: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 38: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 39: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 40: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 41: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from

Page 42: Novel mechanism of chronic exposure of oleic acid-induced ...jpet.aspetjournals.org/content/jpet/early/2006/06/06/jpet.106.105759.full.pdf · Furthermore, FFA-induced inhibition of

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on June 6, 2006 as DOI: 10.1124/jpet.106.105759

at ASPE

T Journals on February 26, 2020

jpet.aspetjournals.orgD

ownloaded from