A Link between Insulin Resistance and Hyperinsulinemia: Inhibitors of ...

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0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 9 Printed in U.S.A.

A Link between Insulin Resistance and Hyperinsulinemia: Inhibitors of Phosphatidylinositol 3-Kinase Augment Glucose-Induced Insulin Secretion from Islets of Lean, But Not Obese, Rats* WALTER S. ZAWALICH

AND

KATHLEEN C. ZAWALICH

Yale University School of Nursing, New Haven, Connecticut 06536-0740 ABSTRACT Wortmannin (5–100 nM), a specific phosphatidyinositol 3-kinase inhibitor, augmented 8 mM glucose-induced insulin secretion from control Sprague Dawley rat islets in a dose-dependent manner. This effect persisted after its removal from the perifusion medium; however, this augmenting effect was reduced by the calcium channel inhibitor nitrendipine or by lowering the glucose level to 3 mM. Wortmannin amplified insulin release induced by the combination of 6 – 8 mM glucose plus 1 ␮M carbachol; however, it had no effect on phorbol ester- or ␣-ketoisocaproate-induced insulin secretion. The potentiating action of wortmannin on 8 mM glucose-induced release was duplicated by LY294002. Wortmannin had no effect on glucose usage rates or inositol phosphate accumulation in [3H]inositol-prelabeled

A

SENSITIVE and dynamic balance between tissue sensitivity to insulin and the prevailing insulin concentration exists. In situations (prediabetes, obesity, and type 2 diabetes) characterized by the development of insulin resistance (1–3), a compensatory secretory response of the ␤-cell occurs, and plasma levels of insulin are increased. Although the development of insulin resistance in peripheral tissues such as liver, muscle, and adipose cells is thought to be the result of a reduction in insulin signaling via phosphatidyinositol 3-kinase (PI3K) (4, 5), the nature of the stimulus for the enhanced secretion of insulin has yet to be established. Of particular significance, perhaps, are the observations that insulin receptors (6), insulin receptor messenger RNA (7), insulin receptor substrate-1 (7), and PI3K have been identified in ␤-cells (8). This raises the possibility that the same biochemical pathways that determine insulin sensitivity in peripheral tissues may be involved in ␤-cell secretory activity and participate in the adaptive response of these cells to insulin resistance. In an attempt to address this issue, studies were conducted with wortmannin, a fungal metabolite that specifically inhibits signaling via the PI3K pathway (9 –11), using islets isolated from control Sprague Dawley rats. Additional experiments were conducted with LY294002, a PI3K inhibitor Received February 22, 2000. Address all correspondence and requests for reprints to: Dr. Walter S. Zawalich, Yale University School of Nursing, 100 Church Street South, New Haven, Connecticut 06536-0740. E-mail: [email protected] yale.edu. * This work was supported by NIH Grant 41230 and a grant from the American Diabetes Association.

islets. Of particular significance, although 50 nM wortmannin potentiated 8 mM glucose-induced secretion from islets of lean Zucker control rats, the fungal metabolite had little effect on 8 mM glucoseinduced release from islets of insulin-resistant Zucker fatty rats. These findings support the concept that the same biochemical process, inhibition of phosphatidyinositol 3-kinase, that causes peripheral tissue insulin resistance enhances ␤-cell sensitivity to glucose and produces a compensatory increase in insulin secretion from these cells. The efficacy of wortmannin depends on the in vivo status of the donor’s insulin signaling pathways. This elegant biochemical control mechanism in ␤-cells ensures the maintenance of glucose homeostasis despite a reduction in insulin action on peripheral tissues. (Endocrinology 141: 3287–3295, 2000)

structurally distinct from wortmannin. Finally, we determined whether the in vivo status of the donor’s insulin signaling pathways played any role in the in vitro islet responses to wortmannin by performing parallel studies using islets isolated from obese, insulin-resistant, hyperinsulinemic Zucker fatty rats and their lean counterparts. Materials and Methods Islet isolation The detailed methodologies employed to assess insulin output from collagenase-isolated islets have been previously described (12). Male Sprague Dawley rats (350 – 475 g), lean control Zucker rats (220 –240 g), and fa/fa Zucker fatty rats (260 –310 g) were purchased from Charles River Laboratories, Inc. (Wilmington, MA). All animals were treated in a manner that complied with the NIH Guidelines for the Care and Use of Laboratory Animals (NIH Publication 85–23, revised 1985). The animals were fed ad libitum. After ip Nembutal (pentobarbital sodium, 50 mg/kg; Abbott Laboratories, North Chicago, IL)-induced anesthesia, islets were isolated by collagenase digestion and handpicked, using a glass loop pipette, under a stereo microscope. They were free of exocrine contamination.

Perifusion studies Groups of 14 –18 isolated islets were loaded onto nylon filters (Tetko, Inc., Briarcliff Manor, NY) and perifused in a Krebs-Ringer bicarbonate (KRB) buffer at a flow rate of 1 ml/min for 30 min, usually with 3 mm glucose, to establish basal and stable insulin secretory rates. In experiments with ␣-ketoisocaproate, glucose was omitted during the first 30 min of the perifusion. After this 30-min stabilization period they were then perifused with the appropriate agonist or agonist combinations as indicated in the figure legends and Results. Wortmannin or LY294002 were dissolved in dimethylsulfoxide, and comparable amounts of this diluent were added to control solutions. Perifusate solutions were

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gassed with 95% O2-5% CO2 and maintained at 37 C. Insulin released into the medium was measured by RIA (13).

Islet labeling for inositol phosphate (IP) studies After isolation, groups of 18 –26 islets were loaded onto nylon filters, placed in a small glass vial, and incubated for 3 h in a myo-[2-3H]inositolcontaining KRB solution made up as follows. Myo-[2-3H]inositol (10 ␮Ci; SA, 16 –23 Ci/mm) was placed in a 10 ⫻ 75-mm culture tube. To this aliquot of tracer 250 ␮l warmed (to 37 C) and oxygenated (KRB) medium supplemented with 5.0 mm glucose were added. After mixing, 240 ␮l of this solution were gently added to the vial with islets. The vial was capped with a rubber stopper, gassed for 10 sec with 95% O25%CO2, and incubated at 37 C. The vials were again gently oxygenated after 90 min. After the labeling period, the islets still on nylon filters were washed with 5 ml fresh KRB.

IP measurements After washing, the islets on nylon filters were placed in small glass vials. Added gently to the vial was 400 ␮l KRB supplemented with 10 mm LiCl to prevent IP degradation and the appropriate agonists as indicated. The vials were capped and gently gassed for 5 sec with 95% O2-5% CO2. After a 60-min incubation with the indicated agonists, the generation of IPs was stopped by adding 400 ␮l 20% perchloric acid. Total IPs formed were then measured using Dowex (Bio Rad Laboratories, Hercules, CA) columns as described previously (14, 15).

Glucose utilization rates The usage of glucose was measured by determining the rate of 3H2O formation from [5-3H]glucose using methods previously described (16). These islets were incubated in 0.125 ml 8 mm glucose with or without 50 nm wortmannin solution supplemented with tracer [5-3H]glucose. The 3H2O formed during a 1-h incubation was separated from the unused [3H]glucose as described previously (16).

Reagents Hanks’ solution was used for the islet isolation. The perifusion medium consisted of 115 mm NaCl, 5 mm KCl, 2.2 mm CaCl2, 1 mm MgCl2, 24 mm NaHCO3, and 0.17 g/dl BSA. The 125I-labeled insulin for the insulin assay, [5-3H]glucose, and the 3H2O for the glucose usage studies were purchased from New England Nuclear (Boston, MA). [3H]Inositol was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). BSA (RIA grade), glucose, wortmannin, carbachol, phorbol 12-myristate 13-acetate (PMA), ␣-ketoisocaproate, LY294002, and the salts used to make the Hanks’ solution and perifusion medium were purchased from Sigma (St. Louis, MO). Nitrendipine was the gift from A. Scriabine (Miles Institute for Preclinical Pharmacology, Elkhart, IN). Rat insulin standard (lot 615-ZS-157) was a gift from Dr. Gerald Gold (Eli Lilly & Co., Indianapolis, IN). Collagenase (type P) was obtained from Roche Molecular Biochemicals (Indianapolis, IN).

Statistics Statistical significance was determined using Student’s t test for unpaired data or ANOVA in conjunction with the Newman-Keuls test for unpaired data. P ⬍ 0.05 was taken as significant. Values presented in the figures and results represent the mean ⫾ se of at least three observations.

Results Effects of wortmannin on glucose-induced secretion from control Sprague Dawley rats

Rat islets were collagenase isolated and perifused. After a 30-min perifusion in the presence of 3 mm (54 mg/100 ml) glucose to establish stable basal rates of insulin secretion, the response to 8 mm (144 mg/100 ml) glucose was determined. When compared with prestimulatory secretory rate of 40 ⫾ 3 pg/islet䡠min (n ⫽ 14) in the presence of 3 mm glucose alone,

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the insulin secretory rate increased slowly when the perifusate glucose concentration was increased to 8 mm (Fig. 1, left). Forty minutes after the onset of 8 mm glucose stimulation, insulin release rates had increased to 184 ⫾ 18 pg/islet䡠min (n ⫽ 14). Extending the stimulatory period to 60 min resulted in a response of 202 ⫾ 19 pg/islet䡠min. Including wortmannin (50 nm) in the perifusate together with 8 mm glucose markedly augmented insulin release from perifused rat islets (Fig. 1, left). Most dramatic were its effects during the final 30 – 60 min of the perifusion period. For example, although release rates from control islets perifused with 8 mm glucose alone averaged 160 ⫾ 16, 184 ⫾ 18, and 201 ⫾ 15 pg/islet䡠min 30, 40, or 50 min after the onset of stimulation with 8 mm glucose alone, the addition of 50 nm wortmannin significantly increased release rates to 325 ⫾ 42, 504 ⫾ 63, and 596 ⫾ 92 pg/islet䡠min (n ⫽ 5) at these time points, respectively. A level (50 nm) of wortmannin that increased 8 mm glucoseinduced release 3-fold had no additional effect when release was stimulated by a maximally effective 20-mm glucose stimulus (Fig. 1, right). Dose-response studies (Fig. 2) revealed that 5 nm wortmannin was sufficient to significantly amplify the modest insulin stimulatory action of 8 mm glucose. However, levels of wortmannin as high as 50 nm had no effect on release in the presence of 3 mm glucose (results not shown). Calcium influx and insulin secretion

The influx of calcium into the ␤-cell is essential for glucoseinduced secretion and also for the amplifying effect of 50 nm wortmannin (Fig. 1, left panel). For example, 30 or 60 min after the onset of stimulation with the combination of 8 mm glucose plus 50 nm wortmannin, the release rate from islets averaged 325 ⫾ 42 or 567 ⫾ 51 pg/islet䡠min (n ⫽ 5). Inclusion of 500 nm of the calcium channel inhibitor nitrendipine attenuated the stimulatory effect of 8 mm glucose plus 50 nm wortmannin on secretion from isolated islets, with the release rate averaging 88 ⫾ 15 or 95 ⫾ 4 pg/islet䡠min (n ⫽ 3) 30 or 60 min after the onset of stimulation, respectively. Pretreatment with wortmannin accelerates its potentiation of glucose-induced secretion

When simultaneously perifused with 8 mm glucose plus 50 nm wortmannin, a potentiated insulin secretory response was evident after a delay of 20 min (Fig. 1, left panel). In an attempt to accelerate the effects of wortmannin, islets were pretreated with the fungal metabolite during the 30-min perifusion with 3 mm glucose. As shown in Fig. 3, a significant potentiating effect of wortmannin was observed within minutes after exposure to 8 mm glucose. Reversibility of wortmannin action on the ␤-cell

Previous studies have demonstrated that in other tissues wortmannin is a specific and irreversible inhibitor of PI3K (10, 17). In the next series of studies we examined whether wortmannin’s potentiating effect persisted after its removal from the perifusion medium. Islets were stimulated for 40 min with 8 mm glucose plus 50 nm wortmannin, during which time secretion was dramatically amplified from these islets compared with that from islets stimulated with 8 mm

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FIG. 1. Wortmannin potentiates 8 mM, but not 20 mM, glucose-induced insulin secretion. Groups of 14 –18 rat islets were isolated and perifused. For the initial 30 min all islets were maintained with 3 mM glucose (G3) to establish basal and stable insulin secretory rates. Left panel, Islets were then perifused (indicated by the vertical line) for 60 min with 8 mM glucose (G8) alone (open circles; n ⫽ 14), 8 mM glucose plus 50 nM wortmannin (closed circles, solid line; n ⫽ 5) or 8 mM glucose, 50 nM wortmannin, plus 500 nM nitrendipine (closed circles, dashed line; n ⫽ 4). Right panel, Islets were then perifused (indicated by the vertical line) for 60 min with 20 mM glucose (G20) alone (n ⫽ 9) or 20 mM glucose plus 50 nM wortmannin (n ⫽ 5). The mean ⫾ SE are given in this and subsequent figures. Note the change in insulin secretion scale between left and right panels. The asterisks indicate a significant (P ⬍ 0.05) difference between release values at this time. This and subsequent perifusion figures have not been corrected for the dead space in the perifusion apparatus, 2.5 ml or 2.5 min with a flow rate of 1 ml/min.

FIG. 2. Dose-response of wortmannin on 8 mM glucose-induced insulin release. Groups of islets were perifused for 30 min with 3 mM glucose and for an additional 60 min with 8 mM glucose alone or in the additional presence of the indicated wortmannin concentrations. The data presented here are the mean ⫾ SE insulin secretion rates measured during the final 30 min of stimulation (60 –90 min) during the perifusion. The asterisk indicates significance between release rates measured in the presence and absence of wortmannin. At least four separate experiments were conducted under each condition.

glucose alone (compare Fig. 1, left, open circles, with Fig. 4). For the final 20 min of the perifusion, wortmannin was removed from the medium in one group (closed circles, solid line), whereas the glucose level was maintained at 8 mm. Before the removal of wortmannin from the perifusion medium, release rates averaged 606 ⫾ 127 pg/islet䡠min (n ⫽ 4).

FIG. 3. Wortmannin pretreatment accelerates its potentiating effect on 8 mM glucose-induced secretion. Two groups of islets were studied. They were perifused for 30 min with 3 mM glucose alone (open circles) or 3 mM glucose plus 50 nM wortmannin (closed circles). For an additional 40 min (indicated by the vertical line), the islets were stimulated with 8 mM glucose alone (open circles) or 8 mM glucose plus 50 nM wortmannin (closed circles). Three experiments were conducted under each condition, and the asterisks indicate significance between release rates measured in the presence and absence of wortmannin.

Release persisted at high rates for the remainder of the perifusion. For example, 20 min after removal of the fungal metabolite release rates still averaged 513 ⫾ 94 pg/islet䡠min. In additional studies, the glucose level was decreased to 3 mm, whereas wortmannin was maintained at 50 nm for the final 20 min. Under this condition, and consistent with the

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inability of wortmannin to augment release at substimulatory glucose levels, release rates fell dramatically upon removal of 8 mm glucose (Fig. 4).

FIG. 4. Examining the reversibility of wortmannin’s effect on glucose-induced release. Two groups of islets were studied. They were both perifused for 30 min with 3 mM glucose and for an additional 40 min with 8 mM glucose plus 50 nM wortmannin (indicated by the vertical line). For the final 20 min one group was perifused with 8 mM glucose alone (closed circles, solid line), and the second group was perifused with 3 mM glucose plus 50 nM wortmannin (closed circles, dashed line). Note the rapid decline in secretion when the glucose level was lowered to 3 mM, but not when wortmannin was omitted from the medium in the continued presence of 8 mM glucose. The asterisks indicate a significant (P ⬍ 0.05) difference between release values at this time. Four experiments were conducted under each condition.

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Effects of wortmannin on glucose- plus carbacholinduced secretion

Postprandial increments in the prevailing insulin level are thought to be a result of the interactions between both fuel and neurohumoral signals, especially vagally derived acetylcholine, acting in concert on the ␤-cell (18 –21). We next explored the effect of 50 nm wortmannin on secretion evoked by glucose-carbachol combinations. When stimulated with 6 mm glucose plus 1 ␮m carbachol, insulin secretory rates remained relatively constant at about 40 –50 pg/islet䡠min (Fig. 5, left panel). The inclusion of 50 nm wortmannin during the stimulatory phase together with 6 mm glucose plus 1 ␮m carbachol amplified release. This effect was most prominent during the final 30 min of stimulation (Fig. 5, left panel), and significance (P ⬍ 0.05) was achieved during the final 20 min of the study. Elevating the glucose level to 8 mm and maintaining carbachol at 1 ␮m were accompanied by a more robust insulin secretory response from control islets compared with the response to 6 mm glucose plus 1 ␮m carbachol (Fig. 5, right panel). The addition of 50 nm wortmannin together with 8 mm glucose plus 1 ␮m carbachol further amplified secretion (Fig. 5, right panel). For the final 45 min of the perifusion, the differences were significant (P ⬍ 0.05) for each time point. Effects of wortmannin on glucose usage and IP accumulation

There is little question that glucose metabolism is a primary component of its insulin stimulatory action. To determine whether the potentiating effect of wortmannin on glucose-induced release is mediated by any impact on metabolism, glucose usage rates were measured in the presence or absence of 50 nm wortmannin. At 8 mm, islets use glucose

FIG. 5. Wortmannin amplifies release in response to glucose plus carbachol combinations. Groups of islets were perifused for 30 min with 3 mM glucose. Left panel, For the next 60 min (onset indicated by vertical line) islets were stimulated with 6 mM glucose plus 1 ␮M carbachol (open circles; n ⫽ 6) or 6 mM glucose, 1 ␮M carbachol, plus 50 nM wortmannin (closed circles, n ⫽ 6). Right panel, For the next 60 min islets were stimulated with 8 mM glucose plus 1 ␮M carbachol (open circles; n ⫽ 11) or 8 mM glucose, 1 ␮M carbachol, plus 50 nM wortmannin (closed circles; n ⫽ 10) The asterisks indicate a significant (P ⬍ 0.05) difference between release values at this time.

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at rates of 108 ⫾ 7 pmol/islet䡠h (n ⫽ 4). The additional presence of 50 nm wortmannin during the 1-h incubation was without any significant effect; usage rates now averaged 114 ⫾ 6 pmol/islet䡠h (n ⫽ 5). An increase in the glucose level bathing them is accompanied by significant dose-dependent increases in IP accumulation in [3H]inositol-prelabeled rat islets (22–24). We next determined whether the response to wortmannin could be accounted for by enhanced glucose-induced activation of phospholipase C, using IP accumulation as the biochemical marker of this event. IP accumulation in response to 8 mm glucose stimulation was comparable regardless of whether 50 nm wortmannin was included in the incubation medium (Table 1). Specificity of wortmannin’s stimulatory effect on insulin secretion

In addition to glucose, a variety of structurally distinct compounds also possess the capacity to augment release TABLE 1. Effects of wortmannin (wort) on inositol phosphate accumulation in rat islets of Langerhans Stimulatory condition

Inositol phosphate accumulation (cpm/40 islet䡠h)

8 mM glucose 8 mM glucose ⫹ Wort

16,954 ⫾ 2,017 14,981 ⫾ 2,329

Groups of rat islets were collagenase isolated. They were then incubated for 3 h in KRB solution supplemented with 5 mM glucose plus tracer [3H]inositol (10 ␮Ci). After washing with fresh KRB to remove unincorporated label, they were incubated for 60 min with 8 mM glucose alone (n ⫽ 7) or 8 mM glucose plus 50 nM wortmannin (n ⫽ 6). Also included during the 60-min stimulatory period was 10 mM LiCl to prevent dephosphorylation of the inositol phosphates. The mean ⫾ SE are given. No significant differences were observed between the groups.

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independent of an elevation in the glucose concentration. Studies were then conducted with two such compounds to determine the specificity of wortmannin on islet responses. As shown in Fig. 6, a level of wortmannin that amplifies 8 mm glucose-induced release approximately 3-fold had no effect on release stimulated by 10 mm ␣-ketoisocaproate (left panel), an amino acid derivative metabolized within the mitochondrion, or 500 nm PMA (right panel), a protein kinase C (PKC) activator (25, 26). Effects of LY294002, a PI3K inhibitor structurally distinct from wortmannin, on 8 mM glucose-induced release

If wortmannin’s potentiating effect on glucose-induced release is a result of PI3K inhibition, then it might be predicted that LY294002, another commonly used PI3K inhibitor, should mimic the effect of the fungal metabolite. At a level of 10 ␮m, LY294002 addition to islets perifused with 8 mm glucose resulted in a potentiated insulin secretory response (Fig. 7). Effect of wortmannin on 8 mM glucose-induced release from islets of insulin-resistant Zucker fatty rats or their lean counterparts

In vivo insulin resistance in a variety of animal models is thought to be a result at least in part of a reduction in insulin signaling via PI3K-dependent processes (4, 27, 28). It has been demonstrated previously that significant reductions in the expression of the regulatory subunits of PI3K occur in livers of Zucker fatty rats, a commonly used model of insulin resistance and hyperinsulinemia (29). We next examined the effects of wortmannin on islets isolated from Zucker fatty rats. As shown in Fig. 8 (top) and consistent with the actions of

FIG. 6. Wortmannin fails to potentiate secretion in response to ␣-ketoisocaproate (KIC) or PMA. Left panel, Two groups of islets were perifused for 30 min in the absence of any added fuel and for an additional 60 min (indicated by vertical line) with 10 mM KIC alone (n ⫽ 6) or 10 mM KIC plus 50 nM wortmannin (n ⫽ 5). Right panel, Two groups of islets were perifused for 30 min with 3 mM glucose. For the next 60 min both groups were stimulated by the further addition of 500 nM PMA alone (n ⫽ 8) or with 500 nM PMA plus 50 nM wortmannin (n ⫽ 5).

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FIG. 7. LY294002 potentiates 8 mM glucose-induced insulin secretion. Groups of 14 –18 rat islets were isolated and perifused. For the initial 30 min all islets were maintained with 3 mM glucose (G3) to establish basal and stable insulin secretory rates. Islets were then perifused (indicated by the vertical line) for 60 min with 8 mM glucose (G8) alone (open circles; n ⫽ 14; these are the same data as in Fig. 1) or 8 mM glucose plus 10 ␮M LY294002 (closed circles; n ⫽ 3). The asterisks indicate a significant (P ⬍ 0.05) difference between release values at this time.

wortmannin on islets isolated from Sprague Dawley rats, the fungal metabolite significantly enhanced 8 mm glucoseinduced release from lean control Zucker rats. For the final 30 min of the perifusion, insulin release rates were significantly greater in the simultaneous presence of 50 nm wortmannin. For example, 40, 50, or 60 min after the onset of stimulation with 8 mm glucose plus 50 nm wortmannin, insulin release rates averaged 322 ⫾ 65, 409 ⫾ 39, or 463 ⫾ 37 pg/islet䡠min, respectively. The release rates to 8 mm glucose alone from lean Zucker rats averaged 163 ⫾ 14, 216 ⫾ 16, or 252 ⫾ 22 pg/islet䡠min at these times. We then proceeded to explore the impact of 50 nm wortmannin on 8 mm glucose-induced release from Zucker fatty rats. Compared with islets isolated from lean Zucker rats and in accord with the established hyperresponsiveness of Zucker fatty rat islets to glucose stimulation (30), the responses to 8 mm glucose alone were significantly greater in fatty Zucker rats islets than in lean Zucker rat islets (compare open circles, Fig. 8, top and bottom panels). The effects of wortmannin on Zucker fatty rat islets were minimal. During the final 30 min of the perifusion, time points when its impact on 8 mm glucose-induced release from lean islets is maximal, there was no significant enhancing effect of the fungal metabolite on rates of insulin release from islets of Zucker fatty rats (Fig. 8, bottom panel). At only one time point during the entire 60-min stimulatory period was significance achieved. Finally, to allay the criticism that islets isolated from Zucker fatty rats are secreting insulin at their maximum capacity and are unresponsive to wortmannin for this reason, these islets were stimulated for 20 min with 5 ␮m forskolin in the presence of 8 mm glucose. Release rates increased significantly in response to the addition of forskolin. For example, compared

FIG. 8. Effect of wortmannin on 8 mM glucose-induced insulin release from islets of lean Zucker rats or Zucker fatty rats. Top panel, Two groups of 14 –18 islets were isolated from lean Zucker rats and perifused for 30 min with 3 mM glucose. One group (open circles; n ⫽ 9) was then stimulated with 8 mM glucose alone for 60 min. The second group (closed circles; n ⫽ 9) was stimulated with 8 mM glucose plus 50 nM wortmannin. The asterisks indicate a significant (P ⬍ 0.05) difference between release values at this time. Bottom panel, Two groups of 14 –18 islets were isolated from Zucker fatty rats and perifused for 30 min with 3 mM glucose. One group (open circles; n ⫽ 6) was then stimulated with 8 mM glucose alone for 60 min. The second group (closed circles; n ⫽ 5) was stimulated with 8 mM glucose plus 50 nM wortmannin. The asterisks indicate a significant (P ⬍ 0.05) difference between release values at this time.

with the release rate of 416 ⫾ 89 pg/islet䡠min during the final 5 min of stimulation with 8 mm glucose plus 50 nm wortmannin, the addition of 5 ␮m forskolin to 8 mm glucose increased the secretory rate from these islets to 766 ⫾ 100 pg/islet䡠min after 10 min of stimulation. Discussion

In the present series of experiments we used the fungal metabolite wortmannin to inhibit PI3K, an enzyme normally activated by insulin in several insulin-dependent tissues including muscle, liver, and fat cells and whose impaired activation has been implicated in the pathogenesis of type 2 diabetes (4, 31). The inactivation of PI3K by wortmannin was not directly monitored. However, the levels used in our islet studies are well within the range of concentrations employed

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by many investigators to inhibit this enzyme in insulin-sensitive target tissues (11, 32–34). Moreover, direct inhibition of PI3K in isolated islets and MIN cells by wortmannin levels identical to those used in our studies has been reported (8, 35). Furthermore, the salient observation reported here for wortmannin and 8 mm glucose-induced secretion was duplicated using a structurally distinct PI3K inhibitor, LY294002. It seems reasonable to conclude that a reduction in PI3K-dependent processes accounts at least in part for these results. Using perifused rat islets, islets whose secretory responsiveness is comparable to that found in the perfused pancreas preparation (36, 37), we observed that 50 nm wortmannin inclusion together with 8 mm glucose markedly and persistently amplified insulin secretion during a dynamic perifusion. The potentiating effect was most pronounced during the final 30 min of the perifusion. Dose-response studies revealed that in the presence of 8 mm glucose, the threshold for this action was around 5 nm, with a maximal effect observed at about 100 nm. Secretion fell somewhat at the highest level (1 ␮m) employed. A level (50 nm) of wortmannin that increased 8 mm glucose-induced release about 3-fold had no additional effect when islets were stimulated by a maximally effective 20 mm glucose stimulus. Wortmannin had no effect on release in the presence of 3 mm glucose, a finding suggesting that it amplifies or prolongs in some manner a glucose-derived stimulatory signal normally attenuated by insulin signaling. Several other characteristics of wortmannin’s effects on islet cell responses were also established. First, release in response to the combination of 8 mm glucose plus 50 nm wortmannin was largely abolished by reducing calcium influx into the ␤-cell with nitrendipine. This finding would seem to preclude nonspecific damage to the ␤-cell and the subsequent leakage of insulin into the perifusion medium as a cause of its dramatic potentiating action. Second, consistent with its largely irreversible effect on PI3K documented in other tissues (17), insulin release persisted at high rates despite the removal of wortmannin from 8 mm glucosecontaining perifusion medium. In contrast, lowering the glucose level to 3 mm even in the continued presence of wortmannin abruptly curtailed secretion. Finally, pretreatment with 50 nm wortmannin during the 30-min prestimulatory phase of the perifusion with 3 mm glucose markedly accelerated its potentiating effect on 8 mm glucose-induced secretion. As postprandial insulin release is regulated by both modest increments in the prevailing glucose level and increases in cholinergic stimulation (18), we examined how wortmannin influenced secretion induced by glucose plus carbachol combinations. Even in the presence of 6 mm (108 mg/100 ml) glucose and a level of cholinergic agonist similar to that used to study the effects of cholinergic stimulation in islets and other tissues (20, 38), the inclusion of 50 nm wortmannin enhanced insulin secretion. When islets were stimulated with 8 mm glucose, 1 ␮m carbachol, and 50 nm wortmannin, an increase in peak insulin release rates approaching that produced in response to 20 mm glucose alone were noted. We are aware of several other studies in which the effects of wortmannin on glucose-induced insulin release from rat

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islets or mouse insulinoma cells (MIN 6) were studied. In the report by Gao and co-workers (8), levels of wortmannin overlapping those employed in the present experiments had no stimulatory effect on release despite the demonstrated inhibition of PI3K activity. Several major points of departure between this study and ours deserve emphasis. First, in this report (8) the effects of wortmannin were tested in islets stimulated with 3 mm glucose alone or with the combination of 28 mm glucose plus 500 ␮m carbachol. As pointed out above and in agreement with this report (8), we also found no effect of wortmannin in the presence of 3 mm glucose. In our studies no amplifying effect of 50 nm wortmannin was observed if the glucose level was increased to 20 mm. We have repeated studies using islets stimulated with 28 mm glucose plus 500 ␮m carbachol and have confirmed, in agreement with this report (8), that wortmannin has no positive impact on release under this extreme stimulatory condition (unpublished observations). Our findings suggest that under the conditions employed in these studies wortmannin increases the sensitivity of islets to modest glucose or glucose plus carbachol stimulation, but it is not capable of enhancing release evoked by a maximally effective glucose stimulus. One additional difference between this (8) and our study merits further consideration. We monitored secretion using a dynamic perifusion system. Excessive amounts of insulin (39) are not allowed to accumulate in the vicinity of the islet, but are washed away and replenished by fresh medium. In the studies by Gao et al. (8), batch-incubated conditions were employed. This type of methodology allows large amounts of insulin to accumulate in the medium and may influence ongoing ␤-cell responses. Moreover, the failure of batchincubated islets to release insulin at rates comparable to those observed from perifused islets (40) might be a consequence of a negative feedback effect of insulin on its own release, as this and other studies suggest (41, 42). In a series of experiments using the mouse cell line MIN6, Hagiwara and co-workers (35) found that levels of wortmannin identical to those employed in our studies potentiated glucose-induced release and directly inhibited PI3K activity. Our findings fully support these observations made with this cell line and extend them. We could not ascribe the potentiating effect of wortmannin on 8 mm glucose-induced insulin secretion to any enhancing effect of the fungal metabolite on glucose usage rates by the islet. Wortmannin was also without any effect on the activation of phospholipase C, monitored by IP accumulation in islets. Furthermore, of the independent stimulants employed to augment release, the effects of wortmannin were confined to glucose. When used at concentrations that are approximately equipotent to 8 mm glucose, wortmannin had no potentiating effect on either ␣-ketoisocaproate- or phorbol ester-induced insulin release in terms of their ability to stimulate secretion. The inability of wortmannin to augment release in response to ␣-ketoisocaproate or to the PKC activator PMA suggests that biochemical events proximal to the entry of hexose metabolites into the mitochondria and proximal to the activation of PKC are responsible for potentiation. Taken in their entirety, these findings indicate a high degree of specificity for wortmannin and localize its action to early steps in glucose recognition by the ␤-cell.

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The most critical assessment of the potential significance of these findings to the regulation of glucose homeostasis in vivo comes from our studies using a well characterized model of obesity, insulin resistance, and hyperinsulinemia: the Zucker fatty rat. Like Sprague Dawley rats, lean Zucker rats were sensitive to the potentiating effect of wortmannin on 8 mm glucose-induced insulin release. We confirmed (30) that compared with islets isolated from lean Zucker rats, islets isolated from fatty Zucker rats are significantly more responsive to 8 mm glucose stimulation. Most importantly and a result that would have been predicted based on the assumption that reduced PI3K activity documented in peripheral tissues also occurs in the ␤-cell, wortmannin had no appreciable effect on sustained rates of insulin release from islets of Zucker fatty rats. However, these islets were still quite responsive to forskolin, a compound that activates adenylate cyclase, increases cAMP, and potentiates secretion. The simplest interpretation of these findings is that the reduction in PI3K activation that has been established to exist in the peripheral tissues of these animals (29) also exists in their islets, plays a role in their in vivo hyperinsulinemia, persists after their isolation, and also makes them immune in vitro to any further potentiating effect of wortmannin. Complex effects in mice with targeted disruption or with heterozygous null mutations of the insulin receptor, insulin receptor substrate (IRS-1), and/or IRS-2 have been reported (43– 46). Insulin resistance, hyperinsulinemia, and ␤-cell hyperplasia are characteristic of these genetic alterations. Islet responses to glucose and arginine have been studied in mice with tissue-specific knockout of the insulin receptor (47) or from IRS-1 knockout mice (48). ␤-Cell insensitivity to glucose stimulation occurs in cultured islets isolated from these animals (48). In our studies with freshly isolated rat islets, disruption of insulin signaling via PI3K amplifies glucoseinduced secretion, differences that may be due to the acute nature of our studies or to underlying species differences in glucose sensitivity (49 –51). Studies using cultured rat islets, insulinoma cells, or clonal mouse ␤-cells have also demonstrated that insulin stimulates insulin gene transcription via a PI3K-dependent pathway (52, 53). Insulin stimulates PDX-1 DNA binding, and insulin promoter activity, also via a PI3Kdependent pathway (54). Although earlier reports demonstrated that insulin suppresses its own secretion (42, 55, 56), it has been reported that insulin stimulates its own secretion (57). These and other studies (58) suggest that, similar to glucose’s effects, the observed actions of insulin on the ␤-cell may be time dependent, cell line dependent, and complex. These results provide evidence that insulin exerts a negative autocrine feedback effect on its own secretion and uses the same PI3K-dependent pathways that have been characterized in insulin-dependent target tissues such as liver, muscle, and adipose cells. The inhibition of PI3K by wortmannin abolishes this negative feedback effect, dramatically enhances the sensitivity of the ␤-cell to glucose concentrations in the postprandial range, and culminates in a markedly amplified insulin secretory response. The results also suggest that as insulin resistance and potential deterioration of fuel homeostasis develop in peripheral tissues as a consequence of reduced PI3K activation, the same biochemical process regulates a compensatory increase in insulin secretion from

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the ␤-cell. As the same signaling pathway is altered in both ␤-cells and peripheral tissues, the release of insulin is titrated to the developing peripheral tissue insulin resistance, and euglycemia is maintained. However, the euglycemia is maintained at the expense of hyperinsulinemia. Our findings using islets isolated from Zucker fatty rats demonstrate that the in vivo status of the donor’s insulin signaling pathways may be a key determinant of wortmannin’s effect on the ␤-cell; in this animal model of insulin resistance and hyperinsulinemia in which PI3K activity is already reduced, wortmannin has no further effect on ␤-cell responses to glucose stimulation. Finally, these findings may shed some light on the still unsettled issue of which comes first, insulin resistance or hyperinsulinemia (1, 59 – 64), in the pathogenesis of obesity or obesity culminating in type 2 diabetes. Insulin resistance at the level of the ␤-cell may account for the coupled and parallel emergence of hyperinsulinemia seen under a variety of conditions (1, 2). References 1. Warram JH, Martin BC, Krolewski AS, Soeldner JS, Kahn CR 1990 Slow glucose removal and hyperinsulinemia precede the development of type II diabetes in the offspring of diabetic parents. Ann Intern Med 113:909 –915 2. Vaag A, Henrikssen JE, Beck-Nielsen H 1992 Decreased insulin activation of glycogen synthesis in skeletal muscles in young nonobese Caucasian firstdegree relatives of patients with NIDDM. J Clin Invest 89:782–788 3. Perley MJ, Kipnis DM 1967 Plasma insulin responses to oral and intravenous glucose: studies in normal and diabetic subjects. J Clin Invest 46:1954 –1962 4. Virkama¨ki A, Ueki K, Kahn CR 1999 Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. J Clin Invest 103:931–943 5. Dresner A, Laurent D, Marcucci M, Griffin ME, Dufour S, Cline GW, Slezak LA, Anderson DK, Hundal RS, Rothman DL, Petersen KF, Shulman GI 1999 Effects of free fatty acids on glucose transport and IRS-1-associated phosphatidyinositol 3-kinase activity. J Clin Invest 103:253–259 6. Verspohl EJ, Ammon HP 1980 Evidence for the existence of insulin receptors in rat islets of Langerhans. J Clin Invest 65:1230 –1237 7. Harbeck MC, Louie DC, Howland J, Wolf BA, Rothenberg PL 1996 Expression of insulin receptor mRNA and insulin receptor substrate 1 in pancreatic islet ␤-cells. Diabetes 45:711–717 8. Gao Z, Konrad RJ, Collins H, Matschinsky FM, Rothenberg PL, Wolf BA 1996 Wortmannin inhibits insulin secretion in pancreatic islets and B-TC3 cells independent of its inhibition of phosphatidyinositol 3-kinase. Diabetes 45:854 – 862 9. Acaro A, Wymann MP 1993 Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem J 296:297–301 10. Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M 1994 Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes: studies with a selective inhibitor wortmannin. J Biol Chem 269:3568 –3573 11. Yeh J-I, Gulve EA, Rameh L, Birnbaum MJ 1995 The effects of wortmannin on rat skeletal muscle. J Biol Chem 270:2107–2111 12. Zawalich WS, Zawalich KC 1988 Phosphoinositide hydrolysis and insulin release from isolated perifused rat islets. Studies with glucose. Diabetes 37:1294 –1300 13. Albano JDM, Ekins RP, Maritz G, Turner RC 1972 A sensitive, precise radioimmunoassay of serum insulin relying on charcoal separation of bound and free hormone moieties. Acta Endocrinol (Copenh) 70:487–509 14. Berridge MJ, Dawson RMC, Downes CP, Heslop JP, Irvine RP 1983 Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. Biochem J 212:473– 482 15. Zawalich WS, Zawalich KC 1992 Biochemical mechanisms involved in monomethyl succinate-induced insulin secretion. Endocrinology 131:649 – 654 16. Zawalich WS, Rognstad R, Pagliara AS, Matschinsky FM 1977 A comparison of the utilization rates and hormone-releasing actions of glucose, mannose, and fructose in isolated pancreatic islets. J Biol Chem 252:8519 – 8523 17. Yano H, Nakanishi S, Kimura K, Hanai N, Saitoh Y, Fukui Y, Nonomura Y, Matsuda Y 1993 Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells. J Biol Chem 268:25846 –25856 18. Louis-Sylvestre J 1978 Relationships between the two stages of prandial insulin release in rats. Am J Physiol 235:E103–E111

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