Insulin secretion and IP levels in two distant lineages of the genus Mus: comparisons with rat islets

Walter S. Zawalich1, Kathleen C. Zawalich1, Gregory J. Tesz1, John A. Sterpka2, and William M. Philbrick2

1 Yale University School of Nursing, and 2 Section of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06536


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Islet responses of two different Mus geni, the laboratory mouse (Mus musculus) and a phylogenetically more ancient species (Mus caroli), were measured and compared with the responses of islets from rats (Rattus norvegicus). A minimal and flat second-phase response to 20 mM glucose was evoked from M. musculus islets, whereas a large rising second-phase response characterized rat islets. M. caroli responses were intermediate between these two extremes; a modest rising second-phase response to 20 mM glucose was observed. Prior, brief stimulation of rat islets with 20 mM glucose results in an amplified insulin secretory response to a subsequent 20 mM glucose challenge. No such potentiation or priming was observed from M. musculus islets. In contrast, M. caroli islets displayed a modest twofold potentiated first-phase response upon subsequent restimulation with 20 mM glucose. Inositol phosphate (IP) accumulation in response to 20 mM glucose stimulation in [3H]inositol-prelabeled rat or mouse islets paralleled the insulin secretory responses. The divergence in 20 mM glucose-induced insulin release between these species may be attributable to differences in phospholipase C-mediated IP accumulation in islets.

islets; insulin secretion; biphasic response; phospholipase C; evolution


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A COMMON ANCESTOR, Antemus, is thought to have given rise to the two most common murid species used in biomedical research, rats and mice (11). Approximately 10 million years ago (MYA), the phylogenetic tree connecting the genus Rattus and the future genus Mus diverged. Several levels of quasi-synchronous speciation events ~2 MYA further subdivided the Mus species. One branch led ultimately to Mus musculus, the commonly employed laboratory mouse, whereas another, more ancient lineage culminated in Mus caroli.

Our interest in the phylogeny of mice and rats has been stimulated by the observations that pronounced species differences exist in terms of their islet sensitivity to glucose stimulation. For example, over 30 years ago, Malaisse and Malaisse-Lagae (25) reported that, in response to glucose stimulation, less insulin was released from mouse pancreatic pieces than from similarly stimulated rat pancreatic pieces. The advent of more sophisticated methodology employing the perfused rat or mouse pancreas preparation or perifused islets substantiated these early observations (5, 23, 24, 35, 36, 53). The most dramatic deviation between the two most commonly employed species used to study the biochemical mechanisms that regulate insulin secretion occurs during the sustained second-phase insulin secretory response to glucose stimulation (5). A large, rising second-phase insulin secretory response to stimulatory glucose is characteristic of rat islets (5, 15, 21, 22, 29, 30). In quantitative terms, the increments in release rates above basal control values are on the order of 25- to 50-fold. The robust secretory response from rat islets is duplicated by human islets studied by the hyperglycemic clamp technique (18, 19, 38), supporting the concept that the same biochemical mechanisms regulate the responses of rat and human islets. In contrast to these two species, sustained insulin release rates from mouse islets in response to glucose are flat and only modestly elevated above prestimulatory release rates. This pattern has been observed in studies utilizing both the perfused pancreas preparation and perifused islets (5, 23, 24, 35, 36, 53). Failure of mouse islets to respond to high glucose cannot be attributed to any reduction in insulin content, because both species contain comparable amounts of stored insulin (16, 17, 51). Moreover, when stimulated with the combination of glucose plus a cholinergic agonist, a dramatic enhancement of second-phase release rates is observed from mouse islets (20, 53).

The failure of glucose to evoke a rising second-phase secretory response from perfused or perifused mouse islets is not the only anomaly compared with rat islet responses. It is well documented that brief prior exposure of rat or human islets to stimulatory glucose amplifies the subsequent response to the hexose (6, 13, 14, 22, 45). Termed time-dependent potentiation (TDP) or sensitization, the response to a second stimulation, particularly the first phase of release, is markedly enhanced. Mouse islets also fail to display the induction of TDP to a prior glucose stimulus under conditions in which it can be readily detected in rat islets (6, 51). Finally, sustained exposure of rat islets to high glucose desensitizes them to subsequent restimulation (10, 41, 55), termed time-dependent inhibition (TDI) of release or desensitization. Mouse islets are also immune to this adverse effect of chronic high glucose exposure (41).

We have attributed the anomalous behavior of mouse islets compared with rat islets to a reduction in glucose signaling via the phospholipase C (PLC)/protein kinase C (PKC) signaling pathway. In support of this hypothesis are studies in which the PLC-mediated hydrolysis of islet phosphoinositide pools has been monitored by measuring inositol phosphate (IP) accumulation in [3H]inositol-prelabeled islets. In rat islets, exposure to stimulatory glucose results in 500-800% increases in IP accumulation (8, 9, 44, 48). The comparable response from mouse islets is an ~0.5- to 1.0-fold increase (34, 53). Accounting for this biochemical deviation may be the underexpression of several PLC isozymes in mouse islets compared with rat islets (42, 53).

In the present series of experiments, an alternative approach was used to probe the differences in glucose sensitivity that exist between species and to elucidate the potential biochemical mechanisms involved. Comparative studies were conducted using islets isolated from a phylogenetically ancient Mus species, Mus caroli, and with the evolutionarily more recent Mus musculus species, the laboratory mouse. Our findings underscore the potential involvement of PLC/PKC activation in the regulation of islet responses to glucose stimulation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Islet isolation. The detailed methodologies employed to assess insulin output from collagenase-isolated islets have been described previously (46). Young adult male M. musculus mice (CD-1 strain, body wts at time of study 20-36 g), male M. caroli mice (body wts at time of study 14-22 g), or male Rattus norvegicus (Sprague-Dawley) rats (body wts at time of study 350-450 g) were used. The CD-1 mice and the Sprague-Dawley rats were purchased from Charles River (Wilmington, MA). The M. caroli mice were obtained from the colony maintained by Dr. Rosemary Elliott at Roswell Park Memorial Institute (Buffalo, NY). All animals were treated in a manner that complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1985). The animals were fed ad libitum. After anesthesia induced by pentobarbital sodium (Nembutal, 50 mg/kg; Abbott, North Chicago, IL), islets were isolated by collagenase digestion and handpicked by use of a glass loop pipette under a stereomicroscope. They were free of visual exocrine contamination.

Perifusion studies. Groups of 14-18 freshly isolated rat or mouse islets were loaded onto nylon filters (Tetko, Briarcliff Manor, NY) and perifused in a Krebs-Ringer bicarbonate (KRB) buffer at a flow rate of 1 ml/min for 30 min in the presence of 3 mM glucose to establish basal and stable insulin secretory rates. After this 30-min stabilization period, they were then perifused with the appropriate agonist or agonist combinations, as indicated in the text and Figs. 1-5 of RESULTS. Perifusate solutions were gassed with 95% O2-5% CO2 and maintained at 37°C. Insulin released into the medium was measured by RIA (1).


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Fig. 1.   Glucose-induced release from perifused rat or mouse islets. Top: groups of 14-18 rat islets were perifused. For the initial 30 min the islets were maintained with 3 mM glucose (G3) to establish basal and stable insulin secretory rates. Islets were then perifused (indicated by vertical line) for 60 min with 20 mM glucose (G20; black-triangle, solid line, n = 8). Other islets (black-triangle, dashed line, n = 4) were maintained with 3 mM glucose for the entire perifusion. Bottom: Mus musculus (CD-1) mouse islets (open circle , n = 7), or Mus caroli islets (, n = 6) were isolated and perifused. For the initial 30 min, islets were maintained with 3 mM glucose (G3) and then perifused (indicated by vertical line) for 60 min with 20 mM glucose (G20). Values are means ± SE. This and subsequent perifusion figures have not been corrected for dead space in the perifusion apparatus, 2.5 ml or 2.5 min with a flow rate of 1 ml/min. Note change in scale between top and bottom.



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Fig. 2.   Effects of phorbol 12-myristate 13-acetate (PMA) and alpha -ketoisocaproate (KIC) on mouse islets. Groups of 14-18 islets were isolated from Mus musculus (open circle ) or Mus caroli (). After 30 min of perifusion with 3 mM glucose, they were stimulated with 500 nM PMA (left) or with 20 mM KIC (right) in the continued presence of 3 mM glucose. *Significant differences in response to KIC between the 2 species.



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Fig. 3.   Effects of prior short-term stimulation by 20 mM glucose on rat or mouse islets. Groups of 14-18 Rattus norvegicus (left, n = 4), Mus musculus (middle, n = 10), or Mus caroli (right, n = 7) islets were perifused for 30 min with 3 mM glucose, for 20 min with 20 mM glucose, for 15 min with 3 mM glucose, and for an additional 30 min with 20 mM glucose. Values are means ± SE of the first 10 min of the secretory response to a repeated 20 mM glucose stimulus. Solid line, initial stimulation with 20 mM glucose; dashed line, second stimulation with 20 mM glucose. Note the change in scale between left, middle, and right panels.



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Fig. 4.   Enzyme expression patterns in Mus islets. Groups of M. musculus (M.m) and M. caroli (M.c) islets were isolated and subjected to quantitative Western blotting and analysis, as described in METHODS. Left: actual blots of representative experiments; arrows indicate position of molecular weight markers (not shown). Right: expression of protein kinase C (PKC)alpha (n = 3), phospholipase C (PLC)gamma 1 (n = 3), and PLCbeta 1 (n = 3) in M. caroli islets as a percentage of those found in M. musculus islets.



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Fig. 5.   PLCdelta 1 expression in rodent islets. Groups of islets were isolated from R. norvegicus (rat), M. musculus (M.m), or M. caroli (M.c) and probed for PLCdelta 1. A homogenate of rat brain is also shown. Molecular weight markers for protein standards (not shown) are given at right. In 3 separate experiments we could not detect a significant PLCdelta 1 signal in Mus islets, regardless of the species.

Islet labeling for 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]inositol-containing KRB solution made up as follows. Ten microcuries of myo-[2-3H]inositol (specific activity 16-23 Ci/mM) were placed in a 10 × 75-mm culture tube. To this aliquot of tracer were added 250 µl of warmed (to 37°C) and oxygenated KRB medium supplemented with 5.0 mM glucose. After mixing, 240 µl of this solution were gently added to the vial with islets. The vial was capped with a rubber stopper, gassed for 10 s with 95% O2-5% 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 of fresh KRB.

IP measurements. After washing, the islets on nylon filters were placed in small glass vials. Four hundred microliters of KRB, supplemented with 10 mM LiCl to prevent IP degradation, and the appropriate agonists were added gently to the vial. The vials were capped and gently gassed for 5 s with 95% O2-5% CO2. After 30 min, the generation of IPs was stopped by addition of 400 µl of 20% perchloric acid. Total IPs formed were then measured using Dowex columns as described previously (7, 47).

Western blot studies. Freshly isolated islets from M. caroli and M. musculus (CD-1) mice were suspended in a 2:1 ratio of islets per microliter of homogenization buffer (1 mM dithiothreitol, 0.1 mM leupeptin, 5 mM benzamidine, 10 µg/ml soybean trypsin inhibitor, 5 µg/ml aprotinin, 2 µM pepstatin A, and 2 mM phenylmethylsulfonyl fluoride in 12.5 mM Tris, 1.25 mM EGTA, 1.25 mM EDTA, and 0.25% Triton X-100, pH 7.6). Islets were then disrupted by sonic oscillation. Duplicate aliquots were analyzed for protein content with the Lowry assay by use of BSA to generate the standard curve and a rat liver preparation in homogenization buffer as an internal standard. Equal amounts of protein were loaded from both CD-1 and M. caroli samples for Western blot analysis. For PKCalpha (15-20 µg), PLCbeta 1 (15-20 µg), PLCdelta 1 (15-25 µg), and PLCgamma 1 (10-25 µg), proteins were separated by SDS-PAGE with a 4% stacking gel with a 7% separating gel at 12 and 16 mA, respectively. Separated proteins were electro-transferred onto a polyvinylidene difluoride transfer membrane with 15 V for 20 h. Membranes were stained with Ponceau S staining solution to confirm transfer. Membranes were then washed with distilled deionized water briefly and then blocked in Tris-buffered saline containing 5% Carnation nonfat dried milk and 0.05% Tween 20 for 1 h and 50 min.

For PKCalpha determinations, membranes were incubated for 45 min with primary antibody, rabbit polyclonal IgG anti-PKCalpha (0.5 µg/ml), washed, incubated for 30 min with anti-rabbit IgG-horseradish peroxide (HRP, 0.5 µg/ml), and then washed again. For PLCbeta 1 determinations, membranes were incubated for 60 min with primary antibody, rabbit polyclonal IgG anti-PLCbeta 1 (0.5 µg/ml), washed, incubated for 30 min with anti-rabbit IgG-HRP (0.5 µg/ml), and then washed again. For PLCdelta 1 determinations, membranes were incubated for 60 min with primary antibody, mouse polyclonal IgG anti-PLCdelta 1 (1.0 µg/ml), washed, incubated for 30 min with anti-mouse IgG-HRP (0.5 µg/ml), and then washed again. For PLCgamma 1 determinations, membranes were incubated for 60 min with primary antibody, mouse polyclonal IgG anti-PLCgamma 1 (0.5 µg/ml), washed, incubated for 30 min with anti-mouse IgG-HRP (0.5 µg/ml), and then washed again. Antigen-antibody complexes were visualized using the New England Nuclear (NEN, Boston, MA) Western Blot Chemiluminescence Reagent Plus system. Membranes were exposed on the Kodak Digital Imaging Station 440cf for 1 min (PKCalpha  + PLCbeta 1), for 1-2 min (PLCgamma 1), and for 2-4 min (PLCdelta 1). Images were captured and transferred to the Kodak 1D Image Analysis software for analysis. Bands were analyzed by net intensity (a measure of the total intensity or sum of the background subtracted pixels within the band). The net intensity of CD-1 mice for the desired enzyme was taken as 100%, which allowed the comparisons of the corresponding M. caroli enzyme to be analyzed as a percentage of the CD-1 enzyme content.

Reagents. Hanks' solution was used for the islet isolation. The perifusion medium consisted of (in mM) 115 NaCl, 5 KCl, 2.2 CaCl2, 1 MgCl2, and 24 NaHCO3, and 0.17 g/dl BSA. Other compounds were added where indicated, and the solution was gassed with a mixture of 95% O2-5% CO2. The 125I-labeled insulin used for the insulin assay was purchased from NEN, and the labeled myo-[2-3H]inositol was from Amersham (Arlington Heights, IL). BSA (RIA grade), carbachol, phorbol 12-myristate 13-acetate (PMA), glucose, and the salts used to make the Hanks' solution and perifusion medium were purchased from Sigma (St. Louis, MO). Antibodies to PLCdelta 1 and PLCgamma 1 were purchased from Upstate Biotechnology (Lake Placid, NY). Antibodies to PLCbeta 1 and PKCalpha were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rabbit IgG and anti-mouse IgG were also obtained from Santa Cruz Biotechnology. Rat insulin standard (lot no. 615-ZS-157) was the generous gift of Dr. Gerald Gold, Eli Lilly (Indianapolis, IN). Collagenase (type P) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN).

Statistics. Statistical significance was determined using the Student's t-test for unpaired data or ANOVA in conjunction with the Newman-Keuls test for unpaired data. A P value <= 0.05 was taken as significant. Values presented in the text, Figs. 1-5, and Table 1 in RESULTS represent means ± SE of at least three observations.

                              
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Table 1.   IP responses of murid islets to 3 mM or 20 mM glucose


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Species differences in response to 20 mM glucose stimulation. The first series of experiments confirmed the existence of species differences in islet responses to sustained 20 mM glucose stimulation. The response of islets isolated from Sprague-Dawley rats was biphasic in character, consisting of an initial spike of secretion followed by a slowly rising second-phase response (Fig. 1, top). Release rates peaked ~30 min after the onset of 20 mM glucose stimulation and averaged 1,063 ± 84 pg · islet-1 · min-1 (n = 8) at this time. Release rates declined to ~50% of peak values as the perifusion with 20 mM glucose progressed, an observation reported previously by Curry (15) in studies with the perfused pancreas. Compared with rates of secretion observed from rat islets maintained for 90 min with 3 mM glucose (24 ± 6 pg · islet-1 · min-1, n = 4), peak second-phase release rates were increased >30-fold. This compares favorably with findings made using the perfused pancreas preparation and indicates that the physiological integrity of our islet preparation was maintained during the isolation procedures.

A different picture developed when islets from M. musculus (CD-1 strain) were stimulated with 20 mM glucose under comparable perifusion conditions (Fig. 1, bottom). An initial spike of secretion, comparable in timing but less in magnitude (111 ± 20 pg · islet-1 · min-1, n = 7) than the response from rat islets (269 ± 45 pg · islet-1 · min-1, n = 8) was observed. Most striking compared with rat islet responses, however, was the failure of M. musculus islets to demonstrate a rising second-phase response to 20 mM glucose. For example, whereas release rates from rat islets increased significantly during the first 30 min of exposure to 20 mM glucose, release rates from CD-1 mouse islets remained flat, averaging ~80 pg · islet-1 · min-1 during the first 30 min of sustained 20 mM glucose stimulation. This failure to respond to glucose alone cannot be attributed to islet damage or lack of releasable insulin stores, because stimulation of these islets with the combination of 20 mM glucose plus 10 µM carbachol was accompanied by a dramatic augmentation of release. For example, 20 min after exposure of these same islets to the combination of glucose plus carbachol, response rates of 437 ± 25 pg · islet-1 · min-1 were measured.

Insulin secretion from M. caroli islets to 20 mM glucose (Fig. 1, bottom), compared with rat or M. musculus islet responses, fell between the responses of these two species. Peak first-phase secretory rates (124 ± 28 pg · islet-1 · min-1, n = 8) were comparable to those noted from M. musculus. However, sustained second-phase responses were considerably larger. For example, whereas release rates from CD-1 islets averaged 85 ± 7, 69 ± 6, or 56 ± 6 pg · islet-1 · min-1 by 30, 40, or 50 min after the onset of 20 mM glucose stimulation, the comparable responses form M. caroli islets were 412 ± 63, 368 ± 56, or 331 ± 60 pg · islet-1 · min-1, respectively. The addition of 10 µM carbachol in the continued presence of 20 mM glucose had little further effect on secretion. For example, 20 min after exposure of these islets to the combination of glucose plus carbachol, responses rates of 369 ± 54 pg · islet-1 · min-1 were measured. When measured at the termination of the perifusion, islet insulin contents averaged 82 ± 3 or 68 ± 5 ng/islet in M. musculus or M. caroli, respectively.

Additional studies were conducted using Mus islets stimulated with the PKC activator PMA or the mitochondrial fuel alpha -ketoisocaproate (KIC). Neither species responded to the phorbol ester alone in the simultaneous presence of 3 mM glucose (Fig. 2, left). Both species responded to stimulation with 20 mM KIC. During the final 45 min of stimulation, the responses of M. caroli islets to the amino acid derivative were significantly greater than those observed from M. musculus islets (Fig. 2, right).

Species differences in the induction of TDP by prior glucose stimulation. In addition to the evocation of a rising second-phase insulin secretory response, a second time-dependent characteristic of rat islet responses to glucose stimulation is the induction of priming, also referred to as sensitization or TDP. The induction of TDP in rat islets by glucose was confirmed. Rat islets were briefly stimulated with 20 mM glucose, the hexose level decreased to 3 mM for 15 min, and a repeat stimulation with 20 mM glucose was conducted. As shown in Fig. 3 (left), this protocol resulted in a marked enhancement of first-phase secretion rates. Compared with the peak first-phase response achieved during the initial stimulation, 217 ± 18 pg · islet-1 · min-1 (n = 4), a dramatic enhancement of the subsequent first-phase response was evident. Peak first-phase release rates now averaged 804 ± 30 pg · islet-1 · min-1.

First reported by Berglund (6) and confirmed more recently (50) is the failure of prior glucose stimulation to induce TDP in either perfused or perifused mouse islets. This aberration of M. musculus islets compared with rat islets is demonstrated in Fig. 3 (middle). Only the first 10 min of the response to 20 mM glucose, not corrected for the dead space in the perifusion medium, are depicted. Brief stimulation with 20 mM glucose resulted in a spike of release that peaked at 130 ± 9 pg · islet-1 · min-1 (n = 10). Release subsided to prestimulatory rates when the glucose level was decreased to 3 mM. Restimulation with 20 mM glucose was characterized by a peak first-phase response of 90 ± 5 pg · islet-1 · min-1, ~30% less than that observed during the initial stimulation with 20 mM glucose.

Islets isolated from M. caroli exhibited TDP when stimulated with 20 mM glucose (Fig. 3, right). The initial peak first response to 20 mM glucose stimulation averaged 79 ± 12 pg · islet-1 · min-1 (n = 7), whereas that observed during restimulation with 20 mM glucose increased to 172 ± 25 pg · islet-1 · min-1.

IP accumulation in rat and mouse islets. An analysis of information flow in the PLC/PKC cycle prompted us to suggest previously that the rising second-phase insulin secretory response and the induction of TDP by glucose were both related to information flow in this signaling pathway (48). In an attempt to provide an explanation for the divergence in second-phase response rates and the induction of TDP in the Mus species investigated, we next examined IP accumulation in [3H]inositol-prelabeled islets. After a 3-h labeling period and a 30-min incubation in 3 mM glucose, levels of labeled IPs were significantly greater in rat islets compared with either of the Mus species investigated (Table 1). We next examined the magnitude of rodent islet IP responses to 20 mM glucose stimulation (Table 1). In agreement with previous reports, a marked enhancement of IP accumulation was observed when rat islets were stimulated with 20 mM glucose. In confirmation of what we previously observed with mouse islets (53) and of a response reported by Sato and Henquin (34), who used this species as well, a minimal IP accumulation in response to 20 mM glucose was observed in M. musculus islets. Lying between these two values were the responses of M. caroli islets to 20 mM glucose.

PKC/PLC isozyme expression patterns in rodent islets. We have proposed previously (42, 53) that the underexpression of a nutrient-sensitive PLC isozyme in mouse islets vs. rat islets may be a contributory factor to the deviation in glucose responsiveness observed between these two species. Additional studies examining the expression patterns of PKCalpha and the major PLC isozymes, beta 1, gamma 1, and delta 1, were conducted. Compared with M. musculus islets, M. caroli islets exhibited two- to threefold increases in the expression of both PKCalpha and PLCbeta 1 (Fig. 4). M. caroli islets expresssed slightly less PLCgamma 1 (Fig. 4) than M. musculus islets, whereas neither species expressed any measurable level of PLCdelta 1 compared with rat islets (Fig. 5).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

It is often assumed that the responses of rodent islets to glucose stimulation are similar, irrespective of the species from which they were isolated. This and at least seven other studies (5, 6, 23, 24, 36, 50, 53) suggest caution in the extrapolation of data between rat and mouse islets when glucose is used as the stimulant. In the former species, glucose alone evokes time-dependent actions on their islets that cannot be readily duplicated using mouse islets. Failure of mouse islets to develop a rising second-phase insulin secretory response to stimulatory glucose and the failure of glucose to induce TDP and TDI of secretion are distinguishing features between rodent islet responses. These differences cannot be explained by any obvious disparity in glucose metabolism, because both species utilize glucose at comparable rates (3, 42). On the basis of an analysis of information flow in the PLC/PKC cycle, we have suggested that the reduced activation of PLC by glucose may account for this species disparity (48). The underexpression of several PLC isozymes in mouse islets compared with rat islets may provide the biochemical explanation for this divergence in species responses. This is not to suggest that mouse islets are incapable of PLC activation, but only that they rely more exclusively on the activation of a non-nutrient-regulated PLC isozyme for the provision of phosphoinositide-derived second messengers.

In the present experiments we used a phylogenetic approach to address how glucose's action on pancreatic beta -cells has been retained or lost in different species of mice over time and compared the beta -cells with rat islet responses. In addition, we stimulated islets with an alternate fuel agonist in an attempt to ascertain whether the established deviation in glucose sensitivity that exists extends to another agonist and species as well. Finally, the expression patterns of the major PLC isozymes and PKCalpha were measured. For these studies we employed M. musculus, the common laboratory mouse, a species that diverged from M. caroli about 1.5 MYA (11). Phylogenetically, this latter species is closer evolutionarily to the rat, but all of these species are thought to share a common historical ancestor, Antemus.

In agreement with numerous studies using the perfused rat pancreas preparation or islets isolated from this species, a robust biphasic insulin secretory response was evoked from perifused rat islets stimulated with 20 mM glucose. Most dramatic is the enhancement of insulin release as the perifusion progressed. Compared with release rates observed in the presence of 3 mM glucose, the increment amounts to an ~30-fold increase. When islets from M. musculus, in this case CD-1 mice, were similarly stimulated, a spike of first-phase secretion was noted. However, these islets failed to develop a rising second-phase response that is characteristic of R. norvegicus islets studied using perfusion or perifusion techniques or Homo sapiens islets studied with the hyperglycemic clamp technique (18, 19, 38). Lying between these responses of two species, but much closer in similarity to the rat than the mouse, were the responses evoked from 20 mM glucose-stimulated M. caroli islets. A rising second-phase response to 20 mM glucose was noted. The failure of M. musculus islets to respond in a manner comparable to R. norvegicus or M. caroli islets cannot be attributable to insufficient insulin stores or damage to the islets during isolation. Comparable amounts of insulin were measured in the two Mus species; furthermore, when M. musculus islets were stimulated with 20 mM glucose plus 10 µM carbachol, a dramatic enhancement of sustained insulin secretion was noted.

Two additional stimulants were used to probe the specificity of mouse islet responses. Unlike rat islets (26, 52), neither Mus species responded to stimulation with 500 nM of the PKC activator PMA in the presence of low (<= 3 mM) glucose, a finding previously reported when islets isolated from lean C57BL mice were used (49). Although islets from both species responded to KIC, the secretory responses from M. caroli islets again exceeded those from M. musculus islets. This latter observation would appear to eliminate the early steps in glucose metabolism, such as glycolysis and the pentose cycle, as potential explanations for these deviations. Rather, a common distal response element sensitive to a mitochondrial signal appears to be involved.

The ability of prior 20 mM glucose stimulation to induce TDP in rat islets and the failure of islets isolated from M. musculus to demonstrate a similar response pattern (6, 50) were also confirmed in this report. Like their second-phase responses to 20 mM glucose stimulation, M. caroli islets demonstrated a response to prior glucose stimulation that was similar to the response from rat islets, although of a smaller amplitude. For example, after a brief priming period with 20 mM glucose, peak first-phase responses to 20 mM glucose restimulation were increased about fourfold from rat islets. A twofold amplification was observed from M. caroli islets. M. musculus islet responses to a second 20 mM glucose stimulation fell ~30%.

We measured IP accumulation, a surrogate marker for PLC activation (4, 7, 31, 32), in rat and mouse islets. The results of these studies confirm the minimal stimulatory effect of 20 mM glucose on this parameter of PLC activation in CD-1 mouse islets compared with the response of rat islets (48). Of particular significance, a finding that reinforces the concept that PLC activation may play a pivotal role in the evocation of the time-dependent effects that glucose exerts upon islets is the observation made with M. caroli islets. Paralleling the capacity of glucose to evoke a modest rising second-phase response and to induce TDP of secretion, an effect on IP accumulation intermediate between R. norvegicus and M. musculus was observed in studies with M. caroli islets.

When quantitative Western blotting techniques and analysis were used, the expression of PKCalpha was found to be greater in M. caroli islets than in M. musculus islets. Of the PLC isozymes analyzed, neither species expressed PLCdelta 1 to any measurable degree. PLCgamma 1 was slightly underexpressed in M. caroli islets compared with M. musculus islets. However, a potential candidate for the amplified IP responses to glucose observed from M. caroli islets compared with M. musculus islets is PLCbeta 1, an enzyme expressed in greater amounts in the islets of the former species. This PLC isozyme's activation, although complex, is dependent not only on calcium availability but also on GTP-binding proteins (32, 33). Previous studies with rat islets have proposed that an adequate supply of GTP, dependent on ATP generation by metabolic fuels, is necessary for PLC activation (37).

Our working hypothesis is that the robust activation of PLC by high glucose is in part responsible for the large rising second-phase secretory response in rat islets. When rat islets are stimulated by 20 mM glucose, the multiple cellular effects of PLC-derived second messengers induce cumulative changes in islets that only slowly return to the prestimulatory state. Restimulation during this period results in a potentiated secretory response (45). The fact that the PMA (12) evokes a response reminiscent of the rising second phase of secretion (27, 39) and is an effective inducer of priming in rat islets (28, 52) supports the concept that PKC activation may be involved in both processes. This idea is also supported by results in which other agonists that increase information flow in the PLC/PKC system are used to induce priming (43, 45, 54). In the case of M. musculus islets, the minimal increase in PLC/PKC activation by 20 mM glucose induces minimal changes in islet sensitivity that are poorly maintained and incapable of evoking a large second-phase response or to induce priming. On the other hand, the more significant PLC responses of M. caroli islets, coupled to the increased expression of PKCalpha , may be responsible for a modest (when compared with rat islets) increase in second-phase secretion and the induction of priming.

Several additional issues deserve final comment. First, on the basis of PLCdelta 1's sensitivity to calcium (2) and the obligatory role of calcium in glucose-induced PLC activation (44), we initially speculated that this PLC isozyme might be the one activated by various nutrient secretagogues (40). Unlike in rat islets, we could not detect a significant PLCdelta 1 signal despite the analysis of similar amounts of islet protein. Although this type of analysis might appear to exclude PLCdelta 1 as a nutrient-activated isozyme, it should be remembered that, even though the M. caroli response to glucose was improved compared with M. musculus islets, it was still modest compared with rat islet responses. It is possible that the activation of this enzyme in rat islets contributes to the robust glucose-induced secretory response from this species. Finally, this type of analysis suggests caution in the extrapolation of findings made by use of transgenic mice to probe the regulation of glucose homeostasis. If the phenotypic expression of any observed alteration is a function of glucose-induced insulin release, the species-dependent contribution of the beta -cell should be considered.

In summary, we have confirmed that significant species differences to glucose stimulation exist between R. norvegicus and M. musculus islets, differences possibly due to genetic divergence occurring over 10 million years of evolution (11). A phylogenetic Mus species more closely related evolutionarily to the rat demonstrates responses to glucose stimulation intermediate between R. norvegicus and M. musculus. This difference may be attributable to the retained capacity of glucose to more effectively activate a nutrient-regulated PLC isozyme in the caroli compared with the musculus species. Our findings also suggest that the increased expression of PLCbeta 1 and PKCalpha may be involved in these species-dependent responses of Mus islets to nutrient stimulation. Finally, when we consider that attempts are being made to genetically engineer beta -cells for therapeutic purposes, the elucidation of the enzymes that regulate the magnitude of glucose-induced insulin secretion is of more than passing academic interest.


    ACKNOWLEDGEMENTS

These studies were supported by National Institutes of Health Grant no. 41230 (to W. S. Zawalich) and Diabetes and Endocrinology Research Center Grant DK-45735, and a grant from the American Diabetes Association.


    FOOTNOTES

Address for reprint requests and other correspondence: W. S. Zawalich, Yale Univ. School of Nursing, 100 Church St. So., New Haven, CT 06536-0740 (E-mail: Walter.Zawalich{at}Yale.Edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7 July 2000; accepted in final form 15 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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