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Originally published In Press as doi:10.1074/jbc.M706481200 on December 20, 2007

J. Biol. Chem., Vol. 283, Issue 8, 4560-4567, February 22, 2008
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G{alpha}z Negatively Regulates Insulin Secretion and Glucose Clearance*

Michelle E. Kimple{ddagger}, Jamie W. Joseph§, Candice L. Bailey{ddagger}, Patrick T. Fueger§, Ian A. Hendry, Christopher B. Newgard{ddagger}§||, and Patrick J. Casey{ddagger}||1

From the Departments of {ddagger}Pharmacology and Cancer Biology and ||Biochemistry and Biophysics, and §The Sarah W. Stedman Nutrition and Metabolism Center, Duke University Medical Center, Durham, North Carolina 27710 and the Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 0200 Australia

Received for publication, August 6, 2007 , and in revised form, December 10, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Relatively little is known about the in vivo functions of the {alpha} subunit of the heterotrimeric G protein Gz (G{alpha}z). Clues to one potential function recently emerged with the finding that activation of G{alpha}z inhibits glucose-stimulated insulin secretion in an insulinoma cell line (Kimple, M. E., Nixon, A. B., Kelly, P., Bailey, C. L., Young, K. H., Fields, T. A., and Casey, P. J. (2005) J. Biol. Chem. 280, 31708–31713). To extend this study in vivo, a G{alpha}z knock-out mouse model was utilized to determine whether G{alpha}z function plays a role in the inhibition of insulin secretion. No differences were discovered in the gross morphology of the pancreatic islets or in the islet DNA, protein, or insulin content between G{alpha}z-null and wild-type mice. There was also no difference between the insulin sensitivity of G{alpha}z-null mice and wild-type controls, as measured by insulin tolerance tests. G{alpha}z-null mice did, however, display increased plasma insulin concentrations and a corresponding increase in glucose clearance following intraperitoneal and oral glucose challenge as compared with wild-type controls. The increased plasma insulin observed in G{alpha}z-null mice is most likely a direct result of enhanced insulin secretion, since pancreatic islets isolated from G{alpha}z-null mice exhibited significantly higher glucose-stimulated insulin secretion than those of wild-type mice. Finally, the increased insulin secretion observed in G{alpha}z-null islets appears to be due to the relief of a tonic inhibition of adenylyl cyclase, as cAMP production was significantly increased in G{alpha}z-null islets in the absence of exogenous stimulation. These findings indicate that G{alpha}z may be a potential new target for therapeutics aimed at ameliorating β-cell dysfunction in Type 2 diabetes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin secretion from the β-cells of the pancreas follows a biphasic pattern, in which an initial peak minutes after stimulation by glucose is followed by a lower magnitude sustained phase over the duration of glucose stimulation. This phenomenon is thought to be due to the interaction of two signaling pathways: the triggering pathway and the amplifying pathway (1, 2). In the initial (triggering) phase of insulin secretion, metabolism of glucose in the mitochondria leads to an increase in cytosolic ATP/ADP ratio, which causes the closure of the ATP-sensitive K+ channel (KATP channel)2 and membrane depolarization. The resulting membrane depolarization opens voltage-dependent calcium channels, allowing an influx of extracellular Ca2+ and stimulation of calcium-induced calcium release from the endoplasmic reticulum. These intracellular changes lead to exocytosis of a readily releasable pool of insulin granules, a process that involves ATP-dependent insulin granule priming, granule fusion, and insulin release. The amplifying pathway of insulin secretion is also dependent on glucose but does not elevate the intracellular Ca2+ concentrations and is therefore referred to as the KATP channel-independent pathway (3); the mechanisms underlying this pathway are not as well defined as those of the triggering pathway but may involve an increase in the efficiency of the exocytosis process and/or the replenishment of the readily releasable granule pool.

Heterotrimeric G proteins are involved in both the positive and negative regulation of insulin secretion (4, 5). There are four subfamilies of the GTP-binding G protein {alpha} subunits, classified based on sequence similarities and signaling pathways engaged: G{alpha}q, G{alpha}s, G{alpha}i, and G{alpha}12. In terms of pancreatic β-cell physiology, acetylcholine can activate M3 muscarinic receptors that are coupled to G{alpha}q, leading to Ca2+ release from intracellular stores and activation of protein kinase C, both of which augment insulin secretory granule release (6). Pituitary adenylate cyclase-activating peptide (PACAP)-, glucagon-like peptide (GLP)-, and gastric inhibitory polypeptide (GIP)-specific receptors all couple primarily to adenylyl cyclase-stimulating G{alpha}s (7), leading to an increase in cAMP and activation of its downstream signaling cascades. cAMP action requires glucose and is primarily thought of as impacting the amplifying phase of insulin secretion (8) but has also been shown to have effects on proximal events, such as expression of key genes involved in metabolism and augmentation of the closure of the KATP channel (7). Conversely, G{alpha}i proteins have been shown to block insulin secretion both by inhibition of adenylyl cyclase and by direct regulation of the KATP channel, voltage-dependent calcium channel, and/or the exocytosis process itself (4, 5). Examples of agonists that work through G{alpha}i-coupled receptors are epinephrine, galanin, somatostatin, and E-prostaglandins (5), among several others.

Because of the lack of a required cysteine residue, G{alpha}z is the only member of the G{alpha}i subfamily of G protein {alpha} subunits, the largest of the four G{alpha} subfamilies (9), that is immune to inactivation by pertussis toxin (PTX)-catalyzed ADP-ribosylation (10). Other unique G{alpha}z characteristics are a very low GDP-to-GTP exchange rate that is reduced 20-fold further in the presence of physiologic concentrations of Mg2+ and an extremely low intrinsic GTP hydrolysis rate that is 200-fold lower than that described for other G{alpha} subunits (11). G{alpha}z also displays a limited expression profile, and protein expression has been demonstrated conclusively only in the brain, adrenal medulla, retina, platelets, pituitary, and pancreatic islets (10, 12, 13).

Initial work studying the role of G{alpha}z in regulating insulin secretion took advantage of INS-1-derived 832/13 cells (a clonal pancreatic β-cell line) (14). As expected for a member of the G{alpha}i subfamily, constitutively activated G{alpha}z inhibited GSIS from 832/13 cells (13). More importantly, inhibition of insulin secretion by prostaglandin E1 was PTX-insensitive and could be blocked either by overexpression of the "regulator of G protein signaling" domain of RGSZ1, a G{alpha}z-specific deactivator (15), or by small interfering RNA-mediated knockdown of G{alpha}z expression (13). Together, these data indicated that endogenous G{alpha}z in the 832/13 cells coupled to endogenous G protein-coupled receptors to inhibit insulin secretion. However, the question of whether G{alpha}z was important for regulation of GSIS in vivo remained unanswered.

The generation of a G{alpha}z knock-out mouse has provided an opportunity to study the involvement of this G{alpha} protein in physiology. Thus far, the phenotypes associated with G{alpha}z deletion are moderate consequences on platelet function (16, 17) and altered behavioral and central nervous system responses to serotonin, morphine, apomorphine, amphetamine, and dopamine agonists (17–20); the effect of G{alpha}z knock-out on insulin secretion and glucose homeostasis in these mice has not been determined. We show here that G{alpha}z-null mice have increased insulin secretion after glucose challenge and a corresponding decrease in blood glucose levels. We also show a potentiation of insulin secretion in islets from G{alpha}z-null mice. The findings from these studies may have implications for development of novel therapeutic agents for impaired β-cell function and Type 2 diabetes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of the G{alpha}z Knock-out Mouse and Animal Care—G{alpha}z knock-out mice were originally derived by targeting the gene in the C57BL/6 mouse strain and back-crossing more than 10 times into the Balb/c strain (16). Mice were housed at five or fewer per cage, with a 12-h light/dark cycle and ad libitum access to chow and water. Animals were handled in accordance with the principles and guidelines established by the Duke University Animal Care and Use Committee. G{alpha}z-null and wild-type control mice were generated by heterozygote matings to produce littermate controls.

Glucose Tolerance Tests—Nine- to 11-week-old mice were fasted for 12 h, and an intraperitoneal glucose tolerance test (IPGTT) was performed as previously described (21). Because the glucose values measured during the assay in male mice of both genotypes were influenced more dramatically probably by the stress of the assay (data not shown), all experiments were conducted with female mice only. Plasma insulin was measured at 0 and 5 min during the IPGTT, and blood glucose values were determined at 5, 10, 15, 30, 60, and 120 min postinjection. Glucose readings were taken from tail blood using a BD LogicTM meter (BD Biosciences). The glucose readings were averaged within genotypes at each time point, giving the means ± S.E. Oral glucose tolerance tests (OGTTs) were conducted essentially as described for the IPGTTs, except that the glucose was administered by oral gavage after a 20-h fast, and the postglucose blood sample for insulin determination was taken at 10 min instead of 5 min.

Plasma Insulin ELISAs—During the IPGTTs, blood samples were collected into heparinized tubes and centrifuged at 3,000 x g for 15 min. The clarified plasma layer was transferred to a new tube, snap-frozen, and stored at –80 °C. On the day of the assay, the plasma samples were thawed, and 5 µl was used for each replicate of an insulin ELISA according to the manufacturer's protocol (rat insulin ELISA kit, Crystal Chem Inc., Downers Grove, IL). One to four replicates of each plasma sample were performed, depending on total sample volume; the insulin values for all of the replicates of a single sample were averaged, and the averages for all three experiments were used to calculate the means ± S.E.

Insulin Tolerance Tests—Insulin tolerance tests (ITTs) were performed essentially as previously described (21). Eleven-week-old female mice were fasted for 4 h and then injected intraperitoneally with 2 units/kg, body weight, insulin lispro, using a solution of 0.08 units/ml Humalog® and sterile insulin diluent (Lilly). Glucose readings were taken at 0, 30, 50, 80, and 120 min postinjection. The glucose readings were averaged within genotypes at each time point, giving the means ± S.E.

Pancreatic Islet Isolation and Insulin Secretion Assays—Islet isolation and insulin secretion assays were performed essentially as previously described (22). After overnight incubation to recover secretion capacity, the islets were picked into a dish containing Krebs-Ringer bicarbonate solution (KRB; 4.38 mM KCl, 1.2 mM MgSO4, 1.5 mM KH2PO4, 129 mM NaCl, 5 mM NaHCO3, 10 mM HEPES, 3.11 mM CaCl2, 0.25% bovine serum albumin, pH 7.4) with 2.8 mM D-glucose and incubated at 37 °C, 5% CO2 for 1 h. Twelve islets were picked into a tube containing 250 µl of KRB with the indicated concentrations of glucose for each replicate and incubated at 37 °C, 5% CO2 for 2 h. After the incubation, samples were taken for insulin assay (Coat-A-Count® Insulin; Diagnostic Products Corp., Los Angeles, CA). The insulin secretion values for each glucose concentration replicate (normalized to its DNA content) were averaged, and the averages from all three experiments were used to calculate the means ± S.E.

cAMP Production Assays—Islets were isolated and preincubated in KRB as described above for insulin secretion assays. For each replicate, 20 islets were picked into a tube containing 250 µl of KRB with 0.1 M of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (Sigma) and the indicated concentrations of glucose. When specified, 20 nM exendin-4 (Sigma) was added in addition to stimulatory glucose. After 15 min, the islets were pelleted by a centrifugal pulse, and the KRB was removed. Islets were immediately lysed for cAMP ELISA according to the manufacturer's protocol (cAMP BiotrakTM enzyme immunoassay system; GE Healthcare). Each sample replicate (five replicates per glucose concentration) was measured in triplicate, and the averaged replicates for each concentration were averaged from all three experiments to calculate the means ± S.E.

Immunoblot Analyses—Islets (100–150 of each genotype) were washed in PBS, pelleted at 500 x g for 5 min, resuspended in 1x Laemmli sample buffer, and subjected to 15% SDS-PAGE. Proteins were transferred to nitrocellulose using a tank transfer apparatus. The membrane was incubated with 1:1000 rabbit anti-G{alpha}z (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by 1:3000 goat anti-rabbit horseradish peroxidase conjugate (GE Healthcare) according to standard protocols. Horseradish peroxidase was detected using Western Lightning® chemiluminescence reagent (PerkinElmer Life Sciences), and the membrane was exposed to film. The membrane was stripped with RestoreTM Western blot stripping buffer (Pierce), washed, reblocked, and incubated with 1:1000 mouse anti-{alpha}-tubulin (Santa Cruz Biotechnology) followed by 1:3,000 goat anti-mouse horseradish peroxidase conjugate (GE Healthcare). Horseradish peroxidase was detected as above.

Immunohistochemical Assays—Tissues were dissected, fixed, and sectioned as previously described (13). For insulin immunostaining, the HistoMouse-MAX kit (Invitrogen) was used with prediluted guinea pig anti-insulin primary antibody (Invitrogen) incubated for 1 h at room temperature. The substrate used for colorimetric detection of the horseradish peroxidase-coupled secondary antibody was 3,3'-diaminobenzidine. Sections were lightly counterstained with hematoxylin. Islet area was determined using ImageJ software (National Institutes of Health, Bethesda, MD) on two independent sections from three mice of each genotype.

Statistical Analyses—Data were analyzed using GraphPad Prism version 4 (GraphPad Software Inc., San Diego, CA). An unpaired t test or two-way analysis of variance was used to determine the p value as appropriate. p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Islets from G{alpha}z-null and Wild-type Mice Are Morphologically Identical—Before beginning the task of analyzing the in vivo and in vitro insulin secretory capacity of G{alpha}z-null islets, it was crucial to assess whether any potential differences in secretion that were observed might be due to an overall change in the islet morphology between the two genotypes. To assess potential morphological differences between the pancreatic islets of G{alpha}z-null and wild-type control mice, pancreases from three mice of each genotype were dissected, fixed, paraffin-embedded, and stained for insulin (Fig. 1, A and B). Since insulin-containing β-cells comprise the majority of the mouse islet, measuring the β-cell area as a function of total pancreatic area analyzed is a reliable measure of the islet area. In fact, the measurements of islet area were nearly identical between the two genotypes (Fig. 1C), suggesting that there was no net increase in either β-cell number or size that could account for potential differences in insulin secretion from G{alpha}z-null islets. Finally, extracts were prepared from 100–150 islets isolated from G{alpha}z-null or wild-type control mice, subjected to SDS-PAGE, and transferred to nitrocellulose for immunoblotting. The membrane was probed for G{alpha}z and {alpha}-tubulin content. Islets from G{alpha}z-null mice were completely deficient in G{alpha}z, confirming the loss of G{alpha}z expression as expected (Fig. 1D).


Figure 1
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  		FIGURE 1.
Morphogenic analysis of pancreatic islets of 10–12-week-old wild-type and G{alpha}z-null mice. A and B, representative insulin immunostaining of a pancreas section from a wild-type and G{alpha}z-null mouse, respectively. Pancreases from three mice of each genotype were formalin-fixed and paraffin-embedded, and nonserial sections from each pancreas were stained with an anti-insulin antibody. C, quantification of islet area using insulin-stained sections from wild-type and G{alpha}z-null mice. Two independent pancreas sections from each of three mice of each genotype were used for determination of islet area as a function of pancreas area. D, analysis of G{alpha}z expression in islets isolated from G{alpha}z-null and wild-type mice. Extracts were prepared from 100–150 islets of each genotype, and G{alpha}z expression was assessed by immunoblot analysis. {alpha}-Tubulin levels were probed as control for protein levels. Results are representative of three independent experiments.

 
Gaz-null Mice Display Increased Glucose Clearance Correlating to Increased Plasma Insulin Levels—To determine the effect of knock-out of G{alpha}z on glucose homeostasis, IPGTTs were performed after a 12-h fast on G{alpha}z-null mice and wild-type controls. During the IPGTTs, G{alpha}z-null mice had blood glucose levels that were significantly different at 10, 15, 30, and 60 min after injection of glucose (Fig. 2A). Area under the curve (AUC) analyses on both the G{alpha}z-null and wild-type IPGTT profiles indicated that, although the G{alpha}z-null mice did display slightly lower fasting glucose levels (corresponding to t = 0), the glucose AUC, which is inversely proportional to the glucose clearance, was significantly lower in the G{alpha}z-null mice (449.0 ± 40.8 mM for 120 min, wild type; versus 264.2 ± 30.3 mM for 120 min, G{alpha}z-null; Table 1). If the differences in blood glucose had remained the same at each time point, identical glucose AUCs would have been observed. Furthermore, the time to peak glucose level was shorter on average in the G{alpha}z-null mice (mean of 17.3 ± 1.8 in wild-type versus 12.3 ± 1.0 in G{alpha}z-null mice; range of 10–30 min versus 5–15 min, respectively), and the peak glucose levels were dramatically lower (about 3 mM less) (Table 1).


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  		TABLE 1
Glucose clearance in G{alpha}z-null versus wild-type mice

The base line was set as the glucose reading at t = 0 min, and AUC analyses were performed on IPGTT data from G{alpha}z-null and wild-type mice. Data are mean ± S.E.

 


Figure 2
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  		FIGURE 2.
Glucose clearance and insulin secretion in G{alpha}z-null mice and wild-type controls. A, IPGTTs of 9–11-week-old wild-type control and G{alpha}z-null mice, performed after a 12-h fast. Blood glucose levels were measured at progressive time points after injection of 2 g/kg D-glucose. n = 14 (wild type) and 11 (G{alpha}z-null). B, plasma insulin levels in wild-type control and G{alpha}z-null mice before glucose injection and 5 min after glucose injection during the IPGTT. Approximately 50–100 µl of blood was massaged from the tail at t = 0 and 5 min after glucose load to generate plasma samples for analysis. n = 8 (wild type) and 6 (G{alpha}z-null). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
To determine whether increased insulin secretion after glucose load could account for the increased glucose clearance of the G{alpha}z-null mice, plasma insulin concentrations were measured from blood samples collected before and during the IPGTTs. G{alpha}z-null mice did not display significantly different fasting plasma insulin levels (corresponding to t = 0 of the IPGTT); however, they did have significantly increased plasma insulin levels in the plasma sample taken 5 min after the glucose load (Fig. 2C). This increased insulin concentration is consistent with the faster time to peak blood glucose levels and the lower peak blood glucose levels observed in the G{alpha}z-null mice (Fig. 2A and Table 1).


Figure 3
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  		FIGURE 3.
Insulin sensitivity of G{alpha}z-null and wild-type mice. ITTs were performed on 9–11-week-old G{alpha}z-null and wild-type control mice to assess whether the G{alpha}z-null mice had increased insulin sensitivity. Blood glucose readings were taken after a 4-h fast and were measured at progressive time points after injection of 2 units/kg insulin lispro. The values were normalized to percentage of base line. n = 6 (wild type) and 6 (G{alpha}z-null).

 
To determine whether decreased glucose levels might be also be due to increased insulin sensitivity, ITTs were performed on 9–11-week-old female G{alpha}z-null mice and wild-type controls. After a 4-h fast, the G{alpha}z-null mice had about 1 mM lower blood glucose levels than wild-type mice (data not shown), similar to the difference observed after a 12-h fast (Fig. 2A, t = 0). Unlike during the IPGTTs, however, this difference remained nearly the same at each time point after injection of 2 units/kg insulin lispro; thus, no differences were observed in the change in blood glucose concentrations over time when plotted as percentage of base line between the wild-type and G{alpha}z-null mice (Fig. 3). The results of this experiment suggest that the decreased blood glucose levels in the G{alpha}z-null mice are not due to increased insulin sensitivity.

Islets Isolated from Gaz-null Mice Display an Increased GSIS Response—To determine whether increased plasma insulin levels after glucose load in the G{alpha}z-null mice were due to increased pancreatic islet insulin secretion, islets were isolated from G{alpha}z-null mice and wild-type littermate controls. After an overnight recovery in islet medium, insulin secretion assays were performed at various concentrations of glucose. Although the insulin secretion from G{alpha}z-null and wild-type islets was nearly indistinguishable at 2.8 and 5.6 mM glucose, the G{alpha}z-null islets began to secrete more insulin at stimulatory concentrations of glucose (8.4–16.7 mM; Fig. 4A). Curve fit analyses of insulin secretion data from G{alpha}z-null and wild-type islets reflected these observations; the base-line insulin secretion and EC50 of glucose for insulin secretion of the two genotypes were almost indistinguishable, whereas the maximal response was significantly higher in G{alpha}z-null islets (Table 2). The results from these isolated islet secretion studies are consistent with both the improved glucose clearance and the increased plasma insulin levels observed in G{alpha}z-null mice during the IPGTTs (Fig. 2, A and B; Table 1).


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  		TABLE 2
Curve fit parameters from analyses of dose-response insulin secretion of islets from G{alpha}z-null and wild-type mice

Data are mean ± S.E.

 
To confirm that the differences observed were not due to increased islet number, size, or insulin content, DNA and insulin were extracted from each experimental replicate (12 islets/replicate), and their levels were measured. Consistent with the absence of an effect of loss of G{alpha}z expression on islet morphology (Fig. 1), there were no significant differences in the DNA or insulin content of the islets isolated from G{alpha}z-null and wild-type mice (Fig. 4, B and C). Together, these results provide compelling evidence that the differences observed between the GSIS response of G{alpha}z-null and wild-type islets is due to an effect on GSIS itself and not an artifact of a change in gross islet morphology or physiology.


Figure 4
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  		FIGURE 4.
G{alpha}z-null mice display an increased GSIS response. A, insulin secretion of islets isolated from 10–12-week-old G{alpha}z-null and wild-type control mice was elicited using increasing concentrations of glucose and measured using an insulin radioimmunoassay. G{alpha}z-null mice demonstrated significantly higher insulin secretion at stimulatory glucose concentrations. B, DNA content per islet for G{alpha}z-null and wild-type control mice. C, insulin content per islet for G{alpha}z-null and wild-type control mice. Insulin assays were performed and described under "Experimental Procedures," and values were normalized to DNA content (see B). Each experiment was performed on islets pooled from two mice of each genotype (six mice each total), with 3–6 replicates for each glucose concentration. n = 3. *, p < 0.05.

 


Figure 5
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  		FIGURE 5.
cAMP levels in G{alpha}z-null versus wild-type control mice. cAMP levels in islets isolated from 16-week-old G{alpha}z-null and wild-type control mice were determined as described under "Experimental Procedures." Islets were incubated in buffer containing 0.1 M 3-isobutyl-1-methylxanthine and 2.8 or 11.1 mM glucose for 15 min, after which the medium was removed, and islets were lysed for measurement of cAMP. Each experiment was performed on islets pooled from 2–3 mice of each genotype (eight wild-type and seven G{alpha}z-null mice total), with 3–5 replicates for each glucose concentration. n = 3. *, p < 0.05.

 
A Tonic Inhibition of Adenylyl Cyclase Is the Probable Mechanism for G{alpha}z Inhibition of GSIS—G{alpha}z is a member of the G{alpha}i subfamily of G{alpha} subunits, whose classical signaling mechanism is a decrease in cAMP production resulting from the inhibition of adenylyl cyclase. Furthermore, cAMP is a known modulator of multiple steps in the GSIS pathway. As such, an effect on cAMP levels seemed a logical mechanism for the action of G{alpha}z, and loss of this effect could underlie the GSIS phenotype observed in G{alpha}z-null mice. In order to determine whether an impact on modulation of cAMP levels could be the mechanism through which deletion of G{alpha}z impacts on insulin secretion, cAMP levels were determined in islets isolated from G{alpha}z-null and wild-type mice. An observed increase in cAMP production in G{alpha}z-null islets would explain the correspondingly higher magnitude of insulin secretion in these islets (Figs. 2B and 4A). In fact, G{alpha}z-null islets displayed significantly higher cAMP production in both low and stimulatory glucose concentrations (Fig. 5). These data suggest that genetic deletion of G{alpha}z relieves a tonic inhibition of adenylyl cyclase, independent of stimulation with glucose or other agonists. Since cAMP does not itself stimulate insulin secretion but rather only potentiates GSIS, this finding also explains why increased insulin secretion is only observed in G{alpha}z-null mice and islets under conditions of stimulatory glucose concentrations (Figs. 2B and 4A).

We also performed an OGTT analysis in order to examine the role of G{alpha}z in maintaining glucose homeostasis in a more typical physiologic situation. Incretins, such as GLP-1 and GIP, which activate Gs-coupled receptors in the β-cell and might oppose the actions of G{alpha}z, are released from the gastric mucosa upon gastrointestinal absorption of glucose. Similar to that observed with IPGTT, during the OGTT, G{alpha}z-null mice had blood glucose levels that were significantly different at 10, 20, and 30 min after injection of glucose (Fig. 6A). These glucose levels during the OGTT were almost indistinguishable from those observed during the IPGTT, as can be seen by overlay of the two sets of curves (Fig. 6B). The peak glucose levels occurred ~10 min later and 5 min later in the OGTT as compared with the IPGTT for wild-type and G{alpha}z-null mice, respectively, which is consistent with glucose having to be absorbed through the gastrointestinal tract. Besides the lower starting glucose levels due to a longer fast in the OGTT, there were no significant differences between the data obtained during IPGTT and during OGTT within a genotype, except at 10 min for the G{alpha}z-null mice (p < 0.05), a reflection of the faster time to peak glucose. These results suggest that if G{alpha}z plays a role in regulating incretin-potentiated insulin secretion, it is likely to be modest. This is supported by a similar increase in plasma insulin levels within each genotype during the OGTT as compared with the IPGTT (Fig. 6C). Furthermore, the stable GLP-1 analog exendin-4 elicited a similar augmentation of cAMP levels in islets isolated from wild-type and G{alpha}z-null mice (data not shown).


Figure 6
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  		FIGURE 6.
The effect of incretins on glucose clearance and insulin secretion in G{alpha}z-null mice and wild-type controls. A, OGTTs of G{alpha}z-null mice and wild-type controls. Blood glucose levels were measured at progressive time points after administration of 2 g/kg D-glucose by OG to 20-week-old wild-type control and G{alpha}z-null mice after a 20-h fast. n = 8 (wild type) and 8 (G{alpha}z-null). B, blood glucose levels observed during the OGTT as compared with the IPGTT, as represented by overlay of the mean blood glucose levels from Fig. 2A (IPGTT) and A (OGTT). C, the increase in insulin observed in the plasma of G{alpha}z-null mice and wild-type controls with oral administration of glucose (OGTT) as compared with intraperitoneal injection (IPGTT). *, p < 0.05; ***, p < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It is currently appreciated that both β-cell dysfunction and insulin resistance are early and necessary events in the development of Type 2 diabetes (23). The present study focused on the role of G{alpha}z in regulating insulin secretion in a mouse model system. G{alpha}z has been previously shown to modulate an endogenous GSIS inhibitory pathway in an insulinoma cell line (13); therefore, we hypothesized that G{alpha}z-null mice would display increased GSIS and correspondingly decreased blood glucose levels as compared with wild-type control mice. These phenotypes were indeed observed and point to G{alpha}z as a critical component of the cellular mechanisms responsible for β-cell homeostasis.

G{alpha}z is the least understood member of the G{alpha}i subfamily of heterotrimeric G proteins. Previous rodent models have been used to study the potential involvement of other G{alpha}i subfamily members in insulin secretion. Injection of rats with PTX (originally named "islet-activating protein") or incubation of isolated rat islets with PTX augmented the effects of various secretagogues and blocked the ability of G{alpha}i-coupled receptor agonists, such as somatostatin and epinephrine, to inhibit GSIS (24). Interestingly, however, no insulin secretion phenotype has been reported in mouse lines deficient in the PTX-sensitive G{alpha}i subfamily members, which include G{alpha}o, G{alpha}i1, G{alpha}i2, and G{alpha}i3. A likely explanation is that there exists some level of redundancy of these G{alpha} isoforms in islet cell biology, as has been shown in other systems (9). G{alpha}o knock-out mice do display changes in the nervous system structure and aberrant Ca2+ channel signaling in the heart but are normoglycemic (25). Interestingly, loss of G{alpha}i2 expression does not impact insulin secretion but instead leads to insulin resistance (26), whereas expression of constitutively active G{alpha}i2 in mouse liver and adipose tissue leads to enhanced glucose tolerance (27). This occurs because G{alpha}i2 signals to GLUT4 and regulates glucose uptake into peripheral tissues (28).

In contrast to G{alpha}i2-null mice (28), there is no effect of loss of G{alpha}z on insulin sensitivity (Fig. 3). This is not unexpected, since G{alpha}z is not expressed in the liver, adipose tissue, or skeletal muscle (11, 29, 30). Conversely, we did find that G{alpha}z-null mice do have lower blood glucose levels and increased glucose clearance, as demonstrated by glucose tolerance tests (Fig. 2A). These effects appear to be due to a direct impact on insulin secretion, since G{alpha}z-null mice have higher plasma insulin levels after glucose load than wild-type mice (Fig. 2B), and islets isolated from G{alpha}z-null mice secrete more insulin at stimulatory glucose concentrations (Fig. 4). Hence, although under conditions of ectopic expression, constitutively active forms of all G{alpha}i subfamily members can inhibit GSIS in vitro, only loss of G{alpha}z impacts GSIS in vivo, possibly because of the aforementioned abilities of the others to compensate for each other. This is of particular relevance in regard to the inhibition of adenylyl cyclase; G{alpha}z displays specificity for adenylyl cyclases I and V, whereas all three G{alpha}i isoforms block adenylyl cyclases V and VI (31). The possibility remains, however, that one or more of the PTX-sensitive G{alpha}i proteins is required for inhibition of GSIS through a specific G protein-coupled receptor agonist (e.g. somatostatin, epinephrine, etc.).

A significant increase in GSIS was observed in isolated G{alpha}z-null islets, although no G protein-coupled receptors had been specifically engaged in the tissue treatments. This is not entirely unexpected, since the slow GTP hydrolysis rate of G{alpha}z leads to a significant amount of active, GTP-liganded protein even under basal conditions (11), in contrast to the rapid hydrolysis rates of the other G{alpha}i subfamily members. In support of this hypothesis, cAMP production was significantly increased in G{alpha}z-null islets in the absence of exogenous stimuli (Fig. 5), suggesting that G{alpha}z tonically inhibits adenylyl cyclase in wild-type islets and that in G{alpha}z-null islets, this inhibitory constraint has been removed. cAMP augments insulin secretion dependent on the glucose concentration being above a threshold of ~5.6–7 mM (2, 32, 33). This is consistent with our observations that G{alpha}z-null islets do not secrete more insulin at 2.8 or 5.6 mM glucose and only begin to show increased insulin secretion at higher levels of glucose. Furthermore, increases in cAMP levels at suprathreshold glucose concentrations have been shown to correlate directly with augmentation of insulin secretion (32). At 11.1 mM glucose, the cAMP production in G{alpha}z-null islets is ~1.9-fold that of wild-type islets (Fig. 5), which correlates well with a 1.5-fold increase in insulin secretion at this glucose concentration (Fig. 4A).

cAMP has been shown to augment insulin secretion by numerous mechanisms, including increasing glucokinase expression and/or activity, augmenting KATP channel closure, augmenting voltage-dependent calcium channel opening, enhancing calcium-induced calcium release, and directly enhancing the exocytosis process itself (4, 34). In addition, G{alpha}i proteins have been implicated in regulation of the KATP channel and voltage-dependent calcium channels, leading to hyperpolarization of the membrane and inhibition the influx of extracellular Ca2+ (4, 5). Furthermore, G{alpha}i proteins have been localized to various degrees on secretory granules from pancreatic {alpha}- and β-cells (35, 36) and to the trans-Golgi network in the exocrine pancreas (37). G{alpha}i subfamily members have also been implicated in directly inhibiting exocytosis from β-cells (4), which, together with their subcellular localization, indicates a role for G{alpha}i proteins in the regulation of a distal step in the stimulated secretion pathway. Taken together, the regulation of cAMP levels seems a likely explanation for the effects of G{alpha}i proteins on insulin secretion, in particular those of the subfamily member that is the subject of the current study, G{alpha}z.

Agents that impact cAMP levels are being tested and/or currently being used for the treatment of β-cell dysfunction in Type 2 diabetes. The incretin GLP-1, which activates Gs-coupled receptors and thereby increases cAMP levels, is rapidly degraded, but the stable GLP-1 analogs exendin-4 and liraglutide have been shown to be clinically effective Type 2 diabetes treatments (34, 38). In addition, inhibition of the enzyme that degrades GLP-1 and GIP, dipeptidyl peptidase-IV, also leads to improved β-cell function and insulin sensitivity in Type 2 diabetes patients (34, 38). Inhibition of PDE3B, the enzyme responsible for degradation of cAMP in the β-cell, leads to increased insulin secretion, although its expression in the liver and adipose tissues prevents inhibitors from decreasing systemic blood glucose levels (34).

G{alpha}z-null mice do not appear to have further augmented insulin secretion during the OGTT, suggesting that G{alpha}z does not play a major role in the regulation of incretin-potentiated insulin secretion (Fig. 6, A–C). This is not entirely unexpected, since GLP-1-stimulated Gs appears to couple to ACVIII (39), which is not inhibited by G{alpha}z. Since G{alpha}z and GLP-1 probably signal through different upstream pathways, it may be possible that G{alpha}z inhibition can be used in combination with a GLP-1 analogue in vivo. Overall, the significant effect of knock-out of G{alpha}z on improving GSIS and glucose clearance, without significant negative systemic effects, makes G{alpha}z worthy of further study, possibly as a potential target for impaired β-cell function therapeutics toward Type 2 diabetes.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK67799 (to M. E. K.), DK42583 (to C. B. N.), and GM55717 (to P. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Duke University Medical Center, Box 3813, Durham, NC 27710-3813. Tel.: 919-613-8613; Fax: 919-613-8642; E-mail: casey006{at}mc.duke.edu.

2 The abbreviations used are: KATP channel, ATP-sensitive K+ channel; AUC, area under the curve; GSIS, glucose-stimulated insulin secretion; IPGTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test; KRB, Krebs-Ringer bicarbonate; OGTT, oral glucose tolerance test; PTX, pertussis toxin; ELISA, enzyme-linked immunosorbent assay. Back


   ACKNOWLEDGMENTS
 
We thank the members of the Casey Laboratory, especially Patrick Kelly and Rainbo Hultman; members of the Newgard laboratory, especially Dr. Hans Hohmeier, Dr. Danhong Lu, and Helena Winfield; and Dr. Andrew Nixon and Mark Starr for technical advice and productive discussions. We also thank Missy Infante for general laboratory assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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