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. Author manuscript; available in PMC: 2015 Oct 8.
Published in final edited form as: Cell Rep. 2015 Sep 24;13(1):157–167. doi: 10.1016/j.celrep.2015.08.072

Adenylosuccinate is an insulin secretagogue derived from glucose-induced purine metabolism

Jessica R Gooding 1,*, Mette V Jensen 1,*, Xiaoqing Dai 2, Brett R Wenner 1, Danhong Lu 1, Ramamani Arumugam 1, Mourad Ferdaoussi 2, Patrick E MacDonald 2,#, Christopher B Newgard 1,#
PMCID: PMC4598307  NIHMSID: NIHMS720189  PMID: 26411681

Abstract

Pancreatic islet failure, involving loss of glucose-stimulated insulin secretion (GSIS) from islet β-cells, heralds the onset of type 2 diabetes (T2D). To search for mediators of GSIS, we performed metabolomics profiling of the insulinoma cell line 832/13, and uncovered significant glucose-induced changes in purine pathway intermediates, including a decrease in inosine monophosphate (IMP) and an increase in adenylosuccinate (S-AMP), suggesting a regulatory role for the enzyme that links the two metabolites, adenylosuccinate synthase (ADSS). Inhibition of ADSS or a more proximal enzyme in the S-AMP biosynthesis pathway, adenylosuccinate lyase, lowers S-AMP levels and impairs GSIS. Addition of S-AMP to the interior of patch-clamped human β-cells amplifies exocytosis, an effect dependent upon expression of sentrin/SUMO-specific protease 1 (SENP1). S-AMP also overcomes the defect in glucose-induced exocytosis in β–cells from a human donor with T2D. S-AMP is thus an insulin secretagogue capable of reversing β-cell dysfunction in T2D.

Keywords: Pancreatic islet β-cells, insulin secretion, diabetes, metabolomics, purine metabolism

Graphical abstract

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Introduction

Both type 1 and type 2 diabetes are diseases of insulin deficiency, albeit with different etiologies. Type 1 diabetes (T1D) occurs when pancreatic islet β-cells are destroyed by the host immune system. Type 2 diabetes (T2D) involves loss of key β-cell functions such as glucose-stimulated insulin secretion (GSIS), coupled with gradual depletion of β-cell mass by non-autoimmune mechanisms (Muoio and Newgard, 2008). A clearer understanding of β-cell function is critical for ultimate success in creation of “surrogate” cells for insulin replacement therapy in T1D, and for development of better drugs for enhancing β-cell function in T2D. Here we have focused on metabolic coupling mechanisms in the β-cell, and how they are altered when islets fail in diabetes.

For over 30 years, one particular model of glucose stimulus/secretion coupling has gained wide acceptance. In this construct, glucose-driven increases in ATP:ADP ratio inhibit ATP-sensitive K+ channels (KATP channels), resulting in plasma membrane depolarization, activation of voltage-gated Ca2+ channels, and influx of extracellular Ca2+, which serves to activate granule exocytosis (Bryan et al., 2005; Henquin et al., 2003; Newgard and Matschinsky, 2001). The KATP channel-dependent mechanism appears to be particularly important in the first, acute phase of insulin release. In the second and sustained phase of insulin secretion, closure of KATP channels is required as an initiating event, but does not fully explain the amplification of insulin secretion after initial glucose stimulation (Gembal et al., 1992; Sato et al., 1992; Straub and Sharp, 2002). In fact, amplifying signals contribute as much as 60% of total GSIS (Henquin et al., 2003), but mechanisms driving this component of the secretory response are still incompletely understood. Growing evidence implicates non-oxidative, anaplerotic metabolism of glucose as an important regulator of GSIS. In particular, the full GSIS response requires pyruvate carboxylase activity, efficient export of citrate and/or isocitrate from the mitochondria to the cytosol, and engagement of isocitrate with the cytosolic, NADP-dependent isocitrate dehydrogenase (IDH1 or ICDc) (Gooding et al., 2015; Ferdaoussi, et al. 2015; Jensen et al., 2006; Jensen et al., 2008; Joseph et al., 2006; Lu et al., 2002; Ronnebaum et al., 2006).

It has been recognized for some time that glucose stimulation of β-cells causes broad-scale changes in an array of nucleotides and their biosynthetic precursors (Detimary et al., 1996). In the current study, we have applied a targeted LC-MS/MS-based method to probe this response in more detail. We find that exposure of the robustly glucose responsive INS-1-derived insulinoma cell line 832/13 to stimulatory glucose concentrations results in a striking increase in adenylosuccinate (S-AMP) levels. Furthermore, molecular or pharmacological manipulation of S-AMP levels results in alterations in insulin secretion that are consistent with a regulatory role for this metabolite. Using patch clamped human β-cells and insulinoma cells, we are able to add S-AMP to the cell interior via the patch pipette. S-AMP enhances insulin granule exocytosis in 832/13 cells and non-diabetic human β-cells, and when applied to β-cells from a human donor with T2D, S-AMP completely reverses the defect in the glucose-dependent amplification of exocytosis. We also present evidence that S-AMP-mediated facilitation of granule exocytosis requires sentrin/SUMO-specific protease 1 (SENP1). These studies identify S-AMP as a metabolic intermediate that couples glucose metabolism to insulin secretion and that is able to reverse β-cell dysfunction in T2D.

Results

Glucose-induced changes in purine pathway intermediates and nucleotides

We applied a targeted LC-MS/MS assay method for measuring glucose-induced changes in 37 nucleotides and purine pathway intermediates in islet β-cells. Our goal was to determine if any regulated metabolites could be ascribed a role in GSIS. Stimulation of the robustly glucose responsive rat insulinoma cell line 832/13 with 12 mM glucose for 2 h caused an increase in PRPP (1.9-fold, p=0.038), but no detectable changes in other metabolites of early de novo purine synthesis (acadesine, ZMP) relative to cells treated with basal glucose (2.5 mM glucose) (Figure 1). Glucose stimulation significantly changed the concentrations of intermediates later in the pathway, including inosine monophosphate (IMP) (77% decrease, p = 1.3x10−8), S-AMP (3.4-fold increase, p = 4.0 x10−5), hypoxanthine (73% decrease, p = 0.024), and ATP (18% decrease, p = 0.013). Other purine (AMP, ADP, XMP, GMP, GDP, GTP) and pyrimidine (CMP, CDP, CTP, UMP, UDP, UTP) nucleotides did not change significantly in response to stimulatory glucose. Oxidized pyridines tended to decrease in concentration (NAD 16%; NADP 27%), whereas their reduced forms increased significantly (NADH 2.4-fold, p = 0.009; NADPH 1.8-fold, p = 0.05) in response to stimulatory glucose. Nucleotide conjugates GDP-mannose (3.7-fold increase, p = 4.1x10−6) and 5′-methylthioadenosine (MTA) (35% decrease, p = 0.046) also changed dynamically with glucose.

Figure 1. Targeted nucleotide profiling of 37 metabolites in 832/13 cells.

Figure 1

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832/13 cells were harvested after 2 h exposure to either 2.5 mM (grey bars) or 12 mM (black bars) glucose and analyzed by LC-MS/MS for nucleotide content. Data are expressed as average ± SE of 5 independent experiments performed in triplicate. + p < 0.05 relative to 2.5 mM glucose; @ p < 0.01 relative to 2.5 mM glucose as calculated by Wilcoxon rank sum test. Full compound names and acquisition parameters for LC-MS/MS analyses are shown in Table S1.

MPA inhibition of GSIS from 832/13 cells is rescued by provision of guanine

Inosine monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) catalyzes the NAD-dependent conversion of IMP to XMP, and is considered to be the rate-limiting step in the de novo biosynthesis of guanine nucleotides. Two IMPDH isoforms are expressed in mammalian cells, encoded by distinct genes that share 84% amino acid identity and with similar catalytic activity (Carr et al., 1993; Hager et al., 1995; Natsumeda et al., 1990). qRT-PCR analysis of IMPDH mRNA levels in rat islets and 832/13 cells reveals that IMPDH2 is the more abundant isoform in both settings, being 6.7 ± 1.2-fold more abundant than IMPDH1 in rat islets (n = 4 independent islet samples, each measured in triplicate) and 9.7 ± 2.3-fold higher in 832/13 cells (n = 7 independent samples, each measured in duplicate). To test the role of the guanine arm of purine biosynthesis in control of GSIS, we applied mycophenolic acid (MPA), a selective, reversible and noncompetitive inhibitor of both isoforms of IMPDH (Kitchin et al., 1997), to 832/13 cells. MPA inhibited GSIS in a dose dependent manner (Figure 2A). Co-culture with 100 μM guanine fully reversed the strong inhibitory effect of 2 μg/mL MPA on GSIS (Figure 2B), whereas 250 μM adenine caused only a minimal improvement.

Figure 2. Guanine, but not adenine, rescues the inhibitory effects of mycophenolic acid on GSIS and purine metabolites.

Figure 2

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Insulin secretion from 832/13 cells measured (A) in the presence or absence of mycophenolic acid (MPA) addition ON, during preincubation and GSIS; (B) with or without adenine (250 μM) or guanine (100 μM) addition in combination with MPA. Data are expressed as average ± SE of 3 independent experiments performed in triplicate. * p < 0.05 relative to no MPA; # p < 0.05 relative to + MPA. (C) 832/13 cells were treated with MPA (2 μg/mL) in the presence of adenine (A) or guanine (G), or neither (−) ON, during preincubation and glucose stimulation (12 mM). Purine concentrations were quantified by LC-MS/MS. Data are expressed as average ± SE in nmol metabolite/mg protein of 3 independent experiments performed in triplicate. See Figure S1 for quantification of additional metabolites.

Effects of MPA on nucleotide levels

To further understand the inhibitory effect of MPA on GSIS and the discrete restorative effects of guanine versus adenine addition, we explored the effects of these agents on nucleotide pools in 832/13 cells exposed to 12 mM glucose (Figure 2C and Figure S1). As expected, treatment with 2 μg/mL MPA caused increases in upstream purine pathway intermediates such as PPRP, acadesine, ZMP and IMP (all p ≤ 0.05). Also as expected, metabolites in the guanine nucleotide pathway, including guanosine, GMP, GDP and GTP decreased in response to inhibition of IMPDH with MPA (all p < 0.05). Surprisingly, MPA-treated cells also had lower levels of adenine metabolites, including S-AMP, AMP, ADP and ATP (all p < 0.05), demonstrating that a block in the guanine metabolic pathway impacts production of intermediates of adenine metabolism.

Consistent with their divergent effects on rescue of GSIS in MPA-treated cells, guanine and adenine addition had discrete effects on purine and nucleotide metabolites when added in the presence of MPA. Under these conditions, both guanine and adenine addition lowered the levels of the precursor metabolites PRPP, acadesine, and ZMP, back to levels observed in control cells or lower. In the presence of MPA + adenine, guanosine, GMP, GDP, GTP, S-AMP, AMP, ADP, and ATP were all lower than in control cells (no MPA, no nucleobase addition), with p < 0.01 (Figure 2C). In contrast, all of the foregoing analytes were equal to or greater than control in presence of MPA + guanine. In other words, guanine, but not adenine was able to reverse the effects of MPA to lower these metabolites, in concert with guanine’s unique ability to rescue GSIS.

Pharmacologic inhibition and siRNA-mediated suppression of ADSS impairs GSIS

One potential explanation for the surprising effect of guanine to rescue adenine nucleotide pools and GSIS is that GTP is a cofactor for adenylosuccinate synthase (ADSS; EC 6.3.4.4.), which catalyzes conversion of IMP and aspartic acid to S-AMP. In mammals, two isozymes are present. ADSS1 is highly abundant in cardiac and skeletal muscle tissues and is thought to play a role in energy metabolism via the purine nucleotide cycle. ADSS2 is present at low levels in most tissues and functions in de novo AMP biosynthesis (Stayton et al., 1983). Interestingly, ADSS2 is more abundant than ADSS1 in rat islets and 832/13 cells, with 26 ±5-fold more ADSS2 than ADSS1 mRNA in rat islets, and 15 ±1-fold more in 832/13 cells. To test the role of ADSS in GSIS, we treated 832/13 cells with siRNA duplexes specific for ADSS1 and ADSS2, resulting in 71±3 % and 84±3% decreases in mRNA expression, respectively (Figure 3A). These manipulations had no effect on basal insulin output (Figure 3B) but knockdown of ADSS2 caused 38% and 29% decreases in insulin secretion at 12 mM glucose compared to mock transfected (ND) cells or cells treated with a control siRNA duplex (siCont), respectively (p < 0.01 for both controls). In cells treated with siADSS1, insulin secretion at 12 mM glucose was reduced relative to ND (p < 0.01) but did not reach significance relative to siCont (p = 0.094). Simultaneous knockdown of ADSS1 and ADSS2 (by 63±8% and 78±4% compared to ND, respectively) suppressed GSIS to the same extent as knockdown of ADSS2 alone. These findings were confirmed with a second set of siRNA duplexes targeting ADSS1 and ADSS2 (data not shown).

Figure 3. Pharmacologic or molecular inhibition of ADSS expression impairs GSIS.

Figure 3

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832/13 cells were mock transfected (ND), or transfected with either a control siRNA (siCont) or siRNA duplexes targeting ADSS1 or 2, and kept in culture for 72h prior to (A) qRT-PCR analyses or (B) insulin secretion analyses. * p < 0.01 relative to ND and siCont; # p < 0.01 relative to ND. Insulin secretion from primary rat islets (C) or 832/13 cells (D) incubated with various doses of alanosine (alano) for 20 h in the presence (+A) or absence (−) of adenine. + p < 0.05 relative to no alanosine control; @ p < 0.01 relative to no alanosine control. Data in A-D represent the average ± SE of 3–4 independent experiments performed in triplicate. (E) 832/13 cells were treated with alanosine (20 μg/mL) in the presence (A) or absence (−) of adenine (100 μM) ON, during preincubation and glucose stimulation (12mM), as indicated. Purine concentrations were quantified by LC-MS/MS. Data are expressed as average ± SE in nmol metabolite/mg protein for 2 independent experiments performed in triplicate. See Figure S2 for quantification of additional metabolites.

When added to cells, alanosine is converted by SAICAR synthase to 5-amino-4-imidazole carboxylic acid, a powerful and selective inhibitor of ADSS (Ki ~0.3 μM) (Jayaram et al., 1979). We therefore determined if the inhibitory effects of siADSS duplexes on GSIS were recapitulated by alanosine exposure. Treatment of 832/13 cells with 20 μg/mL alanosine significantly reduced insulin secretion at 12 mM glucose (p < 0.001) without affecting basal insulin secretion (p = 0.7) (Figure 3D). Alanosine also had a dose-dependent effect to inhibit GSIS in primary rat islets, with a 15% decrease at 20 μg/ml, and a 40% decrease at 100 μg/mL at stimulatory glucose (16.7 mM) (Figure 3C). Provision of adenine completely reversed the inhibitory effect of 20 μg/mL alanosine on GSIS in 832/13 cells (p < 0.01).

Effects of alanosine on nucleotide levels

To explore the mechanism of alanosine inhibition on GSIS, we performed nucleotide profiling in 832/13 cells treated overnight and during the insulin secretion assay with 20 μg/mL alanosine (Figure 3E and Figure S2). As anticipated, inhibition of ADSS with alanosine caused increases in upstream metabolites such as PRPP, ZMP, acadesine, and IMP (all p < 0.05). ADSS inhibition also decreased the concentration of its enzyme product, S-AMP, by 52% (p = 0.0022), as well as the pools of downstream products adenosine, AMP, ADP, and ATP (all p < 0.01). Some products of the guanine pathway tended to increase in alanosine-treated cells (XMP 4.3-fold p = 0.26; GMP 1.9-fold p = 0.0022), indicating that ADSS inhibition may cause some diversion of IMP through the IMPDH reaction.

The addition of 100 μM adenine to alanosine-treated cells lowered the concentrations of upstream metabolites (PRPP, ZMP, acadesine) back to or below the level of controls, and raised downstream metabolite concentrations (S-AMP, adenosine, AMP, ADP, ATP) back to control levels.

S-AMP is rapidly induced by glucose and augments depolarization-induced exocytosis in 832/13 cells

In evaluating our nucleotide profiling data, we were particularly intrigued with the 3.4-fold increase in S-AMP in response to stimulatory glucose, because this increase was in the opposite direction of metabolites directly upstream (IMP 77% decrease, aspartate 59% decrease) in its metabolic pathway, and in the same direction as its degradation product fumarate (Ferdaoussi et al., 2015; Jensen et al., 2006). These findings implicate S-AMP as a candidate stimulus-secretion coupling factor. Also supporting this idea, GSIS is impaired in response to molecular or pharmacologic suppression of ADSS, the enzyme that generates S-AMP (Figure 3). Moreover, S-AMP levels were rescued by provision of guanine in MPA-treated cells, and adenine in alanosine-treated cells, in concert with rescue of GSIS in both cases (Figures 2 and 3).

As a first step in evaluating S-AMP as a potential coupling factor for insulin secretion, we sought better understanding of the time course of its response to glucose, since all of the nucleotide profiling shown in Figures 1–3 was performed after 2 hours of exposure to basal or stimulatory glucose. If S-AMP is relevant as an insulin secretagogue, its levels should increase at time points at which insulin secretion is initiated, earlier than 2 hours. We therefore performed measurements of GSIS and S-AMP levels at 30 minutes and 120 minutes in 832/13 cells incubated at basal (2.5 mM) and stimulatory (12 mM) glucose. We observed robust stimulation of GSIS (Figure 4A), in concert with strong and significant increases in S-AMP at both time points (Figure 4B). These data show that S-AMP levels are responsive to glucose over a timeframe that is consistent with a role for this metabolite in regulation of GSIS.

Figure 4. Time course of glucose-stimulated S-AMP changes and effect of S-AMP to augment the exocytotic response of 832/13 cells to membrane depolarization.

Figure 4

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GSIS (A) and changes in S-AMP levels (B) in 832/13 cells incubated at basal (2.5 mM) or stimulatory (12 mM) glucose for 30 or 120 minutes. (n = 2 independent experiments, each assayed in triplicate and expressed as average ± SE; * p < 0.001 compared to basal glucose). Cumulative exocytosis over 10 depolarizations (C) and the total exocytotic response (D) in 832/13 cells following acute glucose (10 mM) exposure, or upon infusion of S-AMP (10 μM), AMP (10 μM) or fumarate (10 μM) (n=22, 24, 26, 19, and 20 cells, respectively). Data are expressed as average ± SE; * p < 0.01 and # p < 0.05 compared to basal glucose. @ p < 0.05 compared to 10 mM glucose. ns: not significant.

S-AMP is not expected to be membrane permeant, so in order to test its potential role as an insulin secretagogue, we performed patch clamp studies on 832/13 cells, using membrane capacitance as a measure of insulin granule exocytosis (Kanno et al., 2004). With this system, compounds such as S-AMP can be readily introduced to the interior of the cell via the patch pipette. In patch-clamped 832/13 cells, we observed a 2.9-fold amplification of exocytosis in cells pre-treated with 10 mM compared with 1 mM glucose (Figures 4C, 4D; n=22–24 cells, p < 0.001). Remarkably, when tested in the presence of 1 mM glucose, 10 μM S-AMP was an even more potent facilitator of exocytosis than 10 mM glucose, resulting in a 4.1-fold increase in the exocytotic response relative to 1 mM glucose alone (Figures 4C, 4D; n = 26 cells, p < 0.001). SAMP is further metabolized to yield AMP and fumarate by adenylosuccinate lyase (ADSL). Consistent with a specific effect of S-AMP on exocytosis, 10 μM AMP had no significant effect on exocytosis (n = 19 cells, p = 0.9) whereas 10 μM fumarate increased exocytosis 1.8-fold over basal levels (n = 19 cells, p < 0.05), a significant but clearly lesser effect compared to glucose or S-AMP (p < 0.01; Figures 4C,D).

Suppression of adenylosuccinate lyase expression lowers S-AMP and impairs GSIS

Adenylosuccinate lyase (ADSL) plays two important roles in nucleotide metabolism, serving first to catalyze a proximal step in the purine biosynthetic pathway, the conversion of SAICAR to AICAR (ZMP), and then also acting to degrade S-AMP to AMP and fumarate more distally. Since knockdown of ADSL could either slow biosynthesis of S-AMP and/or inhibit its degradation, with no certain net outcome, we decided to test the effect of siRNA-mediated suppression of the enzyme directly. Accordingly, we used an siRNA duplex to suppress ADSL mRNA levels by approximately 50% in 832/13 cells, resulting in clear impairment of GSIS (Figure 5A). Consistent with a dominant role of ADSL in proximal steps of the purine pathway, ADSL knockdown also caused a clear decrease in S-AMP levels at stimulatory glucose levels (Figure 5B). Unlike inhibition of IMPDH or ADSS, ADSL knockdown did not result in decreases in other adenine nucleotides (AMP, ADP, ATP; Figure 5B) or GTP (data not shown), and in fact caused small increases in AMP, ADP and ATP levels (Figure 5B). These data are consistent with a regulatory role of S-AMP in GSIS that is independent of other nucleotides.

Figure 5. Effect of ADSL knockdown on GSIS, S-AMP and adenine nucleotide levels.

Figure 5

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832/13 cells were treated with an siRNA duplex specific for ADSL (siAdsl), resulting in a ~50% decrease in ADSL mRNA levels compared to cells treated with a nontargeting siRNA (siCont) or untreated cells (ND). (A) GSIS. (B) Changes in S-AMP, AMP, ADP, and ATP in ND, siCont, and SiAdsl cells. For all panels, n = 2 independent experiments, each assayed in triplicate and expressed as average ± SE. * p < 0.05 compared to siCont for the same glucose level.

Effects of S-AMP on exocytosis in β-cells from non-diabetic human donors

Next we investigated if the effect of S-AMP on exocytosis in 832/13 cells extrapolates to primary human β-cells. In dispersed and reaggregated islet cells from normal (non-diabetic) donors, insulin secretion was increased by 3.3-fold in response to 16.7 mM glucose relative to 2.5 mM glucose (Figure 6A). Patch clamping of β-cells from the same donors following an acute (~10–15 minutes) increase of glucose from 1 to 10 mM revealed a 2.2-fold increase in depolarization-induced exocytosis (n=15–17 cells from 3 donors; Figure 6B). Intracellular dialysis with 10 μM S-AMP amplified the exocytotic response to membrane depolarization-induced Ca2+ entry by 3-fold compared to 1 mM glucose alone (Figure 6B; n=20 cells from 3 donors, p < 0.01). Taken together, our studies identify S-AMP as a glucose-regulated intermediate of nucleotide metabolism that enhances insulin granule exocytosis stimulated by membrane depolarization in human β-cells and rodent insulinoma cells.

Figure 6. S-AMP increases the exocytotic response of human β-cells to membrane depolarization in a SENP1-dependent manner and rescues impaired exocytosis in human T2D ββ β-cells.

Figure 6

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(A) GSIS from islet cell aggregates from non-diabetic human donors. (B) The total exocytotic response of non-diabetic human β-cells following acute glucose (10 mM) stimulation, or upon infusion of 10 μM S-AMP (n=15, 17, and 20 cells, from 3 donors). (C) The total exocytotic response of non-diabetic human β-cells transfected with control duplex (siScrambled; n=14, 17, and 25 cells, from 3 donors), or a duplex targeting SENP1 (siSenp1, n=13, 16, and 20 cells, from 3 donors) following acute glucose (10 mM) stimulation, or after administration of 10 μM S-AMP. (D) GSIS from islet cell aggregates from a human donor with T2D. (E) The total exocytotic response of human β-cells from a T2D donor following acute glucose (10 mM) stimulation, or upon infusion of 10 μM S-AMP (n=9, 8, and 8 cells, from 1 donor). Data are expressed as average ± SE; * p < 0.01 compared with the basal glucose condition. # p < 0.01 compared to siScrambled/10mM glucose. @ p < 0.05 compared to siScrambled/S-AMP.

The amplifying effect of S-AMP on exocytosis is dependent on SENP1

Cellular proteins can be modified by SUMOylation, the covalent attachment of small ubiquitin-like modifier (SUMO) peptides. These processes are reversed by the deSUMOylation enzymes known as sentrin/SUMO-specific proteases (SENPs) (Yeh, 2009). In β-cells, insulin granule exocytosis is impaired by SUMOylation, and nutrient stimulation activates SENP1 to remove these modifications and activate secretion (Dai et al., 2011; Vergari et al., 2012; Ferdaoussi, et al, 2015). To determine if the amplifying effect of S-AMP on exocytosis is dependent on SENP1 activity, we used siRNA to suppress SENP1 expression in human β-cells from non-diabetic donors, leading to a 62% decrease in SENP1 mRNA levels relative to siCont-treated cells (n=6 and 6 cells, respectively, from 3 donors, p < 0.01). This treatment resulted in a 58% decline in the exocytotic response following 10 mM glucose treatment compared to siCont-treated cells (Figure 6C; n=17 and 16 cells, respectively from 3 donors, p < 0.01). Similarly, knockdown of SENP1 reduced the S-AMP-dependent facilitation of exocytosis by 50% (Figure 6C; n=25 and 20 cells from 3 donors, p < 0.01), indicating that the full amplifying effect of S-AMP requires SENP1 activity.

S-AMP rescues impaired exocytosis in human T2D β-cells

Given the potent effects of SAMP to enhance exocytosis in 832/13 cells and β-cells from non-diabetic human donors, we investigated the effects of S-AMP in β-cells from a human T2D donor. As expected, the GSIS response measured in islet cell aggregates from the T2D donor was impaired relative to secretion from human islet cell aggregates from non-diabetic donors (1.6-fold versus 3.3-fold, compare Figures 6A and 6D). Similarly, 10 mM glucose failed to potentiate depolarization-induced exocytosis compared to 1 mM glucose in β-cells from the T2D donor (n=8–9 cells, p = 0.6) (Figure 6E). However, dialysis of 10 μM S-AMP induced an approximate 2.5-fold amplification of depolarization-induced exocytosis in the T2D β-cells (Figure 6E; n=8 cells, p < 0.01), similar to the effect of S-AMP or stimulatory glucose in normal human β-cells (Figure 6B). Thus our data demonstrate that in addition to amplifying the exocytotic response in non-diabetic human β-cells, S-AMP rescues dysfunctional exocytosis in human T2D β-cells.

Discussion

Glucose metabolism contributes to purine and nucleotide biosynthesis via entry of glucose into the pentose monophosphate shunt to form ribose-5-P, on which the ring structures of nucleotides are synthesized from glycine, glutamine, aspartate, and 10-formyltetrahydrofolate. Several recent studies have used metabolomics to document changes in an array of purine and nucleotide pathway intermediates in response to glucose challenge in the INS-1-derived 832/13 β-cell line created in our laboratory (Huang and Joseph, 2014; Lorenz et al., 2013; Spegel et al., 2011). These findings provide correlative data suggesting that some of the metabolites could function as stimulus/secretion coupling factors, but direct experiments to test individual candidate mediators have not appeared. To delve deeper into this possibility, we employed a targeted, quantitative LC-MS/MS method for survey of purine and nucleotide pathway intermediates. Using this method, we found a decrease in inosine monophosphate (IMP), and an increase in adenylosuccinate (S-AMP) in glucose-stimulated 832/13 cells, suggesting that the enzyme linking the two metabolites, adenylosuccinate synthase (ADSS), could play a regulatory role in β-cell glucose sensing. Although the glucose-induced decrease in IMP has been reported previously (El-Azzouny et al., 2014), this is the first finding of changes in levels of S-AMP in β-cells in response to glucose stimulation. Consistent with the concept that ADSS and its product S-AMP have a stimulus-secretion coupling role, pharmacologic or molecular suppression of ADSS caused impairment of GSIS with attendant decreases in S-AMP, effects that could be overcome by addition of adenine. Moreover, siRNA-mediated suppression of ADSL, a more proximal enzyme in the S-AMP biosynthetic pathway, impairs GSIS and lowers S-AMP levels, independent of decreases in other adenine nucleotides or GTP. Strikingly, infusion of S-AMP into patch-clamped 832/13 cells or β-cells from non-diabetic human subjects stimulated exocytosis at least as well as glucose, and S-AMP also rescued exocytosis in glucose-insensitive β-cells from human T2D subjects.

Recent studies report that inhibition of the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (G6PDH) by dehydroepiandrosterone (DHEA) (Spegel et al., 2013), or suppression of the subsequent step in the pathway, 6-phosphogluconate dehydrogenase (6PGDH) by siRNA or the chemical inhibitor 6-AN (Goehring et al., 2011), results in impaired GSIS. The effects of 6PGDH inhibition were ascribed to accumulation of early pentose shunt pathway intermediates and activation of p-ERK (Goehring et al., 2011), whereas DHEA treatment was reported to cause a decrease in reduced glutathione (GSH) levels, presumably secondary to impaired NADPH production, although the effect of DHEA on NADPH was not reported (Spegel et al., 2013). Here we show that a specific, glucose-regulated product of the pentose shunt and the purine pathway, S-AMP, has a direct effect to augment exocytosis in normal islets, presenting an alternative explanation for these prior findings.

Our studies suggest that manipulation of S-AMP levels may provide a means of enhancing secretion in otherwise dysfunctional islets in T2D. Currently, there is very little information available about the impact of T2D on the purine pathway in general, or on S-AMP levels more specifically. Earlier reports showed the inhibitory effect of MPA on GSIS and guanine nucleotide levels, but did not fully recognize the impact of MPA on the adenine branch of nucleotide metabolism, and did not identify S-AMP as a regulated metabolite (Li et al., 2000). Another report describes altered adenine nucleotide metabolism in the diabetic heart in the BB/Wistar and streptozotocin diabetes rat models, with increases in the AMP catabolic enzymes 5′-nucleotidase, adenosine deaminase, and AMP deaminase, and apparent compensatory induction of the nucleotide cycle enzymes ADSS and ADSL (Jenkins et al., 1988), but islet studies were not reported. More recently, a study of the thiazolidinedione (TZD)/PPARγ activators rosiglitazone and pioglitazone in normal rats revealed increases in cardiac IMP and AMP levels in response to drug treatment, accompanied by transcriptional upregulation of G6PDH, the PRPP synthesizing enzyme Prps1, and ADSS (Liu et al., 2013), but again, S-AMP was not measured and islet studies were not performed. Interestingly, interaction of PPARγ with the ADSS promoter was reported, and TZDs are known to be protective against development of β-cell failure and diabetes in the Zucker diabetic fatty (ZDF) (Shimabukuro et al., 1998; Sreenan et al., 1996). Further studies will be required to define potential changes in expression of key S-AMP generating and degrading enzymes in islets in rodent models and human T2D islets, and the possible restorative effects of TZD or adenine supplementation in those preparations. Conversely, one potential explanation for the impact of ADSS suppression on GSIS could have been a secondary perturbation of genes known to regulate GSIS, or “disallowed genes” associated with β-cell secretory dysfunction (Thorrez et al., 2011). However, we found no changes in expression of GLUT2, glucokinase, Kir6.2 (component of the KATP channel), the cytosolic, NADP-dependent isoform of isocitrate dehydrogenase (ICDc), or lactate dehydrogenase (representative of the “disallowed genes”) in response to ADSS1 or ADSS2 knockdown in 4 separate biological samples, each assayed in duplicate or triplicate (data not shown).

The classical model of GSIS holds that glucose-induced increases in ATP:ADP ratio trigger insulin secretion via inhibition of KATP channels, causing membrane depolarization and activation of voltage-gated Ca2+ channels. Evidence for KATP channel-independent, complementary metabolic signaling pathways in control of GSIS has been mounting steadily (Jensen et al., 2008; MacDonald et al., 2005; Prentki et al., 2013, Gooding, et al, 2015). Anaplerotic metabolism of glucose in mitochondria appears to control one such complementary pathway by affecting β-cell redox status, GSH:GSSG ratio, and activity of SENP1 (Ferdaoussi et al., 2015; Ivarsson et al., 2005; Jensen et al., 2008; Reinbothe et al., 2009). Here we show that glucose-induced flux through de novo purine synthesis and ADSS2 drive increases in S-AMP levels in β-cells, causing enhanced insulin granule exocytosis. During glucose stimulation, the β-cell has multiple sources of anaplerotic substrates, including pyruvate entering through pyruvate carboxylase and conversion of glutamine/glutamate to α-ketoglutarate, thus generating sufficient levels of TCA cycle intermediates to allow their exit from the mitochondria for engagement in cytosolic, signal-inducing pathways, most notably the pyruvate-isocitrate pathway (Ferdaoussi et al., 2015; Jensen et al., 2008; Ronnebaum et al., 2006). In contrast, in skeletal muscle during exercise, anaplerotic substrates are limited, and activation of the nucleotide cycle through ADSS1 and ADSL is thought to be an important source of the anaplerotic metabolite fumarate, thus sustaining the TCA cycle and ATP production. The data presented herein are consistent with the idea that S-AMP synthesis is an important functional role of the nucleotide cycle in islets, allowing S-AMP to act as a glucose-induced amplifying signal for insulin granule exocytosis (See Figure 7 for schematic summary).

Figure 7. Schematic summary of the role of purine/nucleotide pathways in coupling of glucose metabolism and insulin secretion.

Figure 7

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The purine metabolic pathway is shown with metabolites that increase (red) or decrease (blue) in response to glucose stimulation in the 832/13 cell-line. Abbreviations : ADA: adenosine deaminase; ADSL: adenylosuccinate lyase; ADSS: adenylosuccinate synthase; AK: adenylate kinase; AMPDA: AMP deaminase; Fum: fumarate; IMP: inosine monophosphate; IMPDH: inosine 5-monophosphate dehydrogenase ; MPA: mycophenolic acid; S-AMP: adenylosuccinate.

Given the important role assigned to changes in ATP:ADP ratio in control of GSIS in the historical literature, can we separate the proposed signaling role of S-AMP from changes in these other nucleotides? Several of our observations support a specific role for S-AMP, including: 1) Alanosine-mediated inhibition of ADSS causes decreases in levels of S-AMP, ADP, and ATP, while also causing impairment of GSIS. The suppression of GSIS can be rescued by provision of adenine, which causes S-AMP, ADP, and ATP levels to return to normal (Figure 3). The [ATP]/[ADP] ratio in both the suppressed and rescued states is similar, arguing against a key role for this variable in mediating the observed changes in GSIS under these conditions; 2) As shown in Figure 5, silencing of ADSL results in a specific lowering of S-AMP in concert with impairment of GSIS, with no decreases in ADP or ATP levels or changes in ATP:ADP ratio; 3) In studies with patch-clamped rat insulinoma and human β-cells shown in Figures 4 and 6, ATP levels are fixed and KATP channel regulation does not occur because membrane potential is clamped in this experimental system. Despite this, S-AMP exhibits a strong effect to amplify insulin granule exocytosis in the patch-clamped cells. We also note that other metabolic intermediates in addition to ATP, ADP and changes in the ratios of these nucleotides have been considered as mediators of GSIS. Our study focused on the purine/nucleotide pathway, but the reader is referred to a recent review for discussion of other implicated metabolites and pathways (Gooding, et al, 2015).

The fundamental question of how S-AMP interacts with the downstream secretory machinery to affect granule exocytosis remains to be resolved. Insulin granule exocytosis is impaired by attachment of small ubiquitin-like modifier (SUMO) peptides to secretory granules, and nutrient stimulation activates SENP1 to remove these modifications and activate secretion (Dai et al., 2011; Vergari et al., 2012). We have recently demonstrated that GSH/glutaredoxin interact with cysteine residues required for SENP1 catalytic function, and that the stimulatory effects of isocitrate or GSH on amplification of exocytosis require SENP1 (Ferdaoussi et al., 2015). Here we show that the amplifying effect of S-AMP on exocytosis is also dependent on SENP1 activity. Whether the S-AMP/SENP1 signaling pathway represents an independent or convergent pathway relative to the pyruvate-isocitrate-GSH-SENP1 pathway remains to be investigated. In sum, our studies have identified a potent glucose-derived secretagogue from the purine biosynthesis pathway. Future work will be focused on probing this pathway as a potential source of targets for improving β-cell function in T2D.

Experimental Procedures

Reagents, cell lines and primary islets

All reagents and solutions were obtained from Sigma-Aldrich unless otherwise indicated. The rat insulinoma cell line, 832/13 was cultured as described (Hohmeier et al., 2000). Rat islets were harvested from male Sprague-Dawley rats weighing ~300 g under a protocol approved by the Duke University Institutional Animal Care and Use Committee and cultured as described (Ronnebaum et al., 2008). Human islets were isolated from pancreata of non-diabetic organ donors (n=6) and a donor with T2D (n=1) at the Alberta Diabetes Institute Islet Core as previously described (Kin and Shapiro, 2010). All studies with human islets were approved by the Human Research Ethics Board (Pro00001754) at the University of Alberta. All organ donors provided informed consent for use of pancreatic tissue in research (Pro00013094).

Glucose-stimulated insulin secretion

Insulin secretion from 832/13 cells and rat islets (~20 islets per incubation) was measured as described previously (Jensen et al., 2006) by RIA (Siemens) or Elisa (ALPCO, Salem, NH). For GSIS from human islets, human islet cell aggregate cultures were prepared and used as described in Supplemental Methods.

IMPDH and ADSS inhibition experiments

For inosine monophosphate dehydrogenase (IMPDH) inhibition experiments in confluent 832/13 cells, 0.1% ethanol or mycophenolic acid (MPA) was added to the culture media overnight and was included during preincubation and GSIS as well, with or without adenine (250 μM), guanine (100 μM) or 0.125 N NaOH. For adenylosuccinate synthase (ADSS) inhibition, L/D-alanosine (Santa Cruz) was dissolved in 50 mM Hepes, pH 8.6 at several concentrations and added to confluent 832/13 cells or whole rat islets, in the presence and absence of 100 μM adenine, overnight and during performance of the GSIS assays. Controls were treated with equal volumes of 50 mM Hepes, pH 8.6.

siRNA-mediated gene silencing of ADSS1, ADSS2, and ADSL and quantitative real time PCR analyses

Methods and siRNA duplex sequences used for silencing of ADSS1,ADSS2 and ADSL in 832/13 cells, as well as methods used for quantitative real time PCR analyses (qRT-PCR), are provided in Supplemental Experimental Procedures.

Cell harvest for nucleotide extraction

After 2 h of GSIS, media was removed and 832/13 cells were scraped from plates in secretion buffer into a 15 mL conical tube and centrifuged. The cell pellet was flash frozen in a dry-ice ethanol bath. A cold solution of methanol (200 μL) containing nine internal standards (13C10, 15N5-AMP (90 μM), 13C10, 15N5-GMP (30 μM), 13C10, 15N2-UMP (15 μM), 13C9, 15N3-CMP (15 μM), 13C10-GTP (30 μM), 13C10-UTP (45 μM), and 13C9-CTP (15 μM), 13C10-ATP (300 μM) and nicotinamide-1,N6-ethenoadenine dinucleotide (eNAD) (75 μM) in methanol) was added and the pellets were allowed to thaw on ice before resuspension by pipetting. Chilled water (100 μL), and then hexane (300 μL) was added to each tube, the tubes were vortexed vigorously, and then centrifuged. The bottom aqueous layer was collected and centrifuged again. The supernatants were transferred to a 96-well plate for analysis by LC-MS/MS.

LC-MS/MS

The method is a modification of a previously published approach (Cordell et al., 2008). Chromatographic separations were performed using an Agilent Technologies (Santa Clara, CA) 1200 HPLC system and a Chromolith FastGradient RP-18e 50-2mm column (EMD Millipore, Billerica, MA, USA). Injection volume was 2 μL. Mobile phase A was 95% water, 5% methanol and 5 mM dimethylhexylamine adjusted to pH 7.5 with acetic acid. Mobile phase B was 20% water, 80% methanol and 10 mM dimethylhexylamine. Flow rate was set to 0.3 mL/min and column temperature was 40 °C. A 22 min gradient method (t=0, %B=0; t=1.2, %B=0; t=22, %B=40) was run followed by a 3 min wash and 7 min equilibration. Flow was directed to an Agilent 6410 Triple Quadrupole MS (Santa Clara, CA) and source conditions were set to 4000 V capillary voltage, 350 °C gas temperature, 12 L/min gas flow, and 30 psi nebulizer flow. All nucleotides were detected in negative ion MRM mode based on a characteristic fragmentation reaction (see Table S1). Quantitation of metabolites used 9 isotope labeled internal standards and an external calibration using serial dilution of nucleotide standards.

Single-cell measurement of β-cell exocytosis

For patch clamping experiments, islets were cultured in low glucose (5.5 mM) DMEM with L-glutamine, 110 mg/l sodium pyruvate, 10% FBS and 100 U/mL penicillin/streptomycin. For SENP1 siRNA studies in human β-cells, siRNA duplexes were transfected into dissociated human islet cells with Dharmafect Transfection Reagent 1. siSenp1 was obtained from Life Technologies (s26615) and a scrambled siRNA (siScrambled; Allstars Negative control siRNA, Qiagen; Toronto, ON) was used as the control. Dissociated human islet cells or 832/13 cells, plated on 35-mm dishes overnight or for 72 hours in the case of SENP1 knockdown experiments. Prior to patch-clamping, cells were pre-incubated in DMEM (human) or RPMI-1640 (832/13) media with 1 mM glucose for 1 hour and then exposed to patch-clamp bath solution with either 1 mM or 10 mM glucose for ~15 minutes. Whole-cell patch-clamp was performed after this, according to previously published methods (Dai, et al., 2011; Ferdaoussi, et al., 2015) and as outlined in the Supplementary Materials.

Statistical analyses

For insulin secretion, relative expression, and exocytosis experiments, statistical significance was determined by two-sample, unpaired equal variance Student’s t test. The statistical significance of metabolite concentrations was analyzed using the Wilcoxon rank sum test to allow for a non-normal distribution of values due to experimental batch effects (Bauer, 1972). The Wilcoxon test was completed using the wilcox.test function in R (R-Core Team, 2014). In all cases p-values of < 0.05 were considered significant.

Supplementary Material

1
2

Acknowledgments

The authors thank the Human Organ Procurement an Exchange (HOPE) program and Trillium Gift of Life Network (TGLN) for their efforts in procuring human organs for research, Mr. James Lyon (University of Alberta) for his efforts in human islet isolation, Ms. Helena Winfield for assistance with rat islet isolation, and Drs. Lisa Norquay, Jeff Trimmer, and Julia Brosnan (Pfizer Cardiovascular and Metabolic Disease Unit) for sharing methods for studying human islet cell aggregate cultures. This work was funded by NIH grant DK046492-22 (to CBN), NIH grant K01GM109320-02 (to JRG) and CIHR grant MOP 244739 (to PEM). PEM holds a Canada Research Chair in Islet Biology.

Abbreviations

ADSL

adenylosuccinate lyase EC 4.3.2.2

ADSS

adenylosuccinate synthase EC 6.3.4.4

Alano

alanosine

GSIS

glucose-stimulated insulin secretion

IMP

inosine monophosphate

IMPDH

inosine monophosphate dehydrogenase EC 1.1.1.205

KATP channels

ATP-sensitive K+ channels

LC-MS/MS

liquid chromatography - tandem mass spectrometry

MPA

mycophenolic acid

S-AMP

adenylosuccinate

SENP1

sentrin/SUMO-specific protease-1

T1D

Type-1 diabetes

T2D

Type-2 diabetes

qRT-PCR

Quantitative Real Time PCR Analyses

ZMP

5-Aminoimidazole-4-carboxamide ribonucleotide. See supplemental table for full metabolite names

Footnotes

Author contributions. JRG, MVJ, PEM, and CBN designed the study. JRG and BRW performed metabolomics analyses. MVJ performed gene knockdown and insulin secretion studies in 832/13 cells, rat islets, and human islet cell cultures. DL isolated and purified rat islets and RA helped develop human islet cell culture techniques and assisted MVJ in studies with such preparations. XD and MF performed patch clamp studies and SENP1 knockdown studies in 832/13 cells and human islets, including T2D islets. JRG, MVJ, PEM, and CBN analyzed data. JRG, MVJ and CBN wrote the paper, and JRG, MVJ, PEM, and CBN edited and finalized the paper.

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Supplementary Materials

1
NIHMS720189-supplement-1.pdf (557.7KB, pdf)
2
NIHMS720189-supplement-2.docx (174.8KB, docx)

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