Encoding acyl-CoA thioesterase-7 (Acot7) is one of ∼60 genes expressed ubiquitously across tissues but relatively silenced, or disallowed, in pancreatic β-cells. The capacity of ACOT7 to hydrolyze long-chain acyl-CoA esters suggests potential roles in β-oxidation, lipid biosynthesis, signal transduction, or insulin exocytosis. We explored the physiological relevance of β-cell–specific Acot7 silencing by re-expressing ACOT7 in these cells. ACOT7 overexpression in clonal MIN6 and INS1(832/13) β-cells impaired insulin secretion in response to glucose plus fatty acids. Furthermore, in a panel of transgenic mouse lines, we demonstrate that overexpression of mitochondrial ACOT7 selectively in the adult β-cell reduces glucose tolerance dose dependently and impairs glucose-stimulated insulin secretion. By contrast, depolarization-induced secretion was unaffected, arguing against a direct action on the exocytotic machinery. Acyl-CoA levels, ATP/ADP increases, membrane depolarization, and Ca2+ fluxes were all markedly reduced in transgenic mouse islets, whereas glucose-induced oxygen consumption was unchanged. Although glucose-induced increases in ATP/ADP ratio were similarly lowered after ACOT7 overexpression in INS1(832/13) cells, changes in mitochondrial membrane potential were unaffected, consistent with an action of Acot7 to increase cellular ATP consumption. Because Acot7 mRNA levels are increased in human islets in type 2 diabetes, inhibition of the enzyme might provide a novel therapeutic strategy.

Maintained secretion of insulin is essential for normal blood glucose homeostasis, and both the loss and the dysfunction of pancreatic β-cells, the sole source of the circulating hormone in man, are implicated in type 2 diabetes (T2D) (1). Glucose sensing by β-cells involves a number of gene products, such as GLUT2 and glucokinase, whose expression is restricted to these and only a few other cell types and that ensure that elevated blood glucose concentrations are converted into enhanced glycolytic and then citrate cycle flux, stimulating respiratory chain activity and, ultimately, ATP production by mitochondria. The resulting rise in cytosolic ATP/ADP ratio closes ATP-sensitive K+ (KATP) channels, and this in turn leads to plasma membrane depolarization and Ca2+ influx through voltage-gated calcium channels, triggering dense-core secretory granule exocytosis (1).

In addition to β-cell signature genes, a small group of housekeeping genes is relatively repressed in β-cells compared with other cell types (24). Of these, the monocarboxylate (lactate/pyruvate) transporter MCT-1 (SLC16A1) and lactate dehydrogenase (LDHA) are particularly weakly expressed in the β-cell but strongly expressed elsewhere in the body (5). This configuration appears to prevent inappropriate insulin secretion in response to circulating pyruvate derived from muscle during exercise (57). Thus, patients with activating mutations in the SLC16A1 promoter display exercise-induced hyperinsulinism (6), a situation mimicked in mice by overexpression of MCT-1 selectively in the adult β-cell (7). Systematic comparisons of the transcriptome of mouse islets versus other tissues (2,3) have revealed an additional 64 genes similarly suppressed (or disallowed) in β-cells, of which a core of 11 genes (4) was common in two independent studies. Although evidence exists for a role for the suppression of some of these genes in the function or survival of β-cells (notably, MCT-1 as just described, as well as LDHA [8] and PDGFRA [9]), for the remainder, the biological rationale for β-cell–selective repression is obscure (4).

Acyl-CoA thioesterase 7 (Acot7) has been identified by our group as a member of the β-cell disallowed genes group (3). ACOT7 is a member of a family of 13 enzymes responsible for the hydrolysis of acyl-CoA (10,11). The murine Acot7 gene comprises 13 exons and undergoes differential splicing to generate cytosolic and mitochondrial variants (12). ACOT7 acts on acyl-CoAs with a range of chain lengths (10) and is particularly highly expressed in the brain and testis (12,13). Additionally, ACOT7 is implicated in the hydrolysis of arachidonoyl-CoA (14), which furnishes free arachidonic acid for the synthesis of prostaglandins. Others have suggested a role for ACOT7 in the brain in the maintenance of low, nontoxic acyl-CoA levels (13,15).

Suggesting a role in β-cell decompensation in T2D, levels of Acot7 are increased in Zucker diabetic fatty rat islets (16) and in microdissected β-cell–enriched tissue from patients with T2D (17). Given the importance of intracellular lipids in the control of many β-cell functions, including membrane trafficking, ion channel activity, and insulin exocytosis (18), we explored the impact of Acot7 overexpression in these cells both in vitro and in vivo.

Generation of Constructs Overexpressing ACOT7 Isoforms

The murine Acot7 coding sequences were amplified by RT-PCR from liver and kidney RNA and ligated into P3XFLAG-CMV-14 in-frame with the COOH-terminal 3xFLAG epitope tag. The coding sequences of each isoform, complete with epitope tag, were then amplified and ligated into the pBI-L vector, generating two plasmids with a bidirectional tetracycline-regulated promoter that simultaneously drives the expression of both firefly luciferase and FLAG:Acot7_mit (pBI-LTet FLAG::Acot7_mit) or FLAG:Acot7_cyt (pBI-LTet FLAG::Acot7_cyt).

Generation and Maintenance of Acot7 Transgenic Mice

The expression cassette was excised from pBI-LTet FLAG::Acot7_mit and used for pronuclear microinjection into C57BL/6J oocytes at the Imperial College London/Medical Research Council Transgenics and Embryonic Stem Cell Facility. Successful integrants (F9, F15, and F26) were identified by PCR and backcrossed with C57BL/6 wild-type mice for at least three generations. The resulting heterozygous Acot7_mit mice were crossed with homozygous RIP7rtTA mice (C57BL/6 background) to produce littermate Acot7 transgenic (Acot7 Tg) mice (Acot7_mit+/−, Rip7rtTA positive, heterozygous, 1:2 ratio) and controls (Acot7_mit−/−, Rip7rtTA positive, heterozygous, 1:2 ratio). All the animals were administered doxycycline in the drinking water (0.5 g/L) from the age of 5 weeks. High-fat diet (HFD) (DIO Rodent Purified Diet w/60% Energy From Fat, DIO-58Y1) was administered from the age of 5 weeks when indicated. All in vivo procedures were performed at the Imperial College Central Biomedical Service and approved by the U.K. Home Office Animals (Scientific Procedures) Act of 1986 (PPL 70/7349).

Acot7 Expression and Protein Analysis

RNA-Seq read data were downloaded for a range of mouse tissues (accession numbers SRA056174: β-cells; GSE21860: islets; GSE36026: other tissues). Reads were mapped to the mouse genome (Ensembl NCBIM37) using Bowtie:TopHat, and genes were quantified with Cufflinks (19). Isolation of total RNA, quantitative RT-PCR (RT-qPCR), and Western (immuno-) blot were performed as previously described (20).

Human Growth Hormone Release Assay

Human growth hormone (hGH) secretion assays were performed as previously described (21). To reach a final experimental concentration of 0.5 mmol/L, a 2:1 unsaturated:saturated mix of fatty acids (FAs), which in the presence of 1% BSA results in unbound free FA concentrations in the nanomolar range (22), oleate, and palmitate (Sigma-Aldrich) were incubated with BSA for 2 h at 37°C.

Immunocytochemistry and Immunohistochemistry

MIN6 and INS1(832/13) cells, cotransfected with pTet-off and pBI-LAcot7_mit or pBI-L-Acot7_cyt, and isolated pancreata were fixed, stained, and visualized as previously described (23). Cells and slides were visualized with an LSM 780 microscope (Zeiss, Welwyn Garden City, U.K.) and an Axiovert 200M microscope (Zeiss), respectively. ImageJ software was used to calculate the mean intensity of FLAG (Acot7 transgene) in the β-cell area surrounded by glucagon-positive (α-cell) area. For β-cell mass estimation, we determined the percentage of pancreatic surface that was insulin positive, as measured in whole-pancreas sections separated by 25 μm in the z-axis.

Intraperitoneal Glucose and Insulin Tolerance Tests and In Vivo Insulin Secretion

Intraperitoneal glucose and insulin tolerance tests were performed as previously described (20). For insulin secretion, animals were fasted overnight, and glucose (3 g/kg) was administered intraperitoneally. Plasma insulin was measured by ELISA (Crystal Chem).

Isolation and Analysis of Mouse Islets

Islets were isolated by digestion with collagenase as described elsewhere (24). Insulin secretion, Ca2+ (Fura-2-AM; Invitrogen) and ATP/ADP (Perceval) imaging were performed as previously described (23,25). Insulin release during perifusion was monitored by using a custom-built device that uses 50 islets and a perifusion rate of 500 μL ⋅ min−1 at 37°C. Insulin was quantified by using a homogeneous time-resolved fluorescence-based assay (Cisbio) in a PHERAstar reader (BMG LABTECH).

Electrophysiology was performed as previously described (26) in perforated patch-clamp configuration by using an EPC9 patch-clamp amplifier controlled by PULSE acquisition software (HEKA Elektronik, Pfalz, Germany). Data were filtered at 1 kHz and sampled at 2 kHz.

Seahorse experiments were performed as previously described (27). Briefly, 50 islets per well (matched size) were placed in 24-well islet plates in Krebs-Ringer bicarbonate buffer in 0.1% BSA containing 3 mmol/L glucose and preincubated at 37°C without CO2 for 1 h. Islets were then incubated at 3 mmol/L glucose followed by 17 mmol/L glucose and oligomycin (5 μmol/L) to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).

Transfection, ATP/ADP, and Changes in Mitochondrial Membrane Potential Analysis in INS1(832/13) Cells

Cells were transfected for 24 h with pBI-LTet FLAG::Acot7_mit or a control empty vector plus pEGFP-C1 (Addgene) (secretion and tetramethylrhodamine ethyl ester [TMRE] experiments) or GW1-PercevalHR (Addgene) (ATP/ADP experiments, ratiometric mode) using Lipofectamine 2000, with 50–60% efficiency. Cells were preincubated for 1 h at 3 mmol/L glucose before stimulation. ATP/ADP and TMRE dynamics were monitored by using a widefield microscope (Olympus IX70) equipped with a Polychrome IV illumination system (TILL Photonics), a heating stage, and a 40×/1.35NA objective. ATP/ADP measurements were performed as previously described (28). For TMRE experiments, INS1(832/13) was loaded with 10 nmol/L TMRE in imaging buffer (28) for 45 min and re-equilibrated with 2 nmol/L for 10 min before recordings. TMRE (2 nmol/L) was present throughout and excited at 550 nm (0.25 Hz). Data were analyzed using ImageJ with a purpose-designed macro.

Lipidomics

Lipids extraction from isolated islets (150–300 per animal and sample) and analysis were conducted by the Lipidomics Mass Spectrometry Facility at Babraham Institute (29).

Statistical Analysis

Statistical analysis was performed with GraphPad Prism 6.0 software. Statistical significance was evaluated as indicated in the figure legends. All data are shown as mean ± SEM. P < 0.05 was considered statistically significant.

Acot7 Expression Is Weak in Mouse Islets Compared With Other Tissues

Interrogation of publicly accessible RNA-Seq databases (Fig. 1A) and RT-qPCR (Fig. 1B) confirmed lower levels of mRNA encoding all Acot7 mRNA variants in mouse islets compared with all other tissues examined. Of note, RNA-Seq data showed the lowest Acot7 expression in purified β-cells (Fig. 1A). Western blot analysis confirmed the strongest expression at the protein level in brain, lung, and testis. ACOT7 immunoreactivity was undetectable in islets (Fig. 1C). As observed by relative RT-qPCR, other members of the Acot family were also poorly expressed in mouse islets (Fig. 1D).

Overexpression of Acot7 in β-Cell Lines Impairs Glucose Plus FA-Induced Insulin Secretion

We first determined whether Acot7 overexpression may affect glucose-stimulated insulin secretion (GSIS) or secretion in response to depolarization of the plasma membrane with KCl. We generated two DNA constructs expressing the cytosolic (Acot7_cyt) or the mitochondrially targeted (Acot7_mit) isoforms. Experiments were performed using either isoform co-overexpressed in β-cell lines alongside hGH as a reporter of regulated secretion from the transfected cell population. The latter peptide is costored and released with insulin from dense-core granules (21).

Both Acot7_cyt and Acot7_mit were expressed with the appropriate subcellular localization in both murine MIN6 (30) and INS1(832/13) (31) cells (Fig. 2A). Thus, in each case, the epitope tag displayed a cytosolic localization (Acot7_cyt) or was closely colocalized with the mitochondrial marker TOM20 (Acot7_mit) (Fig. 2A).

Examined in MIN6 cells, overexpression of either isoform affected hormone release neither in response to 30 versus 3 mmol/L glucose nor in response to 30 mmol/L KCl (Fig. 2B). However, because Acot7 action is likely to lead to the degradation of acyl-CoA derived from exogenous FAs, we next asked whether secretion in response to a mixture of glucose plus FAs may be susceptible to Acot7 overexpression. Confirming this possibility, secretion in response to 20 mmol/L glucose plus a mixture of palmitate and oleic acid (0.5 mmol/L oleate:palmitic acid [2:1]/1% [weight for volume] BSA), was significantly reduced in MIN6 and INS1(832/13) cells upon overexpression of either Acot7_cyt (16% and 13%, respectively) or Acot7_mit (17% and 13%, respectively) (Fig. 2C).

β-Cell–Selective Overexpression of Mitochondrial Acot7 in Adult Mice Impairs Glucose Tolerance

To explore the potential role in vivo for the suppression of insulin release in clonal β-cells, we generated three mouse lines re-expressing the mitochondrial form of Acot7 by using the Tet-ON system (32). The in vitro results suggested that both isoforms inhibited secretory function to a similar extent, but given the central role of mitochondria in β-cell stimulus-secretion coupling (1), we focused on mitochondrial ACOT7.

Genomic qPCR analysis demonstrated the presence of 4, 7, and 70 copies of the transgene in three lines originating from three different founders (F9, F26, and F15 respectively; data not shown). After establishing stable integration at a single site by the inheritance of the same number of copies for three generations, offspring were subsequently crossed to RIP7rtTA mice expressing the reverse tetracyclin-regulated transactivator selectively in β-cells (32). Islets isolated from animals bearing or lacking the Acot7 transgene were incubated with doxycycline for 48 h to induce the expression of the transgene. Alternatively, animals were administered doxycycline for 2–4 weeks to induce expression before islet isolation. RT-qPCR revealed an increase of ∼14-, 28-, and 100-fold over endogenous Acot7 mRNA levels, respectively, in the three lines (F15, F9, and F26) when doxycycline was administered in vivo, with comparable levels of induction observed in vitro in cultured islets (Fig. 3A). By contrast, Acot7 mRNA levels remained unchanged in the hypothalamus (a potential site of RIP2 activity [33]) or the liver of the mice with maximum Acot7 overexpression in islets (F26) (Supplementary Fig. 1A). Additionally, the ratio of Acot7:β-actin mRNA in islets from mice displaying the strongest overexpression of the thioesterase in islets (F26) was in the same range as that observed in the hypothalamus (Supplementary Fig. 1B), indicating that levels of overexpression in β-cells were still within the normal physiological range in the context of other cell types. Expression of the other Acot family members was identical in transgenic versus control islets, eliminating the possibility of compensatory changes in the expression of the latter (Supplementary Fig. 1C).

Measurements of expression by Western blot confirmed a degree of induction of Acot7 in the three lines essentially proportional to changes at the mRNA level (Fig. 3B). Immunocytochemical analysis of pancreata revealed immunoreactivity corresponding to the incorporated FLAG tag within the islets in lines F9 and F26 (Fig. 3C and D). Immunoreactivity was below the level of detection in pancreata from F15 mice (Fig. 3C and D). Examined in the most strongly expressing line F26, the pattern of immunoreactivity was punctate, consistent with mitochondrial targeting, and was exclusively restricted to the insulin-positive β-cell population (Fig. 3E). Although we noticed little interanimal variation in expression levels in animals from lines F26 and F15, F9 offspring expressed Acot7 at highly variable levels (for both mRNA and protein). The reasons for this variability are unclear.

Animals from line F26 displayed indistinguishable weight gain and random-fed glycemia compared with wild-type littermates (Supplementary Fig. 2). However, intraperitoneal glucose tolerance was significantly impaired in the transgenic animals at 17, 21, and 25 weeks, with females showing a stronger phenotype (Fig. 4A and B). Similar, but less marked changes were also obtained for lines F15 and F9 (Supplementary Fig. 3).

To determine whether the effects on glucose tolerance in the Acot7 Tg line F26 may be further exacerbated during β-cell stress, we maintained these mice on an HFD (60% total calories) for 4 weeks (34), sufficient to engender insulin resistance and significant increases in peak glycemia in control nontransgenic mice of the same age (Fig. 4C and D). Differences in glucose tolerance between nontransgenic animals and transgenic littermates became apparent in younger animals (9 weeks) under these conditions than those on a normal chow diet (in which the phenotype was not observed until the age of 17 weeks [12 weeks for females]), especially in male mice (Fig. 4A). Comparable results were obtained for the lines with F15 and F9 (Supplementary Fig. 3). Whether examined in animals on an HFD or regular chow diet, the observed defects in glucose tolerance were milder in mice from line F15, with lowest Acot7 expression, indicating a dose-dependent effect of Acot7 overexpression.

To exclude insulin insensitivity as the cause of the glucose intolerance detected in Acot7 Tg mice, we evaluated insulin tolerance in 9-week-old F26 female mice fed an HFD and 25-week-old F15 female mice fed a chow diet (Supplementary Fig. 4A). No differences between transgenic and control mice were observed (Supplementary Fig. 4A). β-Cell mass was also similar for control and transgenic mice from line F26 kept on a chow diet or an HFD (Supplementary Fig. 4B and C).

Overexpression of Mitochondrial Acot7 in the β-Cell Impairs Glucose-Induced Increases in Cytosolic ATP/ADP Ratio, Membrane Depolarization, Free Ca2+, and Insulin Secretion

To explore the mechanisms underlying the glucose intolerance after Acot7 overexpression, we measured plasma insulin levels in fasted F26 mice (previously kept on regular chow diet or HFD). This revealed a nonsignificant tendency toward elevated insulin levels in Acot7 Tg mice versus littermate controls, but strikingly, no further increase in plasma insulin was observed after glucose (3 g/kg) challenge (Fig. 5A). This result indicates a reduced capacity of the β-cells to secrete insulin in response to increased glucose concentration. Consistent with this in vivo observation, insulin secretion from transgenic mouse isolated islets revealed a marked inhibition in release in response to 17 mmol/L glucose or 8 mmol/L glucose plus FAs versus nontransgenic islets (Fig. 5B). As observed in MIN6 cells (Fig. 2B), no differences in insulin secretion were detected in response to depolarization of the plasma membrane with KCl (Fig. 5C).

In contrast to our observations in β-cell lines, Acot7 overexpression reduced secretion in response to high glucose, even in the absence of supplementation with exogenous FA. Because the requirement for exogenous FAs may reflect a depletion of endogenous lipid stores after 24 h, INS1(832/13) cells expressing mitochondrial Acot7 and then precultured for 1 h in 3 mmol/L glucose displayed lower insulin secretion in response to 17 mmol/L glucose than control plasmid-transfected cells (Fig. 6A). Dynamic measurements of insulin secretion in transgenic islets revealed changes in both phases of secretion in response to high glucose, with the second (sustained) phase being particularly strongly inhibited (Fig. 5D and E).

These observations suggest that signaling by glucose to exocytosis (1) may become defective in Acot7 Tg β-cells. To test this hypothesis, we measured the metabolic response to high glucose by following changes in the cytosolic ATP/ADP ratio with the recombinant fluorescent probe Perceval (35,36). The glucose-mediated ATP/ADP rise, largely reflective of changes in β-cells (rather than other cell types), was strongly reduced in Acot7 Tg islets versus controls (Fig. 7A). We also assessed ATP/ADP changes in INS1(832/13) cells by using the ratiometric sensor PercevalHR to provide a measure of this ratio that was independent of the level of probe expression (28). Overexpression of Acot7 had no effect on basal ATP/ADP ratios but significantly lowered the speed and extent of the ATP/ADP increases in response to 17 mmol/L glucose (Fig. 6B and C). By contrast, glucose-induced increases in mitochondrial membrane potential (Δψm), assessed with TMRE, were identical in each case (Fig. 6D).

Correspondingly, glucose-induced changes in intracellular free cytosolic Ca2+, imaged with the intracellular fluorescent dye Fura-2-AM (37), were also significantly reduced in Acot7 Tg islets (Fig. 7B) consistent with impaired downstream closure of KATP channels (38,39). The aforementioned defects were not explained by changes in the expression of genes essential for GSIS, including the glucose transporter GLUT2 (Slc2a2), glucokinase (Gck), the KATP channel subunits Kir6.2 (Kcnj11) and SUR1 (Abcc8), and the ATP synthase ATP5 (Atp5a1), in Acot7 Tg islets (Supplementary Fig. 5).

To investigate a direct role for defective KATP channel closure, we used perforated patch-clamp electrophysiology (40) to measure Δψm in single β-cells. These experiments were performed in animals maintained on an HFD for 4 weeks, where differences in glucose tolerance between transgenic and wild type were already apparent (Fig. 4). Transgenic β-cells showed significantly weaker membrane depolarization in response to high glucose than those from control mice (Fig. 7C), which is expected if a reduction in the closure of KATP channels occurs as a consequence of lowered ATP/ADP increases (Fig. 7A).

To shed more light onto the mechanisms underlying the lowered ATP/ADP increases after Acot7 overexpression, we assessed mitochondrial oxidative metabolism upstream of ATP generation. Monitored with a Seahorse XF Analyzer, no significant differences in OCR were observed between control and transgenic islets (Fig. 7D), although a tendency was observed toward higher basal OCR in transgenic islets. This difference persisted in the presence of oligomycin, an inhibitor of ATP synthase, arguing against increased uncoupling in transgenic islets. Of note, the fold increase in OCR in response to 17 mmol/L glucose (percent of basal) (Fig. 7E) tended to be lower in transgenic islets. Although not significantly different, the ECAR tended to be higher in Acot7 Tg islets at high glucose, possibly reflecting a tendency toward increases in Ldha but not Slc16a1 (MCT-1) expression (Supplementary Fig. 6A).

Overexpression of Acot7 in β-Cells Results in Reduced Fatty-Acyl-CoA Generation but Not in Significantly Altered FA Levels

To determine whether an action of ACOT7 to hydrolyze FA-CoA esters into free FA and CoA (41) might contribute to impaired metabolic signaling, we performed complete lipidomic profiling of islets isolated from transgenic or control mice maintained on an HFD for 4 weeks using electrospray ionization mass spectrometry. Islets were incubated overnight at 11 mmol/L glucose, starved (3 mmol/L glucose) for 1 h, and subsequently incubated at high glucose (17 mmol/L) for 30 min. A clear reduction in FA-CoA was observed in Acot7 Tg islets, as expected (Fig. 8A and C). A tendency toward an increase in the levels of FAs of a particular chain length, notably C18:1 (most probably oleic acid), was also detected (Fig. 8B). No significant changes were observed in the levels of monoacylglycerol (MG), diacylglycerol, or other lipids (Fig. 8A). A strong tendency (P = 0.06) toward increased fatty-acyl-carnitine levels was also observed in transgenic islets.

Although at least 11 genes are selectively repressed in the β-cell (4), the physiological relevance, if any, of this tissue-specific suppression has only been examined for Ldha (8), Slc16a1 (MCT-1) (7), and Pdgfra (9). We show first, using overexpression in β-cell lines, that low ACOT7 levels are required for normal secretory responses to glucose in the presence of adequate FA levels. Extending these data to the in vivo setting, we also demonstrate that β-cell–specific overexpression in mice of mitochondrial ACOT7 causes a marked and dose-dependent impairment in glucose tolerance that reflects altered β-cell function rather than reduced β-cell mass (42). No direct evidence was obtained for changes in insulin sensitivity in this model, although clamp studies would be needed to exclude this possibility entirely.

Insulin secretion in response to glucose (and glucose plus FA) was strongly impaired in Acot7 Tg mouse islets, likely explaining the impairment in glucose tolerance and insulin secretion observed in vivo. Although only islets overexpressing the mitochondrial isoform of Acot7 were interrogated, increased levels of fatty-acyl-carnitine in transgenic islets suggest increased shuttling of acyl-CoA into the mitochondria in this model expected to result in a decrease of cytosolic FA-CoA.

Consistent with the current results, Klett et al. (43) found that small interfering RNA–mediated reduction of Acsl4 (acyl-CoA synthetase isoform 4), responsible for the conversion of certain FAs into FA-CoA, reduced insulin secretion in response to glucose and, more markedly, glucose plus FA in INS1(832/13) cells. In contrast with the current findings and suggesting a different underlying mechanism, the effects occurred without changes in acyl-CoA species. On the contrary, Acsl4 depletion resulted in reduced epoxyeicosatrienoic acids. Although measurement of eicosanoids in transgenic islets would be necessary to discard their contribution to the observed phenotype in Acot7 Tg mice, Acsl4 inhibition also affected KCl-mediated insulin secretion, whereas ACOT7 overexpression did not. Furthermore, Ellis et al. (15) did not observe changes in prostaglandin D2 or E2 in Acot7-depleted brains or, more surprisingly, in acyl-CoA levels. Thus, lipid composition and availability may be critical determinants of Acot7 action in different cell types (44).

FA-CoA has been shown to modulate GSIS through a range of mechanisms, including the control of secretory granule exocytosis, protein acylation, and modulation of the ATP sensitivity of KATP channels (44). Most recently, Prentki et al. (18) provided evidence that MGs play a particularly important role in stimulus-secretion coupling in β-cells, possibly acting through Munc13-1 to enhance a late event in exocytosis downstream of cytosolic Ca2+ increases. In the current study, we did not observe an effect of ACOT7 overexpression on depolarization-stimulated secretion or observe significant differences in the levels of MG, arguing against a role for Acot7 in MG-dependent regulation of GSIS. Of note, ACOT7 overexpression reduced both phases of GSIS, arguing against a preferential effect on a plasma membrane–proximal, readily releasable granule pool (45).

On the other hand, we observed a strong reduction in glucose-mediated ATP/ADP and Ca2+ rises in Acot7 Tg animals, suggesting that low ACOT7 levels are necessary to ensure effective KATP channel closure. The exact mechanisms underlying the impaired ATP/ADP increases remain unclear. However, preserved OCRs in islets (Fig. 5D) and Δψm glucose-mediated increases (Fig. 6D) in INS1(832/13) cells after Acot7 overexpression add weight to the view that increased ATP consumption, rather than impaired mitochondrial ATP generation, is involved. This may conceivably involve a futile cycle wherein increased acyl-CoA synthesis occurs in the face of an accelerated breakdown of this lipid. Consistent with this view, secretion stimulated by glutamine plus leucine, which allows flux into the tricarboxylic acid cycle downstream of citrate, was slightly reduced in INS1(832/13) cells overexpressing Acot7 (Supplementary Fig. 6B). Further analysis will be needed, however, to determine whether this reflects enhanced acyl-CoA breakdown/resynthesis.

Of note, all members of the Acot family were poorly expressed in mouse islets, stressing the importance of maintaining acyl-CoA hydrolysis to a minimum in these cells. Furthermore, expression of Acot7 is upregulated in the islets of Zucker diabetic fatty rat (16) and in β-cell–enriched microdissected tissue from patients with T2D (17). Pharmacotherapeutic approaches that correct this defect might thus be useful in some forms of T2D.

Acknowledgments. The authors thank Professor Gerhard Christofori, University of Basel, Basel, Switzerland, for providing the RIP7rtTA mouse and Professor David Carling, Medical Research Council Clinical Sciences Centre, Imperial College London, for use of the Seahorse XF Analyzer.

Funding. T.J.P. is a Diabetes Research & Wellness Foundation post-doctoral fellow (SCA/01/F/12). This work was supported by grants to G.A.R. from the Wellcome Trust (WT098424AIA), the Medical Research Council, U.K. (project G0901521 and programme MR/J0003042/1), and the Biotechnology and Biological Sciences Research Council (BB/J015873/1). G.A.R. is a Royal Society Wolfson Research Merit Award holder.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. A.M.-S. designed and conducted the in vitro and in vivo studies and contributed to the Seahorse experiments, lipidomic analyses, and writing of the manuscript. T.J.P. generated the constructs for Acot7 expression and transgene production and performed analyses of published RNA-Seq data. P.C. contributed to the imaging studies. Q.Z. contributed to the lipidomic analyses. E.H. contributed to the electrophysiology studies. M.C.C. performed the islet perifusion studies. M.-S.N.-T. contributed to the in vivo experiments. S.R.S. contributed to the Seahorse experiments. G.A.R. conceived the study and contributed to the writing of the manuscript. G.A.R. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in abstract form at the Diabetes UK Professional Conference, Glasgow, U.K., 2–4 March 2016, and the Islet Biology: From Cell Birth to Death (X5) Keystone Symposia on Molecular and Cellular Biology, Keystone, CO, 13–17 March 2016.

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Supplementary data