Ablation of AMPKα1 and α2 from pancreatic beta cells and RIP.Cre neurons suppresses insulin release in vivo

Abstract

Aims/Hypothesis

AMP-activated protein kinase (AMPK) is an evolutionarily-conserved enzyme and a target of antihyperglycemic agents including metformin. However, the precise role(s) of the enzyme in controlling insulin secretion remains uncertain.

Methods

The catalytic α1 and α2 subunits of AMPK were ablated selectively in pancreatic beta cells and hypothalamic neurons by breeding AMPKα1 null mice, bearing flox’d AMPKα2 alleles, with animals expressing Cre recombinase under the rat insulin promoter. The latter promoter was used to express constitutively-activated AMPK selectively in beta cells in transgenic mice. Food intake, body weight and urinary catecholamines were measured using metabolic cages. Glucose and insulin tolerance were determined after intraperitoneal injection. Beta cell mass and morphology were analysed by optical projection tomography and confocal immunofluorescence microscopy, respectively. Granule docking, insulin secretion, membrane potential, and intracellular free Ca²⁺ were measured with standard techniques.

Results

Trigenic βAMPKdKO mice, lacking both AMPK α subunits in the beta cell, displayed normal body weight and increased insulin sensitivity, but were profoundly insulin deficient. Secreted catecholamine levels were unchanged. Total beta cell mass was unaltered whilst mean islet and beta cell volume were reduced. AMPK-deficient beta cells displayed normal glucose-induced changes in membrane potential and intracellular free Ca²⁺ whilst granule docking and insulin secretion were enhanced. Conversely, βAMPK transgenic mice were glucose-intolerant and displayed defective insulin secretion.

Conclusions/Interpretation

Inhibition of AMPK activity within the beta cell is necessary, but not sufficient, for the stimulation of insulin secretion by glucose. AMPK activation in extrapancreatic RIP.Cre-expressing cells might also influence insulin secretion in vivo

Introduction

AMP-activated protein kinase is an evolutionarily-conserved fuel-sensitive protein kinase implicated in the control of glucose homeostasis and with roles in both insulin-sensitive tissues [1-3] and in the pancreatic beta cell [4-7]. Whilst the stimulation of AMPK activity in muscle and liver is now seen as a likely mechanism through which glucose-lowering agents, including metformin and thiozilidenediones, act to improve insulin sensitivity [8], the long-term effects of these agents on pancreatic beta cell survival and insulin release are less clear [7].

Mammalian AMPK is a trimeric protein comprising a catalytic α-subunit, encoded by one of two separate genes (α1 and α2), a scaffold β- (β1 or β2) subunit and a regulatory γ-subunit (γ1, γ2 or γ3)[9,10]. The existence of two separate AMPKα subunit genes has so far hindered investigations of role of AMPK activity in controlling glucose homeostasis in mammals since the unconditional deletion of both isoforms leads to early embryonic lethality in mice [11]. By contrast, animals with a global inactivation of the α1 isoform do not display significant metabolic abnormalities [11,12]. Deletion of the AMPKα2 gene leads to insulin resistance and glucose intolerance, in part due to increased parasympathetic tone [13]. Whereas insulin secretion was normal in islets isolated from whole-body α2 knockout mice, insulin release in vivo appeared to be diminished when measured at a single time point during oral glucose tolerance tests. No measurements were made, however, during intraperitoneal glucose tolerance tests, where the complicating effects of potentially-altered incretin release could be excluded. Importantly, since complexes containing the α1 isoform are substantially (>10-fold) more abundant in beta cell lines than AMPKα2 complexes [5], increases in the expression of the latter may also, in part, have compensated for the loss of AMPKα2.

The cell-permeant AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) diminishes glucose-stimulated insulin secretion from clonal beta cells and islets [4,6], an effect mimicked by the biguanide metformin [14] or by the expression of a constitutively-active form of AMPK [6]. Moreover, over-expression of constitutively-active AMPK diminished the performance of islets transplanted into streptozotocin-diabetic mice [15]. These actions may be due in part to a blockade of secretory granule transport to the cell surface [16] as a result of kinesin-1 light chain phosphorylation [17]. In addition, AMPK activation decreases beta cell viability [18,19] possibly by phosphorylating the cell cycle regulator, p53 [20]. By contrast, inhibition of AMPK activity with dominant-negative forms of the enzyme tends to increase insulin secretion at low glucose concentrations [6], without affecting release at elevated glucose concentrations [21], consistent with the inactivation of the enzyme as a result of AMP depletion under these conditions.

AMPK is also thought to play an important role in the central control of feeding and glucose homeostasis. Thus, forced changes in AMPK in the ventromedial hypothalamus (VMH) achieved by stereotactic injection of viral vectors led to marked changes in food intake and body weight [22] and in hypoglycaemia sensing [23,24]. Moreover, the deletion of AMPKα2 in pro-opiomelanocortin- (POMC) or agouti-related peptide-(AgRP) expressing neurons leads respectively to increased or decreased food intake in mice, [25]. Finally, manipulation of AMPK activity ex vivo was found to affect glucose-induced changes in the electrical activity and of Ca²⁺ transients in isolated AgRP-(glucose-inhibited), but not POMC (glucose-responsive) neurons in culture [26,27].

We have previously demonstrated distinct roles in the pancreatic beta cell for AMPK complexes containing differing catalytic subunits [5]. Thus, the α2 subunit, which displayed substantial nuclear localisation [4,5], was implicated in the control of gene expression whereas α1-containing complexes, whose total activity exceeded by 5-10 fold that of α2-containing complexes [5], were almost exclusively cytosolic. The role of the latter remains unclear but may include the regulation of plasma membrane ion channels, as proposed in oxygen-sensing cells in the carotid body [28].

In order to assess the role in of AMPK in insulin-expressing cells, we have generated trigenic mice, globally inactivated for AMPKα1 and with AMPKα2 deleted selectively in pancreatic beta cells and in a small population of hypothalamic neurons using the rat insulin promoter (RIP2) [29], and transgenic mice expressing activated AMPK selectively in beta cells. Using these models we highlight multiple novel mechanisms through which AMPK controls insulin production and glucose homeostasis in mammals.

METHODS

Generation of mutant mice lacking AMPKα1 globally and selectively lacking AMPKα2 in pancreatic beta cells and RIP.Cre neurons

AMPK α1^+/−,α2^fl/fl (fl/fl) mice were first crossed with wild type C57BL/6 mice to generate double heterozygous AMPKα1^+/−,α2^fl/+ mice. Offspring were then crossed with heterozygous RIP.Cre⁺ transgenic mice (expressing Cre recombinase under the rat insulin 2 promoter; Jackson Laboratory, Maine, USA). The resulting triple heterozygous AMPKα1^+/−,α2^fl/+,Cre⁺ mice were interbred with their siblings. Since RIP.Cre transgenic mice have been suggested to show glucose intolerance and impaired insulin secretion [30,31], Cre⁺ mice were always used as a negative control. Due to the low probability (1:64) of obtaining double knockout mice through heterozygote crossing, two different breeding strategies were used to obtain double AMPK knockout mice and their littermate controls: to generate AMPKα1^−/−,α2^fl/fl,Cre⁺ (βAMPKdKO) mice and their heterozygous AMPKα1^+/−,α2^fl/+,Cre⁺ mice (Het) controls, AMPKα1^−/−,α2^fl/+,Cre^- and AMPKα1^+/−, α2^fl/fl,Cre⁺ mice were crossed. To produce heterozygous AMPKα1^+/−,α2^fl/+,Cre⁺ and their wild-type AMPKα1^+/+,α2^+/+,Cre⁺ littermate controls, AMPK α1^+/−,α2 ^fl/+,Cre⁺ and wild-type mice were crossed. All mice were kept on a C57BL/6 background and offspring genotypes were obtained in the expected Mendelian ratios.

Generation of mutant mice selectively over-expressing AMPK.CA or -DN in pancreatic beta cells and RIP.Cre neurons

An expression vector containing the RIP2 promoter fragment (600bp), c-myc-tagged rat AMPKα1³¹².T172D (CA) or AMPKα2.D157A (DN) cDNA and an SV40 poly (A) cassette (Fig. 6a) was excised with BssHII and microinjected into the male pronucleus of fertilized C57BL/6 oocytes. The injected zygotes were re-implanted into pseudo-pregnant female C57BL/6 mice (GenOway, France). We obtained three AMPK.CA (named C1, C2 and C10) and two AMPK.DN (D1 and D2) founder mice that stably transferred the corresponding transgenes to their offspring. Founder mice were crossed with wild type C57BL/6 mice to achieve F1 generation. Distributions of genotypes in the offspring followed a Mendelian pattern. All AMPK transgenic mice were kept heterozygous. F3 and later generations and their littermate wild type controls were used for experiments. All lines were maintained on a pure C57BL/6 background.

Fig. 6.

Mouse maintenance and diet

Mice were housed at 2-5 animals per cage in a pathogen-free facility with 12 h light/dark cycle. Mice were fed ad libitum with a standard mouse chow diet or a high fat diet (60 % (w/w) fat content; Research Diet, UK). As indicated, six-week old mice were transferred onto a high fat diet for a maximum of 18 weeks. All in vivo procedures stated were performed at the Imperial College Central Biomedical Service (CBS) and approved by the UK Home Office Animals Scientific Procedures Act, 1986.

Body weight and food intake

Fed mouse weights were monitored weekly after six weeks of age. Food intake was measured daily for three consecutive days using a metabolic cage.

In vivo physiological studies

Intraperitoneal glucose tolerance test (IPGTT)

Mice fasted for 15h (water allowed) were intraperitoneally injected with 1 g glucose/kg mouse weight. Blood from the tail vein was obtained at 0, 15, 30, 60, 90 and 120 min after injection. Blood glucose levels were measured with an automatic glucometer (Accuchek, Roche, UK). To study the effect of α-adrenergic antagonist on glucose tolerance, mice were intraperitoneally injected with 10 mg/kg mouse weight phentolamine 30 min. before glucose challenge.

Plasma insulin measurement

Mice fasted for 15 h were intraperitoneally injected with 3 g glucose/kg mouse weight. Blood from mice tail veins was collected into a heparin coated tube (Sarstedt, UK) at 0, 15 and 30 min after injection. Plasma was separated by centrifuging the blood at 2000 g for 5 min. Plasma insulin levels were measured using an ultrasensitive mouse insulin ELISA kit (Mercodia, Uppsala, Sweden). Normal fed plasma insulin levels were measured from blood collected from 12-week-old mice tail veins between 10:00-11:00 am.

Insulin tolerance test (ITT)

Bovine insulin (Sigma, UK; 0.75U/kg) was intraperitoneally injected into fed mice at 13.00-14.00. Blood glucose levels were measured at 0, 15, 30 and 60 min. after injection.

Urine collection and catecholamine measurement

Daily urine collection from single mouse for a period of three days was performed using metabolic cages. Catecholamine levels in urine were determined by reverse-phase HPLC.

Other methods

Details of islet isolation and insulin secretion, electron microscopy, electrophysiology, optical projection tomography, Ca²⁺ imaging, RNA extraction and RT-PCR, AMPK assay, antibodies and immunocytochemistry are provided under Supplementary Methods.

Statistical analysis

Data are expressed as means±SEM. Significance was tested by Student’s two samples unpaired or paired Student’s t-test using Excel, or ANOVA using Graphpad4.0. p<0.05 was considered significant.

RESULTS

βAMPKdKO mice have normal body weight but are hyperglycemic

mRNAs encoding both AMPKα1 and α2 subunits were present in highly purified [32,33] wild-type mouse beta cells (Supp. Fig. 1). AMPKα1 mRNA was ~15-fold more abundant than that encoding the α2 subunit, in line with previous AMPK activity measurements in clonal beta cells [5] and with distinct roles for each isoform in these cells [5]. Since mice deleted globally for either subunit display essentially normal insulin release in vitro [13], we generated trigenic mice inactivated for both α1 and α2 subunits selectively in beta cells and a small population of hypothalamic neurons. Mice globally inactivated for AMPKα [11] were first crossed with animals bearing flox’d AMPKα2 gene alleles (Fig. 1a, left). Crossing with RIP2.Cre mice [29] led to a selective loss of the catalytic domain of the AMPKα2 subunit (aa189-260) from islets and hypothalamus (Fig. 1a, right). Consistent with the abundant expression in islets of the Cre transgene (Fig. 1b, left), and deletion of the both catalytic subunits selectively from beta cells [34], the crossing of AMPKα2 flox’d and RIP.Cre mice resulted in a decrease in islet AMPKα2 mRNA of 60-70% (Fig. 1b, right). Assessed at low glucose concentrations to near-maximally stimulate the enzyme [14], total islet AMPK activity was decreased by 93% in AMPKα1^−/− versus AMPKα1^+/− (α2^fl/fl.Cre^- in each case) mice, and by 95% in βAMPKdKO mice versus AMPKα1^+/− (Fig. 1c).

Fig. 1.

Male βAMPKdKO mice displayed entirely normal growth and normal food intake (Fig. 1d,e) whilst females displayed a small reduction in body weight up to ten weeks of age (Supp. Fig. 2). Assessed at three (Fig. 2) and six months (not shown) of age, βAMPKdKO mice of either sex, but not mice deleted for either AMPKα1 [12] or α2 (Supp. Fig. 3) alone, displayed markedly elevated plasma glucose and decreased plasma insulin levels Fig. 2a,b; Supp. Fig. 3). Correspondingly, double knockout mice displayed abnormal glucose tolerance and insulin release in vivo, despite increased insulin sensitivity (Fig. 2c-e; Supp Fig. 2). No differences in glucose tolerance, insulin release or sensitivity were observed between AMPKα1^+/+,α2^+/+,Cre⁺ and AMPK,α1^+/−,α2^fl/+,Cre⁺ control mice (not shown).

Fig. 2.

Since mice with global homozygous deletion of AMPKα2 display abnormal insulin secretion in vivo and elevated catecholamine levels [13], we determined whether the latter parameter may contribute to abnormal insulin secretion in βAMPKdKO mice. Indicating that this was not the case, we detected no alterations in the levels of urinary catecholamines (Fig. 3a), and abnormal glucose tolerance was still observed, though diminished in extent, in the presence of the α-adrenoreceptor blocker, phentolamine (Fig.3b)[35].

Fig. 3.

βAMPKdKO mice have normal beta cell mass but smaller beta cells and islets

Changes in relative beta cell mass, which might have explained the marked decrease in insulin release in βAMPKdKO animals, were not observed in βAMPKdKO mice, as assessed by optical projection tomography (OPT) of whole pancreata [36] (Fig. 4a-c; videos betaAMPKhet and betaAMKPdKO) or through analysis of pancreatic slices (Supp. Fig. 4). However, the distribution of islet sizes between heterozygous and βAMPKdKO animals, as assessed by OPT, revealed a significant ~40% decrease in the average volume of individual islets (Fig.4b,d).

Fig. 4.

By contrast, no differences were apparent in the ratio of alpha to beta cells within individual islets, nor with the relative disposition of the two cell types (Fig. 5a). Similarly, despite proposed roles for AMPK and the upstream kinase LKB1 in the control beta cell polarity [37-39](Sun et al, unpublished), we observed no abnormalities in the formation of adherens (anti-E-cadherin antibodies) (Fig. 5b) or tight (anti-zona occludins-1, ZO-1, antibody) junctions (Fig. 5c) and microfilament and microtubule structure was unchanged in islets from βAMPKdKO mice (not shown).

Fig. 5.

AMPK is a known regulator of the mTOR complex, acting to phosphoryate the mTORC1 components Raptor and the upstream regulator tuberous sclerosis complex-2 (TSC2) [40]. Since mTOR is involved in the regulation of cell size [41] we assessed the size of individual beta cells, using anti-E-cadherin (Fig. 5d) or anti-Glut2 (not shown) antibodies to label the plasma membrane. This revealed an ~18% decrease in the average area of beta cells, corresponding to a decrease in volume of ~36%, closely in line with the reduction in average islet size. However, no changes in the phosphorylation state of the downstream targets of mTOR, ribosomal S6 subunits were detected (not shown) arguing against changes in the activity of the latter pathway as responsible for the decrease in beta cell volume. Unexpectedly, beta cell proliferation was also substantially (>2-fold) increased, as assessed by ki67 staining (Fig. 5e). No changes in the low level of apoptosis could be detected by caspase-3 or in situ TUNEL staining (not shown), possibly reflecting rapid clearance of apoptotic cells.

Glucose-induced insulin secretion is enhanced in islets from βAMPKdKO mice but beta cell electrical activity and intracellular free Ca²⁺ changes are normal

The above analyses revealed a profound compromise in the capacity of an unchanged mass of morphologically normal beta cells to release insulin in vivo. To determine whether this reflected a change in the intrinsic properties of beta cells, we next undertook analyses of insulin release from isolated islets and glucose-sensing by dissociated beta cells. In contrast to what was observed in vivo, islets from βAMPKdKO mice displayed significantly elevated rates of glucose-stimulated insulin release compared with control (heterozygote) islets (Fig. 6a) arguing for a restraining effect of AMPK on hormone release. Unexpectedly, the previously-described inhibitory effects of AMPK activation with the AMPK activator and AMP analogue 5-aminoimiazole-4-carboxamide-1-beta-d-ribofuranoside, AICAR [4,6] were still preserved in βAMPKdKO mouse islets, indicating effects of this activator were largely independent of AMPK.

To determine whether beta cells from βAMPKdKO mouse islets may show altered glucose sensing, as observed after LKB1 deletion (Sun et al, unpublished) we used electrophysiological approaches. Whilst glucose-induced changes in the conductance of ATP-sensitive K⁺ channels (G_KATP) revealed lower conductance of these channels at low (3 mmol/l) but not elevated (16.7 mmol/) glucose in βAMPKdKO beta cells versus heterozygous controls, this was not translated into a difference in membrane potential changes (Fig. 6b). Likewise, no differences were apparent in the extent of glucose or depolarisation- (KCl−) induced increases in intracellular free Ca²⁺ ions in heterozygous versus βAMPKdKO beta cells (Fig. 6c). Correspondingly, again in contrast to the impact of LKB1 deletion (Sun et al, unpublished), no differences were observed in the levels or plasma membrane association of the liver/ beta cell glucose transporter Glut2 (Fig. 6d) [42]. By contrast, the number of morphologically-docked granules was significantly increased in βAMPKdKO beta cells (Fig. 6e).

We next determined whether AMPK present in beta cells and RIP.Cre neurons may contribute to the deleterious effects of a high fat diet (HFD) on insulin release and glucose homeostasis [43]. No differences in body weight gain were apparent between heterozygous and βAMPKdKO mice maintained on HFD (Fig. 7a). However, after six weeks on HFD, sufficient to cause profound abnormalities in glucose-induced insulin secretion in C57BL/6 mice [43], the differences in glycemia observed between heterozygote and βAMPKdKO mice observed on a normal diet (Fig. 1e) were abolished (Fig. 7b). This change reflected a more dramatic increase in glucose levels in the heterozygous mice on HFD versus normal chow (compare Fig. 7b and 2a). Likewise, the difference in glucose tolerance between βAMPKdKO and heterozygous mice observed on normal chow (Fig. 2c, ~5 mmol/l at 60 min) was substantially reduced when the comparison between genotypes was performed in mice maintained on HFD (Fig. 7c; ~ 2 mmol/l at 60 min.). By contrast, the enhanced insulin sensitivity of βAMPKdKO mice (Fig. 2e; ~20 % of the initial glucose level 30 min. post insulin injection) was maintained or enhanced on HFD (Fig. 7d; ~ 30 % of initial glucose). Strikingly, the 3.5-fold decrease in glucose-stimulated insulin secretion observed in islets from heterozygous mice maintained on HFD versus normal chow (from ~0.7 to 0.2 %/30 min; Fig. 6a vs Fig 7e) was reduced in βAMPKdKO mouse islets to ~0.6 fold (from ~1.3 to 0.8%/30 min.). Hence, βAMPKdKO mice were still susceptible to the effects of HFD, albeit to a lesser extent than controls.

Fig. 7.

Over-expression of constitutively-active AMPK in beta cells causes glucose intolerance

The above results suggested that, at extrapancreatic sites of RIP.Cre expression, notably in the mediobasal hypothalamus, AMPK activity is permissive for insulin secretion in vivo.

To determine whether increases in AMPK imposed selectively in beta cells may affect insulin secretion in vivo, we generated transgenic mice in which the constitutively active enzyme (“AMPK.CA”) [6] was expressed under the control of the insulin promoter (Fig. 8a). Of two founder mouse lines generated (Fig. 8b), we examined one line carrying two transgene copies in detail. Over-expression of the mRNA was clearly evident in islets (Fig. 8c), and under the control of glucose ex vivo (as expected for expression under the insulin promoter; Fig. 8d) but barely (<0.001% of the islet level) in the hypothalamus or other tissues (Fig. 8c,d), reflecting the more restricted expression of the RIP2 transgene to the beta cell in adult mice [44].

Fig. 8.

Islets from transgenic mice over-expressing AMPK.CA (AMPK.CA Tg) displayed a significant increase in total (α1 + α2 complex) AMPK activity at elevated (16.7 mM) glucose concentrations, but not at low (2.8 mM) glucose concentrations, where the endogenous enzyme was strongly activated (Fig. 8e) [14]. Conversely, AMPK activity was reduced at low glucose concentrations in islets from RIP2.AMPK.DN transgenic mice, generated in parallel with βAMPK.CA mice. Assuming approximately equal endogenous AMPK levels in beta versus islet non-beta cells (Supp. Fig.1) and an islet beta cell content of 60% [34], AMPK activity was increased by 36%, and reduced by 50%, in beta cells by over-expression of AMPK.CA or AMPK.DN respectively.

Maintained on normal chow, AMPK.CA and AMPK.DN transgenic displayed normal body weight increases (Fig. 9a). Whereas male AMPK.CA Tg mice displayed abnormal glucose tolerance at three (Fig. 9b), but not six (Fig. 9c) months, no abnormalities were seen in AMPK.DN Tg animals (Fig. 9b,c) nor in female AMPK.CA mice (not shown). The abnormalities in the AMPK.CA Tg mice were not associated with any alterations in insulin sensitivity (Fig. 9d) but with decreased fasting and stimulated plasma insulin levels (Fig. 9e) and a 25% decrease in beta cell area (0.09±0.02% in wild-type versus 0.068±0.04% in transgenic littermates) (data not shown). No significant changes in these parameters were observed in AMPK.DN Tg mice.

Fig. 9.

To determine whether the alterations in insulin secretion in AMPK.CA mice may also result from defects at the level of the beta cell we performed studies with isolated islets. Insulin release stimulated by 16.7 mM glucose was decreased by >60 % (Fig. 9f), a similar change to that observed after maintenance of mice for 18 weeks on HFD. Indeed, islets from AMPK.CA Tg mice maintained on HFD displayed no further diminution in GSIS compared to wild type littermates on the same diet (Fig. 9f). Conversely, glucose-stimulated insulin release was significantly enhanced in islets from AMPK.DN Tg mice (Fig. 9f), reminiscent of the effect of deletion of α1 and α2 subunits (Fig.6a), though in this case the improved secretion was eliminated in islets from animals fed a HFD.

DISCUSSION

The principal aim of this study was to determine the physiological impact of the complete and selective loss of both catalytic isoforms of AMPK activity from pancreatic beta cells. This has appeared to us an important question, given the likely role of AMPK as a target for antihyperglycemic drugs and uncertainties surrounding the role of AMPK in the beta cell [5,6,15,18,21,45,46]}.

Cell autonomous roles of AMPK within the beta cell

Examined here in isolated islets and beta cells, total inactivation of AMPK led to a potentiation of glucose-induced insulin secretion. We also noted an increase in ATP-sensitive K⁺ (K_ATP) channel activity at low glucose concentrations (Fig.4b), perhaps reflecting altered trafficking of channel subunits [47]. However, glucose-induced K_ATP channel closure and increases in intracellular Ca²⁺ were unaltered, suggesting that the loss of an inhibitory effect of AMPK-mediated phosphorylation of kinesin light chains [16,17], and increased granule translocation to the plasma membrane (Fig. 6e), possibly underlies the enhanced secretion. Conversely, activation of AMPK selectively in beta cells in βAMPK.CA transgenic mice decreased glucose-stimulated insulin secretion. These findings are consistent with previous results involving the over-expression in islets of AMPK.CA in vitro [21] as well as with the effects of pharmacological activation of AMPK in clonal beta cells and isolated islets using AICAR or metformin [4,6,14,48]. Furthermore, a diminished number of docked granules was observed in MIN6 cells over-expressing AMPK.CA [16].

We also show here that AMPK deletion leads to a decrease in cell volume, an effect strikingly different from that of deleting the upstream kinase, LKB1. Indeed, beta cells lacking LKB1 as a result of either RIP.Cre (Sun et al, unpublished) or PDX-1-CreER [38,49] -mediated excision are larger than wild type beta cells. LKB1 and AMPK appear therefore to engage substantially distinct downstream signalling pathways in beta cells, the former acting at least to a large extent via Par1b/MARK2 [38,49].

In the present study, inactivation of AMPK in RIP.Cre neurons exerted no apparent effect on body mass or food intake. However, αAMPKdKO mice displayed defective glucose homeostasis due to abnormalities in the capacity of a preserved beta cell mass to secrete insulin in response to hyperglycemia. This deficiency was only partially compensated by an increase in insulin sensitivity, likely due to increased insulin receptor levels or enhanced downstream signalling in target tissues [50]. Importantly, we used intraperitoneal injection of glucose, rather than oral administration, in order to achieve increases in blood glucose concentration in the absence of a substantial release of incretins including GLP-1. In this way, we sought to compare the effects of an increase in circulating glucose in vivo with changes imposed on isolated islets. Nonetheless, we noted a dramatic decrease in insulin release in βAMPK.dKO mice in vivo which was not apparent in vitro.

We have considered the possibility that a decrease in beta cell and islet size may contribute to this deficiency in insulin secretion in vivo. However, the absence of an impairment in insulin release from islets in vitro (in fact, the opposite was observed) makes it seem unlikely that these changes are responsible for the drastically impaired insulin release observed in vivo in βAMPKdKO mice. It seems likely that the absence of the “opposite” in vivo phenotype (i.e. improved glucose intolerance) in mice over-expressing activated AMPK under the same promoter (Fig. 9a-e) most likely reflects the predominant expression of the transgene in the pancreatic beta cell (rather than the hypothalamus) in adult mice. One possible explanation for these data is that a signal(s) emanating from RIP.Cre neurons controls the activity of beta cells in vivo. Indeed, central administration of leptin has previously been shown to inhibit insulin secretion in vivo [51] and it is conceivable that this involves RIP.Cre neurons and intracellular signalling pathways modulated by AMPK. Nevertheless, further and more definitive studies are needed to ascertain whether, and by what means, RIP.Cre neurons may influence insulin secretion.

A further interesting finding of the present studies is that mice lacking AMPK in the beta cell are somewhat less susceptible to the deleterious effects of HFD on glucose metabolism and insulin secretion in vitro. These results are consistent with the previously-demonstrated role for AMPK [18] in the actions of cytokines [19] on beta cell function and mass.

Conclusions

The results presented here suggest that activation of AMPK in hypothalamic neurons and in pancreatic beta cells may play distinct roles in the control of insulin release in vivo. Our findings should inform the use and development of agents which act through AMPK to control glycemia.

Abbreviations

AICAR

5-aminoimidazole-4-carboxamide ribonucleotide

AMPK

AMP-activated protein kinase

HFD

high fat diet

OPT

optical projection tomography

Tg

transgenic

VMH

ventromedial hypothalamus