Loss of AMP-activated protein kinase alpha 2 subunit in mouse beta-cells impairs glucose-stimulated insulin secretion and inhibits their sensitivity to hypoglycaemia

AMPK (AMP-activated protein kinase) signalling plays a key role in whole-body energy homoeostasis, although its precise role in pancreatic β-cell function remains unclear. In the present stusy, we therefore investigated whether AMPK plays a critical function in β-cell glucose sensing and is required for the maintenance of normal glucose homoeostasis. Mice lacking AMPKα2 in β-cells and a population of hypothalamic neurons (RIPCreα2KO mice) and RIPCreα2KO mice lacking AMPKα1 (α1KORIPCreα2KO) globally were assessed for whole-body glucose homoeostasis and insulin secretion. Isolated pancreatic islets from these mice were assessed for glucose-stimulated insulin secretion and gene expression changes. Cultured β-cells were examined electrophysiologically for their electrical responsiveness to hypoglycaemia. RIPCreα2KO mice exhibited glucose intolerance and impaired GSIS (glucose-stimulated insulin secretion) and this was exacerbated in α1KORIPCreα2KO mice. Reduced glucose concentrations failed to completely suppress insulin secretion in islets from RIPCreα2KO and α1KORIPCreα2KO mice, and conversely GSIS was impaired. β-Cells lacking AMPKα2 or expressing a kinase-dead AMPKα2 failed to hyperpolarize in response to low glucose, although KATP (ATP-sensitive potassium) channel function was intact. We could detect no alteration of GLUT2 (glucose transporter 2), glucose uptake or glucokinase that could explain this glucose insensitivity. UCP2 (uncoupling protein 2) expression was reduced in RIPCreα2KO islets and the UCP2 inhibitor genipin suppressed low-glucose-mediated wild-type mouse β-cell hyperpolarization, mimicking the effect of AMPKα2 loss. These results show that AMPKα2 activity is necessary to maintain normal pancreatic β-cell glucose sensing, possibly by maintaining high β-cell levels of UCP2

AMPK is essential for energy homeostasis regulation and glucose sensing by POMC and AgRP neurons

Hypothalamic AMP-activated protein kinase (AMPK) has been suggested to act as a key sensing mechanism, responding to hormones and nutrients in the regulation of energy homeostasis. However, the precise neuronal populations and cellular mechanisms involved are unclear. The effects of long-term manipulation of hypothalamic AMPK on energy balance are also unknown. To directly address such issues, we generated POMCa2KO and AgRPa2KO mice lacking AMPKa2 in proopiomelanocortin– (POMC-) and agouti-related protein–expressing (AgRP-expressing) neurons, key regulators of energy homeostasis. POMCa2KO mice developed obesity due to reduced energy expenditure and dysregulated food intake but remained sensitive to leptin. In contrast, AgRPa2KO mice developed an age-dependent lean phenotype with increased sensitivity to a melanocortin agonist. Electrophysiological studies in AMPKa2-deficient POMC or AgRP neurons revealed normal leptin or insulin action but absent responses to alterations in extracellular glucose levels, showing that glucose-sensing signaling mechanisms in these neurons are distinct from those pathways utilized by leptin or insulin. Taken together with the divergent phenotypes of POMCa2KO and AgRPa2KO mice, our findings suggest that while AMPK plays a key role in hypothalamic function, it does not act as a general sensor and integrator of energy homeostasis in the mediobasal hypothalamus

The role of insulin receptor substrate signalling pathways in the regulation of energy homeostasis

Title ofthesis: The Role of Insulin Receptor Substrate Signalling Pathways in the Regulation of Energy Homeostasis Degree: Doctor of Philosophy Leptin and insulin act as adiposity signals signalling in the brain to regulate energy homeostasis. However, in contrast to leptin, the precise details of the cell types and signalling pathways involved in the actions of insulin have not been well defined. The dominant view in the field at the commencement of this work was largely extrapolated from studies on leptin action. It was therefore suggested that insulin exerted its effects on energy balance by inhibiting orexigenic NPY / AgRP and activating anorexigenic PO MC/CART neurons of the arcuate nucleus region of the hypothalamus. thereby coordinately regulating energy homeostasis. Mouse genetic studies have demonstrated that insulin receptor substrate (IRS) 2, a major downstream effector of insulin signalling, plays a key role in the regulation of glucose and energy homeostasis. Mice lacking Irs2 in all tissues exhibit insulin resistance, hyperglycaemia and ~-cell failure. In addition, Irs2 null female mice are hyperphagic, obese and infertile. However, the precise contribution of CNS IRS2 signalling in the actions of insulin and leptin, and the identity of the neuronal circuits in which IRS2 acts to regulate energy homeostasis, are unclear. The PI3K signalling pathway has been implicated in mediating the effects of insulin and leptin, in part acting downstream ofIRS signalling. However, the precise hypothalamic cell types in which PI3K signalling acts also remain to be defined. To address these issues, mice with deletion of Irs2 in all neurons (NesCrelrs2KO), POMC neurons (POMCCrelrs2KO) and AgRP neurons (AgRPCrelrs2KO) were generated. Animals lacking the PI3K pll0~ catalytic subunit in POMC (POMCCrep110~KO) and AgRP neurons (AgRPCrep110~KO) were also generated. NesCrelrs2KO animals were obese, hyperphagic and long, suggesting altered melanocortin function. Despite hyperleptinaemia, NesCrelrs2KO animals were leptin sensitive suggesting that IRS2 pathways are not required for leptin action. Reproductive function in NesCreIrs2KO females was normal. In addition, NesCrelrs2KO mice 4 displayed hyperglycaemia, mild glucose intolerance and hyperinsulinaemia. In contrast, POMCCrelrs2KO and AgRPCrelrs2KO mice exhibited normal hypo thalamic function and glucose homeostasis. AgRPCrep 11 O~K 0 mice were lean and hypophagic. Conversely, POMCCrep110~KO animals demonstrated increased adiposity and are hyperphagia. Taken together, these studies highlight a key role for CNS IRS2 pathways in the regulation of energy homeostasis but demonstrate that CNS IRS2 pathways act in neuronal populations distinct from POMC and AgRPINPY neurons to regulate energy homeostasis. In contrast, p 11 O~-mediated signals in POMC and AgRP neurons play a key role in the regulation of energy homeostasis. Overall, these studies have provided new insights into the role insulin receptor substrate signalling mechanisms in the hypo thalamic regulation of energy homeostasis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Genetic deletion of S6k1 does not rescue the phenotypic deficits observed in the R6/2 mouse model of Huntington’s disease

AbstractHuntington’s disease (HD) is a fatal inherited autosomal dominant neurodegenerative disorder caused by an expansion in the number of CAG trinucleotide repeats in the huntingtin gene. The disease is characterized by motor, behavioural and cognitive symptoms for which at present there are no disease altering treatments. It has been shown that manipulating the mTOR (mammalian target of rapamycin) pathway using rapamycin or its analogue CCI-779 can improve the cellular and behavioural phenotypes of HD models. Ribosomal protein S6 kinase 1 (S6K1) is a major downstream signalling molecule of mTOR, and its activity is reduced by rapamycin suggesting that deregulation of S6K1 activity may be beneficial in HD. Furthermore, S6k1 knockout mice have increased lifespan and improvement in age-related phenotypes. To evalute the potential benefit of S6k1 loss on HD-related phenotypes, we crossed the R6/2 HD model with the long-lived S6k1 knockout mouse line. We found that S6k1 knockout does not ameliorate behavioural or physiological phenotypes in the R6/2 mouse model. Additionally, no improvements were seen in brain mass reduction or mutant huntingtin protein aggregate levels. Therefore, these results suggest that while a reduction in S6K1 signalling has beneficial effects on ageing it is unlikely to be a therapeutic strategy for HD patients.</jats:p

Brain Deletion of Insulin Receptor Substrate 2 Disrupts Hippocampal Synaptic Plasticity and Metaplasticity

Diabetes mellitus is associated with cognitive deficits and an increased risk of dementia, particularly in the elderly. These deficits and the corresponding neurophysiological structural and functional alterations are linked to both metabolic and vascular changes, related to chronic hyperglycaemia, but probably also defects in insulin action in the brain. To elucidate the specific role of brain insulin signalling in neuronal functions that are relevant for cognitive processes we have investigated the behaviour of neurons and synaptic plasticity in the hippocampus of mice lacking the insulin receptor substrate protein 2 (IRS-2).). mice, with a concomitant loss of metaplasticity, the modulation of synaptic plasticity by the previous activity of a synapse. These plasticity changes are associated with reduced basal phosphorylation of the NMDA receptor subunit NR1 and of downstream targets of the PI3K pathway, the protein kinases Akt and GSK-3β.These findings reveal molecular and cellular mechanisms that might underlie cognitive deficits linked to specific defects of neuronal insulin signalling

Insulin receptor substrate 2 is a negative regulator of memory formation

Insulin has been shown to impact on learning and memory in both humans and animals, but the downstream signaling mechanisms involved are poorly characterized. Insulin receptor substrate-2 (Irs2) is an adaptor protein that couples activation of insulin- and insulin-like growth factor-1 receptors to downstream signaling pathways. Here, we have deleted Irs2, either in the whole brain or selectively in the forebrain, using the nestin Cre- or D6 Cre-deleter mouse lines, respectively. We show that brain- and forebrain-specific Irs2 knockout mice have enhanced hippocampal spatial reference memory. Furthermore, NesCreIrs2KO mice have enhanced spatial working memory and contextual- and cued-fear memory. Deletion of Irs2 in the brain also increases PSD-95 expression and the density of dendritic spines in hippocampal area CA1, possibly reflecting an increase in the number of excitatory synapses per neuron in the hippocampus that can become activated during memory formation. This increase in activated excitatory synapses might underlie the improved hippocampal memory formation observed in NesCreIrs2KO mice. Overall, these results suggest that Irs2 acts as a negative regulator on memory formation by restricting dendritic spine generation.</jats:p

NMDA receptor-mediated short-term plasticity (STP) is impaired in adult <i>NesCreIrs2KO</i> mice.

<p><b>A</b>: High-intensity theta burst stimulation (H-TBS) was applied to Schaffer collateral-commissural fibres following at least 15 minutes of stable baseline recording of synaptic activity in slices from 5–10 month old mice that had previously undergone behavioural training. In the absence of GABA receptor inhibitors, H-TBS induced robust long-term potentiation (LTP) of similar magnitude in both control (+/+; 160±7%, N = 7, n = 10) and <i>NesCreIrs2KO</i> mice (−/−; 152±6%, N = 5, n = 7), when measured 60 min following induction. Recordings made following H-TBS commence at time 0. <b>B</b>: Graph illustrates the same data as in <b>A</b>, but on an expanded time scale. The STP measured 2 min following H-TBS was significantly reduced in adult <i>NesCreIrs2KO</i> mice (−/−; average EPSP slope change: 110±5%, N = 5, n = 7) compared with that recorded from littermate controls (+/+; average EPSP slope change: 134±6%, N = 7, n = 10, p<0.01) during the same post-stimulus period. Insets illustrate typical EPSP traces (average of 4 consecutive sweeps) recorded immediately prior to, and either 60 min (<b>A</b>) or 2 min (<b>B</b>) following H-TBS in either control (+/+) or <i>NesCreIrs2KO</i> (−/−) mice. <b>C</b>: No significant change in paired-pulse facilitation (PPF) was observed following H-TBS in either control (pre H-TBS: 1.7±0.1; post H-TBS: 1.6±0.1; N = 5, n = 6) or <i>NesCreIrs2KO</i> mice (pre H-TBS: 1.6±0.2; post H-TBS: 1.5±0.1; N = 4, n = 5). Similarly, during the 2 min immediately following H-TBS, there was no significant difference in PPF between genotypes (+/+: 1.6±0.1; −/−: 1.5±0.1). <b>D</b>: Representative PPF traces (average of 4 consecutive sweeps) taken from single experiments carried out on a control (+/+) and <i>NesCreIrs2KO</i> (−/−) slice. EPSP traces are averages of 2 min recording immediately prior to, and following H-TBS. <b>E</b>: In the presence of the NMDA receptor antagonist DL-AP5 (50 µM), STP, measured 2 min following H-TBS, was significantly impaired in control mice (+/+ AP5; average EPSP slope change: 89±10%, N = 3, n = 5) compared with values obtained in the absence of DL-AP5 (+/+ control; average EPSP slope change: 134±6%, N = 7, n = 10, p<0.01). <b>F</b>: DL-AP5 had no significant effect on STP in <i>NesCreIrs2KO</i> mice (−/−) (−/− AP5; average EPSP slope change: 111±12%, N = 3, n = 5), compared with STP values obtained in the absence of DL-AP5 (−/− control; average EPSP slope change: 110±4%, N = 5, n = 6). Arrows indicate application of H-TBS.</p