The role of insulin receptor substrate 2 in hypothalamic and β cell function

Insulin receptor substrate 2 (Irs2) plays complex roles in energy homeostasis. We generated mice lacking Irs2 in β cells and a population of hypothalamic neurons (RIPCreIrs2KO), in all neurons (NesCreIrs2KO), and in proopiomelanocortin neurons (POMCCreIrs2KO) to determine the role of Irs2 in the CNS and β cell. RIPCreIrs2KO mice displayed impaired glucose tolerance and reduced β cell mass. Overt diabetes did not ensue, because β cells escaping Cre-mediated recombination progressively populated islets. RIPCreIrs2KO and NesCreIrs2KO mice displayed hyperphagia, obesity, and increased body length, which suggests altered melanocortin action. POMCCreIrs2KO mice did not display this phenotype. RIPCreIrs2KO and NesCreIrs2KO mice retained leptin sensitivity, which suggests that CNS Irs2 pathways are not required for leptin action. NesCreIrs2KO and POMCCreIrs2KO mice did not display reduced β cell mass, but NesCreIrs2KO mice displayed mild abnormalities of glucose homeostasis. RIPCre neurons did not express POMC or neuropeptide Y. Insulin and a melanocortin agonist depolarized RIPCre neurons, whereas leptin was ineffective. Insulin hyperpolarized and leptin depolarized POMC neurons. Our findings demonstrate a critical role for IRS2 in β cell and hypothalamic function and provide insights into the role of RIPCre neurons, a distinct hypothalamic neuronal population, in growth and energy homeostasis

5-ht inhibition of rat insulin 2 promoter cre recombinase transgene and proopiomelanocortin neuron excitability in the mouse arcuate nucleus

A number of anti-obesity agents have been developed that enhance hypothalamic 5-HT transmission. Various studies have demonstrated that arcuate neurons, which express proopiomelanocortin peptides (POMC neurons), and neuropeptide Y with agouti-related protein (NPY/AgRP) neurons, are components of the hypothalamic circuits responsible for energy homeostasis. An additional arcuate neuron population, rat insulin 2 promoter Cre recombinase transgene (RIPCre) neurons, has recently been implicated in hypothalamic melanocortin circuits involved in energy balance. It is currently unclear how 5-HT modifies neuron excitability in these local arcuate neuronal circuits. We show that 5-HT alters the excitability of the majority of mouse arcuate RIPCre neurons, by either hyperpolarization and inhibition or depolarization and excitation. RIPCre neurons sensitive to 5-HT, predominantly exhibit hyperpolarization and pharmacological studies indicate that inhibition of neuronal firing is likely to be through 5-HT1F receptors increasing current through a voltage-dependent potassium conductance. Indeed, 5-HT1F receptor immunoreactivity co-localizes with RIPCre green fluorescent protein expression. A minority population of POMC neurons also respond to 5-HT by hyperpolarization, and this appears to be mediated by the same receptor-channel mechanism. As neither POMC nor RIPCre neuronal populations display a common electrical response to 5-HT, this may indicate that sub-divisions of POMC and RIPCre neurons exist, perhaps serving different outputs

The effects of adenosine and guanine nucleotides on the ATP-activated K+ channel in rat isolated hypothalamic neurones

No abstract available

Activation by intracellular ATP of a potassium channel in neurones from rat basomedial hypothalamus

1. Cell-attached recordings from isolated glucose-sensitive hypothalamic neurones show that on removal of extracellular glucose there is an increased action current frequency concomitant with decreased single-channel activity. Conversely activation of single K+ channels was observed when extracellular glucose was increased. Isolation of membrane patches into the inside-out configuration following cell-attached recording demonstrated the presence of an ATP-activated K+ channel. 2. The ATP-activated K+ channel was characterized by a mean single-channel conductance of 132 pS in symmetrical 140 mM KCl solutions. Single-channel open-state probability (Po) was not calcium dependent, and the presence of calcium did not prevent activation of the channel by ATP. 3. Activation of the channel by ATP was concentration dependent and the Po of the ATP-activated channel was unaffected by membrane voltage, regardless of the degree of activation elicited by ATP. 4. Open and closed time histograms were constructed from inside-out and cell-attached recordings and were consistent with a single open and two closed states. Channel openings were grouped in bursts. Application of ATP, in isolated patches, and glucose, in cell-attached patches, increased the burst duration and number of bursts per second and decreased the slow closed-state time constant. In neither case was there a significant change in the fast closed-state time constant nor the open-state time constant. 5. The non-hydrolysable ATP analogue adenylylimidodiphosphate (AMP(PNP)) and ‘Mg2(+)-free’ ATP produced little change in the Po of the ATP-activated K+ channel when applied to the intracellular surface of excised patches. These results suggest that activation of this channel is via an enzymic mechanism. 6. ADP, GTP and GDP also activated the channel in a Mg(2+)-dependent manner. ADP and ATP activated the channel in an additive manner and neither GTP nor GDP inhibited channel activity induced by ATP. 7. It is concluded that the ATP-activated K+ channel observed in isolated inside-out patches from hypothalamic neurones is the same as the channel activated by an increase in the concentration of extracellular glucose in cell-attached recordings from glucose-sensitive neurones

Characterization of an ATP-modulated large conductance Ca2+-activated K+ channel present in rat cortical neurones

1. Single channel current recordings were used to study the characteristics of a large conductance Ca(2+)-activated K+ (BKCa) channel present in neurones acutely dissociated from the rat motor cortex. Application of ATP to the intracellular surface of excised inside-out patches produced a large, concentration-dependent increase in BKCa channel activity. 2. This ATP-mediated activation was dependent upon the presence of Mg2+ in the intracellular bathing solution and was diminished by the phosphatases 2,3-butanedione monoxime (BDM) or alkaline phosphatase and by the protein kinase inhibitors staurosporine, H-7 and PKI. 3. ADP stimulated BKCa channel activity in a Mg(2+)-dependent manner, an action also inhibited by the concomitant application of PKI or BDM. The effect of ADP was reduced by application of hexokinase and glucose or by application of the adenylate kinase inhibitor Ap5A. 4. Of other nucleotides tested, only CTP consistently activated BKCa channel activity. 5. Using the cell-attached configuration, bath application of forskolin or dibutyryl cAMP stimulated BKCa channel activity. 6. It is concluded that BKCa channel activity in the rat motor cortex is subject to modulation by the activity of a closely associated kinase. The ability of cAMP activators to stimulate BKCa channel activity in the intact cell suggests that this system may be of physiological importance

Down-regulation of the α- and β-subunits of the calcium-activated potassium channel in human myometrium with parturition

Large-conductance, calcium-dependent potassium (BKCa) channels are implicated in maintaining uterine quiescence during pregnancy. The mechanisms whereby calcium sensitivity of the BKCa channel is dramatically removed at parturition remain unknown. The aim of the present study was to investigate whether this loss of calcium sensitivity of the BKCa channel with the onset of labor is associated with changes in the protein expression of the a- and/or ß-subunit or arises from a physical dissociation of the a-subunit from the ß-subunit. The ß-subunit is a key determinant of BKCa-channel Ca2+ sensitivity. Western blot analysis, using a- and ß-subunit-specific antibodies, detected bands of 110-125 and 36 kDa, respectively. Protein expression levels of the a-subunit in term labor myometrium were significantly reduced compared with term pregnancy without labor. Furthermore, a-subunit levels at term pregnancy were significantly increased relative to the nonpregnant state, whereas levels at preterm gestations were unchanged. Densitometric analysis demonstrated significantly decreased ß-subunit levels in term and preterm labor samples compared with term nonlabor samples. Immunoprecipitation studies revealed the presence of both the a- and ß-subunits in samples taken before or after the onset of labor. We conclude that during labor, the a-subunit is not physically uncoupled from the ß-subunit, but a decline occurs in the level of ß-subunit protein, which may underlie the loss of calcium and voltage sensitivity of the BKCa channel with labor. Furthermore, reduced ß-subunit protein in preterm labor myometrium implies that ion channels may also contribute to pathophysiological labor.</p

The effects of trypsin on ATP-sensitive potassium channel properties and sulfonylurea receptors in the CRI-G1 insulin-secreting cell line

The effects of the proteolytic enzyme trypsin upon ATP-sensitive potassium (KATP) channel activity were examined in the CRI-G1 insulin-secreting cell line. Trypsin activated channels only when applied to the intracellular surface of the cell membrane. The activation could be prevented by the concomitant application of trypsin inhibitor or by heat inactivation of the enzyme. The trypsin-induced change in channel activity was accompanied by a reduction in the rate of channel rundown. However, trypsin did not affect the mean single channel conductance (55.2 pS), the ionic selectivity, or rectification of the KATP channel. Concentration response curves for various KATP channel inhibitors were constructed in the presence and absence of intracellular trypsin. The EC50 for tolbutamide was shifted from 30.0 +/- 4.5 microM, with 100 micrograms/ml heat-inactivated trypsin present to 9.7 +/- 1.0 mM with active trypsin in the intracellular solution. Treatment of the cells' external surface with 1 mg/ml trypsin did not alter the potency of tolbutamide. Intracellular trypsin also produced a significant fall in the potency of glibenclamide, meglitinide, and phentolamine but did not alter the effectiveness of thiopentone. Radioligand binding studies demonstrated a total loss of 3H-labeled glibenclamide binding when the intracellular surface of the cells was exposed to trypsin. In contrast, 3H-labeled glibenclamide binding was not affected when the enzyme was applied to the external surface. Trypsin treatment, therefore, alters a number of characteristics of KATP channel pharmacology, and we suggest that this is due to action at possibly more than one site but includes the functional cleavage of the sulfonylurea receptor from the KATP channel

Supplementary Material for: Anorexigenic and Orexigenic Hormone Modulation of Mammalian Target of Rapamycin Complex 1 Activity and the Regulation of Hypothalamic Agouti-Related Protein mRNA Expression

Activation of mammalian target of rapamycin 1 (mTORC1) by nutrients, insulin and leptin leads to appetite suppression (anorexia). Contrastingly, increased AMP-activated protein kinase (AMPK) activity by ghrelin promotes appetite (orexia). However, the interplay between these mechanisms remains poorly defined. The relationship between the anorexigenic hormones, insulin and leptin, and the orexigenic hormone, ghrelin, on mTORC1 signalling was examined using S6 kinase phosphorylation as a marker for changes in mTORC1 activity in mouse hypothalamic GT1-7 cells. Additionally, the contribution of AMPK and mTORC1 signalling in relation to insulin-, leptin- and ghrelin-driven alterations to mouse hypothalamic agouti-related protein (AgRP) mRNA levels was examined. Insulin and leptin increase mTORC1 activity in a phosphoinositide-3-kinase (PI3K)- and protein kinase B (PKB)-dependent manner, compared to vehicle controls, whereas increasing AMPK activity inhibits mTORC1 activity and blocks the actions of the anorexigenic hormones. Ghrelin mediates an AMPK-dependent decrease in mTORC1 activity and increases hypothalamic AgRP mRNA levels, the latter effect being prevented by insulin in an mTORC1-dependent manner. In conclusion, mTORC1 acts as an integration node in hypothalamic neurons for hormone-derived PI3K and AMPK signalling and mediates at least part of the assimilated output of anorexigenic and orexigenic hormone actions in the hypothalamus

Chronic hyperglycaemia increases the vulnerability of the hippocampus to oxidative damage induced during post-hypoglycaemic hyperglycaemia in a mouse model of chemically induced type 1 diabetes

Aims/hypothesis Chronic hyperglycaemia and recurrent hypoglycaemia are independently associated with accelerated cognitive decline in type 1 diabetes. Recurrent hypoglycaemia in rodent models of chemically induced (streptozotocin [STZ]) diabetes leads to cognitive impairment in memory-related tasks associated with hippocampal oxidative damage. This study examined the hypothesis that post-hypoglycaemic hyperglycaemia in STZ-diabetes exacerbates hippocampal oxidative stress and explored potential contributory mechanisms.Methods The hyperinsulinaemic glucose clamp technique was used to induce equivalent hypoglycaemia and to control post-hypoglycaemic glucose levels in mice with and without STZ-diabetes and Nrf2(-/-) mice (lacking Nrf2 [also known as Nfe2l2]). Subsequently, quantitative proteomics based on stable isotope labelling by amino acids in cell culture and biochemical approaches were used to assess oxidative damage and explore contributory pathways.Results Evidence of hippocampal oxidative damage was most marked in mice with STZ-diabetes exposed to post-hypoglycaemic hyperglycaemia; these mice also showed induction of Nrf2 and the Nrf2 transcriptional targets Sod2 and Hmox-1. In this group, hypoglycaemia induced a significant upregulation of proteins involved in alternative fuel provision, reductive biosynthesis and degradation of damaged proteins, and a significant downregulation of proteins mediating the stress response. Key differences emerged between mice with and without STZ-diabetes following recovery from hypoglycaemia in proteins mediating the stress response and reductive biosynthesis.Conclusions/interpretation There is a disruption of the cellular response to a hypoglycaemic challenge in mice with STZ-induced diabetes that is not seen in wild-type non-diabetic animals. The chronic hyperglycaemia of diabetes and post-hypoglycaemic hyperglycaemia act synergistically to induce oxidative stress and damage in the hippocampus, possibly leading to irreversible damage/modification to proteins or synapses between cells. In conclusion, recurrent hypoglycaemia in sub-optimally controlled diabetes may contribute, at least in part, to accelerated cognitive decline through amplifying oxidative damage in key brain regions, such as the hippocampus.Data availability The datasets generated during and/or analysed during the current study are available in ProteomeXchange, accession no. 1-20220824-173727 (www.proteomexchange.org). Additional datasets generated during and/or analysed during the present study are available from the corresponding author upon reasonable request