PAS kinase is a nutrient and energy sensor in hypothalamic areas required for the normal function of AMPK and mTOR/S6K1.

The complications caused by overweight, obesity and type 2 diabetes are one of the main problems that increase morbidity and mortality in developed countries. Hypothalamic metabolic sensors play an important role in the control of feeding and energy homeostasis. PAS kinase (PASK) is a nutrient sensor proposed as a regulator of glucose metabolism and cellular energy. The role of PASK might be similar to other known metabolic sensors, such as AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR). PASK-deficient mice resist diet-induced obesity. We have recently reported that AMPK and mTOR/S6K1 pathways are regulated in the ventromedial and lateral hypothalamus in response to nutritional states, being modulated by anorexigenic glucagon-like peptide-1 (GLP-1)/exendin-4 in lean and obese rats. We identified PASK in hypothalamic areas, and its expression was regulated under fasting/re-feeding conditions and modulated by exendin-4. Furthermore, PASK-deficient mice have an impaired activation response of AMPK and mTOR/S6K1 pathways. Thus, hypothalamic AMPK and S6K1 were highly activated under fasted/re-fed conditions. Additionally, in this study, we have observed that the exendin-4 regulatory effect in the activity of metabolic sensors was lost in PASK-deficient mice, and the anorexigenic properties of exendin-4 were significantly reduced, suggesting that PASK could be a mediator in the GLP-1 signalling pathway. Our data indicated that the PASK function could be critical for preserving the nutrient effect on AMPK and mTOR/S6K1 pathways and maintain the regulatory role of exendin-4 in food intake. Some of the antidiabetogenic effects of exendin-4 might be modulated through these processes.

PAS kinase as a nutrient sensor in neuroblastoma and hypothalamic cells required for the normal expression and activity of other cellular nutrient and energy sensors.

PAS kinase (PASK) is a nutrient sensor that is highly conserved throughout evolution. PASK-deficient mice reveal a metabolic phenotype similar to that described in S6 kinase-1 S6K1-deficient mice that are protected against obesity. Hypothalamic metabolic sensors, such as AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR), play an important role in feeding behavior, the homeostasis of body weight, and energy balance. These sensors respond to changes in nutrient levels in the hypothalamic areas involved in feeding behavior and in neuroblastoma N2A cells, and we have recently reported that those effects are modulated by the anorexigenic peptide glucagon-like peptide-1 (GLP-1). Here, we identified PASK in both N2A cells and rat VMH and LH areas and found that its expression is regulated by glucose and GLP-1. High levels of glucose decreased Pask gene expression. Furthermore, PASK-silenced N2A cells record an impaired response by the AMPK and mTOR/S6K1 pathways to changes in glucose levels. Likewise, GLP-1 effect on the activity of AMPK, S6K1, and other intermediaries of both pathways and the regulatory role at the level of gene expression were also blocked in PASK-silenced cells. The absence of response to low glucose concentrations in PASK-silenced cells correlates with increased ATP content, low expression of mRNA coding for AMPK upstream kinase LKB1, and enhanced activation of S6K1. Our findings indicate that, at least in N2A cells, PASK is a key kinase in GLP-1 actions and exerts a coordinated response with the other metabolic sensors, suggesting that PASK might play an important role in feeding behavior.

Insulin-receptor substrate-2 (irs-2) is required for maintaining glucokinase and glucokinase regulatory protein expression in mouse liver.

Insulin receptor substrate (IRS) proteins play important roles in hepatic nutrient homeostasis. Since glucokinase (GK) and glucokinase regulatory protein (GKRP) function as key glucose sensors, we have investigated the expression of GK and GKRP in liver of Irs-2 deficient mice and Irs2(-/-) mice where Irs2 was reintroduced specifically into pancreatic ß-cells [RIP-Irs-2/IRS-2(-/-)]. We observed that liver GK activity was significantly lower (p<0.0001) in IRS-2(-/-) mice. However, in RIP-Irs-2/IRS-2(-/-) mice, GK activity was similar to the values observed in wild-type animals. GK activity in hypothalamus was not altered in IRS-2(-/-) mice. GK and GKRP mRNA levels in liver of IRS-2(-/-) were significantly lower, whereas in RIP-Irs-2/IRS-2(-/-) mice, both GK and GKRP mRNAs levels were comparable to wild-type animals. At the protein level, the liver content of GK was reduced in IRS-2(-/-) mice as compared with controls, although GKRP levels were similar between these experimental models. Both GK and GKRP levels were lower in RIP-Irs-2/IRS-2(-/-) mice. These results suggest that IRS-2 signalling is important for maintaining the activity of liver GK. Moreover, the differences between liver and brain GK may be explained by the fact that expression of hepatic, but not brain, GK is controlled by insulin. GK activity was restored by the ß-cell compensation in the RIP-Irs-2/IRS-2 mice. Interestingly, GK and GKRP protein expression remained low in RIP-Irs-2/IRS-2(-/-) mice, perhaps reflecting different mRNA half-lives or alterations in the process of translation and post-translational regulation.

Glucagon-like peptide 1 (GLP-1) can reverse AMP-activated protein kinase (AMPK) and S6 kinase (P70S6K) activities induced by fluctuations in glucose levels in hypothalamic areas involved in feeding behaviour.

The anorexigenic peptide, glucagon-like peptide-1 (GLP-1), reduces glucose metabolism in the human hypothalamus and brain stem. The brain activity of metabolic sensors such as AMP-activated protein kinase (AMPK) responds to changes in glucose levels. The mammalian target of rapamycin (mTOR) and its downstream target, p70S6 kinase (p70S6K), integrate nutrient and hormonal signals. The hypothalamic mTOR/p70S6K pathway has been implicated in the control of feeding and the regulation of energy balances. Therefore, we investigated the coordinated effects of glucose and GLP-1 on the expression and activity of AMPK and p70S6K in the areas involved in the control of feeding. The effect of GLP-1 on the expression and activities of AMPK and p70S6K was studied in hypothalamic slice explants exposed to low- and high-glucose concentrations by quantitative real-time RT-PCR and by the quantification of active-phosphorylated protein levels by immunoblot. In vivo, the effects of exendin-4 on hypothalamic AMPK and p70S6K activation were analysed in male obese Zucker and lean controls 1 h after exendin-4 injection to rats fasted for 48 h or after re-feeding for 2-4 h. High-glucose levels decreased the expression of Ampk in the lateral hypothalamus and treatment with GLP-1 reversed this effect. GLP-1 treatment inhibited the activities of AMPK and p70S6K when the activation of these protein kinases was maximum in both the ventromedial and lateral hypothalamic areas. Furthermore, in vivo s.c. administration of exendin-4 modulated AMPK and p70S6K activities in those areas, in both fasted and re-fed obese Zucker and lean control rats.

Effects of glucose and insulin on glucokinase activity in rat hypothalamus.

In an attempt to study the role of glucokinase (GK) and the effects of glucose and peptides on GK gene expression and on the activity of this enzyme in the hypothalamus, we used two kinds of biological models: hypothalamic GT1-7 cells and rat hypothalamic slices. The expression of the GK gene in GT1-7 cells was reduced by insulin (INS) and was not modified by different glucose concentrations, while GK enzyme activities were significantly reduced by the different peptides. Interestingly, a distinctive pattern of GK activities between the ventromedial hypothalamus (VMH) and lateral hypothalamus (LH) were found, with higher enzyme activities in the VMH as the glucose concentrations rose, while LH enzyme activities decreased at 2.8 and 20 mM glucose, the latter effect being prevented by incubation with INS. These effects were produced only by d-glucose and the modifications found were due to GK, but not to other hexokinases. In addition, GK activities in the VMH and the LH were reduced by glucagon-like peptide 1, leptin, orexin B, INS, and neuropeptide Y (NPY), but this effect was only statistically significant for NPY in LH. Our results indicate that the effects of both glucose and peptides occur on GK enzyme activities rather than on GK gene transcription. Moreover, the effects of glucose and INS on GK activity suggest that in the brain GK behaves in a manner opposite to that in the liver, which might facilitate its role in glucose sensing. Finally, hypothalamic slices seem to offer a good physiological model to discriminate the effects between different areas.

Effects of novel maturity-onset diabetes of the young (MODY)-associated mutations on glucokinase activity and protein stability.

Glucokinase acts as the pancreatic glucose sensor and plays a critical role in the regulation of insulin secretion by the beta-cell. Heterozygous mutations in the glucokinase-encoding GCK gene, which result in a reduction of the enzymatic activity, cause the monogenic form of diabetes, MODY2 (maturity-onset diabetes of the young 2). We have identified and functionally characterized missense mutations in the GCK gene in diabetic families that result in protein mutations Leu165-->Phe, Glu265-->Lys and Thr206-->Met. The first two are novel GCK mutations that co-segregate with the diabetes phenotype in their respective families and are not found in more than 50 healthy control individuals. In order to measure the biochemical effects of these missense mutations on glucokinase activity, we bacterially expressed and affinity-purified islet human glucokinase proteins carrying the respective mutations and fused to GST (glutathione S-transferase). Enzymatic assays on the recombinant proteins revealed that mutations Thr206-->Met and Leu165-->Phe strongly affect the kinetic parameters of glucokinase, in agreement with the localization of both residues close to the active site of the enzyme. In contrast, mutation Glu265-->Lys, which has a weaker effect on the kinetics of glucokinase, strongly affects the protein stability, suggesting a possible structural defect of this mutant protein. Finally, none of the mutations tested appears to affect the interaction of gluco-kinase with the glucokinase regulatory protein in the yeast two-hybrid system.

The cytoplasmic domain close to the transmembrane region of the glucagon-like peptide-1 receptor contains sequence elements that regulate agonist-dependent internalisation.

In order to gain better insight into the molecular events involved in the signal transduction generated through glucagon-like peptide-1 (GLP-1) receptors, we tested the effect of deletions and point mutations within the cytoplasmic tail of this receptor with a view to establishing relationships between signal transduction desensitisation and receptor internalisation. Wild-type and truncated (deletion of the last 27 amino acids (GLPR 435R) and deletion of 44 amino acids (GLPR 418R)) GLP-1 receptors bound the agonist with similar affinity. Deletion of the last 27 amino acids decreased the internalisation rate by 78%, while deletion of 44 amino acids containing all the phosphorylation sites hitherto described in this receptor decreased the internalisation rate by only 47%. Binding of the ligand to both receptors stimulated adenylyl cyclase. In contrast, deletion of the region containing amino acids 419 to 435 (GLPR 419delta435) increased the internalisation rate by 268%, and the replacement of EVQ(408-410) by alanine (GLPR A(408-410)) increased this process to 296%. In both receptors, the efficacy in stimulating adenylate cyclase was decreased. All the receptors studied were internalised by coated pits, except for the receptor with a deletion of the last 44 amino acids, which also had a faster resensitisation rate. Our findings indicate that the neighbouring trans-membrane domain of the carboxyl-terminal tail of the GLP-1 receptor contains sequence elements that regulate agonist-dependent internalisation and transmembrane signalling.

Substitution of the cysteine 438 residue in the cytoplasmic tail of the glucagon-like peptide-1 receptor alters signal transduction activity.

Several G-protein-coupled receptors contain cysteine residues in the C-terminal tail that may modulate receptor function. In this work we analysed the substitution of Cys438 by alanine in the glucagon-like peptide-1 (GLP-1) receptor (GLPR), which led to a threefold decrease in cAMP production, although endocytosis and cellular redistribution of GLP-1 receptor agonist-induced processes were unaffected. Additionally, cysteine residues in the C-terminal tail of several G-protein-coupled receptors were found to act as substrates for palmitoylation, which might modify the access of protein kinases to this region. His-tagged GLP-1 receptors incorporated 3H-palmitate. Nevertheless, substitution of Cys438 prevented the incorporation of palmitate. Accordingly, we also investigated the effect of substitution of the consensus sequence by protein kinase C (PKC) Ser431/432 in both wild-type and Ala438 GLP-1 receptors. Substitution of Ser431/432 by alanine did not modify the ability of wild-type receptors to stimulate adenylate cyclase or endocytosis and recycling processes. By contrast, the substitution of Ser431/432 by alanine in the receptor containing Ala438 increased the ability to stimulate adenylate cyclase. All types of receptors were mainly internalised through coated pits. Thus, cysteine 438 in the cytoplasmic tail of the GLP-1 receptor would regulate its interaction with G-proteins and the stimulation of adenylyl cyclase. Palmitoylation of this residue might control the access of PKC to Ser431/432.

The expression of GLP-1 receptor mRNA and protein allows the effect of GLP-1 on glucose metabolism in the human hypothalamus and brainstem.

In the present work, several experimental approaches were used to determine the presence of the glucagon-like peptide-1 receptor (GLP-1R) and the biological actions of its ligand in the human brain. In situ hybridization histochemistry revealed specific labelling for GLP-1 receptor mRNA in several brain areas. In addition, GLP-1R, glucose transporter isoform (GLUT-2) and glucokinase (GK) mRNAs were identified in the same cells, especially in areas of the hypothalamus involved in feeding behaviour. GLP-1R gene expression in the human brain gave rise to a protein of 56 kDa as determined by affinity cross-linking assays. Specific binding of 125I-GLP-1(7-36) amide to the GLP-1R was detected in several brain areas and was inhibited by unlabelled GLP-1(7-36) amide, exendin-4 and exendin (9-39). A further aim of this work was to evaluate cerebral-glucose metabolism in control subjects by positron emission tomography (PET), using 2-[F-18] deoxy-D-glucose (FDG). Statistical analysis of the PET studies revealed that the administration of GLP-1(7-36) amide significantly reduced (p < 0.001) cerebral glucose metabolism in hypothalamus and brainstem. Because FDG-6-phosphate is not a substrate for subsequent metabolic reactions, the lower activity observed in these areas after peptide administration may be due to reduction of the glucose transport and/or glucose phosphorylation, which should modulate the glucose sensing process in the GLUT-2- and GK-containing cells.

Expression of glucose transporter isoform GLUT-2 and glucokinase genes in human brain.

The glucose transporter isoform-2 (GLUT-2) and glucokinase are considered to be components of a glucose sensor system controlling several key processes, and hence may modulate feeding behaviour. We have found GLUT-2 and glucokinase mRNAs in several brain regions, including the ventromedial and arcuate nuclei of the hypothalamus. GLUT-2, glucokinase and glucokinase regulatory protein mRNAs and proteins were present in these areas as determined by biochemical approaches. In addition, glucose-phosphorylating activity with a high apparent Km for glucose that displayed no product inhibition by glucose-6-phosphate was observed. Increased glycaemia after meals may be recognized by specific hypothalamic neurones due to the high Km of GLUT-2 and glucokinase. This enzyme is considered to be the true glucose sensor because it catalyses the rate-limiting step of glucose catabolism its activity being regulated by interaction with glucokinase regulatory protein, that functions as a metabolic sensor.

Evidence that glucokinase regulatory protein is expressed and interacts with glucokinase in rat brain.

Our previous description of functional glucokinase isoforms in the rat brain has opened new questions concerning the presence of glucokinase regulatory protein in the brain and the functional role of its interactions with glucokinase. In this study, we found glucokinase regulatory protein mRNA in rat brain, pancreatic islets and liver. In addition, we found two other variant splicing isoforms, both identified in hypothalamus, pancreatic islets and liver. In situ hybridization studies revealed the presence of glucokinase regulatory protein mRNA, the highest number of positive cells being found in the paraventricular nucleus of the hypothalamus. Glucokinase regulatory protein gene expression gave rise to a protein of 69 kDa mainly in nuclear and soluble cell fractions. Glutathione S-transferase protein fused either to rat liver or human pancreatic islet glucokinase were able to precipitate glucokinase regulatory protein from liver or hypothalamic extracts in the presence of fructose-6-phosphate, the amount of protein co-precipitated being decreased with fructose-1-phosphate. These findings suggest that the presence of glucokinase and glucokinase regulatory protein in the rat brain would facilitate the adaptation of this organ to fluctuations in blood glucose concentrations, and both proteins may participate in glucose-sensing and metabolic regulation in the central nervous system.