Dietary supplementation of clinically utilized PI3K p110α inhibitor extends the lifespan of male and female mice.

Diminished insulin and insulin-like growth factor-1 signaling extends the lifespan of invertebrates1-4; however, whether it is a feasible longevity target in mammals is less clear5-12. Clinically utilized therapeutics that target this pathway, such as small-molecule inhibitors of phosphoinositide 3-kinase p110α (PI3Ki), provide a translatable approach to studying the impact of these pathways on aging. Here, we provide evidence that dietary supplementation with the PI3Ki alpelisib from middle age extends the median and maximal lifespan of mice, an effect that was more pronounced in females. While long-term PI3Ki treatment was well tolerated and led to greater strength and balance, negative impacts on common human aging markers, including reductions in bone mass and mild hyperglycemia, were also evident. These results suggest that while pharmacological suppression of insulin receptor (IR)/insulin-like growth factor receptor (IGFR) targets could represent a promising approach to delaying some aspects of aging, caution should be taken in translation to humans.

Phosphoinositide 3-kinases and membrane traffic.

Phosphoinositide 3-kinases (PI 3-kinases) and their 3-phosphoinositide products were identified initially as components of intracellular signalling pathways emanating from cell surface receptors. A new role for 3-phosphoinositides in the constitutive movement o f proteins from one intracellular compartment to another was proposed with the discovery of homology between the product of a yeast gene important for vacuolar sorting, Vps34p, and a mammalian PI 3-kinase. Recent studies have implicated PI 3-kinase as an essential component in membrane traffic at specific steps o f the trans-Golgi-network-endosomal pre-lysosomal system. Evidence largely emerging from the insulin-stimulated glucose transport system suggests that PI 3-kinase may also mediate the effects o f growth factors on membrane traffic events. These studies suggest a possible link between growth-factor-stimulated and constitutive membrane traffic in the endosomal system.

Atypical antipsychotic drugs induce derangements in glucose homeostasis by acutely increasing glucagon secretion and hepatic glucose output in the rat.

AIMS/HYPOTHESIS: Use of the second-generation antipsychotic drugs (SGAs) results in the development of obesity and a type 2 diabetes-like syndrome. We hypothesised that, in addition to the insulin resistance associated with the obesity, the SGAs might have acute effects on glucose metabolism that could contribute to the derangements in glucose metabolism. METHODS: We investigated the effects of therapeutically relevant levels of three different antipsychotic medications (haloperidol, quetiapine and clozapine) on glucose tolerance, measures of insulin resistance and hepatic glucose production, and on insulin and glucagon secretion in rats. RESULTS: We found that these drugs induce impaired glucose tolerance in rats that is associated with increased insulin secretion (clozapine>quetiapine>haloperidol) but is independent of weight gain. However, Akt/protein kinase B activation is normal, and at these levels of drug there was no effect on insulin action in fat cells or soleus muscle, and no effect on insulin sensitivity as evaluated by insulin tolerance tests. We show that clozapine induces increased glucose levels following pyruvate and glycerol challenges, indicating an increase in hepatic glucose output (HGO). Increased HGO would in turn increase insulin release and would explain the apparent phenotype mimicking insulin resistance. We provide evidence that this effect could at least in part be mediated by a stimulation of glucagon secretion. CONCLUSIONS/INTERPRETATION: Our findings indicate that SGAs can cause acute derangements in glucose metabolism that are not caused by a direct induction of insulin resistance but act via an increase in glucagon secretion and thus stimulation of hepatic glucose production.

Insulin and leptin acutely regulate cholesterol ester metabolism in macrophages by novel signaling pathways.

Leptin is produced in adipose tissue and acts in the hypothalamus to regulate food intake. However, recent evidence also indicates a potential for direct roles for leptin in peripheral tissues, including those of the immune system. In this study, we provide direct evidence that macrophages are a target tissue for leptin. We found that J774.2 macrophages express the functional long form of the leptin receptor (ObRb) and that this becomes tyrosine-phosphorylated after stimulation with low doses of leptin. Leptin also stimulates both phosphoinositide 3-kinase (PI 3-kinase) activity and tyrosine phosphorylation of JAK2 and STAT3 in these cells. We investigated the effects of leptin on hormone-sensitive lipase (HSL), which acts as a neutral cholesterol esterase in macrophages and is a rate-limiting step in cholesterol ester breakdown. Leptin significantly increased HSL activity in J774.2 macrophages, and these effects were additive with the effects of cAMP and were blocked by PI 3-kinase inhibitors. Conversely, insulin inhibited HSL in macrophages, but unlike adipocytes, this effect did not require PI 3-kinase. These results indicate that leptin and insulin regulate cholesterol-ester homeostasis in macrophages and, therefore, defects in this process caused by leptin and/or insulin resistance could contribute to the increased incidence of atherosclerosis found associated with obesity and type 2 diabetes.

Human small intestine facilitative fructose/glucose transporter (GLUT5) is also present in insulin-responsive tissues and brain. Investigation of biochemical characteristics and translocation.

A recent study by C.F. Burant et al. (13) demonstrates that GLUT5 is a high-affinity fructose transporter with a much lower capacity to transport glucose. To characterize the potential role of GLUT5 in fructose and glucose transport in insulin-sensitive tissues, we investigated the distribution and insulin-stimulated translocation of the GLUT5 protein in human tissues by immunoblotting with an antibody to the COOH-terminus of the human GLUT5 sequence. GLUT5 was detected in postnuclear membranes from the small intestine, kidney, heart, four different skeletal muscle groups, and the brain, and in plasma membranes from adipocytes. Cytochalasin-B photolabeled a 53,000-M(r) protein in small intestine membranes that was immunoprecipitated by the GLUT5 antibody; labeling was inhibited by D- but not L-glucose. N-glycanase treatment resulted in a band of 45,000 M(r) in all tissues. Plasma membranes were prepared from isolated adipocytes from 5 nonobese and 4 obese subjects. Incubation of adipocytes from either group with 7 nM insulin did not recruit GLUT5 to the plasma membrane, in spite of a 54% insulin-stimulated increase in GLUT4 in nonobese subjects. Thus, GLUT5 appears to be a constitutive sugar transporter that is expressed in many tissues. Further studies are needed to define its overall contribution to fructose and glucose transport in insulin-responsive tissues and brain.

Insulin-like growth factors require phosphatidylinositol 3-kinase to signal myogenesis: dominant negative p85 expression blocks differentiation of L6E9 muscle cells.

Phosphatidylinositol 3 (PI 3)-kinases are potently inhibited by two structurally unrelated membrane-permeant reagents: wortmannin and LY294002. By using these two inhibitors we first suggested the involvement of a PI 3-kinase activity in muscle cell differentiation. However, several reports have described that these compounds are not as selective for PI 3-kinase activity as assumed. Here we show that LY294002 blocks the myogenic pathway elicited by insulin-like growth factors (IGFs), and we confirm the specific involvement of PI 3-kinase in IGF-induced myogenesis by overexpressing in L6E9 myoblasts a dominant negative p85 PI 3-kinase-regulatory subunit (L6E9-delta p85). IGF-I, des(1-3)IGF-I, or IGF-II induced L6E9 skeletal muscle cell differentiation as measured by myotube formation, myogenin gene expression, and GLUT4 glucose carrier induction. The addition of LY294002 to the differentiation medium totally inhibited these IGF-induced myogenic events without altering the expression of a non-muscle-specific protein, beta1-integrin. Independent clones of L6E9 myoblasts expressing a dominant negative mutant of the p85-regulatory subunit (delta p85) showed markedly impaired glucose transport activity and formation of p85/p110 complexes in response to insulin, consistent with the inhibition of PI 3-kinase activity. IGF-induced myogenic parameters in L6E9-delta p85 cells, ie. cell fusion and myogenin gene and GLUT4 expression, were severely impaired compared with parental cells or L6E9 cells expressing wild-type p85. In all, data presented here indicate that PI 3-kinase is essential for IGF-induced muscle differentiation and that the specific PI 3-kinase subclass involved in myogenesis is the heterodimeric p85-p110 enzyme.

High level overexpression of glucose transporter-4 driven by an adipose-specific promoter is maintained in transgenic mice on a high fat diet, but does not prevent impaired glucose tolerance.

High fat feeding is associated with impaired insulin action, an obese body composition, and down-regulation of glucose transporter-4 (GLUT4) expression in adipocytes. We recently showed that overexpression of GLUT4 selectively in adipocytes of transgenic mice using the aP2 (fatty acid-binding protein) promoter/enhancer results in enhanced glucose tolerance and adipocyte hyperplasia. Here, we fed these GLUT4-overexpressing transgenic mice a high fat (55%) or a low fat (10%) diet for 13-15 weeks to determine the role of alterations in GLUT4 expression in adipocytes in the development of insulin resistance and obesity, which are characteristic of high fat consumption. In nontransgenic mice, high fat feeding results in 45-50% reduction of GLUT4 levels in white and brown adipose tissue, with a parallel decrease in insulin-stimulated glucose transport. In transgenic mice receiving the low fat diet, GLUT4 is overexpressed 20-fold in white and 4-fold in brown adipose tissue. Glucose transport in epididymal adipocytes is increased 20-fold in the basal state and 6-fold in the insulin-stimulated state. Even after transgenic mice are fed a high fat diet, GLUT4 expression and glucose transport in their adipocytes remains 14- to 30-fold greater than that in nontransgenic mice receiving the same diet. Despite these marked effects at the adipose cell level, glucose tolerance is not improved, probably due to insulin resistance in skeletal muscle and liver, where the transgene is not expressed. During the low fat diet, transgenic mice have 80% more body lipid than nontransgenics. High fat feeding increases body lipid 76% and adipocyte size 65% in nontransgenic mice, but has no effect in transgenic mice. Thus, overexpression of GLUT4 selectively in adipocytes protects against a further increase in adiposity. Furthermore, by using a heterologous promoter, high level overexpression of GLUT4 can be maintained even under metabolic conditions where it is normally down-regulated in adipocytes. This overexpression results in markedly increased glucose transport at the cellular level, but adipose-specific GLUT4 overexpression does not prevent the decrease in glucose tolerance associated with high fat feeding.

The alpha-isoform of class II phosphoinositide 3-kinase is more effectively activated by insulin receptors than IGF receptors, and activation requires receptor NPEY motifs.

Little is known about the physiological role and mechanism of activation of class II phosphoinositide 3-kinases (PI3Ks), although it has been shown that the PI3K-C2alpha isoform is activated by insulin. Using chimaeric receptor constructs which can be activated independently of endogenous receptors in transfected cells, we found that PI3K-C2alpha activity was stimulated to a greater extent by insulin receptors than IGF receptors in 3T3-L1 adipocytes. Activation of PI3K-C2alpha required an intact NPEY motif in the receptor juxtamembrane domain. We conclude that PI3K-C2alpha is a candidate for participation in insulin-specific intracellular signalling.

Controlled-release indomethacin and sustained-release diclofenac sodium in the treatment of osteoarthritis: a comparative controlled clinical trial in general practice.

A double-blind, crossover study was undertaken in general practice to compare the efficacy and tolerability of a new controlled-release indomethacin with sustained-release diclofenac sodium in patients with osteoarthritis. Eighty-four patients were randomly allocated to receive controlled-release indomethacin tablets (75 mg) or sustained-release diclofenac sodium tablets (100 mg) at night for a period of 4 weeks before being crossed-over to receive the alternative treatment for a further 4 weeks. Pain scores for day and night, duration of morning stiffness, requirement for escape analgesia, and treatment preference were similar for both treatments. There was no significant difference between treatments for incidence and severity of side-effects. It was concluded that controlled-release indomethacin tablets (75 mg) given as a single night-time dose were as efficacious and well tolerated as sustained-release diclofenac sodium (100 mg).

Glucose tolerance factor potentiation of insulin action in adipocytes from rats raised on a torula yeast diet cannot be attributed to a deficiency of chromium or glucose tolerance factor activity in the diet.

The nature of the dietary component responsible for adipocytes having the ability to respond to Glucose Tolerance Factor (GTF) was investigated. Rats were raised on either a control diet or one of three diets differing only in the protein source (torula yeast, brewer's yeast, or casein). Only in adipocytes from rats fed the torula yeast diet did a GTF fraction prepared from brewer's yeast potentiate the action of suboptimal concentrations of insulin in the incorporation of label from D-[1-14C]-glucose and D-[U-14C]-glucose into CO2 and fatty acids. It was concluded that this potentiation was not the result of a deficiency of GTF activity in torula yeast, because a GTF fraction prepared from torula yeast had similar insulin potentiating activity. Differences in response among diets were not owing to differences in levels of amino acids or owing to concentrations of 22 (Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mo, Na, Ni, P, Pb S, Se, Si, Sn, Sr, Zn) of the 23 trace elements investigated. The level of Mn was low in all diets, but particularly low in the torula yeast diet. Mn deficiencies have previously been implicated in perturbations of glucose metabolism, so that it is possible that this deficiency may be responsible for the effects attributed to the torula yeast diet.

The activation of the Akt/PKB signalling pathway in the brains of clozapine-exposed rats is linked to hyperinsulinemia and not a direct drug effect.

The second generation antipsychotic drug clozapine is a much more effective therapy for schizophrenia than first generation compounds, but the reasons for this are poorly understood. We have previously shown that one distinguishing feature of clozapine is its ability to raise glucagon levels in animal models and thus causes prolonged hyperinsulinemia without inducing hypoglycaemia. Previous studies have provided evidence that defects in Akt/PKB and GSK3 signalling can contribute to development of psychiatric diseases. Clozapine is known to activate Akt/PKB in the brain, and some studies have indicated that this is due to a direct effect of the drug on the neurons. However, we provide strong evidence that elevated insulin levels induced by clozapine are in fact the real cause of the drug's effects on Akt/PKB and GSK3 in the brain. This suggests that the elevated levels of insulin induced by clozapine may contribute to this drug's therapeutic efficacy.

Clozapine directly increases insulin and glucagon secretion from islets: implications for impairment of glucose tolerance.

Second generation antipsychotics cause derangements in glucose metabolism that are often interpreted as insulin resistance. In previous studies we have shown that this is not classical insulin resistance but the drugs were actually inducing a hyperglycaemic state associated with elevated hepatic glucose output (HGO) and increased levels of glucagon and insulin. However, it remains unclear whether these effects are directly elicited by drug actions in the liver and pancreas, or whether they are indirectly mediated. Here we investigated if clozapine is capable of inducing insulin resistance in the liver or enhancing insulin and glucagon secretion from the pancreas. It was observed that insulin signalling was elevated in livers from animals treated with clozapine indicating there was no insulin resistance in the early steps of insulin signalling. To explore whether the defects arise at later stages of insulin action we used an isolated perfused liver system. In this model, clozapine had no direct effect on insulin's counter regulatory effect on epinephrine-induced HGO. In isolated mouse islets clozapine significantly increased glucose-stimulated insulin secretion while simultaneously blocking glucose-induced reductions in glucagon secretion. We also show that the non-peptidic glucagon receptor like peptide-1 (GLP-1) receptor agonist Boc5 was able to overcome the inhibitory effects of clozapine on glucose metabolism. Taken together these results suggest that clozapine does not have any direct effect on glucose metabolism in the liver but it simultaneously stimulates insulin and glucagon secretion, a situation that would allow for the concurrent presence of high glucose and high insulin levels in treated animals.

Olanzapine effects on body composition, food preference, glucose metabolism and insulin sensitivity in the rat.

The atypical antipsychotic drug olanzapine induces weight gain and defects in glucose metabolism in patients. Using a rat model we investigated the effects of acute and long term olanzapine treatment on weight gain, food preference and glucose metabolism. Olanzapine treated rats fed a chow diet grew more slowly than vehicle controls but olanzapine treated animals fed a high fat/sugar diet grew faster than control animals on the same diet. These changes in weight were paralleled by changes in fat mass. Olanzapine also induced a strong preference for a high fat/high sugar diet. Acute exposure to olanzapine rapidly induced severe impairments of glucose tolerance and increased insulin secretion but did not impair insulin tolerance. These results indicate the defect in glucose metabolism induced by acute olanzapine treatment was most likely due to increased hepatic glucose output associated with a reduction in active GLP-1 levels and correspondingly high glucagon levels.

Both p110alpha and p110beta isoforms of phosphatidylinositol 3-OH-kinase are required for insulin signalling in the hypothalamus.

Both insulin and leptin action in the brain are considered to involve activation of phosphoinositide 3-kinase (PI3K), although the roles of different PI3K isoforms in insulin signalling in the hypothalamus are unknown. In the present study, we characterised the roles of these isoforms in hypothalamic insulin and leptin signalling and investigated the cross-talk of both hormones. To evaluate PI3K levels in the hypothalamus, PI3K was immunoprecipitated using an antibody directed against the p85 subunit, and then total PI3K activity was measured in the presence of novel isoform-selective pharmacological inhibitors of each isoform of PI3K. Subsequently, these inhibitors were administered into the lateral ventricle of male Sprague-Dawley rats, followed by vehicle, insulin, leptin or both hormones 45 min later. PI3K activity was determined by immunohistochemical detection of phosphorylated AKT (S473). In a separate study, the effects of the inhibitors on the anorexigenic action of insulin and leptin were determined. Hypothalamic insulin signalling was specifically mediated by the combined actions of the class Ia isoforms p110alpha and p110beta. Total hypothalamic PI3K activity was inhibited 65% by a p110alpha inhibitor, and 35% by a p110beta inhibitor, with a combination of inhibitors being equally effective as the broad-spectrum PI3K inhibitor wortmannin. Individual i.c.v. administration of p110alpha and p110beta inhibitors partly prevented insulin-induced phosphorylated AKT (S473) in the arcuate nucleus, whereas simultaneous application completely blocked insulin action. Unlike insulin, leptin did not induce phosphorylated AKT in the hypothalamus, as detected by immunohistochemistry, and the anorectic effects of leptin were not affected by pre-treatment with a combination of p110alpha and p110beta inhibitors. The enhanced anorectic effect of a combined i.c.v. application of both insulin and leptin could be prevented by pre-treatment with the combination of p110alpha and p110beta inhibitors. The data suggest that p110alpha and p110beta isoforms of PI3K are necessary to mediate insulin action in the hypothalamus. The role of PI3K in leptin action is less clear, but it may be involved by means of an insulin-dependent sensitisation of leptin action.

Caffeine and theophylline block insulin-stimulated glucose uptake and PKB phosphorylation in rat skeletal muscles.

AIM: Caffeine and theophylline inhibit phosphatidylinositol 3-kinase (PI3-kinase) activity and insulin-stimulated protein kinase B (PKB) phosphorylation. Insulin-stimulated glucose uptake involves PI3-kinase/PKB, and the aim of the present study was to test the hypothesis that caffeine and theophylline inhibit insulin-stimulated glucose uptake in skeletal muscles. METHODS: Rat epitrochlearis muscles and soleus strips were incubated with insulin and different concentrations of caffeine and theophylline for measurement of glucose uptake, force development and PKB phosphorylation. The effect of caffeine was also investigated in muscles stimulated electrically. RESULTS: Caffeine and theophylline completely blocked insulin-stimulated glucose uptake in both soleus and epitrochlearis muscles at 10 mm. Furthermore, insulin-stimulated PKB Ser(473) and Thr(308) and GSK-3beta Ser(9) phosphorylation were blocked by caffeine and theophylline. Caffeine reduced and theophylline blocked insulin-stimulated glycogen synthase activation. Caffeine stimulates Ca(2+) release and force development increased rapidly to 10-20% of maximal tetanic contraction. Dantrolene (25 microm), a well-known inhibitor of Ca(2+)-release, prevented caffeine-induced force development, but caffeine inhibited insulin-stimulated glucose uptake in the presence of dantrolene. Contraction, like insulin, stimulates glucose uptake via translocation of glucose transporter-4 (GLUT4). Caffeine and theophylline reduced contraction-stimulated glucose uptake by about 50%, whereas contraction-stimulated glycogen breakdown was normal. CONCLUSION: Caffeine and theophylline block insulin-stimulated glucose uptake independently of Ca(2+) release, and the likely mechanism is via blockade of insulin-stimulated PI3-kinase/PKB activation. Caffeine and theophylline also reduced contraction-stimulated glucose uptake, which occurs independently of PI3-kinase/PKB, and we hypothesize that caffeine and theophylline also inhibit glucose uptake in skeletal muscles via an additional and hitherto unknown molecule involved in GLUT4 translocation.