Insulin receptor deficits in schizophrenia and in cellular and animal models of insulin receptor dysfunction

Schizophrenia Research 84 (2006) 1 – 14 www.elsevier.com/locate/schres

Insulin receptor deficits in schizophrenia and in cellular and animal models of insulin receptor dysfunction Zhong Zhao a,b, Hanna Ksiezak-Reding a,b,*, Silvana Riggio b, Vahram Haroutunian b, Giulio M. Pasinetti a,b a

b

Neuroinflammation Research Laboratories, Mount Sinai School of Medicine and Bronx Veterans Affairs Medical Center, New York, NY, United States Department of Psychiatry, Mount Sinai School of Medicine and Bronx Veterans Affairs Medical Center, New York, NY, United States Received 22 August 2005; received in revised form 30 January 2006; accepted 3 February 2006 Available online 11 April 2006

Abstract Schizophrenia is associated with abnormalities in glucose metabolism that may lead to insulin resistance and a 3 fold higher incidence of type II diabetes mellitus. The goal of the present studies was to assess the role of insulin-dependent Akt signaling in schizophrenia and in animal and cellular models of insulin resistance. Our studies revealed a functional decrease in insulin receptor (IR)-mediated signal transduction in the dorsolateral prefrontal cortex (BA46) of medicated schizophrenics relative to control patients using post-mortem brain material. We found ~ 50% decreases in the content and autophosphorylation levels of IRh and ~ 76–78% decreases in Akt content and activity (pSer473-Akt). The inhibition of IRh signaling was accompanied by an elevated content of glycogen synthase kinase (GSK)-3a and GSK-3h without significant changes in phospho-Ser21/9 GSK-3a/h levels. A cellular model of insulin resistance was induced by IRh knockdown (siRNA). As in schizophrenia, the IRh knockdown cells demonstrated a reduction in the Akt content and activity. Total GSK-3a/h content remained unaltered, but phospho-Ser21/9 GSK-3a/h levels were reduced indicating a net increase in the overall enzyme activity similar to that in schizophrenia. Insulin resistance phenotype was induced in mice by treatment with antipsychotic drug, clozapine. Behavioral testing showed decreases in startle response magnitude in animals treated with clozapine for 68 days. The treatment resulted in a functional inhibition of IRh but the Akt activation status remained unaltered. Changes in GSK-3a/h were consistent with a net decrease in the enzyme activity, as opposed to that in schizophrenia. The results suggest that alterations in insulin-dependent Akt signaling in schizophrenia are similar to those observed in our cellular but not animal models of insulin resistance. In animal model, clozapine ameliorates IRh deficits at the GSK-3a/h level, which may justify its role in treatment of schizophrenia. Our studies suggest that aberrant IR function may be important in the pathophysiology of schizophrenia. D 2006 Elsevier B.V. All rights reserved. Keywords: Schizophrenia; Prefrontal cortex; Insulin resistance; Insulin receptor; Akt; Glycogen synthase kinase 3a; Glycogen synthase kinase 3h

* Corresponding author. Department of Psychiatry, Rm. 3F-24, Bronx VA Medical Center, 130 West Kingsbridge Road, Bronx, NY 10468, United States. Tel.: +1 718 584 9000x1841; fax: +1 718 561 4880. E-mail address: [email protected] (H. Ksiezak-Reding). 0920-9964/$ - see front matter D 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2006.02.009

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1. Introduction Schizophrenia is a disorder characterized by a 20% higher mortality rate than the general population. Contributing factors may be a number of medical conditions, including an increased risk for Type II diabetes mellitus (Dynes, 1969; Felker et al., 1996; Mukherjee et al., 1996). Schizophrenia has been associated with impaired glucose metabolism such as glucose intolerance and insulin resistance. For example, a family history of type II diabetes has been found in 18–19% of schizophrenic patients (Mukherjee et al., 1989) as compared to 1.2–6.3% in the population at large (Adams and Marano, 1995). Review of current literature also underlines prevalence of diabetes and impaired glucose tolerance in patients with schizophrenia (Bushe and Holt, 2004). Many atypical antipsychotic drugs, including clozapine and olanzapine, interfere with glucose metabolism (Dwyer et al., 2001, 2003; Lindenmayer et al., 2003). They reduce glucose transport in blood cells and neuron-like PC12 cells (Dwyer et al., 1999). They may also induce diabetes (Henderson et al., 2005a). Clozapine- and olanzapine-treated subjects display a significant insulin resistance and impairment of glucose effectiveness as compared to risperidonetreated patients (Henderson et al., 2005b). Conventional drugs are less likely to cause metabolic syndrome and weight changes in schizophrenia or psychiatric patients (Mackin et al., 2005). It has been speculated that the glycemic state of schizophrenic patients contributes to their psychotic symptoms or modulates the incidence of drug side effects. A study using a large national PORT database found that prevalence of diabetes in schizophrenia exceeds that in the general population well before the widespread use of the new (atypical) antipsychotic drugs (Dixon et al., 2000). More recent studies confirm this observation and demonstrate impaired fasting glucose tolerance and higher insulin resistance in drug-naı¨ve patients with schizophrenia (Ryan et al., 2003). These findings suggest that impaired glucose metabolism is associated with schizophrenia rather than only a sideeffect of drug treatment. Stimulation of insulin receptors triggers phosphorylation of tyrosine receptor kinase and activation of a downstream signal transduction pathway coupled to phosphatidylinositol 3-kinase (PI3K) and protein

kinase B/Akt (Akt) (Fig. 1). Akt is a multifunctional kinase regulating anti-apoptotic activities, cellular growth and glucose metabolism (Chang et al., 2003; Coffer et al., 1998; Franke et al., 1997; Hemmings, 1997). Akt regulates glucose metabolism by phosphorylation and downregulation of GSK-3a/h, which stimulates glycogen and protein synthesis. Moreover, it couples glucose metabolism to oxidative phosphorylation via mitochondria-bound hexokinase (Gottlob et al., 2001). It also phosphorylates tau protein (KsiezakReding et al., 2003), which may play a role in microtubule-based axonal transport (Mandelkow et al., 2003). GSK-3h is a multifunctional Ser/Thr kinase and a key regulator of several signaling pathways involved in cellular growth and development, including insulinand Wnt-dependent signaling (Dajani et al., 2003; Doble and Woodgett, 2003; Jope and Johnson, 2004). GSK-3h is constitutively active in resting cells and becomes inhibited in response to external signals. Insulin inhibits GSK-3 via Akt-dependent phosphorylation at Ser21 (GSK-3a) and Ser9 (GSK-3h). GSK-3 is also phosphorylated by unidentified kinase(s) or autophosphorylation at Tyr279 (GSK-3a) and Tyr216

Fig. 1. Schematic of insulin-dependent Akt signaling. Insulin activates tyrosine kinase receptors, which leads to stimulation of phosphatidylinositol 3-kinase (PI3K). This promotes phosphorylation and activation of Akt by phosphatidylinositol kinases (PDK-1 and PDK-2) at Thr308 and Ser473, respectively. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a major lipid phosphatase and a key negative regulator of PI3K/Akt signaling. Activated Akt downregulates the glycogen synthase kinase 3a/h (GSK-3a/h) activity by phosphorylation at Ser21/Ser9. Inhibition of GSK-3a/h promotes glycogen synthesis. Akt also promotes oxidative glucose metabolism via mitochondrial hexokinase (Gottlob et al., 2001) and may play a role in tau-mediated axonal trafficking (Ksiezak-Reding et al., 2003; Mandelkow et al., 2003).

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(GSK-3h). The role of Tyr phosphorylation implicated in GSK-3 activation is not totally clear since the mutation of Tyr216 to phenylalanine does not impair GSK-3h activity (Itoh et al., 1995). Studies linking insulin receptor function with Akt signaling in schizophrenia have been limited. Recently, convergent evidence has demonstrated a decrease in the total Akt1 protein in brain of schizophrenic patients and identified Akt1 as a potential susceptibility gene (Emamian et al., 2004). In the present studies, we examined the relationship between the insulin receptor and Akt signaling in postmortem brain tissue from schizophrenic patients, in a cellular model of insulin receptor knockdown and in mice with aberrant function of brain insulin receptors. Our results revealed a significant downregulation of insulin receptors and inhibition of Akt activity suggesting drastically suppressed insulin-dependent Akt signaling in schizophrenia.

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Table 1 Data on subjects from which post-mortem brain material was used in the present studies Patients

N

Male/female

Mean age F S.D.

Controla Schizophrenia

11 12

4/7 11/1

85 F 11 75 F 12b

Schizophrenia patients

Antipsychotic drugsc

1 2 3 4 5 6 7 8 9 10 11 12

Conventional Conventional Conventional Conventional Conventional Conventional None Conventional Conventional Conventional Conventional+atypical Conventional+atypical

Others

Lithium Insulin

Insulin Lithium

a

Two control patients were treated with insulin. p N 0.05 (NS) Student’s t-test. c Conventional agents included haloperidol, chlorpromazine and phenothiazine derivatives among others. Patients 11 and 12 received atypical agent, risperidone. b

2. Materials and methods 2.1. Human brain samples Brain tissue was obtained from the Brain Bank of the Mount Sinai Medical Center and of affiliated Bronx Veterans Administration Medical Center. Postmortem brain material was from 12 schizophrenia patients of whom 11 were treated and one not treated with antipsychotic agents prior to autopsy, and 11 control patients without known neurologic impairment (Table 1). All samples derived from dorsolateral prefrontal cortex (Brodmann’s area 46) and were dissected while frozen. Dissected samples were homogenized in cell lysis buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5mM sodium pyrophosphate, 1 mM h-glycerophosphate, 1 mM Na3VO4, 1 Ag/ml leupeptin and 1mM phenylmethyl sulphonyl fluoride) and centrifuged (15,000g, 10 min). Supernatants (lysates) were used for Western blotting and immunoprecipitation. 2.2. Insulin receptor Insulin receptor h subunit (IRh) was detected on Western blots with the anti-total IRh antibodies (Santa Cruz), C-19 and 29B4. To detect phospho-Tyr IRh,

total IRh was immunoprecipitated from lysates (300 Ag protein) following incubation with 1Ag of C-19 in the final volume of 500 Al at 4 8C overnight. The immune-complexes were captured using 10 Ag of immobilized protein A Sepharose 4B beads (Sigma) and washed extensively. Captured proteins were solubilized in sodium dodecyl sulfate (SDS) sample buffer (Laemmli) and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In the immunoprecipitates, the content of total IRh and phosphoTyr1162/1163-IRh was detected by Western blotting using 29B4 and anti-phospho tyrosine (pY20; 1:1000, BD Transduction Lab) antibodies, respectively. 2.3. IRb gene silencing by siRNA Human embryonic kidney 293 (HEK) cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 1% pen/strep. The double-strand siRNA was synthesized and annealed by Qiagen Corp. The target sequence for IRh was AAG GAG CCC AAT GGT CTG ATC.

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The non-silencing scrambled sequence was AAT TCT CCG AAC GTG TCA CGT. HEK cells were transfected with siRNA using TransMessenger Transfection Reagent (Qiagen) according to the manufacturer’s recommendations. For immunoblotting, the cells were collected in Laemmli sample buffer 24h post-transfection. 2.4. Akt, GSK-3a/b and Western blotting For Western blotting, 35 Ag of lysate was loaded per lane. Following separation by SDS-PAGE, proteins were transferred onto nitrocellulose paper. We studied the content and activity of Akt1 isoform referred to as Akt. The total and active forms of Akt were determined using anti-pSer473-Akt1 (1:1000, Cell Signaling) and anti-total Akt1 (1:1000, Sigma). GSK-3a/h was examined using anti-pSer21/9-GSK3a/h (1:1000, Cell Signaling), anti-pTyr279/216-GSK3a/h (1:1000, Upstate) and anti-total GSK-3a/h (1:1000, Cell Signaling). Antibody against actin was from Sigma. The secondary antibodies were conjugated to horseradish peroxidase (Santa Cruz). The specific protein signals were detected using enhanced chemiluminescence supersignal substrates (Amersham) followed by exposure to Kodak X-ray film and quantification using Bioquant software package (Bio-Rad, ver 2.5, Biometrics). Results were expressed relative to total protein and normalized to actin. h-Actin immunoreactivity controlled selectivity of changes. Statistical analysis was performed using two-tailed Student’s t-test. 2.5. Clozapine treatment and insulin resistance in mice Three-month-old male C57BL/6J mice (Jackson Labs, Bar Harbor, ME) were treated with clozapine (Sigma; 2 mg/kg/day) in drinking water changed every 3days (Spurney et al., 1999; Turner et al., 2003). After 3days, 25days and 68 days of treatment, mice were assessed for the development of insulin resistance phenotype as described earlier (Ho et al., 2004). This included among others post-prandial intraperitoneal glucose tolerance test performed as described (Takeda et al., 2003). Briefly, following an overnight fast, mice were given a single dose of glucose (2 g/kg body weight) administered intra-

peritoneally. Blood was collected from tail-vein periodically over a 2-h period. Serum glucose content was determined using OneTouch LifeScan System (LifeScan, Milpitas, CA), following the manufacturer’s instructions. Mice were sacrificed at ~ 5 months of age and the cerebral cortex samples were prepared as described above for human brain tissue. The Institutional Animal Care and Use Committee of the Mount Sinai School of Medicine approved all the experiments. 2.6. Startle response and prepulse inhibition Behavioral testing was performed on mice treated for 68 days with clozapine and wild type counterparts as described (Dirks et al., 2003; Gould et al., 2004; Tarantino et al., 2000). Briefly, startle chamber (SRLab System, San Diego Instruments, San Diego, CA) contained a clear, nonrestrictive Plexiglas cylinder resting on a platform and a high frequency loudspeaker that produced a continuous background noise of 65dB and various acoustic stimuli. Whole-body startle responses of the mouse caused vibrations of the Plexiglas cylinder. These vibrations were converted to analog signals, digitized and stored by a computer. Calibrations of the chamber assured the accuracy of all acoustic stimuli and measurements. Startle trials. The initial 45-min acclimation period (65 dB white noise alone) was followed by eight blocks of trials, with an average intertrial interval of 15s. The first and the last blocks consisted of six startle trials presented as 40-ms broadband of 120 dB bursts. Six middle blocks consisted of a pseudo-randomized sequence of 40-ms broadband of 65, 70, 80, 90, 100, 110 and 120 dB (startle) bursts. Startle magnitude was expressed in arbitrary units as the average response to the startle trials presented in the middle six blocks of the session. Prepulse inhibition test. After initial acclimation, eight blocks of trials were performed with an average intertrial interval of 15s. The first and the last blocks consisted of six startle trials (40-ms 120 dB broadband bursts). Six middle blocks consisted of a pseudo-randomized sequence of 40-ms broadband 65dB (background), 120 dB burst (startle) and four distinct intensities of 20-ms prepulse trials. Prepulse trials consisted of a 20-ms long prepulse (broadband bursts of 70, 74, 82 or 90 dB) and the startle stimulus (a 40-ms 120 dB broadband burst).

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The test session lasted for about 23 min and contained 48 trials. Only data from the middle six blocks were used for statistical analysis. Prepulse inhibition was calculated as a percentage score.

3. Results Results of the present studies were summarized in Table 2. 3.1. Schizophrenia patients We examined insulin receptors in post-mortem brain material from 12 schizophrenia patients, relative to 11

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age-matched controls with normal neurological status (Table 1). The average age of the patients was 75 years in the schizophrenia and 85 years in the control groups ( p N 0.05, NS). Male patients dominated in schizophrenia (11 males) vs. controls (4 males). All but one schizophrenia patients were medicated and received conventional antipsychotic treatment that included haloperidol, chlorpromazine or phenothiazine derivatives among others. Two of those patients were also treated with an atypical antipsychotic medication (risperidone) and two received lithium. Insulin was administered to two schizophrenia patients. In a group of control patients, also two patients received insulin. Insulin binding to its receptors (a-subunit) induces activation of the intrinsic tyrosine kinase activity in

Fig. 2. Insulin signaling in patients with schizophrenia (dorsolateral prefrontal cortex). (A) Total IRh and Tyr-phosphorylated IRh. (B) Total and active (pSer473) forms of Akt. (C) Total and phosphorylated forms of GSK-3a. (D) Total and phosphorylated forms of GSK-3h. Results of quantitative Western blot analysis from twelve schizophrenic (black bars) and eleven control (white bars) patients are presented. Values are means F S.E.M. (n = 11–12). *p b 0.05, **p b 0.005, ***p b 0.0005.

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the h-subunit, which is followed by autophosphorylation within distinct regions of IRh. The activated IRh then recruits adaptor proteins that mediate the biological effects of insulin receptor activation via the PI3K/Akt pathway. We found that total IRh content was significantly reduced by 50% in schizophrenic as compared to control patients (Fig. 2A). The content of Tyr-phosphorylated IRh expressed as

a ratio vs. total IRh was downregulated by 58%, indicating a suppressed insulin receptor activity in schizophrenia. We then examined intermediates of the insulin-dependent signaling cascade, including Akt and GSK-3a/h. We found a severely reduced (by 76–78%) content of both the total and active forms of Akt (Fig. 2B). Total GSK-3a content was upregulated ~ 2-fold but phospho-Ser21- and phospho-Tyr279

Fig. 3. In vitro model of the selective down regulation of IRh (IRh knockdown) using siRNA technology. HEK cells treated with IRh Si-RNA (black bars) or scrambled RNA (negative control, white bars) for 2 days before Western blot analysis. (A) Total IRh and pTyr-IRh. (B) Total Akt and phospho-Ser473 Akt. (C) Total GSK-3a content and phospho-Ser21 GSK-3a. (D) Total GSK-3h and phospho-Ser9 GSK-3h. Values are means F S.E.M. (n = 3). *p b 0.05, **p b 0.005.

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GSK-3a remained unaltered. Total GSK-3h content was also upregulated ~ 2-fold. Both phospho-Ser9and phospho-Tyr216-GSK-3h remained undetectable by Western blotting. The alterations in GSK-3a/h reflected increases in the net activity of these enzymes. Overall, our results are consistent with drastically reduced insulin-dependent Akt signaling in schizophrenia, resulting in a low Akt activation status and upregulated GSK-3a/h activity. We next evaluated the relationship of these changes to experimentally induced insulin resistance in cellular and animal models. 3.2. Insulin receptor knockdown in tissue cultures Our studies used an in vitro model of insulin receptor knock out generated by Si-RNA technology. HEK cells were treated with IRh Si-RNA or scrambled RNA (Neg. control) for 48 h before

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Western blot analysis. We found that total IRh but not actin were downregulated by 50% (Fig. 3A). The ratio of phospho-Tyr1162/1163 IRh vs. total IRh remained unchanged. With total IRh decreased, the projected functional activity of IRh was also decreased. The selective downregulation of insulin receptors promoted a significant 50% reduction in the Akt content and activity (Fig. 3B). Although total content of GSK-3a and GSK-3h was unaltered, the content of phospho-Ser21 GSK-3a and phospho-Ser9 GSK-3h, both dependent on Akt activity, was downregulated by more than 50%. Since Akt-dependent phosphorylation inhibits GSK-3 activity, changes in the phosphorylation status of GSK-3 were considered as reflecting increases in its enzyme activity. The results suggested that, in our cellular model, there was a direct relationship between downregulation of insulin receptor content and inhibition of downstream Akt-dependent insulin signaling. Our findings were

Fig. 4. Intraperitoneal glucose tolerance test and body weight. Mice were treated with clozapine (CLZ) for (A) 3 days, (B) 24 days and (C, D) 68 days (2 mg/kg/day). Serum glucose was determined at 30-min intervals after intraperitoneal injection of glucose (2 mg/kg) and results presented as % of initial glucose values (time 0). Values are means F S.E.M. (n = 11). *p b 0.05.

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consistent with animal models of insulin receptor knockdown reported by others (Schubert et al., 2004). 3.3. Mouse model of insulin resistance Schizophrenia is associated with extensive deficits in both sensorimotor gating and habituation as shown by diminished prepulse inhibition and acoustic startle habituation, respectively (Braff et al., 1992). Clozapine is an atypical antipsychotic drug that ameliorates some of the neurophysiologic deficits in schizophrenia, including sensorimotor gating and habituation (Swerdlow and Geyer, 1998). It also induces excessive body weight gain and insulin resistance (Henderson et al., 2005a). We evaluated a long-term therapeutic potential of clozapine and its ability to promote insulin resistance in mice. Mice treated with clozapine (2 mg/kg/day) for 3days, 25 days and 68days developed a sustained insulin insensitivity, which resulted in impaired plasma glucose clearance as measured by intraperitoneal glucose tolerance test (Fig. 4A–C). We did not observe,

however, a significant weight gain in these mice, except for a small increase (by 6.3%) on day 7 (Fig. 4D). Since studies have suggested that weight gain and therapeutic response resulting from clozapine treatment may be correlated (Meltzer et al., 2003), we next examined behavioral and biochemical effects of clozapine. We measured startle response and prepulse inhibition of the startle reflex in naı¨ve and 68-day clozapine-treated mice (11mice per group). We found that clozapine treatment significantly reduced the startle response (Fig. 5A). The treatment, however, did not potentiate prepulse inhibition in these mice (Fig. 5B). These results indicate that some but not all therapeutic effects of clozapine were replicated in our paradigm. This may be related to the fact that C57B1/ 6J strain of mice studied has been the least responsive to clozapine as compared to two other strains (DBA/ 2J and 129S6SvEvTac) (Olivier et al., 2001). Moreover, the reversal of schizophrenia-related behaviors in mice by clozapine has been associated with shortterm (3–4 days) but not long-term (21–22 days) treatment in some studies (Zarate et al., 2004).

Fig. 5. Behavioral testing of mice treated with clozapine. Mice examined for startle response (A) and prepulse inhibition (B) after treatment with clozapine (CLZ) for 68 days as described in Section 2.5. Values are means F S.E.M. (n = 11). *p b 0.05, **p b 0.005.

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Brain lysates of 68-day clozapine-treated animals showed ~ 2.3-fold increase in total content of IRh by Western blotting (Fig. 6A). The content of Tyrphosphorylated IRh expressed as a ratio vs. total IRh was downregulated by 75%, indicating a largely suppressed insulin receptor activity in these mice. Total Akt content was depressed by ~50% but phospho-Ser473-Akt was unaltered indicating that the Akt activation status remained unchanged (Fig. 6B). Total GSK-3a and GSK-3h were downregulated by N 60%, but both phospho-Ser21 GSK-3a and phosphoSer9 GSK-3h remained unchanged consistent with the sustained Akt activation status (Fig. 6C and D). Phospho-Tyr279 GSK-3a and -Tyr216 GSK-3h also remained unaltered. Changes in GSK-3a/h indicated decreases in the overall enzyme activity. We concluded that long-term treatment with clozapine downregulated insulin receptor function, which could be responsible for induction of insulin resistance at the receptor level. Downstream effects of clozapine on Akt and GSK-3, however, were inconsistent with the

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cellular model of insulin resistance induced by insulin receptor knockdown.

4. Discussion Our present studies found that insulin receptor content and autophosphorylation as well as insulindependent Akt signaling were severely depressed in a group of 12 schizophrenic patients. Our results suggest that glucose metabolism is seriously compromised in schizophrenia. Although direct studies of insulin receptors in schizophrenia have not been reported previously, recent evidence shows that Akt1 might be a potential susceptibility gene (Emamian et al., 2004). In fact, Akt1 is one of the three genes shown to have strongest association with schizophrenia, two others including neuregulin-1 and dysbindin1 (Arnold et al., 2005). Moreover, Akt is also a crucial mediator of neuroregulin-1 (Li et al., 2003) and dysbindin (Numakawa et al., 2004) function. The

Fig. 6. IRh, Akt and GSK-3a/h in cerebral cortex of insulin resistant mice. (A) Total IRh and Tyr-phosphorylated IRh. (B) Total and active (pSer473) forms of Akt. (C) Total and phosphorylated forms of GSK-3a. (D) Total and phosphorylated forms of GSK-3h. Quantitative Western blot analysis of brain lysates. Values are means F S.E.M. (n = 5–6). *p b 0.05, **p b 0.005, ***p b 0.0005.

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results of our studies provide further biochemical evidence to support the role of Akt in schizophrenia. Insulin receptors are expressed at high levels in many brain areas and different cell types, including glial and neuronal cells (Havrankova et al., 1978). Although insulin receptor defects are uncommon in diabetes, insulin receptor genes remain attractive targets for in vivo studies of insulin resistance. Studies of mice with tissue specific ablation of insulin receptors have indicated that both canonical (e.g., muscle and adipose tissue) and non-canonical (e.g., liver and brain) insulin targets can contribute to insulin resistance (Bruning et al., 2000; Okamoto and Accili, 2003). Based on our findings, it was likely that downregulation of Akt in schizophrenia was due to a reduced insulin receptor content and activity (Table 2). HEK cells with knockdown IRh described in the present studies displayed a severe depletion of Akt and reduced phosphorylation of both Akt and GSK3a/h. In other studies, brain/neuron-specific insulin receptor knock out (NIRKO) mice also have shown markedly reduced phosphorylation of Akt and GSK3h (Schubert et al., 2004). It was less likely that downregulation of Akt alone could directly perturb insulin receptors in schizophrenia by a feedback mechanism. Mice deficient in Akt1 displayed defects in both fetal and postnatal growth but were normal with regard to glucose homeostasis as ascertained by glucose and insulin tolerance tests (Cho et al., 2001). Studies of insulin signaling in cultured cells lacking Akt1 have been fragmentary. Loss of Akt1 alone in cultured 3T3-L1 adipocytes slightly impaired insulin-mediated glucose transport (Jiang et al., 2003). Table 2 Summary of results of the present studies Parameter

Schizophrenia

HEK cells (SiRNA)

CLZ mice

IRh, total protein IRh activitya Akt, total protein Akt activitya GSK-3a, total protein GSK-3a activitya GSK-3h, total protein GSK-3h activitya

Decrease Decrease Decrease Decrease Increase Increase Increase Increase

Decrease Decrease Decrease Decrease No change Increase No change Increase

Increase Decrease Decrease No change Decrease Decrease Decrease Decrease

a Projected values based on total protein levels and phosphorylation status.

Upregulation of GSK-3 content (and activity) in our schizophrenic patients was an unexpected finding (Table 2). Previous studies of 15 schizophrenia patients indicated downregulation of GSK-3h levels in the frontal cortex Brodmann’s area 10 (Kozlovsky et al., 2000) and normal GSK-3 levels in occipital cortex (Kozlovsky et al., 2001). Studies using peripheral tissue (lymphocytes and lymphocyte-derived cell lines) from schizophrenic patients showed no difference in GSK-3a and GSK-3h mRNA levels, GSK-3h protein levels, or total GSK-3 activity relative to control subjects (Nadri et al., 2002). Although we cannot exclude effects of more rigorous extraction procedure in our studies (e.g., 1% Triton X100 vs. 0.1% Nonidet P-40 in studies by Kozlovsky et al., 2000), our findings could reflect variations in regional tissue susceptibility as reported at the protein and gene expression levels (Katsel et al., 2005a,b). For our studies, we selected samples of brain tissue (dorsolateral prefrontal cortex, Brodmann’s area 46) implicated in the pathology of schizophrenia by the pattern of cognitive impairment (Fuster, 2001). Insulin receptor dysfunction could contribute to the particular vulnerability of this region. Upregulation of GSK-3 in schizophrenia was consistent with downregulation of the IRh content and activity we found in these patients (Table 2). It was also consistent with most of experimental models of insulin resistance. For example, results of the present studies showed that the depletion of insulin receptors in HEK cells was associated with upregulation of GSK-3 activity. Similar findings were reported for NIRKO mice (Schubert et al., 2004). In other models, insulin resistance experimentally induced by diabetogenic diet led to a reduced IRh/Akt signaling coincidental with increased GSK-3h activity in the brain (Ho et al., 2004). Despite these similarities, it appears that GSK-3 regulatory mechanisms may vary between insulin resistance models and schizophrenia. In these models, the GSK-3 activity increases due to a lower phosphorylation at Ser21/9, whereas in schizophrenia it increases due to an elevation in the protein level. Most of our schizophrenia patients received conventional treatment and two of them were treated with atypical medication. It is possible that antipsychotic treatment could contribute to, or alone lead to, reduced insulin receptor content in schizophrenia

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patients. Studies of clozapine-treated animals, however, diminished such possibility. Long-term treatment (N 2 months) with clozapine significantly upregulated the concentration of insulin receptors in the mouse brain. From a therapeutic perspective, this effect alone could ameliorate insulin receptor deficiency in schizophrenia. In spite of a robust 2.3-fold increase in the content, however, the functional activity of insulin receptors was largely depressed in these animals. Therefore, the functional inhibition of insulin receptors could be responsible for the development of insulin insensitivity in clozapine-treated mice. Indeed, impaired plasma glucose clearance was observed at day 3 and persisted for the entire 68-day period of clozapine treatment. This suggests that clozapine was not only effective in behavioral changes as measured by the startle response magnitude but also in systemic metabolic alterations. One of our most interesting findings was that the suppression of insulin receptor activity by clozapine caused no changes in the phosphorylation of two downstream effectors, Akt and GSK-3. Their total content however was reduced. These alterations not only preserved the Akt activation status but also downregulated the GSK-3 activity. One of the possible mechanisms could involve PTEN, a major lipid phosphatase negatively regulating PI3K/Akt signaling (Sulis and Parsons, 2003). In addition, clozapine could downregulate synthesis/turnover of both Akt and GSK-3 proteins. It is important to note that clozapine-induced alterations in Akt and GSK-3 in mice were opposite to those seen in schizophrenic patients. These findings suggest that antipsychotic treatment may not contribute to the alterations in Akt and GSK-3 seen in our schizophrenic patients. On the contrary, antipsychotic treatment may ameliorate schizophrenia-induced changes in Akt-dependent signaling. Further biochemical studies of naı¨ve and conventional/atypical drugs treated schizophrenic patients are needed to support our conclusion. The specific effects of clozapine on Akt and GSK3 in mice suggest that insulin resistance phenotype induced by the drug does not entirely conform to biochemical alterations associated with insulin receptor knockdown in vitro (the present studies) and in vivo or with other experimental models of insulin resistance. Instead, downregulation of GSK-3 by

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clozapine appears to mimic effects of insulin in normal, insulin-responsive animals. Our studies suggest that therapeutic use of clozapine in schizophrenia may involve such insulin-like action on GSK-3. It is interesting to note that a similar mechanism of action is indicated for other agents, e.g., lithium and valproate, used in bipolar disorders and schizophrenia. They selectively inhibit GSK-3h activity within the therapeutic range (Chen et al., 1999; Stambolic et al., 1996). A number of studies using treatment with antipsychotic agents in animal models have shown effects on the content and activity of Akt and GSK-3 (Emamian et al., 2004; Kozlovsky et al., 2005; Lu et al., 2004). It becomes clear that antipsychotic agents may ameliorate Akt signaling directly or indirectly through insulin receptor function. The mechanisms of conventional and atypical drug treatments may ultimately converge on lowering GSK-3 activity and allowing GSK-suppressed pro-survival signaling to emerge (Eldar-Finkelman, 2002; Plotkin et al., 2003). Our present studies provide evidence of a link between insulin receptor dysfunction and suppressed Akt signaling in schizophrenia. This may have important biological implications. The PI3K-mediated Akt signaling plays a significant role in neuritic growth, synaptic events and memory formation. This pathway induces elongation of processes in PC12 cells (Kobayashi et al., 1997), increases axon caliber and distal branching in sensory neurons (Markus et al., 2002), and promotes long-term potentiation in the hippocampus (Kelly and Lynch, 2000; Sanna et al., 2002). In primary hippocampal neurons, insulindependent Akt signaling results in rapid translocation of GABA receptors (type A) to the plasma membrane, increasing cell-surface levels of the receptor (Wang et al., 2003). The authors suggest that modulation of intracellular trafficking by Akt may be a common mechanism by which diverse extra- and intracellular signaling pathways modulate neuronal activity in the brain. Our recent studies support this view and suggest that Akt may link tyrosine receptor kinase signaling to tau-mediated neuritic growth and axonal trafficking (Ksiezak-Reding et al., 2003; Mandelkow et al., 2003). This is relevant to schizophrenia, which is associated with cognitive deficits and functional impairment of neurons. Since tau is a potentially important mediator of Akt function, inadequate insulin-dependent Akt signaling could contribute to

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