Insulin Resistance in Brain Alters Dopamine Turnover and Causes Behavioral Disorders

Home , Monoamine oxidase A, Monoamine oxidase B

Insulin resistance in brain alters dopamine turnover and causes behavioral disorders

Andre Kleinriddersa, Weikang Caia, Laura Cappelluccib, Armen Ghazarianb, William R. Collinsc, Sara G. Vienberga, Emmanuel N. Pothosb,c, and C. Ronald Kahna,1

aSection of Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215; and bDepartment of Integrative Physiology and Pathobiology and cGraduate Program in Pharmacology and Experimental Therapeutics, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111

Contributed by C. Ronald Kahn, February 2, 2015 (sent for review December 16, 2014)

Diabetes and insulin resistance are associated with altered brain food reward system, synaptic plasticity, signal transmission, imaging, depression, and increased rates of age-related cognitive and neuroprotective functions (18–22). On the other hand, impairment. Here we demonstrate that mice with a brain-specific knockout of IRS-2 has been shown to have protective effects on knockout of the insulin receptor (NIRKO mice) exhibit brain brain pathology in a mouse model of Huntington’s disease (23). mitochondrial dysfunction with reduced mitochondrial oxidative In addition to the association between diabetes and acceler- activity, increased levels of reactive oxygen species, and increased ated cognitive decline, there is growing support for a link be- levels of lipid and protein oxidation in the striatum and nucleus tween diabetes and mood disorders, especially depression (24–26). accumbens. NIRKO mice also exhibit increased levels of mono- The mechanisms by which T2D influences depression are amine oxidase A and B (MAO A and B) leading to increased not known, but insulin resistance states are associated with in- dopamine turnover in these areas. Studies in cultured neurons and creased inflammation and cytokine production in some brain glia cells indicate that these changes in MAO A and B are a direct regions (27, 28). Furthermore, ablation of insulin receptor in consequence of loss of insulin signaling. As a result, NIRKO mice catecholaminergic neurons attenuates insulin-induced excitabil- develop age-related anxiety and depressive-like behaviors that can be ity in dopaminergic neurons (29), whereas insulin administration reversed by treatment with MAO inhibitors, as well as the tricyclic into the central nervous system (CNS) of rats has been shown to antidepressant imipramine, which inhibits MAO activity and reduces increase dopamine transporter protein expression (30). The latter oxidative stress. Thus, insulin resistance in brain induces mitochon- may be important, because alterations in the activity of dopamine drial and dopaminergic dysfunction leading to anxiety and depres- and/or serotonin systems have been linked to depression (31–33). sive-like behaviors, demonstrating a potential molecular link between Mechanistically, one potential link between insulin action and central insulin resistance and behavioral disorders. changes in brain function might be alterations in mitochondrial function. Insulin resistance and type 2 diabetes are well docu- insulin receptor | diabetes | mitochondrial function | monoamine oxidase | mented to be associated with mitochondrial dysfunction in dopamine signaling classical metabolic tissues, such as muscle (34) and liver (35), and we have recently demonstrated that obesity-induced insulin re- s life expectancy in humans has increased, we are faced with sistance is also associated with altered mitochondrial function in Aa worldwide epidemic of age-related diseases such as type the hypothalamus (36). Patients with major depression have also 2 diabetes (T2D) and Alzheimer’s disease (1). These parallel been shown to exhibit mitochondrial dysfunction in the brain epidemics may not be coincidental. Indeed, studies have dem- (37). Although it is still unknown how mitochondrial dysfunction onstrated an association between diabetes and a variety of brain might be linked to depression, it is worth noting that the two enzymes degrading monoamine neurotransmitters, monoamine alterations including depression, age-related cognitive decline, Alzheimer’s disease, and Parkinson’s disease (2, 3). In addition, individuals with both type 1 and type 2 diabetes have been shown Significance MEDICAL SCIENCES to have a variety of abnormalities in brain imaging, including altered brain activity and connectivity by functional MRI (4, 5), Both types 1 and 2 diabetes are associated with increased risks altered microstructure by diffusion tensor imaging (6, 7), and al- of age-related decay in cognitive function and mood disorders, tered neuronal circuitry in the striatum (8). Conversely, patients especially depression. Insulin action has been shown to regu- with Alzheimer’s disease show signs of central insulin resistance late neuronal signaling and plasticity. Here we investigate with increased insulin receptor substrate (IRS) 1 serine phosphor- whether brain-specific knockout of insulin receptor (NIRKO) in ylation in the brain and decreased insulin concentrations in the mice causes behavioral changes and how these are mechanis- cerebrospinal fluid (9, 10). Furthermore, pilot clinical trials of in- tically linked. We find that NIRKO mice exhibit age-related tranasal insulin administered to individuals with Alzheimer’sdis- anxiety and depressive-like behavior. This is due to altered mitochondrial function, aberrant monoamine oxidase (MAO) ease suggest decreased rates of cognitive decline (11). These observations in humans have been mechanistically sup- expression, and increased dopamine turnover in the mesolimbic system, and can be reversed by treatment with Mao ported by studies in rodents and cultured cells, which have inhibitors. Thus, brain insulin resistance alters dopamine turn- shown that insulin receptor signaling in brain has an important over and induces anxiety and depressive-like behaviors. These role in central regulation of metabolism and may also be crucial findings demonstrate a potential molecular link between cen- for proper brain function (12–14). We have previously demon- tral insulin resistance and behavioral disorders. strated that mice with insulin resistance in brain due to targeted

deletion of the insulin receptor (NIRKO mice) develop hyper- Author contributions: A.K., E.N.P., and C.R.K. designed research; A.K., W.C., L.C., A.G., phagia, mild obesity, reduced fertility, and decreased counter- W.R.C., S.G.V., and E.N.P. performed research; A.K., E.N.P., and C.R.K. analyzed data; and regulatory response to hypoglycemia (15, 16). NIRKO mice also A.K. and C.R.K. wrote the paper. display glycogen synthase kinase 3 beta (GSK3-beta) activation, The authors declare no conflict of interest. resulting in hyperphosphorylation of tau protein, a hallmark of 1To whom correspondence should be addressed. Email: [email protected]. ’ early Alzheimer s disease (17). Other animal studies have dem- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. onstrated that insulin has direct effects on the hypothalamic 1073/pnas.1500877112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1500877112 PNAS | March 17, 2015 | vol. 112 | no. 11 | 3463–3468 Downloaded by guest on September 30, 2021 oxidase (MAO) A and B, whose dysregulation has been linked to A B depressive behaviors, reside in the outer mitochondrial membrane Control * (38–40). These data suggest that the relationship between mood 250 NIRKO 250 * disorders and diabetes could be the result of altered insulin regu- 200 200 lation of mitochondrial function and monoamine homeostasis. 150 150 100 100

In the present study, we demonstrate that insulin receptor 50 50

deficiency in the brain results in brain mitochondrial dysfunction. 0 0 Im m obility tim e (s) Im m obility tim e (s) 10 month 17 month Control NIRKO Whereas initially this is not accompanied by behavioral changes, Tail Suspension Test Forced Swimming Test with aging, NIRKO mice exhibit signs of anxiety and depressive- like behaviors. These changes are secondary to decreased do- CDE * ** * pamine signaling in the striatum and nucleus accumbens, which 250 20 (s) 100 r

200 e 80 15 t

in turn is a consequence of increased levels of MAO A and B, est n 150 60 leading to increased dopamine turnover. In vitro data indicate 10 100 40 that this is due to a loss of insulin effect on expression of MAO A oce 5 NSF T

50 et 20 Dark Box Light Dark Light Box and MAO B in neuronal and glial cells, and is further supported im 0

0 0 t Control NIRKO Control NIRKO Control NIRKO time spent in dark (s) by the finding that these depressive behaviors are reversed by number of transitions treatment with the MAO inhibitors. Thus, central insulin re- FG sistance causes altered dopamine turnover and age-related be- * 80 havioral changes, creating a direct link between mood disorders 15 * 60 and insulin-resistant states like type 2 diabetes. 10 40 5 Results 20 Open Field Test Open Field Test 0 0 number of entries Age-Dependent Anxiety and Depressive-Like Behavior in NIRKO Mice. Control NIRKO Latency to enter (s) Control NIRKO NIRKO mice were created by breeding IRlox/lox mice and Nestin- Cre transgenic mice as previously described (15). Quantitative Fig. 1. Age-dependent anxiety and depressive-like behavior in NIRKO mice. PCR (qPCR) analysis of 4-mo-old mice revealed a 95% re- (A and B) Assessment of depressive-like behavior. (A) Immobility time was duction of Ir mRNA throughout the brain, including in isolated assessed using the tail suspension test for 10- and 17-mo-old female control = = hypothalamus (HTM), hippocampus (HCA), prefrontal cortex (n 7) and NIRKO mice (n 9). (B) Immobility time during a forced swimming test in 17-mo-old female control (n = 6) and NIRKO mice (n = 6). Values (PFC), striatum, nucleus accumbens (NAC), and ventral teg- in this panel and all subsequent figures are mean ± SEM; *P ≤ 0.05; **P ≤ mental area (VTA), which was paralleled by decreases in insulin 0.01; ***P ≤ 0.001, Student’s t test, unless otherwise stated. (C and D)As- receptor protein (Fig. S1A). Although NIRKO mice show mild sessment of anxiety via light/dark box. (C) Time spent in dark compartment obesity and metabolic syndrome early in life (15), by 10 mo of and (D) number of transitions during light/dark box test for 17-mo-old fe- age, NIRKO mice exhibited no significant differences in body male control (n = 11) and NIRKO mice (n = 12). (E) Assessment of conflict- weight, blood glucose levels, or food intake compared with controls based anxiety as time to enter the center during novelty suppressed feeding (Fig. S1 B–F), and this continued until at least 24 mo of age. test for 17-mo-old female control (n = 6) and NIRKO mice (n = 6). (F and G) To determine whether insulin receptor deficiency in the brain Assessment of exploratory drive. (F) Number of entries and (G) latency to enter might lead to alterations in behavior, we performed a panel of the center during open field test for 17-mo-old female control (n = 10) and = behavioral tests on 10- and 17-mo-old NIRKO mice, representing NIRKO mice (n 14). middle age and older animals. Consistent with previous studies examining brain function in 4–6 mo old mice (17), the tail sus- and male NIRKO mice exhibited increased anxiety levels as pension test, which assesses depressive-like behavior as indicated by demonstrated by a twofold increase in the time to enter the disk immobility time, revealed no difference in 10-mo NIRKO male and containing food (Fig. 1E and Fig. S2 A–C). Likewise, an open female mice compared with control. However, by 17 mo of age, ± field test, which assesses changes in exploratory drive, revealed female NIRKO mice exhibited a 33 9% increase in immobility ± time in the tail suspension test and a 30 ± 5% increase in immobility that 17-mo-old NIRKO mice had 45 5% fewer entries to the center and a 3.6-fold increase in latency in entering the center time in the forced swimming test, both of which are considered signs < of depressive behavior (both P < 0.05, Fig. 1 A and B). As with their compared with age-matched control littermates (all P 0.05) metabolic abnormalities, male NIRKO mice showed a milder (Fig. 1 F and G and Fig. S2 D and E). Age- and sex-matched phenotype with a 35 ± 13% increase in immobility in the tail sus- mice carrying only the Nestin-Cre transgene had no alterations – pension test and a 16 ± 8% increase in immobility time during the in any of these parameters (Fig. S3 A D). Thus, using multiple forced swimming test, neither of which quite reached statistical tests, 17-mo-old NIRKO mice of both genders showed clear significance (P = 0.06 and P = 0.15, Fig. S1 G and H). signs of increased anxiety and depressive-like behavior. Older NIRKO mice also exhibited signs of increased anxiety and stress response. Using a dark/light box, we found that 17-mo-old NIRKO Mice Display Central Mitochondrial Dysfunction. It has been female NIRKO mice had increased anxiety as evidenced by staying shown that insulin resistance in the brain (36), like insulin re- in the dark compartment 19 ± 6% longer and exhibiting 40 ± 6% sistance in skeletal muscle (34), is associated with mitochondrial fewer transitions between compartments than control mice (Fig. 1 C dysfunction, and mitochondrial dysfunction has been associated and D). Both male and female NIRKO mice (17 mo old) also with neurological disorders (41). Examination of isolated mito- exhibited increased serum corticosterone levels after restraint stress chondria from brains of control and NIRKO mice using a Sea- compared with control, and in females corticosterone was signifi- horse XF24 Extracellular Flux Analyzer revealed a 28 ± 9% cantly increased even in the basal state (Fig. S1 I and J). reduction in basal oxygen consumption rate (OCR) compared To further analyze behavioral changes in NIRKO mice, we with control (P < 0.05), even by 4 mo of age. This defect was assessed anxiety using the novelty-suppressed feeding test. In this further exacerbated in 24-mo-old NIRKO mice, with a 38 ± 6% test, mice are fasted for 16 h and then placed in a large animal reduction compared with control (P < 0.05) (Fig. 2 A and B and box containing a white disk with food in its center. They are Fig. S4A). To investigate whether reduced mitochondrial func- therefore challenged to choose between going for the food and tion was a result of decreased mitochondrial biogenesis, we an- avoiding the unusual white disk on which it rests. Delay in en- alyzed key transcriptional regulators of mitochondrial biogenesis tering the center of the field is considered to have increased in dorsal striatum and NAC, regions implicated in anxiety and anxiety. Again, in this test, 17-mo-old, but not 10-mo-old, female depressive disorders. This analysis revealed overall unaltered

3464 | www.pnas.org/cgi/doi/10.1073/pnas.1500877112 Kleinridders et al. Downloaded by guest on September 30, 2021 expression of Pgc1a (peroxisome proliferator-activated receptor ABC* gamma coactivator 1), Pgc1b,andTfam in young and old NIRKO 5000 5000 * IR β mice, with the exception of temporarily increased mRNA levels of 4000 4000 CV- ATP5A Pgc1b in striatum of NIRKO mice at 4 mo of age (Fig. S4 B–G). 3000 3000 CIII- UQCRC2 2000 2000 Western blot analysis of striatal samples from 4-mo-old control CIV-MTCQ1 AUC OCR AUC OCR 1000 1000

and NIRKO mice also revealed reduced levels of complex I 0 0 CII-SDHB subunit NDUFB8, complex II subunit SDHB, complex III sub- Control NIRKO Control NIRKO unit UQCRC2, complex IV subunit MTCQ1, and complex V D CI-NDUFB8 ATP synthase subunit alpha (Fig. 2C and Fig. S4H). Analysis of Control NIRKO mitochondrial morphology in the striatum using electron mi- β-actin croscopy revealed a 53 ± 8% reduction in average mitochondrial E Control NIRKO cross-sectional area and a 32 ± 4% increase in mitochondrial number (Fig. 2 D and E), with no change in the level of ex- ** pression of enzymes involved in mitochondrial fusion and fission 800 0.4 * (Opa1, Mfn1, Mfn2, Drp1, and Mtfr1) or phosphorylation of Drp1 600 0.3 at serine 616 and 637 (Fig. S5 A–H). Thus, NIRKO mice exhibit 400 0.2 altered brain mitochondrial morphology and mitochondrial 200 0.1 0 0.0 dysfunction with reduced levels of proteins of the electron (um2) Area Mito. ControlNIRKO ControlNIRKO

transport chain and reduced basal respiration early in life before um2 Number/100 Mito. any behavioral changes can be detected. Fig. 2. NIRKO mice display mitochondrial dysfunction. Mitochondrial activity was measured using isolated brain mitochondria in a Seahorse Flux NIRKO Mice Suffer from Oxidative Stress. Mitochondrial dysfunc- Analyzer as described in Materials and Methods. Basal respiration mea- tion is often associated with oxidative stress. Consistent with this surements, displayed as area under the curve, of isolated brain mitochondria association, there was a 1.4-fold increase in lipid peroxidation in from (A) 4-mo-old and (B) 24-mo-old control and NIRKO mice using the XF24 brains of NIRKO mice as determined by thiobarbituric acid re- Extracellular Flux Analyzer from Seahorse Bioscience. A total of six control active substances (TBARS) as early as 4 mo of age, and this and six NIRKO mice were used. (C) Determination of striatal protein levels of increase persisted at 24 mo of age (Fig. 3 A and B). Protein electron transport chain complexes. Representative Western blots are shown oxidation, as assessed by derivatization of protein carbonyl groups of nuclear- and mitochondrial-encoded genes (IR beta, CV-ATP5A, CIII- UQCRC2, CIV-MTCQ1, CII-SDHB, and CI-NDUFB8) using striatal samples from with 2,4-dinitrophenylhydrazine (DNPH), was also increased in = = β NIRKO brains (Fig. 3C). Interestingly, although mitochondrial 4-mo-old female control (n 6) and NIRKO mice (n 6). -Actin served as a loading control. (D) Transmission electron microscopy of brain striatum dysfunction is often associated with an inflammatory response showing mitochondria of 4-mo-old female control and NIRKO mice. This is and increased cellular apoptosis, the mild oxidative stress in a representative example of 12 fields in four animals of each genotype. Note NIRKO mice did not induce inflammation or apoptosis by mul- the decreased mitochondrial diameter. Original magnification 1:10,000. tiple measures (Fig. S6 A–D). (E) Morphometric analysis of electron microscopy using ImageJ. Average mitochondrial area and total mitochondrial number of control and NIRKO mice NIRKO Mice Exhibit Decreased Dopamine Signaling. The monoamine (n = 4 mice of each genotype with 100–200 mitochondria analyzed). hypothesis of depression postulates that the deficiency of neurotransmitters, such as dopamine, serotonin, and catechol- amines, in the brain is responsible for the manifestations of decrease in the average width of the evoked dopamine signal and depression (42). Assessment of the levels of serotonin and cat- a53± 9% decrease in area under the peak (Fig. 4B), whereas echolamines, as well as their degradation products, in the brains recording in the prefrontal cortex, which is not a part of the of 17-mo-old NIRKO mice revealed no differences from control mesolimbic system, did not reveal alterations in dopamine sig- (Fig. S7 A–D). Likewise, blood pressure and heart rate were naling (Fig. S8 I and J). Likewise, catecholamine release from unchanged in control and NIRKO mice, suggesting similar ac- the adrenal glands of NIRKO mice was unaltered compared with

tivation of the sympathetic nervous system (Fig. S7 E–G). There control (Fig. S9C). These data indicate that the increase in do- MEDICAL SCIENCES was a small increase in tryptophan hydroxylase 2, the rate-lim- pamine turnover in NIRKO mice is restricted to the dorsal iting enzyme in the synthesis of serotonin, in the raphe nucleus of striatum and NAC and not part of a general defect in neurose- 4-mo-old NIRKO mice, but this level returned to normal in older cretory granule release or turnover. NIRKO animals and was not observed in the other brain regions The reduced dopamine signaling in the dorsal striatum and analyzed (Fig. S7 H and I). Furthermore, there was no consistent NAC could be the result of increased reuptake by the dopamine alteration in the expression of the serotonin reuptake transporter reuptake transporter (DAT) or increased degradation by mito- Slc6a4 nor in serotonin receptor 4, both genes important for chondrial Mao A and B. Whereas the levels of Dat mRNA in serotonin signaling and regulation of behavior (Fig. S7 J and K). striatal and NAC samples from NIRKO mice were unaltered Dopamine signaling in the striatum and mesolimbic system has (Fig. S9 D and E), mRNA levels of Maoa (P < 0.05) and Maob been shown to be particularly important in depression (32, 33). (did not quite reach statistical significance, P = 0.09) were in- Because there was no change in the levels of mRNA for tyrosine creased in striatal and MAO A levels in NAC samples (Fig. S9 hydroxylase, the rate-limiting enzyme for dopamine synthesis F and G)by40–50%. This finding was confirmed by Western (see Fig. S9 A and B), we further explored this pathway by as- blot analysis, which showed a twofold increase of MAO A protein sessment of electrically evoked dopamine release using carbon (P < 0.01) and a 1.3-fold increase in MAO B protein (P = 0.05) in fiber amperometry. This assessment revealed no change in the the striatum of NIRKO mice by 4 mo of age (Fig. 4 C–E). peak amplitude of electrically evoked dopamine release in brains To test whether increased MAO A and B levels were a direct of 10- and 17-mo-old NIRKO mice in the dorsal striatum (Fig. effect of reduced insulin signaling in NIRKO mice, we assessed S8 A–G). However, there was a 40 ± 9% decrease in the average the effect of insulin on neurons in vitro. Stimulation of the hy- width of the evoked dopamine signal and a 44 ± 10% decrease in pothalamic neuronal GT1–7 cell line with 100 nM insulin for t1/2 (duration of the signal at 50% of its peak amplitude) in the 24 h caused 23 ± 2% and 62 ± 2% reductions in Maoa and Maob dorsal striatum as measured by the peak width at half height mRNA levels and 46 ± 8% and 73 ± 2% reductions of MAO A (both P < 0.05) (Fig. 4A and Fig. S8H). This resulted in a 39 ± and B protein, respectively (all P < 0.05, Fig. 4F and Fig. S9H). 14% decrease in area under the peak in 17-mo-old NIRKO mice. A similar effect of insulin on Maoa and Maob expression was Similarly, recordings obtained from NAC revealed a 53 ± 8% observed in primary neurons (Fig. S9I). Glial cells can also

Kleinridders et al. PNAS | March 17, 2015 | vol. 112 | no. 11 | 3465 Downloaded by guest on September 30, 2021 AB CDNPH staining (schematizedinFig.5C). Thus, a decrease in insulin action in Control NIRKO the brain can directly alter dopamine turnover and lead to ein depressive behaviors via effects at the mitochondrial level. 2.0 * 1.5 prot * The loss of insulin receptor signaling in the brain affects 1.5 1.0

Striatum mitochondrial function in at least three ways: (i) decreased mi- 1.0 0.5 tochondrial activity due to decreased expression of electron 0.5

Snmol/mg transport chain proteins; (ii) increased monoamine oxidase lev-

R 0.0 0.0

ControlNIRKO ControlNIRKO Nucleus els due to a loss of insulin action to suppress MAO gene ex- accumbens TBARS nmol/mg protein TBA pression; and (iii) changes in the morphology of mitochondria in Fig. 3. NIRKO mice suffer from oxidative stress. (A and B) Lipid perox- brain, with smaller and more numerous mitochondria. Whereas idation, as a measure of oxidative stress, was measured using thiobarbituric the first two ways appear to be direct effects of a loss of insulin acid reactive substances (TBARS). Quantification of lipid peroxidation using action, the exact mechanism for the third is unclear. Diet- TBARS assay of (A) 4-mo-old and (B) 24-mo-old control (n = 6) and NIRKO induced obesity can reduce mRNA expression of mitofusin 2, a mitochondria (n = 6). (C) Protein carbonylation, as a measure of oxidative key enzyme in mitochondrial fusion, in proopiomelanocortin stress, was assessed by antibody staining against DNP. Representative im- neurons in the hypothalamus, suggesting that central insulin re- munohistochemical staining of protein carbonylation in the striatum and = = sistance can affect mitochondrial dynamics (48). However, we nucleus accumbens of NIRKO mice (n 4) compared with control (n 4). did not detect any differences in expression levels of regulators of mitochondrial fission/fusion in brains of NIRKO mice. Serine participate in dopamine degradation, and insulin stimulation of phosphorylation of Drp1, a regulator of mitochondrial fission, alters primary glial cells in vitro resulted in 52 ± 4% reduction in Maoa its activity and thereby regulates mitochondrial fission. However, mRNA levels, but did not affect expression levels of Maob (Fig. 4 G NIRKO mice exhibit no changes in phosphorylation of Drp1 and H). Thus, the increased levels of MAO A in brain of NIRKO at serine residue 616, which stimulates mitochondrial fission, or mice appear to be the direct consequence of the loss of insulin re- serine residue 637, which inhibits fission. Recent work has shown ceptorsignalinginbothneuronsandglia,whereastheincreasein that Drp1 can also be phosphorylated at Ser-693 by GSK3-beta MAO B is due primarily to the loss of insulin signaling in neurons. (49), and GSK3-beta is a known downstream target of insulin receptor signaling, such that insulin induced phosphorylation of the Depressive-Like Behavior in NIRKO Mice Is Reversible with Antidepressant enzyme reduces its activity. We have previously shown that NIRKO Treatment. To further explore the relationship between the de- mice display increased GSK3-beta activity (17), but whether this pressive-like behavior, mitochondrial dysfunction, and altered do- affects Ser-693 phosphorylation of Drp1 and mitochondrial dy- pamine signaling in NIRKO mice, we subjected these mice to namics is unclear, because no antibodies specific to this site exist. treatment with the antidepressant imipramine, which has been Mechanistically, with regard to depression and anxiety, it is shown to target MAO A and B, as well as reduce oxidative stress important to note that NIRKO mice show increased rates of secondary to mitochondrial dysfunction (43, 44). Similar to the experiment shown in Fig. 1A, saline-treated NIRKO mice exhibited a 31 ± 13% increase in immobility time during a tail A BCMAO A suspension test compared with saline-treated controls, consistent 8 ** 10 *** β-actin 8 with depressive-like behavior (Fig. 5A). Following imipramine 6 6 Control NIRKO treatment, there was a modest, nonsignificant reduction in im- 4 ± 4 D mobility in controls but a significant 52 9% reduction in in N A c MAO B 2 2 immobility time in NIRKO mice (P < 0.003), such that the in striatum β-actin Peak Width (s) Peak Width (s) 0 0 imipramine-treated NIRKO mice were indistinguishable from ControlNIRKO Control NIRKO Control NIRKO control (Fig. 5A). A similar effect was observed with the irre- 0.05 250 * 300 ** 1.5 E U.) **

versible MAO A and B inhibitor phenelzine (Fig. 5B). Thus, the 200 A. 200 1.0 age-dependent depressive-like behavior in NIRKO mice was 150 on (

100 si reversible with antidepressant treatments, which target dopa- 100 0.5 in N A c OAandB es A r mine turnover and mitochondrial dysfunction. in striatum 50 M 0 0 xp 0.0 Peak Area (pA/s) Peak Area (pA/s) e Discussion ControlNIRKO Control NIRKO MAO A MAO B Both type 1 and type 2 diabetes are associated with a variety of F Basal GHControl Insulin NIRKO CNS complications, including increased rates of cognitive decline, 150 150 altered brain imaging, increased risk of neurodegenerative disease, ** ** ** 150 100 100 100

and increased rates of depression; however, how alterations in in- ontrol) c

sulin signaling might contribute to these complications is still to TBP 50 50 50 poorly understood (5, 45–47). Using mice with a brain-specific %of Maoa mRNA Maob mRNA (% of control) knockout of insulin receptor, we show that depressive-like behavior 0 ( 0 0 eaieExpression Relative Maoa Maob Basal Insulin B asal Insulin and anxiety can be a direct consequence of insulin resistance in the brain. These alterations occur in a progressive, staged process. Fig. 4. NIRKO mice exhibit decreased dopamine signaling. (A and B) In vivo Thus, young NIRKO mice exhibit brain mitochondrial dysfunction determination of dopamine release in the (A) striatum and (B) nucleus and oxidative stress, especially in the dorsal striatum and NAC. accumbens. (Upper) Average peak width and (Lower) average area of elec- NIRKO mice also exhibit increased levels of the dopamine- trically evoked dopamine release of striatal slices using carbon fiber amper- degrading enzymes Mao A and B, and these increased levels result ometry of 17-mo-old female control (slices = 11, recordings = 45, mice = 7) in increased dopamine clearance and decreased dopamine signal- and NIRKO mice (slices = 11, recordings = 43, mice = 6). (C–E) Representative ing. Furthermore, the in vivo and in vitro experiments presented Western blots and densitometric analysis of MAO A and B protein levels in the striatum of 4-mo-old control (n = 5) and NIRKO mice (n = 7). (F)Geneex- here show that altered MAO A and B expression is a direct result of pression analysis of Maoa and Maob in hypothalamic GT1–7 cells under basal a loss of insulin signaling in neurons and glia. As NIRKO mice conditions (n = 4) and following stimulation with 100 nM insulin (n = 4) for age, these alterations in CNS metabolism and dopaminergic 24 h. (G and H)Geneexpressionanalysisof(G) Maoa and (H) Maob in pri- signaling result in multiple signs of anxiety and depressive-like mary glial cells under basal conditions (n = 4) and following stimulation with behavior, which can be reversed by inhibiting MAO activity 100 nM insulin (n = 4) for 24 h.

3466 | www.pnas.org/cgi/doi/10.1073/pnas.1500877112 Kleinridders et al. Downloaded by guest on September 30, 2021 t resistance are associated with hypothalamic inflammatory changes s

A ) *** B e s 0.05 **

( (57, 58). Because old NIRKO mice are neither obese nor hyper- T 200 200 * e Control Control

m glycemic, and have no brain inflammation, but still develop mood 150 NIRKO 150 NIRKO 100 100 disorders, it seems clear that brain insulin resistance, without these pension other abnormalities can lead to behavioral phenotypes. Importantly,

blt ti obility 50 us 50

0 0 this depressive phenotype can be reversed by treatment with anti- mm I Im m obility tim e (s) ail S Saline Im ipram ine Saline Phenelzine T Tail Suspension Test depressive drugs. Previous studies have shown that treating obese, C Healthy Brain Insulin Resistance diabetic mice with the insulin sensitizer rosiglitazone, which has been IR signaling Insulin Resistance shown to reduce reactive oxygen species formation in the brain (59), can also decrease depressive-like behavior (60). Whether this decrease is through increasing insulin sensitivity in the brain or sys- Mao A and B Mao A and B temically remains to be determined. OxidaƟve stress OxidaƟve stress Our data demonstrate that insulin resistance in the brain can lead to alterations in mitochondrial function, increased levels of monoamine oxidases, and increased dopamine clearance (Fig. Dopamine signaling Dopamine signaling 5C). This is consistent with other studies suggesting that mitochondrial dysfunction can contribute to depression (61). Of course, these alterations may also interact with other factors, which can Improved mood Anxiety and depression disorders contribute to depression and anxiety, including genetic suscepti- bility, alterations in circadian rhythms, or changes in content of Fig. 5. Depressive-like behavior in NIRKO mice is reversible with drug treat- monoamines such as serotonin, norepinephrine, and dopamine ment. (A) Immobility time during tail suspension test of 24-mo-old female (62). Thus, the increased incidence of depression in patients with control and NIRKO mice treated with saline or imipramine (16 mg/kg) 1 h diabetes can be a consequence of central insulin resistance. Im- prior to the assessment (each n = 6). (B) Immobility time during a tail suspension test of 24-mo-old female control and NIRKO mice treated with sa- proving central insulin signaling could lead to new therapeutic line or phenelzine (20 mg/kg) for 1 h prior to the assessment (n = 9–11). approaches for treatment of mood disorders, especially in patients Significance was determined by a one-way ANOVA followed by Fisher post with diabetes or metabolic syndrome. hoc analysis. (C) Model of insulin signaling effects on mood and behavior in healthy mice (Left) and mice with brain insulin resistance (Right). Materials and Methods Animal Care. NIRKO mice were generated as previously described (15) and were backcrossed into a C57BL/6J background for at least nine generations dopamine clearance. This is secondary to increases in MAO A before the study. All mice were housed in a mouse facility on a 12-h light/ and B in the striatum and NAC of NIRKO brains. The MAO dark cycle in a temperature-controlled room and maintained on a standard enzymes reside in the outer mitochondrial membrane and have chow diet containing 22% calories from fat, 23% from protein, and 55% also been shown to be increased in human depression (39). from carbohydrates (Mouse Diet 9F 5020; PharmaServ) beginning at ∼4wk Dopamine is predominantly degraded by MAO A in rodents and of age. Mice were allowed ad libitum access to water and food. Animal care by MAO B in humans (50, 51). Based on in vitro studies, it and study protocols were approved by the Institutional Animal Care and Use Committee of Brandeis/Joslin Diabetes Center and were in accordance with appears that insulin can directly down-regulate both MAO A and the National Institutes of Health guidelines. B in neurons and MAO A in glial cells. Thus, reduced insulin action in brain results in up-regulation of MAO A and B, in- Behavioral Tests. To assess depressive-like behavior, we performed the mouse creased dopamine clearance, and decreased dopamine signaling. tail suspension test and forced swimming test on naïve 10-, 17-, and 24-mo- Patients with depression also exhibit decreased half-life of do- old mice. Importantly, depressive-like behavior testing was performed only pamine in the striatum (52), similar to our findings in NIRKO once in a lifetime per mouse to avoid any learning effect in mice. To assess mice. It is unclear why MAO dysregulation was detected in the anxiety, novelty suppressed feeding test, dark/light box test, and stress by restraint test were performed. Open field test was performed to evaluate

striatum and NAC of NIRKO mice, and not in the prefrontal MEDICAL SCIENCES cortex. This may suggest that discrete compartments in the brain exploratory drive. Results of the open field test were quantitated using ANY differ in their response to insulin (53), or that different cellular Maze video tracking software (Stoelting). compositions in discrete brain areas result in different effects of insulin resistance (54). MAO A and B also degrade serotonin, Slice Electrophysiology. Coronal slices from fresh mouse brains were used for thus affecting serotonin signaling. As altered serotonin signaling slice electrophysiology and carbon fiber amperometry as previously described (63). Amperometric peaks were identified as events greater than 3.5 times is also associated with depression, this pathway may also con- the rms noise of the baseline. The event width was the duration between tribute to the pathogenesis of mood disorders (55). We have not (i) the baseline intercept of the maximal incline from the baseline to the first observed any general differences of serotonin metabolite con- point that exceeded the cut off and (ii) the first data point following the centrations in the brain or changes in the rate-limiting enzymes maximal amplitude that registered a value of ≤0 pA. The maximum ampli- for its synthesis in various brain regions. However, we cannot tude (imax) of the event was the highest value within the event. rule out that specifically altered serotonin signaling in distinct brain compartments may also play a role in this scenario. Further High-Performance Liquid Chromatography with Electrochemical Detection. μ research will be needed to address these questions. From each striatal sample, 20 L of a 1:15 dilution with nanopure H20 It is interesting to note that despite the alterations in brain were injected into an amperometric Antec Intro High-Performance Liquid mitochondrial function in the NIRKO mouse, the resulting mi- Chromatography with Electrochemical Detection (HPLC-EC) system (GBC) tochondrial stress does not induce a proinflammatory response with a 10-cm Rainin column and a phosphate mobile phase buffer, which or measurable signs of apoptosis, even in a prooxidative dopa- allows the separation and detection of serotonin, norepinephrine, and the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA). minergic system (56). This finding is consistent with previous studies indicating that ablation of the insulin receptor in dopa- Statistical Methods. Data sets were analyzed for statistical significance using minergic neurons does not itself result in reduced number of a two-tailed unpaired Student’s t test or one- or two-way ANOVA, as ap- neurons (29) and suggests that insulin signaling in the brain acts propriate. P values <0.05 were considered statistically significant. primarily to fine tune brain activity, rather than as a survival Western blot and qPCR analysis, EM, lipid peroxidation, and mitochondrial factor for neurons. This is different from the situation in obese/ respiration assays, analytical procedures, immunohistochemistry, and cell diabetic mice in which obesity, hyperglycemia, and systemic insulin culture procedures are detailed in SI Materials and Methods.

Kleinridders et al. PNAS | March 17, 2015 | vol. 112 | no. 11 | 3467 Downloaded by guest on September 30, 2021 ACKNOWLEDGMENTS. We thank M. Rourk and G. Smyth for animal care; experimental assistance. This work was supported by NIH Grants R01 G. Sankaranarayanan [Joslin Diabetes Center’s Diabetes and Endocrinology DK033201 and R01 DK031036 and the Mary K. Iacocca Professorship Research Center (DERC) Specialized Assay Core], C. Cahill (Joslin’s DERC Ad- (to C.R.K.), DK065872 (to E.N.P.), and P30 DK036836 (Joslin’sDERCCore vanced Microscopy Core), and Maura Mulvey (Joslin’s DERC Physiology Core) Facilities) and Deutsche Forschungsgesellschaft (DFG) Projects KL2399/1-1 for technical assistance; and Elizabeth Hanson and Laura Darnieder for and KL2399/3-1 (to A.K.).

1. Kirkwood TB (2008) A systematic look at an old problem. Nature 451(7179):644–647. 34. Patti ME, et al. (2003) Coordinated reduction of genes of oxidative metabolism in 2. Kodl CT, Seaquist ER (2008) Cognitive dysfunction and diabetes mellitus. Endocr Rev humans with insulin resistance and diabetes: Potential role of PGC1 and NRF1. Proc 29(4):494–511. Natl Acad Sci USA 100(14):8466–8471. 3. Xu Q, et al. (2011) Diabetes and risk of Parkinson’s disease. Diabetes Care 34(4): 35. Zhang D, et al. (2007) Mitochondrial dysfunction due to long-chain Acyl-CoA de- 910–915. hydrogenase deficiency causes hepatic steatosis and hepatic insulin resistance. Proc 4. Bolo NR, et al. (2011) Brain activation during working memory is altered in patients Natl Acad Sci USA 104(43):17075–17080. with type 1 diabetes during hypoglycemia. Diabetes 60(12):3256–3264. 36. Kleinridders A, et al. (2013) Leptin regulation of Hsp60 impacts hypothalamic insulin 5. Musen G, et al. (2012) Resting-state brain functional connectivity is altered in type 2 signaling. J Clin Invest 123(11):4667–4680. diabetes. Diabetes 61(9):2375–2379. 37. Gardner A, Boles RG (2011) Beyond the serotonin hypothesis: Mitochondria, in- 6. den Heijer T, et al. (2003) Type 2 diabetes and atrophy of medial temporal lobe flammation and neurodegeneration in major depression and affective spectrum structures on brain MRI. Diabetologia 46(12):1604–1610. disorders. Prog Neuropsychopharmacol Biol Psychiatry 35(3):730–743. 7. Hoogenboom WS, et al. (2014) Cerebral white matter integrity and resting-state 38. Dlugos AM, Palmer AA, de Wit H (2009) Negative emotionality: Monoamine oxidase B functional connectivity in middle-aged patients with type 2 diabetes. Diabetes 63(2): gene variants modulate personality traits in healthy humans. J Neural Transm 116(10): 728–738. 1323–1334. 8. Ryan JP, Sheu LK, Critchley HD, Gianaros PJ (2012) A neural circuitry linking insulin 39. Meyer JH, et al. (2006) Elevated monoamine oxidase a levels in the brain: An expla- resistance to depressed mood. Psychosom Med 74(5):476–482. nation for the monoamine imbalance of major depression. Arch Gen Psychiatry 9. Bomfim TR, et al. (2012) An anti-diabetes agent protects the mouse brain from de- 63(11):1209–1216. fective insulin signaling caused by Alzheimer’s disease-associated Aβ oligomers. J Clin 40. Villeneuve C, et al. (2013) p53-PGC-1α pathway mediates oxidative mitochondrial Invest 122(4):1339–1353. damage and cardiomyocyte necrosis induced by monoamine oxidase-A upregulation: 10. Talbot K, et al. (2012) Demonstrated brain insulin resistance in Alzheimer’s disease Role in chronic left ventricular dysfunction in mice. Antioxid Redox Signal 18(1):5–18. patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline. 41. Federico A, et al. (2012) Mitochondria, oxidative stress and neurodegeneration. J Clin Invest 122(4):1316–1338. J Neurol Sci 322(1-2):254–262. 11. Craft S, et al. (2012) Intranasal insulin therapy for Alzheimer disease and amnestic 42. Lanni C, Govoni S, Lucchelli A, Boselli C (2009) Depression and antidepressants: Mo- mild cognitive impairment: A pilot clinical trial. Arch Neurol 69(1):29–38. lecular and cellular aspects. Cell Mol Life Sci 66(18):2985–3008. 12. Erion R, Sehgal A (2013) Regulation of insect behavior via the insulin-signaling 43. Nag M (2004) Effect of chlorpromazine, imipramine and lithium on MAO-A and MAO-B pathway. Front Physiol 4:353. activity in rat brain mitochondria. Indian J Exp Biol 42(9):941–944. 13. Plum L, Belgardt BF, Brüning JC (2006) Central insulin action in energy and glucose 44. Réus GZ, et al. (2010) Harmine and imipramine promote antioxidant activities in homeostasis. J Clin Invest 116(7):1761–1766. prefrontal cortex and hippocampus. Oxid Med Cell Longev 3(5):325–331. 14. Kleinridders A, Ferris HA, Cai W, Kahn CR (2014) Insulin action in brain regulates 45. Anthony K, et al. (2006) Attenuation of insulin-evoked responses in brain networks systemic metabolism and brain function. Diabetes 63(7):2232–2243. controlling appetite and reward in insulin resistance: The cerebral basis for impaired 15. Brüning JC, et al. (2000) Role of brain insulin receptor in control of body weight and control of food intake in metabolic syndrome? Diabetes 55(11):2986–2992. reproduction. Science 289(5487):2122–2125. 46. Frölich L, Blum-Degen D, Riederer P, Hoyer S (1999) A disturbance in the neuronal 16. Fisher SJ, Brüning JC, Lannon S, Kahn CR (2005) Insulin signaling in the central nervous insulin receptor signal transduction in sporadic Alzheimer’s disease. Ann N Y Acad Sci system is critical for the normal sympathoadrenal response to hypoglycemia. Diabetes 893:290–293. 54(5):1447–1451. 47. Sandyk R (1993) The relationship between diabetes mellitus and Parkinson’s disease. 17. Schubert M, et al. (2004) Role for neuronal insulin resistance in neurodegenerative Int J Neurosci 69(1-4):125–130. diseases. Proc Natl Acad Sci USA 101(9):3100–3105. 48. Schneeberger M, et al. (2013) Mitofusin 2 in POMC neurons connects ER stress with 18. Figlewicz DP, MacDonald Naleid A, Sipols AJ (2007) Modulation of food reward by leptin resistance and energy imbalance. Cell 155(1):172–187. adiposity signals. Physiol Behav 91(5):473–478. 49. Chou CH, et al. (2012) GSK3beta-mediated Drp1 phosphorylation induced elongated 19. Chiu SL, Chen CM, Cline HT (2008) Insulin receptor signaling regulates synapse mitochondrial morphology against oxidative stress. PLoS ONE 7(11):e49112. number, dendritic plasticity, and circuit function in vivo. Neuron 58(5):708–719. 50. Glover V, Sandler M, Owen F, Riley GJ (1977) Dopamine is a monoamine oxidase B 20. Sanderson TH, et al. (2013) Cytochrome C is tyrosine 97 phosphorylated by neuro- substrate in man. Nature 265(5589):80–81. protective insulin treatment. PLoS ONE 8(11):e78627. 51. Waldmeier PC, Delini-Stula A, Maître L (1976) Preferential deamination of dopamine 21. Gelling RW, et al. (2006) Insulin action in the brain contributes to glucose lowering by an A type monoamine oxidase in rat brain. Naunyn Schmiedebergs Arch Phar- during insulin treatment of diabetes. Cell Metab 3(1):67–73. macol 292(1):9–14. 22. Niswender KD, et al. (2003) Insulin activation of phosphatidylinositol 3-kinase in the 52. Laasonen-Balk T, et al. (1999) Striatal dopamine transporter density in major de- hypothalamic arcuate nucleus: A key mediator of insulin-induced anorexia. Diabetes pression. Psychopharmacology (Berl) 144(3):282–285. 52(2):227–231. 53. Klöckener T, et al. (2011) High-fat feeding promotes obesity via insulin receptor/ 23. Sadagurski M, et al. (2011) IRS2 increases mitochondrial dysfunction and oxidative PI3K-dependent inhibition of SF-1 VMH neurons. Nat Neurosci 14(7):911–918. stress in a mouse model of Huntington disease. J Clin Invest 121(10):4070–4081. 54. Herculano-Houzel S (2014) The glia/neuron ratio: How it varies uniformly across brain 24. Mezuk B, Eaton WW, Albrecht S, Golden SH (2008) Depression and type 2 diabetes structures and species and what that means for brain physiology and evolution. Glia over the lifespan: A meta-analysis. Diabetes Care 31(12):2383–2390. 62(9):1377–1391. 25. Sharma AN, Elased KM, Garrett TL, Lucot JB (2010) Neurobehavioral deficits in db/db 55. Bortolato M, Chen K, Shih JC (2008) Monoamine oxidase inactivation: From patho- diabetic mice. Physiol Behav 101(3):381–388. physiology to therapeutics. Adv Drug Deliv Rev 60(13-14):1527–1533. 26. Stuart MJ, Baune BT (2012) Depression and type 2 diabetes: Inflammatory mecha- 56. Hastings TG (2009) The role of dopamine oxidation in mitochondrial dysfunction: nisms of a psychoneuroendocrine co-morbidity. Neurosci Biobehav Rev 36(1):658–676. Implications for Parkinson’s disease. J Bioenerg Biomembr 41(6):469–472. 27. Könner AC, Brüning JC (2012) Selective insulin and leptin resistance in metabolic 57. Thaler JP, et al. (2012) Obesity is associated with hypothalamic injury in rodents and disorders. Cell Metab 16(2):144–152. humans. J Clin Invest 122(1):153–162. 28. De Felice FG, Ferreira ST (2014) Inflammation, defective insulin signaling, and mito- 58. García-Cáceres C, Yi CX, Tschöp MH (2013) Hypothalamic astrocytes in obesity. chondrial dysfunction as common molecular denominators connecting type 2 di- Endocrinol Metab Clin North Am 42(1):57–66. abetes to Alzheimer disease. Diabetes 63(7):2262–2272. 59. Diano S, et al. (2011) Peroxisome proliferation-associated control of reactive oxygen 29. Könner AC, et al. (2011) Role for insulin signaling in catecholaminergic neurons in species sets melanocortin tone and feeding in diet-induced obesity. Nat Med 17(9): control of energy homeostasis. Cell Metab 13(6):720–728. 1121–1127. 30. Figlewicz DP, Szot P, Chavez M, Woods SC, Veith RC (1994) Intraventricular insulin 60. Sharma AN, Elased KM, Lucot JB (2012) Rosiglitazone treatment reversed depression- increases dopamine transporter mRNA in rat VTA/substantia nigra. Brain Res 644(2): but not psychosis-like behavior of db/db diabetic mice. J Psychopharmacol 26(5): 331–334. 724–732. 31. Heninger GR, Delgado PL, Charney DS (1996) The revised monoamine theory of de- 61. Rezin GT, Amboni G, Zugno AI, Quevedo J, Streck EL (2009) Mitochondrial dysfunc- pression: A modulatory role for monoamines, based on new findings from mono- tion and psychiatric disorders. Neurochem Res 34(6):1021–1029. amine depletion experiments in humans. Pharmacopsychiatry 29(1):2–11. 62. Perona MT, et al. (2008) Animal models of depression in dopamine, serotonin, and 32. Chaudhury D, et al. (2013) Rapid regulation of depression-related behaviours by norepinephrine transporter knockout mice: Prominent effects of dopamine trans- control of midbrain dopamine neurons. Nature 493(7433):532–536. porter deletions. Behav Pharmacol 19(5-6):566–574. 33. Tye KM, et al. (2013) Dopamine neurons modulate neural encoding and expression of 63. Geiger BM, et al. (2009) Deficits of mesolimbic dopamine neurotransmission in rat depression-related behaviour. Nature 493(7433):537–541. dietary obesity. Neuroscience 159(4):1193–1199.

3468 | www.pnas.org/cgi/doi/10.1073/pnas.1500877112 Kleinridders et al. Downloaded by guest on September 30, 2021