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Insulin Receptor Function In Rat Hippocampus

Neural Insulin Resistance And High-Fat Diets


The development of abdominal fleshiness following chronic ingestion of a high-fat diet contributes to peripheral insulin opposition. Although the relationship between peripheral insulin opposition and cognitive damage have been shown, the consequence of high-fat ingestion and the neurofunctional insulin sensitiveness in CA1 hippocampus is ill-defined. We tested the hypothesis that high-fat diet ingestion can take to peripheral insulin opposition and impair neural responses to insulin by utilizing extracellular recording in CA1 hippocampus and the immunoblot technique to find the neural map of insulin receptors ( IRs ) in rats. In 12- hebdomad high-fat-fed ( HF ) rats, peripheral insulin opposition was observed, but was non found in 4-or 8-week HF rats. The neural IR response demonstrated by insulin-mediated long-run depression ( LTD ) in CA1 hippocampus was diminished in 12-week HF rats. This decrease of insulin-mediated LTD correlated with assorted parametric quantities of peripheral insulin opposition ( p & lt ; 0.05 ) . However, the damage of insulin-mediated LTD in hippocampus did non do any alteration in paired-pulse ratio and carbachol-induced LTD between both dietetic groups, proposing that the defect of neural insulin receptors had no consequence on the presynaptic transmittal and did non disrupt other signifiers of synaptic malleability. Furthermore, degrees of phosphorylation of neural IR, neural IR substrate 1 ( IRS-1 ) and neural Akt/PKB in response to insulin were significantly decreased in 12-week HF rats without any alteration in the degree of: IR, IRS-1 and Akt/PKB protein. These findings suggest that neurofunctional insulin opposition can develop at the same clip as peripheral insulin opposition in HF rats. The neural insulin opposition may take to neural ripening as shown in the decrease of nNOS-immunoreactive nerve cells in 12-week HF rats with the damage of neural insulin receptors.


A high-fat diet has been shown as the major cause of fleshiness and insulin opposition ( 1 ) . Surprisingly, sing the dramatic rise of insulin opposition throughout the developed states ( 2, 3 ) and the turning involvement in the function of insulin within the encephalon ( 4-7 ) , there have been merely a few surveies analyzing the effects of this metabolic damage in the cardinal nervous system ( CNS ) . Turning grounds has shown that insulin opposition, defined as a aggressively lessened insulin receptor ( IR ) response to insulin within mark tissues, has grown progressively common in corpulent people ( 8, 9 ) .

The look of insulin receptors ( IRs ) is found throughout the organic structure, in variety meats or cells such as liver, musculus, fat, ruddy blood cells and nerve cells in the CNS ( 10-12 ) . In the CNS, insulin has been shown to modulate neurotransmitter release and synaptic malleability ( 13, 14 ) , and the damage of insulin signaling in the CNS has been shown to associate to neurodegenerative diseases ( 6, 15-19 ) . Evidence from clinical and carnal surveies suggests that insulin/IR signaling may play a function in larning and memory. Zhao and co-workers found that the up-regulation of IR in CA1 hippocampus is associated with short-run memory formation after a spacial acquisition experience ( 20 ) . It has besides been shown that insulin is required to bring forth memory betterment in aged people and patients with Alzheimer ‘s disease ( 21, 22 ) . Furthermore, clinical surveies have shown that cognitive damages are frequently found in association with increased insulin opposition ( 23-25 ) .

The implicit in mechanisms of IRs in acquisition and memory could affect the relationship between IRs and the synaptic malleability in the CNS. Insulin has been shown to play a function in synaptic malleability by moving on alpha-amino-3 hydroxy-5-methylisoxazole-4-propionic acid ( AMPA ) receptor trafficking ( 26-29 ) . It has been demonstrated that insulin facilitates clathrin-dependent internalisation of AMPA receptors, doing long-run depression ( LTD ) of AMPA receptor-mediated synaptic transmittal in hippocampal CA1 nerve cells ( 26, 28 ) . This piece of grounds indicates that the neurofunctional IRs in the CA1 hippocampus is insulin-mediated LTD. Furthermore, the activation of insulin bends on the protein kinase activity of the IR, which triggers Cascadess of signal transduction through its downstream substrate molecules. Several insulin receptor signaling tracts activated by IRs include insulin receptor substrate-1 ( IRS-1 ) ( 30 ) . It has been demonstrated that rats trained in a spacial acquisition undertaking showed the learning-specific addition in IRS-1 in the hippocampal synaptic membranes ( 31 ) . These findings suggest that IR signaling plays a function in larning and memory by modulating activities of synaptic malleability such as insulin-mediated LTD and by triping signal transduction Cascadess such as IR, IRS-1 and Akt/PKB.

Diets have been shown to act upon cognitive maps. In insulin opposition caused by fructose-fed hamsters, it has been shown that hippocampal synaptic malleability, an of import biological mechanism of larning and memory, was impaired ( 32 ) . Excessive fat ingestion has besides been shown to play of import and built-in functions in the development of insulin opposition and type 2 diabetes ( 33 ) . A recent survey demonstrated that ingestion of a high-calorie diet for 32 hebdomads reduced hippocampal synaptic malleability and impaired cognitive map in rats ( 34 ) . Furthermore, several surveies suggest that ingestion of a diet rich in fat for 3 months can develop peripheral insulin opposition and hinder cognitive public presentation ( 6, 7, 35-37 ) . These findings suggest that the development of insulin opposition can intercede the cognitive shortage associated with high-fat diet. However, the consequence of high-fat diet on the neurofunctional IRs is still ill-defined. In add-on, the effects of time-course of high-fat diet ingestion on the neurofunctional IRs have ne’er been investigated. Therefore, in this survey we tested the hypothesis that high-fat diet ingestion for a specific period of clip can do peripheral insulin opposition and can take to impaired neural response to insulin ( or neural insulin opposition ) . We used an electrophysiological survey to look into whether the neural responses to insulin ( insulin-mediated LTD ) are altered by high-fat diet ingestion at different clip class in order to observe the earliest phase of the break of the neurofunctional IRs. We besides examined the change of biochemical activity of insulin receptor tracts: IR? , IRS-1 and Akt/PKB, in the encephalon following each clip class of high-fat diet ingestion. Furthermore, we investigated whether the neural insulin opposition leads to neural ageing utilizing the sum of nNOS-immunoreactive nerve cells as an index of neural ripening.

Materials And Methods

Animals And Dietary Protocols

All experiments were conducted in conformity with an approved protocol from the Faculty of Medicine, Chiang Mai University Institutional Animal Care and Use Committee, in conformity with NIH guidelines. Male Wistar rats weighing ~ 180-200 g were obtained from the National Animal Center, Salaya Campus, Mahidol University, Thailand. All animate beings were separately housed in a temperature-controlled environment with a 12:12 light-dark rhythm. One hebdomad after reaching, rats were indiscriminately assigned to one of the two dietetic groups ( n=43 in high-fat diet group and n=44 in normal diet group ) . The normal-diet ( ND ) group received a standard research lab Zhou, in which 19.7 % of entire energy ( % E ) was from fat, with energy content calculated at 4.02 kcal/g ( Mouse Feed Food No. 082, C.P. Company, Bangkok, Thailand ) . The high-fat ( HF ) group consumed a high-fat diet, incorporating fat, largely from lard ( 59.3 % E ) , with energy content calculated at 5.35 kcal/g, for 12 hebdomads. The animate beings were maintained in single coops with unrestricted entree to nutrient and H2O. Body weight and nutrient consumption were recorded daily. Blood samples were collected from the tail at hebdomads 4, 8 and 12 after fasting for at least 5 hours. Samples for glucose checks were kept on ice in tubings precoated with Na fluoride. Samples for insulin and triglyceride check were taken in tubings with EDTA. Plasma was separated and stored at -80oC for subsequent biochemical analyses. At the terminal of each experimental period ( 4, 8 and 12 hebdomads ) of both dietetic regimens, animate beings were profoundly anesthetized and decapitated. The encephalon was quickly removed for encephalon piece readying and one lobe of liver every bit good as splanchnic fat were removed, weighed and stored at -80oC for farther biochemical analysis.

The unwritten glucose tolerance trial ( OGTT ) was investigated in the 12-week high-fat diet and 12-week normal diet group. After rats being on the dietary for 12 hebdomads, animate beings were fast for 12 hours before they were used in the OGTT. An OGTT consisted of 2 g/kg organic structure weight glucose eating by forced feeding. Blood ( 0.25 milliliter ) was collected from a little cut at the tip of the tail instantly before and at 15, 30, 60 and 120 min after glucose eating. Whole blood was assorted with EDTA and centrifuged at 10,000 revolutions per minute to insulate the plasma. The plasma was stored at -80oC until it was used for glucose analysis with a commercially available kit ( Biotech, Bangkok, Thailand ) .

Analytic Procedure

Fasting plasma glucose and triglyceride concentrations were determined by colorimetric check utilizing commercially available kits ( Biotech, Bangkok, Thailand ) . Fasting plasma insulin degree was measured by Sandwich ELISA kits ( LINCO Research, Missouri, USA ) .

Determination Of Insulin Resistance ( Homa Index )

Insulin opposition was assessed by Homeostasis Model Assessment ( HOMA ) ( 38, 39 ) as a mathematical theoretical account depicting the grade of insulin opposition, calculated from fasting plasma insulin and fasting plasma glucose concentration. A higher HOMA index indicates a higher grade of insulin opposition. The HOMA index was determined by the undermentioned equation:

[ fasting plasma insulin ( µU/ml ) ] ten [ fasting plasma glucose ( mmol/l ) ] 22.5

Analysis Of Liver Triglyceride Concentration

Tissue homogenates were prepared for triglyceride check by a alteration of the method of Frayn and Maycock ( 40 ) . A 100-200 milligram part of liver was minced and put into a glass tubing incorporating 3 milliliter of chloroform-isopropanol 7:11 ( v/v ) . The homogenate was left at room temperature for at least 16 hours. Then, 1 milliliter of homogenate was pipetted into a glass tubing and evaporated to dryness at 40oC for 16 hours. The dried residue was dissolved and mixed in 10 % bovine serum albumen. The triglyceride concentration was analyzed with a commercially available kit ( Biotech, Bangkok, Thailand ) .

Brain Slice Preparation

At the terminal of hebdomads 4, 8 and 12, the animate beings were anesthetized with isoflurance after fasting for at least 5 hours. After being decapitated, the encephalon was removed and immersed in ice-cold “high sucrose” aCSF containing ( millimeter ) : NaCl 85 ; KCl 2.5 ; MgSO4 4 ; CaCl2 0.5 ; NaH2PO4 1.25 ; NaHCO3 25 ; glucose 25 ; sucrose 75 ; kynurenic acid 2 ; ascorbate 0.5, saturated with 95 % O2/5 % CO2 ( pH 7.4 ) . This solution enhanced neural endurance during the slicing process ( 41 ) . Hippocampal pieces were cut utilizing a vibratome ( Vibratome Company, St. Louis, Mo. , USA ) . Following a 30-minute post-slice incubation in high saccharose aCSF, pieces were transferred to a standard aCSF solution incorporating ( millimeter ) : NaCl 119 ; KCl 2.5 ; CaCl2 2.5 ; MgSO4 1.3 ; NaH2PO4 1 ; NaHCO3 26 ; and glucose 10, saturated with 95 % O2/5 % CO2 ( pH 7.4 ) for an extra 30 proceedingss, before being used for the extracellular recordings, immunoprecipitation and immunoblotting.

Extracellular Recording Of Hippocampal Slices

To look into insulin-induced long-run depression ( LTD ) , the hippocampal pieces were transferred to a submergence entering chamber and continuously perfused at 3-4 ml/min with standard aCSF warmed to 25-28°C. Field excitant postsynaptic potencies ( fEPSPs ) were evoked by exciting the Schaffer collateral–commissural tract with a bipolar wolfram electrode, while recordings were gathered from the stratum radiatum of the hippocampal CA1 part with micropipettes ( 3 Mohm ) filled with 2M NaCl. Stimulus frequence was 0.033 Hz. The stimulus strength was adjusted to give a fEPSP of 0.8-1.0 millivolt in amplitude. Hippocampal pieces were perfused with aCSF ( as baseline status ) for 10 proceedingss and so perfused with aCSF plus 500 nM insulin ( as insulin stimulation ) for 10 proceedingss, after which the hippocampal pieces were perfused with aCSF once more ( rinse out ) and recorded for the following 30 proceedingss.

To look into that the decrease of insulin-mediated LTD was the consequence of an change of neural insulin signaling and non a non-specific change of synaptic transmittal following neural insulin opposition, we examined the characterized signifier of synaptic malleability that may non depend upon insulin signaling, by mensurating mated pulse facilitation ( PPF ) . PPF is a measuring of short-run potentiation, which may happen at the presynaptic sites ( 42 ) . PPF occurs following two indistinguishable stimulations, separated by 50-msec, applied to the Schaffer collateral. PPF was shown as the increased response observed in the 2nd stimulation, compared to the first 1.

To look into carbachol-induced LTD ( mLTD ) , the protocol for bring oning mLTD was as following. The stable 10-min baseline of CA1 extracellular field excitatory postsynaptic potencies ( fEPSPs ) were recorded by exciting the Schaffer collateral-commisural tract with bipolar wolframs electrode. Stimulus frequence was 0.1 Hz and stimulus strength was adjusted to give fEPSPs of 0.8-1.0 millivolt amplitude. The cholinergic agonist, carbachol 50 µM ( Calbiochem, San Diego, CA, USA ) , was superfused for 10 proceedingss to bring on mLTD after which the carbachol was washed out and recorded for the following 30 proceedingss.

All informations were filtered at 3 kilohertz, digitized at 10 kilohertz, and stored on a computing machine utilizing pClamp 9.2 package ( Axon Instruments, Foster City, CA, USA ) . The initial incline of the fEPSPs was measured and plotted versus clip utilizing Origin 8.0 package.

Preparation of encephalon homogenates for immunoprecipitation and immunoblotting

To analyze the change of neural insulin-mediated phosphorylation of the IR, the IRS-1 and the Akt/PKB following 4, 8 and 12 hebdomads of two dietetic regimens, six encephalon pieces per animate being were placed into either aCSF or aCSF plus 500 nM insulin ( Humelin R, Eli Lilly, Giessen, Germany ) for 5 proceedingss. Then, three encephalon pieces in each conditioned group were homogenized in 500 ?l of ice-cold encephalon piece lysis buffer [ 1mM EDTA, 1mM EGTA, 1 % NP-40, 1 % Triton X-100 and supplemented with a peptidase inhibitor cocktail, Roche complete mini-tablets, ( Roche Molecular Biochemicals, Indianapolis, IN, USA ) ] . Following, the homogenates were centrifuged at 9,000 g for 30 proceedingss at 4oC and the protein concentration was measured utilizing the Bio-Rad DC Protein assay kit ( Bio-Rad Laboratories, Hercules, CA, USA ) . These homogenates were so stored at -80oC for farther biochemical analysis of the tyrosine phosphorylation of IR, IRS-1 and Akt/PKB.

To find the degree of IR, IRS-1 and Akt /PKB protein look in the encephalon, another set of three encephalon pieces in aCSF were homogenized over ice in non-ionizing lysis buffer containing: 100mM NaCl, 25mM EDTA, 10mM Tris, 1 % Triton X-100, 1 % NP-40 supplemented with a peptidase inhibitor cocktail ( Roche Molecular Biochemicals ) . Then, homogenates were stored at -80oC for farther biochemical analysis of IR and IRS-1.

Immunoprecipitation And Immunoblotting

IRS-1 protein and tyrosine phosphorylation of IR and IRS-1 were immunoprecipitated from encephalon homogenates with polyclonal antibodies against each protein ( 1 ?g antibody/ 500 ?g entire lysate ) . Rabbit anti-IR and rabbit anti-IRS-1 ( Santa Cruz Biotechnology, Santa Cruz, CA, USA ) with protein A agarose beads were used to fix each protein for immunoprecipitation as antecedently described ( 32 ) . After an nightlong incubation at 4oC, all samples were centrifuged and the supernatant was removed. The beads were washed three times with ice-cold phosphate buffer saline ( PBS ) , assorted with sodium dodecyl sulphate sample ( SDS ) buffer and boiled for 5 proceedingss. Then, the proteins were separated by cataphoresis with SDS-Page on 10 % polyacrylamide gels ( Bio-Rad Laboratories ) and transferred to nitrocellulose membranes. After barricading with 5 % non-fat milk/tris-buffer saline with tween-20 ( TBST ) , immunoblotting was conducted with anti IRS-1 coney and phosphotyrosine antibody ( rabbit polyclonal, 1:600 in TBST, Santa Cruz Biotechnology ) to find the alterations in IRS-1 degree and insulin-mediated tyrosine phosphorylation of the IR and IRS-1, severally.

AKt/PKB in both serine 473 and threonine 308 kinases phosphorylation were electrophoresed and immunoblotted with coney antibodies Akt/PKB both serine 473 and threonine 308. Examination of the degrees of IR and Akt/PKB protein was conducted with homogenates prepared from another set of three encephalon pieces. Both proteins were resolved by the immunoprecipitation and immunoblot assay conducted with coney anti-IR and rabbit anti-Akt/PKB ( 1:1,000 in TBST, Santa Cruz Biotechnology ) . For lading control, immunoblotting for each membrane was completed incubation with anti- ?-actin ( 1:400 ; coney polyclonal ; Sigma, Missouri, USA ) .

All membranes for visualising the phosphorylation and the protein degrees of IR, IRS-1 and Akt/PKB were incubated with secondary caprine animal anti-rabbit antibody conjugated with horseradish peroxidase ( 1:8,000 in TBST, Bio-Rad Laboratories ) . The protein sets were visualized on Amersham hyperfilm ECL ( GE Healthcare, Buckinghamshire, UK ) utilizing Amersham ECL western blotting sensing reagents ( GE Healthcare ) . Band strengths were quantified by Scion Image and the consequences were shown in mean signal strength ( arbitrary ) units.

nNOS Immunohistochemistry

After the terminal of 4- , 8- and 12-week of both dietetic intervention ( n = 6/ group ) , all animate beings were profoundly anesthetized with an intraperitoneal injection of Nembutal ( 80mg/kg organic structure weight ) and perfused through the bosom with cold normal saline solution ( 0.9 % ) and so the 400 milliliter of 4 % paraformaldehyde in 0.1 M phosphate buffer ( PBS ) , pH 7.4. Brains were dissected out of the skull, postfixed for 2 hours in the same fixative and so placed overnight in 30 % sucrose solution in PBS until segmenting. The hippocampal countries of the encephalon were cut in the coronal plane at 40 millimeters thickness with the freeze microtome for nNOS immunohistochemistry. The free-floating subdivisions were washed in PBS for 30 proceedingss and so were incubated for 60 proceedingss with 10 % normal caprine animal serum ( Vector Laboratories, Burlingame, CA, USA ) and incubated overnight at 4oC with nNOS coney antibody ( Santa Cruz Biotechnology, Santa Cruz, CA, USA ) diluted 1: 400 in PBS. A biotinylated caprine animal anti-rabbit secondary antibody ( Vector Laboratories ) was so used at a dilution of 1:200 for 60 proceedingss at room temperature. The antigen-antibody reaction was revealed with avidin-peroxidase composite ( Vectastain ABC kit Elite, Vector Laboratories ) for 60 proceedingss. The peroxidase activity was visualized with 3,3-diamino-benzidine ( Sigma, St. Louis, MO, USA ) . All subdivisions were mounted on gelatin-coated slides, air dried, cleared in xylol and screen slipped with Permount. We have besides performed the controls in our stuffs, in which the primary antibody was omitted and replaced with an tantamount concentration of normal caprine animal serum. We selected at least three slides for each CA1 hippocampal country of all animate beings and counted all the nNOS-immunoractive nerve cells per 100 mm2 in each corresponding country of CA1 hippocampus.

Statistical Analysis

Datas were presented as agencies ± SE. All statistical analyses were performed utilizing the statistical plan SPSS ( version 16 ; SPSS, Chicago, Ill. , USA ) . The significance of the difference between the agency was calculated by Student ‘s t-test with P & lt ; 0.05. Simple correlativity analysis was used to find the relationship between the plasma parametric quantities, liver triglyceride content, splanchnic fat, organic structure weight and the per centums of insulin-mediated LTD.


Peripheral insulin opposition found in 12-week high-fat-fed rats

Initial animate being organic structure weight was non different among experimental groups. Animals fed with high-fat diet ( HF ) for four hebdomads had an increased in organic structure weight compared to rats fed with normal-diet ( ND ) ( Table 1 ) . However, the degree of plasma glucose, plasma triglyceride, plasma insulin, liver triglyceride content and HOMA index did non differ between both dietetic groups.

Animals on the 8-week HF diet besides had a important addition in organic structure weight compared to those with ND eating ( Table 1 ) . Fasting plasma glucose degrees in the 8-week HF-fed animate beings were significantly higher than those in the 8-week ND-fed group. In contrast to plasma glucose degree, plasma insulin degree, plasma triglyceride degree, liver triglyceride content and HOMA index were non significantly different between both dietetic groups ( Table 1 ) .

In the rats on the 12-week HF diet, organic structure weight, splanchnic fat, plasma insulin degree, liver triglyceride content and HOMA index were significantly increased compared to those on the normal diet ( p & lt ; 0.05 ; Table 1 ) . However, there was no important difference in fasting plasma glucose and triglyceride degrees between both groups. The glucose responses during the unwritten glucose tolerance trial ( OGTT ) of the 12-week animate beings of both dietetic groups were used to corroborate the peripheral insulin opposition following 12 hebdomads of HF diet eating. The glucose response to the unwritten glucose burden was markedly increased in 12-week HF diet group at 15- , 30- and 60 proceedingss clip points compared to the ND group ( P & lt ; 0.05, Fig. 1A ) . The entire country under the glucose curve ( AUCg ) was besides significantly increased in the 12-week HF diet group ( P & lt ; 0.05, Fig. 1B ) . These consequences indicate the happening of peripheral insulin opposition in the rats on the 12-week HF diet. Rats fed with the HF diet consumed less nutrient per twenty-four hours than those on the normal diet ( 18.97 + 0.45 and 20.51 + 0.45 g/day in HF and ND groups, severally, P & lt ; 0.05 ) . However, when the consumed nutrient weights were converted to caloric consumption, rats fed with HF diet consumed more Calories per twenty-four hours than rats fed with ND ( 102.05 + 2.41 and 84.10 + 1.85 g for HF and ND groups, severally, P & lt ; 0.01 ) .

High-fat eating for 12 hebdomads significantly reduced the ability of insulin to bring on long term depression ( LTD ) of field excitatory postsynaptic potencies ( fEPSPs ) in hippocampal CA1 circuits.

In all clip classs ( 4- , 8- and 12-week ) of ND-fed rats, we found that insulin application to hippocampal pieces reduced the size of the fEPSP responses at 2-3 proceedingss after the start of insulin extract with maximal effects looking over the undermentioned 10-15 proceedingss, and the depression of fEPSPs was outstanding and long lasted for 30-40 proceedingss ( Fig. 2 and 3 ) . In our set-up, 500 nM insulin added to the extract line required about 1 minute to make the pieces.

In HF animate beings, the grade of insulin-mediated LTD observed from pieces of 4-week and 8-week HF animate beings was non significantly reduced compared to the 4-week and 8-week ND animate beings ( Fig. 2 ) . At 30-minute post-insulin stimulation, the per centum decrease of the normalized fEPSP incline from 4-week ND and 8-week ND groups were 78.57 + 6.92 % and 72.19 + 4.74 % of the mean incline entering during the baseline degree, severally ( n=7-8 independent pieces per group, n=6 animals/group ) . The per centum decrease of fEPSPs of 4-week and 8-week HF pieces were 74.86 + 5.98 % and 76.88 + 3.84 % of the values recorded before insulin application, severally ( n=7-8 independent pieces per group, n=6 animals/group ) ( Fig. 2 ) .

In 12-week HF-fed group, the sum of insulin-mediated LTD was significantly diminished ( p & lt ; 0.05 vs. ND group, Fig. 3 ) . At 30-minute post-insulin stimulation, the per centum decrease of the normalized fEPSP incline from 12-week ND was 73.60 + 4.18 % of the mean incline entering during the baseline degree ( n=16 independent pieces, n=14 animals/group ) , while the per centum decrease of fEPSPs of 12-week HF pieces was 9.34 + 3.09 % of the values recorded before insulin application ( n=17 independent pieces, n=13 animals/group ) ( Fig. 3 ) . These consequences indicated that 12-week HF diet eating caused the damage of neural insulin receptor responses by stamp downing the consequence of insulin-mediated LTD. Furthermore, we found that the mean grade of insulin-mediated LTD at all clip classs of both dietetic groups was significantly correlated with several peripheral insulin opposition parametric quantities, such as organic structure weight ( r = -0.717 ) , splanchnic fat ( R = -0.883 ) , plasma insulin degree ( r = -0.440 ) and liver triglyceride content ( r = -0.6.15 ) ( p & lt ; 0.01 ) .

We found no important difference in the pair-pulsed facilitation ( PPF ) , which indicates the change of presynaptic transmittal, of pieces recorded at the baseline between both dietetic groups at all clip classs ( Fig. 4A, P & gt ; 0.05 ) . These findings indicate that the release of neurotransmitter was unchanged undermentioned neural insulin opposition, and that the ability of CA1 hippocampal nerve cells to show short-run alterations was non altered in 12-week HF-fed rats. To corroborate whether the damage of the insulin-mediated LTD in 12-week high-fat diet group could happen without the break of the other signifier of synaptic malleability, the experiment of carbachol-induced LTD was determined. We found that the grade of carbachol-induced LTD in CA1 hippocampus of both 12-week high-fat diet and normal diet groups were similar ( Fig. 4B ) . These findings suggested that the decrease of insulin-mediated LTD in 12-week HF hippocampal pieces was chiefly due to neural insulin opposition happening at the post-synaptic sites but non due to a non-specific change of pre-synaptic transmittal, and that the damage of neural insulin receptors did non interrupt other signifier of synaptic malleability.

Phosphorylation of insulin receptor ( IR ) , insulin receptor substrate- 1 ( IRS-1 ) and Akt/PKB degrees in encephalon pieces was depressed in 12-week high-fat-fed rats.

In order to compare neural insulin receptor signaling within 4- , 8- and 12-week clip classs between HF and ND rats, we, foremost, investigated whether the protein degrees of IR, IRS-1 and Akt/PKB were down-regulated in relation to the decrease of the ability of insulin-mediated LTD in CA1 hippocampus. The sum of IR, IRS-1 and Akt/PKB protein was illustrated via immunoprecipitation and immunoblotting checks. We found that the degrees of the IR, IRS-1 and Akt/PKB from 4- , 8- and 12-week HF encephalon pieces were non significantly different from 4- , 8- and 12-week ND encephalons ( Fig. 5, n=6-8 per group of rats ) . The densitometric quantification of smudges illustrated that the IR/?-actin protein degree in the 4-week HF group was 1.16 ± 0.22 and in the 4-week ND group was 1.26 ± 0.19 arbitrary scanning units ( p=0.74, n=4/group, Fig. 5A ) . The IR/?-actin protein degree in the 8-week HF group was1.22 ± 0.07 and in the 8-week ND group was 1.23 ± 0.08 arbitrary scanning units ( p=0.9, n=6/group, Fig. 5A ) . The IR/?-actin protein degree in the 12-week HF group was 1.32 ± 0.09 and in the 12-week ND group was 1.38 ± 0.06 arbitrary scanning units ( p=0.6, n=8/group, Fig. 5A ) .

The IRS1 protein degree in the 4-week HF group was 134 ± 8 and in the 4-week ND group was 125 ± 14 arbitrary scanning units ( p=0.6, n=5/group, Fig. 5B ) . The IRS1 protein degree in the 8-week HF group was 142 ± 5 and in the 8-week ND group was 141 ±10 arbitrary scanning units ( p=0.9, n=6/group, Fig. 5B ) . Although the IRS-1 protein degrees in the 12-week HF group revealed a 20 % decrease from those pieces observed in the 12-week ND group, it was non significantly different ( 91 ± 12 vs. 114 ± 12 arbitrary scanning units for the 12-week HF and 12-week ND groups, severally, p=0.1, n=8/group, Fig. 5B ) .

The Akt/PKB/ ?-actin protein degree in the 4-week HF group was 1.04 + 0.05 and in the 4-week ND group was 1.07 + 0.08 ( P & gt ; 0.05, n=4/group, Fig. 5C ) . The Akt/PKB/?-actin protein degrees in the 8-week HF group was1.00 + 0.02 and in the 8-week ND group was 1.03 + 0.03 ( P & gt ; 0.05, n=4/group ) . The Akt/PKB/?-actin protein degrees in the 12-week HF group was 0.99 + 0.02 and in the 12-week ND group was 1.00 + 0.02 ( P & gt ; 0.05, n=8/group ) .

The phosphorylation position of IR, IRS-1 and Akt/PKB in the acutely prepared encephalon pieces under radical status following the insulin stimulation is illustrated in Fig. 6. The basal ( non-insulin stimulated ) phosphorylation degrees of IR and IRS-1 from the 4- , 8- and 12-week ND and from the 4- , 8- and 12-week HF groups were weakly detected by immunoprecipitation and phosphotyrosine immunoblotting. However, insulin stimulation resulted in the strong discernible phosphorylation of both IR, IRS-1and Akt/PKB degrees in 4- , 8- and 12-week ND and 12-week HF groups ( Figs. 6A-D ) . In 4- , 8- and 12-week ND-fed rats, the exposure to insulin stimulation resulted in an addition in IR, IRS-1 and Akt/PKB phosphorylation, compared to their ain basal status. However, merely in the 12-week HF group that the insulin stimulation did non change IR, IRS-1 nor Akt/PKB ( serine 473 and threonine 308 ) phosphorylation, compared to their ain basal status. Interestingly, at 12-week eating, the insulin-stimulated IR phosphorylation was significantly lower in the HF group ( 0.96±0.03 ) than in the ND group ( 1.28±0.06 ) , accounting for ~ 25 % decrease ( p=0.00, n=11/group, Fig. 6A ) . Similarly, the insulin-stimulated IRS-1 phosphorylation was significantly lower in the 12-week HF group ( 1.02±0.04 ) than in the 12-week ND group ( 1.20±0.03 ) , accounting for ~ 15 % decrease ( p=0.00, n=10/group, Fig. 6B ) . Furthermore, since the serine/threonine kinase Akt/PKB is one of chief insulin signaling tracts and dramas function in the mediating of endocrine ‘s metabolic consequence ( 43 ) , and that the phosphorylation degrees of the two residues is necessary for Akt/PKB activity, we determined the degrees of serine 473 and threonine 308 in the present survey. From the encephalon pieces of the 12-week HF group, insulin significantly impaired the ability of insulin to do Akt/PKB phosphorylation of serine 473 and threonine 308 ( Fig. 6C and 6D ) . These findings indicate that the unity of neural insulin receptor signaling, the phosphorylation of IR, IRS-1 and Akt/PKB in the 12-week HF-fed rats, and the ability of insulin-mediated LTD in CA1 hippocampus were significantly reduced.

The figure of nNOS-positive nerve cells was significantly reduced in 12-week high-fat diet group.

Insulin signaling tracts: IR, IRS-1 and Akt/PKB, have been shown to play of import functions in neural endurance ( 44 ) . In add-on, old surveies demonstrated that insulin opposition can speed up ageing syndrome ( 45 ) , and that the figure of nNOS immunoreactive nerve cells in intellectual cerebral mantle and hippocampus was significantly decreased in the encephalon of elderly rats ( 46 ) . In the present survey, we found that 12-week HF ingestion significantly reduced neural insulin signaling and peculiarly decreased insulin-mediated Akt/PKB phosphorylation, bespeaking neural insulin opposition. Therefore, we further hypothesized that rats fed with 12-week HF diet have increased neural ripening, compared to 12-week ND group. To prove this hypothesis, we used nNOS-immunoreactivity as a neural ripening marker to look into whether rats with neural insulin opposition caused by HF diet eating for 12 hebdomads would develop neural ripening in CA1 hippocampus faster than rats fed with ND diet.

In the present survey, we found that nNOS-immunoreactive nerve cells in all animate beings were observed in the interneurons of CA1 hippocampus ( Fig. 7A ) . There was no important difference in the figure of nNOS-immunoreactive nerve cells between HF diet and ND groups following 4- and 8- hebdomad of eating ( Fig. 7B ) . However, the figure of nNOS-immunoreactive nerve cells in CA1 hippocampus of 12-week HF group ( 19.9 + 0.8 neurons/ 100 mm2, n=22 encephalon slices/6 animate beings ) were significantly decreased compared to 12-week ND group ( 23.1+ 0.86 neurons/ 100 mm2, n=29 encephalon slices/6 animate beings ; p & lt ; 0.05, Fig. 7B ) . These informations suggest that neural insulin opposition could speed up neural ripening in CA1 hippocampus.


Insulin opposition is rapidly going one of the universe ‘s most prevailing metabolic upsets ( 47-49 ) . Earlier works found that HF diet causes insulin opposition characterized by hyperinsulinemia, lipemia and reduced insulin sensitiveness ( 1, 50 ) . While considerable research has examined both the effects and mechanisms of a lessened insulin response in assorted peripheral tissues, merely a few surveies have investigated the effects of this metabolic break within the CNS, peculiarly the metabolic perturbation following the ingestion of HF diets. In worlds and gnawers, it has been shown that the development of insulin opposition is associated with HF ingestion and is linked to cognitive shortages ( 1, 6, 7 ) . Although much grounds suggests that neural insulin signaling might play a function in neural malleability ( 4, 51, 52 ) , the figure of available studies in this country is still limited.

Turning grounds demonstrates the influence of time-course effects on the acquisition and memory procedures caused by HF diet. Previous surveies demonstrated that rats fed with HF diet for 3 months had cognitive damage ( 53 ) , while rats fed with HF diet for 8 months exhibited impaired acquisition ability and decreased hippocampal synaptic malleability ( 34 ) . Despite these studies, the neurofunctional insulin opposition in hippocampus in different time-course of HF ingestion has non been investigated. To find the being of neurofunctional insulin opposition in the encephalon, we determined the efficaciousness of the neurofunctional insulin receptor characterized as insulin-mediated LTD in CA1 hippocampus and the stirred phosphorylation position of IR, IRS-1 and Akt/PKB in encephalon pieces harvested from control and HF fed rats. We demonstrated a important decrease of the insulin-mediated LTD in CA1 hippocampal pieces and the insulin-mediated phosphorylation of IR, IRS-1 and Akt/PKB in 12-week HF-fed rats. Our findings suggest that the ingestion of HF diet for merely 12 hebdomads can down-regulate the neural insulin receptor sensitiveness every bit good as the peripheral insulin sensitiveness. A old survey on hamsters with peripheral insulin opposition ensuing from a 6-week high-fructose diet has shown decrease of IR, IRS-1 and Akt/PKB phosphorylation in the encephalon and insulin-mediated LTD in hippocampus ( 32 ) . Our survey demonstrates that the HF diet theoretical account required a longer clip than did fructose diet theoretical account to detect the neural insulin opposition.

In the present survey, insulin opposition, characterized by weight addition, increased splanchnic fat, hyperinsulinemia, increased HOMA index and increased OGTT were observed following the 12-week HF diet. However, we found that the fasting plasma triglyceride degree following ingestion of the 12-week HF diet was non changed, whereas the degree of liver triglyceride in the 4- , 8- and 12-week HF-fed rats significantly increased, compared to the 4- , 8- and 12 hebdomad normal diet. This determination is similar to that reported antecedently, in which HF diet-induced fleshiness in rats increased liver triglyceride content without any alteration in plasma triglyceride degree ( 54 ) . A possible account could be that increased consumption of fats preferentially contributed towards the cytosolic pool by increasing liver triglyceride concentration and longer clip is required for the up-regulation and secernment of plasma triglyceride. Furthermore, inordinate consumption of fat leads to an accretion of triglyceride in many tissues, peculiarly in the adipose tissue ( 50 ) . Supporting this is the determination that rats fed with 4- , 8- and 12-week HF diet have dramatically increased splanchnic fat. In add-on, our informations demonstrated that the plasma insulin degree in 12-week HF group significantly increased without any alterations in plasma glucose degree, proposing that rats with 12-week HF diet feeding were in the peripheral insulin opposition position and the unchanged plasma glucose degree was the consequence of the counterbalancing mechanism of hyperinsulinemia.

Long-run depression ( LTD ) is a phenomenon that reflects neural version and is thought to supply a functional measuring of synaptic malleability ( 55 ) . Insulin receptors are dispersed throughout the encephalon with the highest denseness located in the hippocampus, where these receptors may modulate glucose homeostasis and encephalon map such as acquisition and memory ( 52, 56, 57 ) . Insulin has been shown to modulate the endocytosis of AMPA receptors, which causes the depression of excitant synaptic transmittal ( 26-29, 58 ) .

In the present survey, a important decrease of insulin-mediated LTD in hippocampal pieces from the 12-week HF group was observed. The decrease of insulin-mediated LTD was good correlated with other peripheral insulin opposition such as splanchnic fat, weight, plasma insulin degree and liver triglyceride content. These consequences suggest that alterations in metabolic system are linked to the neural map. The weakening of insulin-mediated LTD indicated one of the functional effects of impaired neural insulin signaling. The decreased LTD initiation could be due to the important lessening in insulin-induced tyrosine phosphorylation of the insulin receptor signaling: IR, IRS-1 and Akt/PKB in encephalon pieces of the 12-week HF group ( Fig. 6 ) . Although it is possible that the lessened insulin-mediated LTD in the HF group could be due to alterations in synaptic transmittal in CA1 hippocampus caused by HF, the unchanged PPF hippocampal CA1 parts in both dietetic interventions suggested that HF feeding did non impact presynaptic responses to electrical stimulation. Therefore, it may be concluded that 12 hebdomads of HF eating, which induced peripheral insulin opposition, merely significantly affects the neural insulin receptor signaling map at the post-synaptic sites, but non at the pre-synaptic release in CA1 hippocampus. Carbachol-induced LTD or muscarinic LTD ( mLTD ) in CA1 hippocampus, characterized as the other signifier of synaptic malleability ( 59 ) , is believed to be substrates of acquisition and memory at the molecular degree ( 59 ) . We demonstrated in this survey that the decrease of neural insulin response in the 12-week HF group was unaffected in mLTD, proposing that this signifier of synaptic malleability may non necessitate neural insulin signaling. Our determination was similar with a old survey, demoing that the weakening of insulin-mediated LTD has no consequence upon the initiation and care LTP via high-frequency stimulation ( 32 ) .

In the present survey, the phosphorylation of IR, IRS-1 and Akt/PKB in encephalon pieces was diminished, whereas the degrees of IR, IRS-1 and Akt/PKB in encephalon pieces were non altered by 4-,8- and 12-week HF diet eating ( see Fig. 5 ) . These alterations in phosphorylation of neural insulin signaling confirm the damage of insulin receptor map in the encephalon following 12-week HF eating. The unchanged degrees of IR, IRS-1 and Akt/PKB proteins following HF diet ingestion in the present survey are consistent with those in old studies, in liver ( 60 ) , skeletal musculus ( 61 ) and hippocampus ( 62 ) . Furthermore, the damage of IR, IRS-1 and Akt/PKB tyrosine kinase activity has been demonstrated in skeletal musculus ( 63 ) , fat ( 64 ) and liver tissues ( 65 ) in 10-12 hebdomad ingestion of HF diets. All of these findings suggest that HF diet could do faulty neural insulin receptor map, but non at the degree of protein look.

In add-on to the weakening of neurofunctional insulin receptors and neural insulin signaling, we besides found that 12-wk HF-fed rats had the decrease of nNOS look in hippocampus. Neural NOS look in intellectual cerebral mantle and hippocampus has been shown to correlate with ageing in animate beings ( 66 ) . Therefore, our findings suggest that neural insulin opposition following HF diet ingestion may take to neural ageing similar to ageing syndrome happening following peripheral insulin opposition ( 67 ) .

In drumhead, the present survey demonstrates that a rapid important alteration of of import neural insulin receptor signaling can be induced by a fat-enriched diet. Fed for 12 hebdomads, the HF diet clearly induces neural insulin opposition, which is identified as a important decrease in the ability of insulin to bring on LTD, and a decrease in the stirred phosphotyrosine activity of IR, IRS-1 and AKt/PKB in encephalon pieces. Twelve-week HF eating non merely causes neural insulin opposition, but besides leads to neural ripening. Since the faulty insulin receptor signaling has been shown to tie in with the pathogenesis of Alzheimer ‘s disease, ( 18 ) , cognitive damage ( 6, 7, 53, 68 ) and the presence of cognitive damage in patients with type II diabetes ( 69 ) , the neural insulin opposition developing after 12-week HF ingestion could be responsible for to the damage of knowledge in this carnal theoretical account.


The writers wish to thank Prof. M. Kevin O ‘ Carroll, Professor Emeritus, University of Mississippi School of Dentistry, USA, and Faculty Consultant, Faculty of Dentistry, Chiang Mai University, Thailand, for his column aid. This work is supported by the Thailand Research Fund grants: TRF-RMU5180007 ( SC ) , TRF-RTA5280006 ( NC ) and the Faculty of Medicine Endowment Fund, Chiang Mai University ( WP, AP, NC and SC ) .


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Figure Legends

Fig. 1. Twelve-week HF diet ingestion causes the decrease of insulin sensitiveness. Consequence of 12-week HF diet ingestion on glucose response during unwritten glucose tolerance trial ( A ) and country under the curve of plasma glucose during an unwritten glucose tolerance trial ( AUCg ) ( B ) .

Fig. 2. Four- and eight-week HF diet eating had no effects on the ability of insulin-mediated long-run depression ( LTD ) on fEPSPs in hippocampal CA1 parts. Panel A and B represent responses before and after insulin stimulation in encephalon pieces from ND and HF groups. Panel A: Average normalized fEPSPs ( fEPSPt/fEPSPo with fEPSPs being points at which fEPSP inclines stabilized ) from 4-week-ND-fed ( n=7-8 independent pieces ) and 4-week-HF-fed ( n=7-8 independent pieces ) encephalon pieces Panel Bacillus: Average normalized fEPSPs from 8-week-ND-fed ( n=7-8 independent pieces ) and 8-week-HF-fed ( n=7-8 independent pieces ) encephalon pieces. Both 4-week and 8-week HF diets had no consequence on the ability of insulin to deject fEPSPs. Examples of norms of 20 back-to-back hints taken from a piece treated with aCSF ( basal ) and with 500 nanometers ( insulin ) are shown in the insets of panels A and B.

Fig. 3. Twelve-week-HF diet feeding significantly diminished the ability of insulin-mediated long term depression ( LTD ) in CA1 hippocampus. Panel A: A individual illustration shows that bath application of 500 nM insulin for 10 minute produced a depression of fEPSPs in 12-week-ND encephalon pieces and the fEPSPs did non to the full retrieve after washout of insulin. However, 500 nM insulin-mediated LTD was significantly attenuated by 12-week-HF diets. Panel A inset: Examples of norms of 20 back-to-back hints taken from a piece treated with aCSF ( basal ) and with 500 nanometers ( insulin ) . Panel B: A sum-up of norms of 16-17 experiments shows that the initiation of insulin-mediated LTD was dramatically reduced in the 12-week-HF-fed group, compared to the 12-week-ND-fed group.

Fig. 4 The damage of neural insulin receptors has no consequence on the presynaptic neurotransmitter release. ( A ) Pair-pulse facilitation ( PPF ) within the rat CA1 hippocampus was non affected by the HF diet. Ploting the facilitation of the 2nd fEPSP incline compared with the first reveals no difference between encephalon pieces harvested from rats on neither diet groups at 4, 8 or 12 hebdomads. ( B ) Average normalized fEPSPs ( fEPSPt/fEPSPo with fEPSPs being points at which fEPSP inclines stabilized ) from 12-week-ND-fed ( n=6-8 independent pieces ) and 12-week-HF-fed ( n=6-8 independent pieces ) encephalon pieces. Representative hints were indicated for 12-week ND group and 12-week HF group ( in upper panel ) . Carbachol-induced LTD ( mLTD ) recorded from CA1 hippocampal pieces harvested from 12-week ND and 12-week HF diet, suggests no difference in mLTD happening during the neural insulin opposition. CCh: carbachol

Fig. 5. No alteration in the protein degrees of IR, IRS-1 and AKt/PKB in 4- , 8- and 12- hebdomad HF diet eating. Panels A-C: Representive immunoblots indicate that the entire cellular sum of the IR ( A ) , IRS-1 ( B ) and Akt/PKB ( C ) proteins were unchanged in the 4- , 8- and 12-week HF rat encephalon pieces compared to the 4- , 8- and 12-week ND pieces and densitometric quantitation of smudges from both groups were non different. All immunoblotting lanes were loaded with equal sums of protein ( 40mg/lane ) . ND: normal diet fed group ; HF: high-fat Federal group ; Negative ( Neg ) : no proteins had been loaded ; IP: immunoprecipitation ; IB: immunoblot

Fig. 6. Insulin-induced phosphorylation of both neural insulin receptor ? fractional monetary unit ( IR ) , the substrate protein IRS-1 and Akt/PKB was weakened in the 12-week HF-fed group. Panels A-D: Representive smudges of phosphorylation illustrated a pronounced lessening in the ability of insulin to excite IR ( A ) , IRS-1 ( B ) , serine 473 kinase of Akt/PKB ( C ) and threonine 308 kinase of AKt/PKB ( D ) phosphorylation in encephalon pieces harvested from the 4- , 8- and 12-week HF-fed group compared to the 4-,8- and 12-week-ND-fed group. Densitometric quantitation of smudges from insulin stimulated IR, IRS-1 and Akt/PKB were significantly greater in the 12-week ND-fed group than in the 12-week HF-fed group ( P & lt ; 0.05 ) . All immunoblotting lanes were loaded with equal sums of protein ( 40mg/lane ) . ND, normal diet fed group ; HF, high-fat Federal group ; Negative ( Neg ) , no proteins had been loaded ; – : no insulin stimulation ; + : insulin stimulation ; IP: immunoprecipitation ; IB: immunoblot

Fig. 7. Decrease of nNOS immunorective nerve cells in CA1 hippocampus following 12-week HF diet eating. ( A ) The representation of nNOS immunoreactive nerve cells in 4-,8- and 12-week of both ND and HF diet eating. Scale saloon is equal to 200 micrometer. ( B ) the saloon graph nNOS immunorective nerve cells shows the decrease in its figure of nNOS immunoreactive nerve cells following 12-week HF eating while there is no alteration in nNOS-immunoreactive nerve cells following 4- and 8- hebdomad HF diet eating ( * P & lt ; 0.05 ) . Negative control means that no primary antibody had been added.

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