the growing evidence supports the fact that neuropeptide galanin ( gal ) can regulate energy homeostasis and insulin sensitivity of animals . the high densities of gal - immunoreactivity are found in the hypothalamus , which is an important center in controlling energy metabolism . an injection of gal into paraventricular nucleus ( pvn ) significantly increased caloric intake and body weight of rats , as well as circulating nonesterified fatty acid levels and lipoprotein lipase activity in adipose tissue . gal - knockout mice showed impaired glucose disposal due to reduced insulin response and insulin - independent glucose elimination   and consumed less energy and lost more body weight compared to controls on a high - fat diet , whereas in homozygous gal - transgenic mice the metabolic rates of lipid and carbohydrate were increased due to improved insulin sensitivity . in addition , the results of our and others ' studies revealed that abdominal or intracerebroventricular injection ( i.c.v . ) of m35 , a galanin antagonist , significantly increased insulin resistance and inhibited the glucose transporter 4 ( glut4 ) translocation from intracellular membrane compartments to cell surfaces in skeletal muscle and fat tissue of rats [ 712 ] . all of three gal subtype receptors ( galr1 - 3 ) are g - protein - coupled isoforms . i - gal binding assay showed that galr1 accounted for approximately 90% of all gal binding sites in the pvn . the galr1 mrna is abundantly expressed in many brain regions , including hypothalamus and forebrain regions . its expression level , not galr2 mrna or galr3 mrna , is positively correlated with the gal content in the hypothalamic nuclei of gal - transgenic mice [ 14 , 15 ] . but so far it is unclear whether central galr1 mediates the beneficial effect of gal on insulin sensitivity in fat tissues of animals . injection of a galr1 agonist , m617 on insulin sensitivity , and insulin signaling in the adipose tissue of type 2 diabetic rats were evaluated . because galr1 antagonist can not be found anywhere , we can not survey the role of the antagonist in the current experiment . the synthesis and final cleavage from the resin using fmoc chemistry were carried out as described by sollenberg et al . . , usa . antibodies against glut4 , pakt , akt , pas160 , as160 , phosphorylation of camp response element - binding protein ( pcreb ) , and creb were acquired from santa cruz biotechnology inc . trizol reagent was obtained from gibco invitrogen , usa . rat c - reactive protein ( crp ) and adiponectin elisa kits were obtained from uscn life science , inc . seventy - six 250  10  g male wistar rats , coming from yangzhou university animal center , were kept in the polypropylene cages and under the conditions of temperature 21  2c , humidity 54% , 12-hour light - dark cycles , high - fat diet ( 59% fat , 21% protein , and 20% carbohydrate ) , and water ad libitum . after eight weeks , forty - four of the animals were treated with streptozotocin ( 35  mg / kg i.p . ) in 0.1  mm citrate buffer ( ph 4.5 ) under a fasted state . the tail blood of the rats was weekly taken to determine the blood glucose levels with a glucometer ( hmd biomedical , taiwan ) during the study . after being fed with the high - fat diet for another four weeks , thirty - two animals with fasting glucose concentration over 11.1  mmol / l as well as polydipsia , polyphagia , polyuria , shaggy , cachexia , and weight loss were taken as models of diabetes [ 10 , 17 ] . the diabetic rats were randomly distributed into diabetic control ( n = 16 ) and diabetic m617 ( n = 16 ) groups . in addition , thirty - two nondiabetic rats were evenly attributed to nondiabetic control and nondiabetic m617 groups . the rats in all groups were fed with the high - fat diet during the experiment . all animal procedures used were performed in accordance with the guiding principles for care and use of experimental animals . injection is similar to those described previously in animals . in brief , animals were anesthetized with 3% amobarbital sodium ( 50  mg / kg i.p . ) and stereotaxically implanted with a guide cannula into the lateral ventricle : anterior - posterior ( ap ) , 0.8  mm ; l , 1.4  mm ; and v , 3.3  mm . the cannula was cemented to four jeweler 's screws attached to the skull and closed with an obturator . after recovery for 7  d from surgery , rats in the m617 group were injected with 1  nmol / kg m617 in 5  l artificial cerebrospinal fluid ( in mm : 133.3 nacl , 3.4 kcl , 1.3 cacl2 , 1.2 mgcl2 , 0.6 nah2po4 , 32.0 nahco3 , and 3.4 glucose , ph 7.4 by 0.5  m hydrochloric acid ) at 9:00 am every day for 21 days and rats in both control groups with 5  l vehicle . at the end of the experiment , half of rats in every group ( n = 8) were anesthetized as above after fast 12  h. then 4  ml artery blood and 5  g epididymal fat pad were collected . the blood was centrifugated at 3500 r.p.m . for 10  min to obtain the plasma . the plasma and fat pad were frozen at 80c . in the hyperglycemic clamp tests , the other half of rats in every group ( n = 8) was anesthetized and catheterized in right carotid artery and left jugular vein after fasted 12  h as previously described [ 7 , 9 ] . the animals were infused with insulin at a constant rate of 2 mu / kgmin into the jugular vein until the end of the test . 10% glucose was infused at variable rates as needed to clamp glucose levels at 5  0.5 the glucose infusion rates were calculated corresponding to the last 6 samplings at the clamp level . once above experiments were completed , each rat was euthanized by infusion of amobarbital sodium and saturated potassium chloride . the adiponectin and crp levels were quantified using each competitive elisa kit . to determine the glut4 mrna level , the total rna from 100  mg of the frozen adipose tissue was isolated by trizol the concentration of the rna was calculated by spectrophotometric assays of 260/280  nm , and the integrity of the rna was assessed by running samples on a 1% formaldehyde agarose gel in tae buffer ( 40  mmol / l tris - acetic acid , 1  mmol / l edta ) . the mrna expression levels were determined using real - time fluorescent detection in an exicyclertm 96 pcr machine ( lg company , korea ) . the oligonucleotide primers were as follows : glut4 forward 5-acagggcaaggatggtaga-3 , reverse 5-tggaggggaacaagaaagt-3 , -actin forward 5-ggctgtgttgtccctgtatg-3 , reverse 5-aatgtcacgcacgatttcc-3. amplification condition was as follows : an initial denaturation at 95c for 10  min ; 95c for 15  s ; 62c for 60  s ; 40 cycles . the 2 method was used to analyze the real - time pcr data . briefly , fat pads were washed , homogenized in an ice - cold homogenization buffer ( 250  mmol / l sucrose , 2  mmol / l edta , 2.5  mmol / l tris - hcl , 10  g / ml aprotinin , 10  g / ml leupeptin , and 100  mol / l phenylmethylsulfonyl fluoride , ph 7.4 ) , and then centrifuged at 13,000  g for 20  min at 4c to remove the fat cake . the infranatant was centrifuged at 31,000  g for 1  h to yield the low - density intracellular membranes . the pellet from the first spin was layered over a sucrose cushion and centrifuged at 75,000  g for 1  h. the interphase was removed and spun at 39,000  g for 20  min to yield the plasma membranes . western blot analyses were used to determine glut4 , pakt , akt , pas160 , as160 , and pcreb and creb levels in the adipose tissue . briefly , fifty micrograms of samples was separated via electrophoresis on a 12% sodium dodecyl sulfate - polyacrylamide gel as described before . membranes were blocked in tris - buffered saline ( ph 7.5 ) containing 0.05% tween-20 ( 1  tbst ) and 5% skimmed milk for 2  h and then probed with an antibody overnight at 4c . membranes were washed three times with 1  tbst for 10 minutes and incubated for 2  h with horseradish peroxidase - conjugated secondary antibody . lastly , immunoreactive bands were visualized by chemiluminescence and quantified by densitometry using a quantity one analysis software ( bio - rad ) . the sum of the glut4 concentration in intracellular membranes and in plasma membranes was calculated as the concentration of total cell membranes . comparisons among the groups were analyzed by two - way anova followed by tukey 's multiple - range tests . data about food intake , body weight , plasma insulin , and free fatty acid levels before and after the experiments were compared by paired student 's t - test . data were presented as mean  sem with p < 0.05 as the limit for statistical significance . as showed in table 1 , central administration of m617 enhanced food intake and body weight by 9.0% ( p < 0.05 ) and 5.3% ( p < 0.05 ) in the diabetic m617 group compared with diabetic controls , also by 10.5% ( p < 0.05 ) and 4.6% ( p < 0.05 ) in the nondiabetic m617 group compared with nondiabetic controls , respectively . both indexes after the experiments compared with before were augmented by 13.8% ( p < 0.01 ) and 9.8% ( p < 0.05 ) in the diabetic m617 group and by 13.5% ( p < 0.01 ) and 9.1% ( p < 0.05 ) in the nondiabetic m617 group , while the administration of m617 attenuated plasma insulin and free fatty acid levels by 12.1% ( p < 0.05 ) and 38.7% ( p < 0.01 ) in the diabetic m617 group compared with diabetic controls . compared with nondiabetic controls , the plasma insulin levels were reduced by 16.0% ( p < 0.05 ) , while the free fatty acid levels were nonsignificantly decreased by 7.2% ( p > 0.05 ) in the nondiabetic m617 group . both plasma insulin and free fatty acid levels after the experiments compared with those before were decreased by 11.1% ( p < 0.01 ) and 36.1% ( p < 0.01 ) in the diabetic m617 group and by 19.4% ( p < 0.01 ) and 11.2% ( p < 0.05 ) in the nondiabetic m617 group , respectively . during the clamp tests , figure 1 showed that the infusion rate in the diabetic m617 group was increased by 30.2% ( p < 0.01 ) compared with diabetic controls but reduced by 27.1% ( p < 0.01 ) compared with the nondiabetic m617 group . compared with nondiabetic controls , the rate was increased by 8.8% ( p < 0.05 ) in the nondiabetic m617 group but attenuated by 39.1% ( p < 0.01 ) in the diabetic control group . to determine the effect of activation of central galr1 on crp liberation , we contrasted the plasma crp concentration in the m617 group with corresponding controls . injection of m617 reduced the crp concentration ( f = 62.3 , p < 0.001 ) . the crp concentration in the diabetic m617 group was decreased by 9.9% ( p < 0.05 ) compared with diabetic controls but was elevated by 47.8% ( p < 0.01 ) compared with the nondiabetic m617 group . compared with nondiabetic controls , the concentration was decreased by 13.2% ( p < 0.05 ) in the nondiabetic m617 group but increased by 42.4% ( p < 0.01 ) in the diabetic control group . the results suggest that activation of galr1 in brain may inhibit crp secretion to ameliorate insulin resistance . in this experiment , we observed the impact of central galr1 on plasma adiponectin levels in fat cells . as shown in figure 3 , the central administration of m617 elevated the adiponectin contents ( f = 124.7 , p < 0.001 ) . the adiponectin contents in the diabetic m617 group were enhanced by 7.2% ( p < 0.05 ) compared with the diabetic controls but reduced by 22.7% ( p < 0.01 ) compared with the nondiabetic m617 group . as compared with nondiabetic controls , the levels were increased by 5.5% ( p < 0.05 ) in the nondiabetic m617 group but reduced by 23.9% ( p < 0.01 ) in the diabetic control group . these results suggest that activation of galr1 in brain may increase adiponectin release to ameliorate insulin resistance . in the present study , the central treatment with m617 significantly elevated the glut4 mrna expression in fat cells of rats ( f = 65.7 , p < 0.0001 ) . as shown in figure 4 , the glut4 mrna expression in the diabetic m617 group was increased by 36.3% ( p < 0.01 ) compared with diabetic controls but reduced by 18.6% ( p < 0.01 ) compared with the nondiabetic m617 group . as compared with nondiabetic controls , the glut4 gene expression was enhanced by 11.1% ( p < 0.05 ) in the nondiabetic m617 group but significantly decreased by 33.7% ( p < 0.01 ) in the diabetic control group . the central administration of m617 significantly elevated glut4 protein levels in both total cell membranes ( f = 108.3 , p < 0.0001 ) and plasma membranes ( f = 132.8 , p < 0.0001 ) of adipose cells ( figure 5(a ) ) . the glut4 immunoreactivities in the diabetic m617 group were elevated by 7.2% ( p < 0.05 ) in total cell membranes and 66.9% ( p < 0.01 ) in plasma membranes compared with the diabetic controls but reduced by 22.2% ( p < 0.01 ) in total cell membranes and 29.9% ( p < 0.01 ) in plasma membranes compared with the nondiabetic m617 group . as compared with the nondiabetic control group the glut4 levels in the diabetic control group were reduced by 23.4% ( p < 0.01 ) in total cell membranes and 42.2% ( p < 0.01 ) in plasma membranes but were elevated by 5.6% ( p < 0.05 ) in total cell membranes and 37.8% ( p < 0.01 ) in plasma membranes in the nondiabetic m617 group . moreover , the central treatment with m617 significantly enhanced the ratios of glut4 levels in plasma membranes to total cell membranes ( f[3 , 32 ] = 77.3 , p < 0.0001 ; figure 5(b ) ) . the ratios in the diabetic m617 group were enhanced by 56.1% ( p < 0.01 ) compared with the diabetic control group but attenuated by 9.1% ( p < 0.05 ) compared with the nondiabetic m617 group . as compared with nondiabetic controls , the ratios were elevated by 30.2% ( p < 0.01 ) in the nondiabetic m617 group but reduced by 24.9% ( p < 0.01 ) in the diabetic control group . as shown in figure 6 , the injection of m617 into the brain significantly elevated the ratios of pakt / akt ( f[3 , 32 ] = 10.8 , p < 0.0001 ) and pas160/as160 ( f[3 , 32 ] = 18.9 , p < 0.0001 ) but reduced the ratios of pcreb / creb ( f[3 , 32 ] = 17.9 , p < 0.0001 ) in adipose cells . the ratios of pakt / akt and pas160/as160 in the diabetic m617 group were enhanced by 70.9% ( p < 0.01 ) and 31.8% ( p < 0.05 ) compared with diabetic controls but decreased by 18.4% ( p < 0.05 ) and 24.2% ( p < 0.01 ) compared with the nondiabetic m617 group . the ratios of pcreb / creb in the diabetic m617 group were increased by 36.1% ( p < 0.01 ) compared with the nondiabetic m617 group , while they are not significantly altered ( p > 0.05 ) compared with diabetic controls . as compared with nondiabetic controls , the ratios of pakt / akt and pas160/as160 in the diabetic control group were reduced by 43.1% ( p < 0.01 ) and 33.9% ( p < 0.01 ) , but the ratios of pcreb / creb were elevated by 34.6% ( p < 0.01 ) . compared with nondiabetic controls , the ratios of pakt / akt and pas160/as160 in the nondiabetic m617 group were increased by 17.2% ( p < 0.05 ) and 14.9% ( p < 0.05 ) , while the ratios of pcreb / creb were not significantly altered ( p > 0.05 ) . these results suggest that activation of galr1 in brain may increase pakt and pas160 contents , but not pcreb in adipose cells . adipose tissue is considered not only as an energy storage depot , but also an important endocrine organ to regulate energy homeostasis by releasing adipokines , such as adiponectin [ 18 , 19 ] . although only 10% of insulin - stimulated glucose uptake takes place in the adipose tissue , it is very important to control whole - body energy homeostasis and glucose metabolism , as the glut4 expression level is selectively downregulated in adipose , not in skeletal muscle in insulin resistant states . several studies have implicated that activated galr1 in brain is related to fat intake and body weight of subjects as galanin does . lundstrm and colleagues , m617 can preferentially bind to galr1 receptors and , therefore , is taken as a galr1 preferring agonist . administration of m617 , not galr2 agonist , notably stimulated acute consumption of high - fat milk and cookie mash [ 22 , 23 ] . in line with this , the results obtained in present study provided further evidences that activated galr1 in the brain increased fat intake and body weight of rats , suggesting that galr1 system at least partly mediated the galanin - induced appetitive effects . in addition , it is well known that during the compensated stage of type 2 diabetes , the circulatory insulin level of subjects is enhanced due to hyperglycemia stimuli and insulin resistance . after decompensation , the serum insulin level of subjects is reduced because of heavy damage of -cells in the pancreas . our data showed that the serum insulin levels were higher in the diabetic rats than controls , which might be blunted by central injection of m617 , implicating that the diabetic rats used in this study were in the compensated period of pancreases and activated central galr1 might inhibit insulin release as galanin did . the plasma concentration of the protein in obese subjects and type 2 diabetic patients is decreased [ 25 , 26 ] . prospective longitudinal studies revealed that the plasma adiponectin levels declined at an early phase of obesity and further decreased after the development of type 2 diabetes . heterozygous adiponectin - deficient mice ( adipo ( + / ) ) showed mild insulin resistance , while homozygous adiponectin - deficient mice ( adipo ( / ) ) suffered moderate insulin resistance with glucose intolerance . it has been proved that a common silent t - g exchange in nucleotide 94 ( exon 2 ) of the adiponectin gene associates with increased insulin resistance . intraperitoneal administration of 0.4  mg / kg globular adiponectin can improve insulin resistance and metabolic responses to insulin in male rats fed the high - fat diet via an ampk- and nitric oxide - dependent mechanism . in the current study the central administration of m617 elevated plasma adiponectin levels , suggesting that activated galr1 in brain might stimulate adiponectin secretion from adipose tissues to benefit insulin sensitivity in diabetic rats . discovered from the plasma of patients with acute inflammation first , crp is an independent risk factor for synthesizing macrophage inflammatory factors and developing insulin resistance . crp levels were reversely correlated with high - density lipoprotein contents but directly with plasma triglyceride and glucose concentrations . our current results indicated that the crp levels were markedly decreased by central administration of m617 , suggesting that activation of galr1 in brain might reduce crp discharge to ameliorate insulin resistance . during the hyperinsulinemic - euglycemic clamp test , a constant insulin infusion increases glucose uptake into muscle and fat tissues and inhibits endogenous glucose production by the liver . the amount of exogenous glucose required to maintain plasma glucose at its clamp level is quantified by the glucose infusion rate . thus , an elevation of the glucose infusion rate in the clamp test indicates the increase in insulin sensitivity . our results in the clamp tests showed that the glucose infusion rat in the m617 group was higher than each control , suggesting that activation of galr1 in brain might elevate the insulin sensitivity and benefit glucose clearance . glut4 is responsible for insulin - stimulating glucose transport to keep blood glucose homeostasis . under a basal condition majority of glut4 as stimulation of insulin , the recycle becomes rapid exocytosis and slow endocytosis , resulting in transporting more glut4 onto plasma membranes . only at plasma membranes can glut4 transport glucose into cells . in addition , the gal mrna expressive level was directly correlated with gal synthesis and release . in the gal - transgenic mice , the galr1 mrna expression was increased in discrete areas of the brain . the present experimental results revealed that the central administration of m617 significantly upregulated glut4 protein and mrna expression in adipose cells . moreover , the ratios of glut4 contents in plasma membrane fractions to total cell membranes were higher in the m617 group than controls , implicating that injection of m617 not only enhances glut4 protein and mrna expression , but also accelerates glut4 translocation from intracellular membranes to plasma membranes in adipose cells . a number of studies have addressed the fact that gal regulates glucose uptake and energy consumption at least via two signaling pathways in adipocytes . one is via activation of gi / o receptors to inhibit adenyl cyclase and creb . the other is through upregulation of peroxisome proliferator - activated receptor protein and p2 gene expression to increase pakt and pas160 contents in adipocytes . activation of akt mediates the phosphorylation of as160 , which can promote hydrolysis of rab - gtp to rab - gdp to regulate glut4 translocation . after administration of gal , pas160 inactivates gtpase and increases rab - gtp activity to trigger glut4 translocation to plasma membranes to accelerate glucose uptake [ 3739 ] . we found that central injection of m617 did not change ratios of pcreb / creb but enhanced ratios of pakt / akt and pas160/as160 to boost glut4 translocation and glucose uptake , suggesting that activation of the akt / as160 pathway , not creb , mediated the central galr1 roles to increase glucose uptake . besides galr1 galr1 and galr2 are abundant in the brain of rat , whereas galr3 is the important gal receptor in the brain of human , especially in locus coeruleus of and dorsal raphe nucleus . the ligand binding study revealed that galr1 was the main gal receptor subtype in the rodent brain . in pvn of rat , galr1 accounts for over 90% of all gal binding sites . the present findings showed that the beneficial effect of central m617 on insulin sensitivity was same as that of central gal in adipose tissues [ 810 ] , suggesting that the mitigatory effect of central gal on insulin resistance is mainly mediated by activation of galr1 . in brain , activation of galr1 may be through three possible pathways to regulate insulin sensitivity . first , activation of galr1 can release central transmitters , such as 5-ht [ 41 , 42 ] , npy [ 43 , 44 ] , ach , and ne   in different brain regions to regulate insulin sensitivity of subjects . next , gal - galr1 system in brain can suppress sympathetic firing in a dose - dependent way , which is implicated in alleviation of insulin resistance [ 47 , 48 ] . consistent with this , gal - knockout mice lack the sympathetic inhibition of insulin release . last , gal - galr1 system can alter the activities of central neurons in hypothalamus , amygdale , and arcuate nucleus to regulate energy homeostasis , which is relative to insulin sensitivity [ 49 , 50 ] . the net effect of galr1 deficiency was an impairment of glucose clearance following bolus glucose challenges . nonetheless , more studies are needed using galr1 specific antagonists and galr1 mutant mice to further explore the effect of central galr1 on amelioration of insulin resistance . in summary , the current results in the study suggest that activation of central galr1 facilitates adiponectin release , glucose infusion rates in the hyperinsulinemic - euglycemic clamp test , glut4 mrna expression , and glut4 translocation from intracellular membrane compartments to cell surfaces but inhibits the crp release to benefit insulin sensitivity in adipose cells of type 2 diabetic rats . these effects may be partly due to triggering of the akt / as160 cascade , not creb . this study contributes to our understanding of the central role of galr1 in benefit of glucose uptake and insulin sensitivity , offering a possibility that the galr1 may be taken as a potential therapeutic target for type 2 diabetes mellitus .
our previous studies testified the beneficial effect of central galanin on insulin sensitivity of type 2 diabetic rats . 
 the aim of the study was further to investigate whether central m617 , a galanin receptor 1 agonist , can benefit insulin sensitivity . 
 the effects of intracerebroventricular administration of m617 on insulin sensitivity and insulin signaling were evaluated in adipose tissues of type 2 diabetic rats . 
 the results showed that central injection of m617 significantly increased plasma adiponectin contents , glucose infusion rates in hyperinsulinemic - euglycemic clamp tests , glut4 mrna expression levels , glut4 contents in plasma membranes , and total cell membranes of the adipose cells but reduced the plasma c - reactive protein concentration in nondiabetic and diabetic rats . 
 the ratios of glut4 contents were higher in plasma membranes to total cell membranes in both nondiabetic and diabetic m617 groups than each control . 
 in addition , the central administration of m617 enhanced the ratios of pakt / akt and pas160/as160 , but not phosphorylative camp response element - binding protein ( pcreb)/creb in the adipose cells of nondiabetic and diabetic rats . 
 these results suggest that excitation of central galanin receptor 1 facilitates insulin sensitivity via activation of the akt / as160 signaling pathway in the fat cells of type 2 diabetic rats .