research design and methods are shown in supplement 1 in the online appendix ( 1316 ) ( available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0393/dc1 ) . to examine the effects of nps on body weight and on glucose and lipid metabolism , bnp - tg mice were given a high - fat ( 60 kcal% fat ) diet . the body weight of bnp - tg mice on standard diet tended to decrease compared with that of their littermate wild - type mice ( 4.8% reduction at 18 weeks old , n = 18 per group , p = 0.06 ) ( fig . when fed a high - fat diet from the age of 10 weeks , on the other hand , the weight of the bnp - tg mice at 18 weeks old was significantly lower than that of the wild - type controls ( 38.9  1.0 g for the former and 43.0  0.9 g for the latter ; n = 10 , p < 0.01 ) ( fig . the reduction in body weight of the transgenic mice fed a high - fat diet could be macroscopically observed ( fig . food intake ( kcal / day ) was not significantly different between bnp - tg and wild - type mice whether on standard or high - fat diet , despite the difference in body weight ( fig . wild - type and bnp - tg mice were given high - fat ( 60 kcal% fat ) diet from the age of 10 weeks . a : body weight of the bnp - tg mice on standard diet ( left panel ) and a high - fat diet ( right panel ) ( n = 18 per group on standard diet and n = 10 on high - fat diet ) . b : macroscopic appearance of a wild - type ( wt ) and a bnp - tg mouse fed on high - fat diet at 18 weeks of age . c : food intake on standard diet and high - fat diet ( n = 6 ) . d : blood glucose levels determined with the glucose and insulin tolerance tests ( n = 8) . e : ct images obtained at kidney level of a wild - type ( wt ) and a bnp - tg mouse on standard diet ( upper panel ) and high - fat diet ( lower panel ) . subcutaneous fat ( yellow ) , abdominal fat ( red ) , and muscular region ( blue ) were distinguished . f : macroscopic appearances and weights of epididymal fat ( upper panel ) and mesenteric fat ( lower panel ) . g : microscopic analysis with hematoxylin and eosin staining of epididymal fat in high - fat  fed mice ( left panels ) . adipocyte size of epididymal fat in high - fat  fed mice was estimated from the histological analysis ( right panel ) ( n = 8) . i : macroscopic appearance of the liver of the mice fed on high - fat diet ( upper panel ) . microscopic images of the liver stained with oil red o ( lower panel ) . j and k : triglyceride concentrations in the liver ( j ) and the quadriceps ( k ) ( n = 12 ) . * p < 0.05 ; * * p < 0.01 vs. wild - type ( wt ) mice on the same feeding condition . ( a high - quality color digital representation of this figure is available in the online issue . ) the blood glucose and insulin levels were identical for bnp - tg and wild - type mice on standard diet , both during ad libitum feeding and fasting . however , these levels were significantly lower in bnp - tg mice during ad libitum feeding of the high - fat diet ( table 1 ) . blood glucose levels were also lower in bnp - tg mice fed a high - fat diet after administration of glucose or insulin ( fig . , serum triglyceride and fatty acid levels did not show any significant differences between the two groups , both when feeding ad libitum and fasting ( table 1 ) . the greater increase in the serum fatty acid level after 24-h fasting was observed in bnp - tg mice ( table 1 ) . urinary excretion of catecholamines ( epinephrine and norepinephrine ) was similar between the two groups ( table 1 ) . physical and metabolic parameters of high - fat  fed bnp - tg mice p < 0.05 , compared with wild type . to estimate the fat weight in mice , we used computed tomography ( ct ) and scanned the whole body of mice . the high - fat diet produced a substantial increase in the adipose tissue in both the subcutaneous and visceral area . the total fat weight of bnp - tg mice fed on a high - fat diet was significantly lower ( 26% reduction , n = 6 , p < 0.01 ) ( fig . the relative reduction ratio was similar for both subcutaneous and visceral fat ( 21% reduction for subcutaneous and 29% for visceral fat ) . the high - fat  fed bnp - tg mice had less surgically harvested epididymal and visceral fats than the wild - type mice ( fig . 1f ) , which is a compatible finding with the ct - based fat quantification . on the other hand , lean body mass showed no significant difference ( 26.6  1.3 g in bnp - tg mice fed a high - fat diet and 27.2  1.2 g in wild - type mice ) . to further study the effects of nps on diet - induced lipid accumulation , we examined adipose tissue , liver , and skeletal muscle of bnp - tg mice fed a high - fat diet for a comparison with those of wild - type mice . we found that the histologically examined adipocytes in the epididymal fat were smaller in bnp - tg mice ( fig . finding , serum leptin was found to decrease and adiponectin to increase in bnp - tg mice fed a high - fat diet ( fig . the liver of wild - type mice had a whitish appearance , while that of high - fat  fed bnp - tg mice was reddish and lower in weight ( 1.4  0.1 g in high - fat  fed bnp - tg mice and 1.7  0.1 g in wild - type mice , n = 6 , p < 0.05 ) ( fig . oil red o staining and triglyceride measurements of the liver confirmed that diet - induced lipid accumulation was significantly attenuated in high - fat  fed bnp - tg mice ( 30% decrease in triglyceride concentration , p < 0.05 , n = 12 ) ( fig . 1j ) , and a similar attenuation of lipid accumulation was observed in the skeletal muscle ( 27% reduction , n = 12 , p < 0.05 ) ( fig . these findings indicate that diet - induced ectopic fat accumulation was reduced in bnp - tg mice in addition to the reduction in adipose tissue . the respiratory gas analysis demonstrated that bnp - tg mice on a high - fat diet consumed more oxygen than wild - type mice ( n = 6 , p < 0.01 ) ( fig . mean oxygen consumption for 24 h of bnp - tg mice on standard diet was 64.3  0.9 ml  min  kg body wt , and that of wild - type mice was 62.6  0.9 ml  min  kg body wt . the value of wild - type mice on high - fat diet decreased to 51.8  0.7 ml  min  kg body wt , and that of bnp - tg was 58.7  0.5 ml  min  kg body wt . the value was significantly higher than that of wild - type mice ( n = 6 , p < 0.01 ) ( fig . the rectal temperature was similar between bnp - tg and wild - type mice , whether on standard diet or high - fat diet ( fig . 2c ) . the respiratory quotient showed a significant reduction in high - fat  fed bnp - tg mice , especially during the daytime ( fig . the mean respiratory quotient for 24 h of high - fat  fed bnp - tg mice was 0.80  0.02 , and that of wild - type mice was 0.81  0.01 ( n = 6 , p < 0.05 ) . in line with the reduction in respiratory quotient , mean fat oxidation for 24 h of high - fat  fed bnp - tg mice , estimated from the results of the respiratory gas analysis , was increased to 18.5  0.2 ml  min  kg body wt , while that of wild - type mice was 16.8  0.2 ml  min  kg body wt ( n = 6 , p < 0.01 ) ( fig . the increase in fat oxidation in bnp - tg mice could be augmented by fasting ( fig . high - fat  fed bnp - tg mice exhibit higher oxygen consumption and fat oxidation in association with increased mitochondrial content in the skeletal muscle . total dna and rna were extracted from the brown adipose tissue and the quadriceps , and quantitative pcr analysis was performed . a : oxygen consumption on standard diet ( left panel ) and high - fat diet ( right panel ) ( n = 6 ) . b : mean oxygen consumption for 24 h on standard diet or high - fat diet ( n = 6 ) . c : rectal temperature on standard diet or high - fat diet ( n = 6 ) . d : respiratory quotient of high - fat  fed mice ( n = 6 ) . e : mean fat oxidation estimated from the respiratory gas analysis for 24 h of high - fat  fed mice . g : mitochondrial dna copy number estimated from quantification of mitochondrial and nuclear genome ( n = 8) . h : expressions of genes encoding pgc-1 and ucp1 in the brown adipose tissue and those for pgc-1 and ppar in the skeletal muscle ( n = 8) . high - fat diet :  , wild type ;  , bnp - tg . the values were standardized to those for the control ( wild - type [ wt ] mice fed on standard diet ) in either group . * p < 0.05 ; * * p < 0.01 vs. wild type on the same feeding condition . to further investigate the mechanism for the increase in oxygen consumption of high - fat fed bnp - tg mice , we checked mitochondrial dna copy number in the brown adipose tissue and skeletal muscle , which are the major sites for energy expenditure . quantitative pcr analysis demonstrated a significant increase in the mitochondrial dna copy number in the skeletal muscle of high - fat  fed bnp - tg mice ( fig . the increase in the brown adipose tissue was weak ( fig . 2 g ) , in accordance with unchanged rectal temperature ( fig . in conjunction with these findings , the expressions of the genes encoding peroxisome proliferator  activated receptor ( ppar)- coactivator ( pgc)-1 and uncoupling protein ( ucp ) 1 , which are known to mediate mitochondrial biogenesis and thermogenesis , respectively , were not significantly upregulated in the brown adipose tissue of the tg mice ( fig . 2h ) ; whereas in the skeletal muscle , the expressions of the genes encoding pgc-1 and ppar , which are known to participate in fat oxidation and energy expenditure , were upregulated in high - fat  fed bnp - tg mice ( fig . the expressions of np receptors are regulated by feeding condition , while gca knockdown mice are susceptible to diet - induced obesity and glucose intolerance . total rna was extracted from the quadriceps of wild - type mice , and quantitative pcr analysis for the expressions of np receptors was performed . wild - type and gca heterozygous knockout mice ( gca ) were given a high - fat ( 45 kcal% fat ) diet from the age of 8 weeks . a : expressions of gca , gcb , and c - receptor in the skeletal muscle , brown adipose tissue , and white adipose tissue of wild - type mice for indicated feeding condition ( n = 8) . the values represent the expression levels of each gene compared with that of -actin determined by quantitative pcr analysis . statistical analysis was performed to evaluate the effects of fasting and high - fat diet . * p < 0.05 ; * * p < 0.01 vs. ad libitum eating on the same diet . # p < 0.05 ; # # p < 0.01 vs. standard diet on the same eating condition ( ad libitum or fasting ) . further analysis to clarify whether there were interactions between the effect of fasting and that of high - fat diet was performed as shown in supplemental table s2 . , skeletal muscle ;  , brown adipose tissue ;  , white adipose tissue . b : body weight of wild - type ( wt ) and gca mice fed on standard diet ( dotted lines ) or high - fat diet ( solid lines ) after 8 weeks of age ( n = 8) . d : blood glucose levels determined with the glucose tolerance test ( n = 8) . e : total fat weight estimated from the ct images ( n = 6 ) . * p < 0.05 ; * * p < 0.01 vs. wild type on the same feeding condition . gca , gca heterozygous knockout mice . to determine the effect of cgk - i , a major downstream effector of np / gc / cgmp cascades , on body weight and on glucose and lipid metabolism , we examined the cgk - tg mice with ubiquitously overexpressing human cgk - i . the cgk - tg mice showed a significant reduction in body weight compared with wild - type mice , even on standard diet ( 27.6  0.4 g for cgk - tg and 32.0  0.6 g for wild - type mice , at 18 weeks old on standard diet , n = 8 , p < 0.01 ) ( fig . moreover , high - fat diet  induced weight gain was attenuated in the tg mice , and the reduction in body weight eventually reached > 20% . at 18 weeks of age , their body weight was 35.7  0.4 g , and that of wild - type mice was 43.9  0.6 g ( after 8 weeks of high - fat feeding , n = 8 , p < 0.01 ) ( fig . 4a and b ) . the daily food intake ( kcal / day ) was not noticeably different for cgk - tg and wild - type mice , while it showed a significant increase in cgk - tg mice when it was adjusted for body weight ( kcal  day  g body wt ) ( fig . wild - type and cgk - tg mice were given a high - fat ( 60 kcal% fat ) diet from the age of 10 weeks . a : body weight of cgk - tg mice on standard diet ( left panel ) and high - fat diet ( right panel ) ( n = 810 ) . b : macroscopic appearance of a wild - type ( wt ) and a cgk - tg mouse . c : food intake on standard and high - fat diet ( upper panel ) ( kcal / day , n = 6 ) and the body weight ( bw)-adjusted value ( lower panel ) ( kcal  day  g body wt ) . d : blood glucose levels determined with the glucose and insulin tolerance tests for each genotype on standard diet and high - fat diet ( n = 8) . e : ct images obtained at kidney level of a wild - type ( wt ) and a cgk - tg mouse on standard diet ( upper panel ) and high - fat diet ( lower panel ) . subcutaneous fat ( yellow ) , abdominal fat ( red ) , and muscular region ( blue ) were distinguished . f : microscopic analysis using hematoxylin and eosin staining of epididymal fats in high - fat  fed mice . h : macroscopic appearance of the liver in high - fat  fed mice ( upper panel ) . i and j : triglyceride concentration in the liver ( i ) and the quadriceps ( j ) ( n = 12 ) . * p < 0.05 ; * * p < 0.01 vs. wild type on the same feeding condition . ( a high - quality color digital representation of this figure is available in the online issue . ) the blood glucose levels were significantly lower in cgk - tg mice , both during ad libitum feeding and fasting and on standard diet and high - fat diet ( table 2 ) . the decrease was accompanied by significantly lower insulin levels ( table 2 ) , except for the value for fasting while fed on standard diet . after administration of glucose or insulin , the difference in glucose levels between cgk - tg and wild - type mice became more prominent ( fig . 4d ) . serum triglyceride and fatty acid levels were similar for cgk - tg and wild - type mice , both during ad libitum feeding and fasting , except for the significant increase in fatty acid levels in high - fat  fed cgk - tg mice during fasting , when compared with that of wild - type mice ( table 2 ) . urinary excretion of the catecholamines ( epinephrine and norepinephrine ) was similar for wild - type and cgk - tg mice ( table 2 ) . physical and metabolic parameters of cgk - tg mice fed on standard chow or high - fat diet p < 0.05 compared with wild type on the same feeding condition . in parallel with the reduction in body weight , fat tissues assessed by means of ct analysis were significantly reduced in cgk - tg mice , both on standard and high - fat diet ( fig . hematoxylin - eosin staining and the coulter counter analysis showed that the adipocytes in the epididymal fat were smaller in cgk - tg mice , with mean adipocyte diameters for wild - type and cgk - tg mice of 80 and 60 m , respectively , when on standard diet ( fig . 4f and g ) . the high - fat diet produced an increase in the diameter to 130 m in wild - type mice but to only 80 m in the tg mice ( fig . the liver of high - fat  fed cgk - tg mice weighed less than that of wild - type mice ( 1.3  0.1 g and 1.8  0.1 g , n = 8 , p < 0.01 , respectively ) and had reddish appearance ( fig . the hepatic triglyceride concentration of high - fat  fed cgk - tg mice was significantly reduced when compared with wild - type mice ( 30% decrease at 20 weeks , n = 6 , p < 0.05 ) ( fig . muscular triglyceride concentration of cgk - tg mice was also significantly lower , even on standard diet , as well as in high - fat  fed cgk - tg mice ( 44% decrease in muscle triglyceride at 20 weeks , n = 6 , p < 0.01 ) ( fig . the respiratory gas analysis demonstrated that the oxygen consumption increased in the cgk - tg mice , both when on standard diet and on the high - fat diet ( n = 6 , p < 0.01 ) ( fig . 5a ) . mean oxygen consumption for 24 h of wild - type mice on standard diet was 63.5  0.3 ml  min  kg body wt , and that of cgk - tg was 71.7  0.4 ml  min  kg body wt . the value of wild - type mice on high - fat diet decreased to 43.6  0.2ml  min  kg body wt , and that of cgk - tg was 54.1  0.5 ml  min  kg body wt ( fig . the increase in oxygen consumption in cgk - tg mice was accompanied with a significantly higher rectal temperature ( fig . the respiratory quotient showed a significant reduction in cgk - tg mice ( n = 6 , p < 0.01 ) ( fig . 5d ) , and the reduction was prominent in the light phase ( data not shown ) , similar to the case of bnp - tg mice ( fig . fat oxidation was increased in cgk - tg mice , both when on standard diet and on high - fat diet ( n = 6 , p < 0.01 ) ( fig . 5e ) . mean fat oxidation for 24 h of high - fat  fed cgk - tg mice was 22.8  0.5 ml  min  kg body wt , and that of wild - type mice was 16.8  0.3 ml  min  kg body wt . cgk - tg mice exhibit giant mitochondria in the skeletal muscle , associated with higher oxygen consumption and fat oxidation . total dna and rna were extracted from the brown adipose tissue and the quadriceps , and quantitative pcr analysis was performed . a : oxygen consumption on standard diet ( n = 6 ) . b : mean oxygen consumption for 24 h on standard or high - fat diet . c : rectal temperature on standard or high - fat diet ( n = 12 ) . d : mean respiratory quotient for 24 h on standard or high - fat diet ( n = 6 ) . e : mean fat oxidation estimated from the respiratory gas analysis for 24 h on standard or high - fat diet ( n = 6 ) . f : mitochondrial dna copy number estimated from quantification of mitochondrial and nuclear genome ( n = 8) . the values were standardized to those for the control ( wild - type [ wt ] mice fed on standard diet ) in either group ( n = 12 ) . h : expressions of the genes involved in mitochondrial regulation or fatty acid catabolism in the skeletal muscle ( n = 12 ) . standard diet :  , wild type ( control ) ;  , cgk - tg . high - fat diet :  , wild type ;  , cgk - tg . i : electron microscopic analysis of muscle mitochondria of high - fat  fed cgk - tg mice . * p < 0.05 ; * * p < 0.01 vs. wild - type mice on the same feeding condition . quantitative pcr analysis revealed increased mitochondrial dna copy number in both the brown adipose tissue and the skeletal muscle of cgk - tg mice , both on standard and high - fat diet ( fig . , the expressions of the genes encoding pgc-1 and ucp1 were upregulated in the brown adipose tissue of cgk - tg mice ( fig . , expressions of the genes encoding pgc-1 and ppar were upregulated in cgk - tg mice , both when on standard and high - fat diet , associated with the increase in their downstream target genes involved in mitochondrial oxidative function and fatty acid catabolism ( fig . in addition to pgc-1 and ppar , the expression of atp synthase ( atpsyn ) , cythochrome c oxidase ( cox ) , and carnitine palmitoyl transferase ( cpt ) 1b was significantly enhanced in the skeletal muscle of cgk - tg mice , compared with wild - type mice , both when on standard diet and high - fat diet ; while ucp3 , fatty acid transporter ( fatp ) , and acyl - coa oxidase ( aco ) were upregulated when on the high - fat diet only ( fig . the expression of ppar in the skeletal muscle of cgk - tg mice was not significantly different from wild - type mice , although high - fat diet increased the expression ( data not shown ) . giant mitochondria were densely packed in the skeletal muscle of high - fat  fed cgk - tg mice when it was observed by means of an electron microscope ( fig . schematic representation of the suggested roles for np / cgk cascades , as described in the present and previous studies , is shown in fig . 6c . nps directly increase the expression of pgc-1 and ppar and mitochondrial content in cultured myocytes . c2c12 myocytes were stimulated with the indicated agents for 8 ( a ) or 48 ( b ) hours ( anp 10 mol / l or bnp 10 mol / l , with or without the cgmp antagonist , rp-8-br - cgmp [ rp ] 10 mol / l ) . stimulation of cgk by 8-br - cgmp ( cg ) 10 mol / l was also challenged ( n = 12 ) . a : gene expressions of pgc-1 and ppar in c2c12 cells when treated with anps or bnps . b : mitochondrial mass in c2c12 cells quantified by use of mitotracker green , a fluorescent probe for mitochondria . * p < 0.05 ; * * p < 0.01 vs. control ; # p < 0.05 ; # # p < 0.01 vs. the np ( 10 mol / l)-treated group . rp , rp-8-br - cgmp ( cgmp antagonist ) , cg , 8-br - cgmp ( membrane - permeable cgmp analog ) . previous studies have shown the significant roles for np / cgk cascades in the cardiovascular system that lead to resistance to ischemia , heart failure , and hypertension . in the present study , np / cgk cascades are suggested to promote muscle mitochondrial biogenesis and fat oxidation , as to prevent obesity , fatty liver , and glucose intolerance . to examine the effects of nps on body weight and on glucose and lipid metabolism , bnp - tg mice were given a high - fat ( 60 kcal% fat ) diet . the body weight of bnp - tg mice on standard diet tended to decrease compared with that of their littermate wild - type mice ( 4.8% reduction at 18 weeks old , n = 18 per group , p = 0.06 ) ( fig . when fed a high - fat diet from the age of 10 weeks , on the other hand , the weight of the bnp - tg mice at 18 weeks old was significantly lower than that of the wild - type controls ( 38.9  1.0 g for the former and 43.0  0.9 g for the latter ; n = 10 , p < 0.01 ) ( fig . the reduction in body weight of the transgenic mice fed a high - fat diet could be macroscopically observed ( fig . food intake ( kcal / day ) was not significantly different between bnp - tg and wild - type mice whether on standard or high - fat diet , despite the difference in body weight ( fig . wild - type and bnp - tg mice were given high - fat ( 60 kcal% fat ) diet from the age of 10 weeks . a : body weight of the bnp - tg mice on standard diet ( left panel ) and a high - fat diet ( right panel ) ( n = 18 per group on standard diet and n = 10 on high - fat diet ) . b : macroscopic appearance of a wild - type ( wt ) and a bnp - tg mouse fed on high - fat diet at 18 weeks of age . c : food intake on standard diet and high - fat diet ( n = 6 ) . d : blood glucose levels determined with the glucose and insulin tolerance tests ( n = 8) . e : ct images obtained at kidney level of a wild - type ( wt ) and a bnp - tg mouse on standard diet ( upper panel ) and high - fat diet ( lower panel ) . subcutaneous fat ( yellow ) , abdominal fat ( red ) , and muscular region ( blue ) were distinguished . f : macroscopic appearances and weights of epididymal fat ( upper panel ) and mesenteric fat ( lower panel ) . g : microscopic analysis with hematoxylin and eosin staining of epididymal fat in high - fat  fed mice ( left panels ) . adipocyte size of epididymal fat in high - fat  fed mice was estimated from the histological analysis ( right panel ) ( n = 8) . i : macroscopic appearance of the liver of the mice fed on high - fat diet ( upper panel ) . microscopic images of the liver stained with oil red o ( lower panel ) . j and k : triglyceride concentrations in the liver ( j ) and the quadriceps ( k ) ( n = 12 ) . * p < 0.05 ; * * p < 0.01 vs. wild - type ( wt ) mice on the same feeding condition . ( a high - quality color digital representation of this figure is available in the online issue . ) the blood glucose and insulin levels were identical for bnp - tg and wild - type mice on standard diet , both during ad libitum feeding and fasting . however , these levels were significantly lower in bnp - tg mice during ad libitum feeding of the high - fat diet ( table 1 ) . blood glucose levels were also lower in bnp - tg mice fed a high - fat diet after administration of glucose or insulin ( fig . , serum triglyceride and fatty acid levels did not show any significant differences between the two groups , both when feeding ad libitum and fasting ( table 1 ) . the greater increase in the serum fatty acid level after 24-h fasting was observed in bnp - tg mice ( table 1 ) . urinary excretion of catecholamines ( epinephrine and norepinephrine ) was similar between the two groups ( table 1 ) . physical and metabolic parameters of high - fat  fed bnp - tg mice p < 0.05 , compared with wild type . to estimate the fat weight in mice , we used computed tomography ( ct ) and scanned the whole body of mice . the high - fat diet produced a substantial increase in the adipose tissue in both the subcutaneous and visceral area . the total fat weight of bnp - tg mice fed on a high - fat diet was significantly lower ( 26% reduction , n = 6 , p < 0.01 ) ( fig . the relative reduction ratio was similar for both subcutaneous and visceral fat ( 21% reduction for subcutaneous and 29% for visceral fat ) . the high - fat  fed bnp - tg mice had less surgically harvested epididymal and visceral fats than the wild - type mice ( fig . 1f ) , which is a compatible finding with the ct - based fat quantification . on the other hand , lean body mass showed no significant difference ( 26.6  1.3 g in bnp - tg mice fed a high - fat diet and 27.2  1.2 g in wild - type mice ) . to further study the effects of nps on diet - induced lipid accumulation , we examined adipose tissue , liver , and skeletal muscle of bnp - tg mice fed a high - fat diet for a comparison with those of wild - type mice . we found that the histologically examined adipocytes in the epididymal fat were smaller in bnp - tg mice ( fig . finding , serum leptin was found to decrease and adiponectin to increase in bnp - tg mice fed a high - fat diet ( fig . the liver of wild - type mice had a whitish appearance , while that of high - fat  fed bnp - tg mice was reddish and lower in weight ( 1.4  0.1 g in high - fat  fed bnp - tg mice and 1.7  0.1 g in wild - type mice , n = 6 , p < 0.05 ) ( fig . oil red o staining and triglyceride measurements of the liver confirmed that diet - induced lipid accumulation was significantly attenuated in high - fat  fed bnp - tg mice ( 30% decrease in triglyceride concentration , p < 0.05 , n = 12 ) ( fig . 1j ) , and a similar attenuation of lipid accumulation was observed in the skeletal muscle ( 27% reduction , n = 12 , p < 0.05 ) ( fig . these findings indicate that diet - induced ectopic fat accumulation was reduced in bnp - tg mice in addition to the reduction in adipose tissue . the respiratory gas analysis demonstrated that bnp - tg mice on a high - fat diet consumed more oxygen than wild - type mice ( n = 6 , p < 0.01 ) ( fig . mean oxygen consumption for 24 h of bnp - tg mice on standard diet was 64.3  0.9 ml  min  kg body wt , and that of wild - type mice was 62.6  0.9 ml  min  kg body wt . the value of wild - type mice on high - fat diet decreased to 51.8  0.7 ml  min  kg body wt , and that of bnp - tg was 58.7  0.5 ml  min  kg body wt . the value was significantly higher than that of wild - type mice ( n = 6 , p < 0.01 ) ( fig . the rectal temperature was similar between bnp - tg and wild - type mice , whether on standard diet or high - fat diet ( fig . 2c ) . the respiratory quotient showed a significant reduction in high - fat  fed bnp - tg mice , especially during the daytime ( fig . the mean respiratory quotient for 24 h of high - fat  fed bnp - tg mice was 0.80  0.02 , and that of wild - type mice was 0.81  0.01 ( n = 6 , p < 0.05 ) . in line with the reduction in respiratory quotient , mean fat oxidation for 24 h of high - fat  fed bnp - tg mice , estimated from the results of the respiratory gas analysis , was increased to 18.5  0.2 ml  min  kg body wt , while that of wild - type mice was 16.8  0.2 ml  min  kg body wt ( n = 6 , p the increase in fat oxidation in bnp - tg mice could be augmented by fasting ( fig . high - fat  fed bnp - tg mice exhibit higher oxygen consumption and fat oxidation in association with increased mitochondrial content in the skeletal muscle . total dna and rna were extracted from the brown adipose tissue and the quadriceps , and quantitative pcr analysis was performed . a : oxygen consumption on standard diet ( left panel ) and high - fat diet ( right panel ) ( n = 6 ) . b : mean oxygen consumption for 24 h on standard diet or high - fat diet ( n = 6 ) . c : rectal temperature on standard diet or high - fat diet ( n = 6 ) . d : respiratory quotient of high - fat  fed mice ( n = 6 ) . e : mean fat oxidation estimated from the respiratory gas analysis for 24 h of high - fat  fed mice . g : mitochondrial dna copy number estimated from quantification of mitochondrial and nuclear genome ( n = 8) . h : expressions of genes encoding pgc-1 and ucp1 in the brown adipose tissue and those for pgc-1 and ppar in the skeletal muscle ( n = 8) . high - fat diet :  , wild type ;  , bnp - tg . the values were standardized to those for the control ( wild - type [ wt ] mice fed on standard diet ) in either group . * p < 0.05 ; * * p < 0.01 vs. wild type on the same feeding condition . to further investigate the mechanism for the increase in oxygen consumption of high - fat  fed bnp - tg mice , we checked mitochondrial dna copy number in the brown adipose tissue and skeletal muscle , which are the major sites for energy expenditure . quantitative pcr analysis demonstrated a significant increase in the mitochondrial dna copy number in the skeletal muscle of high - fat  fed bnp - tg mice ( fig . the increase in the brown adipose tissue was weak ( fig . 2 g ) , in accordance with unchanged rectal temperature ( fig . the expressions of the genes encoding peroxisome proliferator  activated receptor ( ppar)- coactivator ( pgc)-1 and uncoupling protein ( ucp ) 1 , which are known to mediate mitochondrial biogenesis and thermogenesis , respectively , were not significantly upregulated in the brown adipose tissue of the tg mice ( fig . 2h ) ; whereas in the skeletal muscle , the expressions of the genes encoding pgc-1 and ppar , which are known to participate in fat oxidation and energy expenditure , were upregulated in high - fat  fed bnp - tg mice ( fig . the results of the study shown in fig . 3 are described in supplement 2 in the online appendix . the expressions of np receptors are regulated by feeding condition , while gca knockdown mice are susceptible to diet - induced obesity and glucose intolerance . total rna was extracted from the quadriceps of wild - type mice , and quantitative pcr analysis for the expressions of np receptors was performed . wild - type and gca heterozygous knockout mice ( gca ) were given a high - fat ( 45 kcal% fat ) diet from the age of 8 weeks . a : expressions of gca , gcb , and c - receptor in the skeletal muscle , brown adipose tissue , and white adipose tissue of wild - type mice for indicated feeding condition ( n = 8) . the values represent the expression levels of each gene compared with that of -actin determined by quantitative pcr analysis . statistical analysis was performed to evaluate the effects of fasting and high - fat diet . * p < 0.05 ; * * p < 0.01 vs. ad libitum eating on the same diet . # p < 0.05 ; # # p < 0.01 vs. standard diet on the same eating condition ( ad libitum or fasting ) . further analysis to clarify whether there were interactions between the effect of fasting and that of high - fat diet was performed as shown in supplemental table s2 . , skeletal muscle ;  , brown adipose tissue ;  , white adipose tissue . b : body weight of wild - type ( wt ) and gca mice fed on standard diet ( dotted lines ) or high - fat diet ( solid lines ) after 8 weeks of age ( n = 8) . d : blood glucose levels determined with the glucose tolerance test ( n = 8) . e : total fat weight estimated from the ct images ( n = 6 ) . * p < 0.05 ; * * p < 0.01 vs. wild type on the same feeding condition . to determine the effect of cgk - i , a major downstream effector of np / gc / cgmp cascades , on body weight and on glucose and lipid metabolism , we examined the cgk - tg mice with ubiquitously overexpressing human cgk - i . the cgk - tg mice showed a significant reduction in body weight compared with wild - type mice , even on standard diet ( 27.6  0.4 g for cgk - tg and 32.0  0.6 g for wild - type mice , at 18 weeks old on standard diet , n = 8 , p < 0.01 ) ( fig . moreover , high - fat diet  induced weight gain was attenuated in the tg mice , and the reduction in body weight eventually reached > 20% . at 18 weeks of age , their body weight was 35.7  0.4 g , and that of wild - type mice was 43.9  0.6 g ( after 8 weeks of high - fat feeding , n = 8 , p < 0.01 ) ( fig . the daily food intake ( kcal / day ) was not noticeably different for cgk - tg and wild - type mice , while it showed a significant increase in cgk - tg mice when it was adjusted for body weight ( kcal  day  g body wt ) ( fig . wild - type and cgk - tg mice were given a high - fat ( 60 kcal% fat ) diet from the age of 10 weeks . a : body weight of cgk - tg mice on standard diet ( left panel ) and high - fat diet ( right panel ) ( n = 810 ) . b : macroscopic appearance of a wild - type ( wt ) and a cgk - tg mouse . c : food intake on standard and high - fat diet ( upper panel ) ( kcal / day , n = 6 ) and the body weight ( bw)-adjusted value ( lower panel ) ( kcal  day  g body wt ) . d : blood glucose levels determined with the glucose and insulin tolerance tests for each genotype on standard diet and high - fat diet ( n = 8) . e : ct images obtained at kidney level of a wild - type ( wt ) and a cgk - tg mouse on standard diet ( upper panel ) and high - fat diet ( lower panel ) . subcutaneous fat ( yellow ) , abdominal fat ( red ) , and muscular region ( blue ) were distinguished . f : microscopic analysis using hematoxylin and eosin staining of epididymal fats in high - fat  fed mice . h : macroscopic appearance of the liver in high - fat  fed mice ( upper panel ) . i and j : triglyceride concentration in the liver ( i ) and the quadriceps ( j ) ( n = 12 ) . * p < 0.05 ; * * p < 0.01 vs. wild type on the same feeding condition . ( a high - quality color digital representation of this figure is available in the online issue . ) the blood glucose levels were significantly lower in cgk - tg mice , both during ad libitum feeding and fasting and on standard diet and high - fat diet ( table 2 ) . the decrease was accompanied by significantly lower insulin levels ( table 2 ) , except for the value for fasting while fed on standard diet . after administration of glucose or insulin , the difference in glucose levels between cgk - tg and wild - type mice became more prominent ( fig . 4d ) . serum triglyceride and fatty acid levels were similar for cgk - tg and wild - type mice , both during ad libitum feeding and fasting , except for the significant increase in fatty acid levels in high - fat  fed cgk - tg mice during fasting , when compared with that of wild - type mice ( table 2 ) . urinary excretion of the catecholamines ( epinephrine and norepinephrine ) was similar for wild - type and cgk - tg mice ( table 2 ) . physical and metabolic parameters of cgk - tg mice fed on standard chow or high - fat diet p < 0.05 compared with wild type on the same feeding condition . in parallel with the reduction in body weight , fat tissues assessed by means of ct analysis were significantly reduced in cgk - tg mice , both on standard and high - fat diet ( fig . 4e ) . hematoxylin - eosin staining and the coulter counter analysis showed that the adipocytes in the epididymal fat were smaller in cgk - tg mice , with mean adipocyte diameters for wild - type and cgk - tg mice of 80 and 60 m , respectively , when on standard diet ( fig . 4f and g ) . the high - fat diet produced an increase in the diameter to 130 m in wild - type mice but to only 80 m in the tg mice ( fig . the liver of high - fat  fed cgk - tg mice weighed less than that of wild - type mice ( 1.3  0.1 g and 1.8  0.1 g , n = 8 , p < 0.01 , respectively ) and had reddish appearance ( fig . the hepatic triglyceride concentration of high - fat  fed cgk - tg mice was significantly reduced when compared with wild - type mice ( 30% decrease at 20 weeks , n = 6 , p < 0.05 ) ( fig . muscular triglyceride concentration of cgk - tg mice was also significantly lower , even on standard diet , as well as in high - fat  fed cgk - tg mice ( 44% decrease in muscle triglyceride at 20 weeks , n = 6 , p < 0.01 ) ( fig . the respiratory gas analysis demonstrated that the oxygen consumption increased in the cgk - tg mice , both when on standard diet and on the high - fat diet ( n = 6 , p < 0.01 ) ( fig . 5a ) . mean oxygen consumption for 24 h of wild - type mice on standard diet was 63.5  0.3 ml  min  kg body wt , and that of cgk - tg was 71.7  0.4 ml  min  kg body wt . the value of wild - type mice on high - fat diet decreased to 43.6  0.2ml  min  kg body wt , and that of cgk - tg was 54.1  0.5 ml  min  kg body wt ( fig . the increase in oxygen consumption in cgk - tg mice was accompanied with a significantly higher rectal temperature ( fig . the respiratory quotient showed a significant reduction in cgk - tg mice ( n = 6 , p < 0.01 ) ( fig . 5d ) , and the reduction was prominent in the light phase ( data not shown ) , similar to the case of bnp - tg mice ( fig . fat oxidation was increased in cgk - tg mice , both when on standard diet and on high - fat diet ( n = 6 , p < 0.01 ) ( fig . mean fat oxidation for 24 h of high - fat  fed cgk - tg mice was 22.8  0.5 ml  min  kg body wt , and that of wild - type mice was 16.8  0.3 ml  min  kg body wt . cgk - tg mice exhibit giant mitochondria in the skeletal muscle , associated with higher oxygen consumption and fat oxidation . total dna and rna were extracted from the brown adipose tissue and the quadriceps , and quantitative pcr analysis was performed . a : oxygen consumption on standard diet ( n = 6 ) . b : mean oxygen consumption for 24 h on standard or high - fat diet . c : rectal temperature on standard or high - fat diet ( n = 12 ) . d : mean respiratory quotient for 24 h on standard or high - fat diet ( n = 6 ) . e : mean fat oxidation estimated from the respiratory gas analysis for 24 h on standard or high - fat diet ( n = 6 ) . f : mitochondrial dna copy number estimated from quantification of mitochondrial and nuclear genome ( n = 8) . the values were standardized to those for the control ( wild - type [ wt ] mice fed on standard diet ) in either group ( n = 12 ) . h : expressions of the genes involved in mitochondrial regulation or fatty acid catabolism in the skeletal muscle ( n = 12 ) . standard diet :  , wild type ( control ) ;  , cgk - tg . high - fat diet :  , wild type ;  , cgk - tg . i : electron microscopic analysis of muscle mitochondria of high - fat  fed cgk - tg mice . * p < 0.05 ; * * p < 0.01 vs. wild - type mice on the same feeding condition . quantitative pcr analysis revealed increased mitochondrial dna copy number in both the brown adipose tissue and the skeletal muscle of cgk - tg mice , both on standard and high - fat diet ( fig . , the expressions of the genes encoding pgc-1 and ucp1 were upregulated in the brown adipose tissue of cgk - tg mice ( fig . , expressions of the genes encoding pgc-1 and ppar were upregulated in cgk - tg mice , both when on standard and high - fat diet , associated with the increase in their downstream target genes involved in mitochondrial oxidative function and fatty acid catabolism ( fig . in addition to pgc-1 and ppar , the expression of atp synthase ( atpsyn ) , cythochrome c oxidase ( cox ) , and carnitine palmitoyl transferase ( cpt ) 1b was significantly enhanced in the skeletal muscle of cgk - tg mice , compared with wild - type mice , both when on standard diet and high - fat diet ; while ucp3 , fatty acid transporter ( fatp ) , and acyl - coa oxidase ( aco ) were upregulated when on the high - fat diet only ( fig . the expression of ppar in the skeletal muscle of cgk - tg mice was not significantly different from wild - type mice , although high - fat diet increased the expression ( data not shown ) . giant mitochondria were densely packed in the skeletal muscle of high - fat  fed cgk - tg mice when it was observed by means of an electron microscope ( fig . the results of the study shown in fig . 6a and b are described in supplement 2 . schematic representation of the suggested roles for np / cgk cascades , as described in the present and previous studies , is shown in fig . 6c . nps directly increase the expression of pgc-1 and ppar and mitochondrial content in cultured myocytes . c2c12 myocytes were stimulated with the indicated agents for 8 ( a ) or 48 ( b ) hours ( anp 10 mol / l or bnp 10 mol / l , with or without the cgmp antagonist , rp-8-br - cgmp [ rp ] 10 mol / l ) . stimulation of cgk by 8-br - cgmp ( cg ) 10 mol / l was also challenged ( n = 12 ) . a : gene expressions of pgc-1 and ppar in c2c12 cells when treated with anps or bnps . b : mitochondrial mass in c2c12 cells quantified by use of mitotracker green , a fluorescent probe for mitochondria . * p < 0.05 ; * * p < 0.01 vs. control ; # p < 0.05 ; # # p < 0.01 vs. the np ( 10 mol / l)-treated group . rp , rp-8-br - cgmp ( cgmp antagonist ) , cg , 8-br - cgmp ( membrane - permeable cgmp analog ) . previous studies have shown the significant roles for np / cgk cascades in the cardiovascular system that lead to resistance to ischemia , heart failure , and hypertension . in the present study , np / cgk cascades are suggested to promote muscle mitochondrial biogenesis and fat oxidation , as to prevent obesity , fatty liver , and glucose intolerance . the findings in this study demonstrate that the activation of np / cgk cascades augments mitochondrial biogenesis and fat oxidation in mice through upregulation of pgc-1 and ppar in the skeletal muscle , thus conferring resistance to obesity and glucose intolerance . bnp - tg mice fed a high - fat diet were protected from obesity and insulin resistance , while cgk - tg mice were lean even on standard diet and showed increased insulin sensitivity , and , surprisingly , giant mitochondria were densely packed in the skeletal muscle . both types of mice showed a reduction in fat tissue and excessive lipid accumulation in the liver and skeletal muscle , in accordance with an increase in fat oxidation . functional np receptors were upregulated during fasting , whereas they were downregulated by ad libitum feeding or high - fat challenge , while whole - body knockdown of the functional receptor , gca , led to promotion of obesity in mice . these findings , together with those that demonstrated np - induced lipolysis , lead us to propose the concept that np / cgk cascades play significant roles in lipid catabolism during fasting or chronic caloric restriction , in addition to their well - known roles in the cardiovascular system , such as in the attenuation of hypertension and congestive heart failure ( fig . 6c ) ( 7,8,17 ) . genetic overexpression of np / cgk in mice attenuated diet - induced obesity and insulin resistance , associated with the increase in muscle mitochondrial content and fat oxidation . we identified the dual upregulation of pgc-1 and ppar in the skeletal muscle of the tg mice as a molecular basis of the increase in mitochondria and confirmed the direct increase in the expression of pgc-1 and ppar and mitochondrial content by both anp and bnp through the experiments using cultured myocytes . pgc-1 is a transcriptional cofactor that acts as a master regulator of mitochondrial biogenesis ( 18 ) . ppar is a transcription factor that binds with pgc-1 and enhances mitochondrial biogenesis and lipid catabolism ( 19 ) . activation of ppar in mice by the treatment with the ppar agonist gw501516 or overexpression of a constitutively active form of ppar were shown to increase muscle mitochondrial content and fatty acid oxidation and , thus , prevent diet - induced obesity and glucose intolerance ( 20,21 ) . however , mice with muscle - specific overexpression of wild - type ppar did not exhibit such a prominent phenotype ( 22 ) . muscle - specific overexpression of pgc-1 has been reported to not prevent diet - induced obesity and insulin resistance in mice , although there was an increase in mitochondrial content ( 23 ) . these results indicate that the coordinated increase in the expression of pgc-1 and ppar , which was observed in the skeletal muscle of bnp- and cgk - tg mice , led to synergistic effects that were beneficial for ameliorating diet - induced obesity and insulin resistance . the expression of pgc-1 and ppar in muscle is augmented by exercise via exercise - sensitive molecules such as amp - activated protein kinase ( ampk ) ( 18,19 ) . the increases in circulating np and cgmp levels during exercise have been demonstrated in previous reports ( 24,25 ) . therefore , we hypothesize that np / cgmp / cgk cascades can interact with the exercise - sensitive molecules in muscle and increase the expression of pgc-1 and ppar. bnp - tg mice gained less body weight than controls when they were fed on high - fat diet . the same tendency was observed even when on standard diet ; however , the magnitude of the change was not so prominent . we interpret the results as the tg mice escaped from diet - induced obesity by increasing muscle mitochondrial content and fat oxidation . the elevation of serum adiponectin levels observed in the high - fat  fed bnp - tg mice , associated with reduced adiposity , might be contributing to the attenuation of insulin resistance ( 26,27 ) . apparent changes in food intake and catecholamine kinetics were not observed . in cgk - tg mice , the increase in mitochondria was evident even on standard diet , resulting in the tg mice being leaner and more insulin sensitive than bnp - tg mice . our data suggested that one reason for the difference in the magnitude of the altered phenotypes between the bnp- and cgk - tg mice was caused by the variations of the target tissues . in the skeletal muscle , similar changes in gene expression were evident in the two lines of tg mice , including upregulation of pgc-1 and ppar. in the brown adipose tissue , on the other hand , the increase in the expression level of pgc-1 and its downstream effector ucp1 was significant in cgk - tg mice , associated with higher body temperature ; however , these changes were not so prominent in bnp - tg mice . we observed that the functional receptors for np , gca , and gcb were abundantly expressed in the brown adipose tissue ; however , the clearance receptor ( c - receptor ) was also rich in the tissue . therefore , the functional receptor  to  c - receptor ratio , which is suggested to correlate with the biological action of nps , was lower in the brown adipose tissue than skeletal muscle . we speculate that the abundant expression of c - receptor and the lower ratio of the functional receptors reduced the effect of np in the brown adipose tissue . high - fat  fed gca heterozygous knockout mice accumulated more fat mass and were glucose intolerant compared with wild - type mice . this phenotype is the opposite to that of bnp - tg or cgk - tg mice so that the effects of nps in terms of the prevention of diet - induced obesity and insulin resistance seemed to be mediated at least in part by gca . we were unable to determine whether gcb also participated because gcb knockout mice were not available . the np receptors were regulated by dietary conditions ; that is , gca and gcb were upregulated by fasting and downregulated during high - fat feeding . therefore , the ratio of functional receptors to c - receptor was lowered by feeding or during chronic high - fat diet ; conversely , fasting increased relative amount of the functional receptors . these results indicate that a high - fat diet can contribute to the development of diet - induced obesity and insulin resistance at least partly by downregulation of gca and upregulation of the c - receptor . the notion that the kinetics of np receptor expression depend on dietary conditions , such as high - fat diet or fasting , is consistent with earlier findings of others . the gca  to  c - receptor ratio was shown to be lowered in obese people than in nonobese ( 28 ) . natriuresis is known to be increased after several hours of fasting , despite the fact that sodium intake is decreased ( 29 ) . the upregulation of functional np receptors in renal tubular cells by fasting and subsequent augmentation of natriuresis might account for the well - known phenomenon of  fasting - induced natriuresis . taken together , the dietary regulation of the receptor expressions indicates that the signal transduction through np / cgk cascades , which is augmented by starvation , might have a role in fasting - induced fat oxidation under physiological conditions . augmented production of nps caused by congestive heart failure plays important roles in compensating for the volume expansion and subsequent decrease in oxygen supply by exerting vasodilating , natriuretic , and neurohumoral - modulating actions ( 30 ) . this compensatory effect in the cardiovascular system has been thought to be the primary role for np / cgk cascades ( 31 ) . in the present study , we reveal the new roles for the cascades on mitochondrial biogenesis and fat oxidation through upregulation of pgc-1 and ppar. the results of the present study are in line with earlier publications from lafontan and colleagues ( 6,7,32,33 ) , which have demonstrated the importance of the np / cgk system in the regulation of adipose tissue lipolysis . their results and ours together support the view that np / cgk cascades have significant roles in lipid catabolism . moreover , the increase in circulating np and cgmp levels during exercise has been demonstrated in previous reports ( 24,25 ) . therefore , np / cgmp / cgk cascades might have significant roles in metabolic adaptations in response to exercise , including mitochondrial biogenesis and fat oxidation . several other vascular hormones that regulate vascular tone , including catecholamines , angiotensin ii , and no , are also implicated in the regulation of cellular metabolism and subsequent change in body weight and glucose tolerance ( 3438 ) . in this context , vascular hormones seem to lie at the crossroad of cardiovascular and metabolic diseases . in a recent report ( 9 ) , the number of risk factors acting as diagnostic markers of metabolic syndrome is shown to inversely correlate with plasma concentrations of nps . the clinical data and the findings of the present study collectively indicate that insufficient activation of np / cgk cascades can be a causative factor of both obesity and hypertension . we speculate that the state of overnutrition would downregulate functional np receptors as to promote both obesity and hypertension . furthermore , we believe that physiological responses of vascular hormones , in accordance with blood pressure , circulating fluid volume , and oxygen demand , would result in coordinated regulation of oxygen supply and consumption , and the coordination is especially important for the adaptive responses to exercise . to summarize , the results of the present study indicate that np / cgk cascades can promote muscle mitochondrial biogenesis and fat oxidation through upregulation of pgc-1 and ppar , as to prevent obesity and glucose intolerance . the results were compatible with our recent report ( 10 ) , which showed that cgmp can increase mitochondrial content and function in cultured myocytes . the pharmaceutical interventions that activate np / cgmp / cgk cascades seem to be potentially beneficial for the treatment of obesity , fatty liver , and glucose intolerance .
objectivenatriuretic peptides ( nps ) have been characterized as vascular hormones that regulate vascular tone via guanylyl cyclase ( gc ) , cyclic gmp ( cgmp ) , and cgmp - dependent protein kinase ( cgk ) . 
 recent clinical studies have shown that plasma np levels were lower in subjects with the metabolic syndrome . 
 the present study was conducted to elucidate the roles for np / cgk cascades in energy metabolism.research design and methodswe used three types of genetically engineered mice : brain np ( bnp ) transgenic ( bnp - tg ) , cgk - tg , and guanylyl cyclase - a ( gca ) heterozygous knockout ( gca+/ ) mice and analyzed the metabolic consequences of chronic activation of np / cgk cascades in vivo . 
 we also examined the effect of nps in cultured myocytes.resultsbnp-tg mice fed on high - fat diet were protected against diet - induced obesity and insulin resistance , and cgk - tg mice had reduced body weight even on standard diet ; surprisingly , giant mitochondria were densely packed in the skeletal muscle . 
 both mice showed an increase in muscle mitochondrial content and fat oxidation through upregulation of peroxisome proliferator  activated receptor ( ppar)- coactivator ( pgc)-1 and ppar. the functional np receptors , gca and guanylyl cyclase - b , were downregulated by feeding a high - fat diet , while gca+/ mice showed increases in body weight and glucose intolerance when fed a high - fat diet . 
 nps directly increased the expression of pgc-1 and ppar and mitochondrial content in cultured myocytes.conclusionsthe findings together suggest that np / cgk cascades can promote muscle mitochondrial biogenesis and fat oxidation , as to prevent obesity and glucose intolerance . 
 the vascular hormone , np , would contribute to coordinated regulation of oxygen supply and consumption .