the study protocol conformed with the declaration of helsinki ( 2008 ) and was approved by the appropriate institutional ethical board . informed consent was obtained from each study participant after explanation of the purpose , nature , and potential complications of the study . after recruitment by means of public advertisement , all participants underwent complete medical history , clinical examination , and laboratory tests to exclude cardiovascular , renal , and thyroid diseases , and viral hepatitis . all volunteers were white and had already participated in our previous study on absolute quantification of liver phosphorus metabolites ( 11 ) . the study excluded patients taking insulin or thiazolidinediones , or presenting with islet cell antibodies and diabetes - related complications . controls had similar body mass and age , but normal glucose tolerance during a 75-g oral glucose tolerance test and had neither a family history of diabetes nor regular drug treatment . of 24 individuals recruited , 3 did not complete the experiment because they refused to continue the mrs session . data sets of three participants could not be used due to low quality resulting from a poor local homogeneity of the static magnetic field . data from one individual could not be used because of a technical problem with the scanner . thus , complete mrs datasets for 17 individuals were available for analyses ( table 1 ) . anthropometric and laboratory parameters data are expressed as mean  sd , unless indicated otherwise . * p < 0.001 , significant differences between groups . type 2 diabetic patients showed acceptable glycemic ( a1c : 7.06  0.98% vs. 5.53  0.27% in controls ; p = 0.001 ) and lipid control on treatment with diet only ( n = 1 ) , sulfonylurea only ( n = 3 ) , or combined with metformin ( n = 5 ) and statins ( n = 7 ) . measurements of fatp and insulin sensitivity were performed on different days under identical conditions of overnight fasting and withdrawal of medications . waist was defined as the midpoint between the highest point of the iliac crest and the lowest part of the costal margin in the midaxillary line ( 13 ) . plasma glucose was measured by the glucose oxidase method ( glucose analyzer ii , beckman instruments inc . , fullerton , ca ) and plasma insulin by double antibody radio immunoassay ( 11 ) . all other parameters were measured in the clinical chemistry laboratory with standardized quality - controlled assays . whole - body glucose disposal ( m , or m related to plasma insulin [ m / i ] ) and egp were measured in seven diabetic subjects ( three women / four men ) and in six controls ( four women / two men ) as described previously ( 7 ) , who exhibited identical demographics as the whole group . studies started at 6:30 a.m. with primed - continuous infusion ( 5 min : 3.6 mg  kg body wt  fasting glucose [ mg / dl]/90 [ mg / dl ] ; 355 min : 0.036 mg  min  kg body wt ) of d-[6,6-h2]glucose ( 98% enriched ; cil , andover , ma ) . the euglycemic ( 5.5 mmol / l)-hyperinsulinemic ( 70 mu / l ) clamp test was started at 10:00 a.m. , with a primed continuous infusion of human regular insulin ( actrapid ; novo nordisk , bagsvaerd , denmark ; 40 mu  [ m body surface area ]  min ) for 240 min . a 20% dextrose infusion labeled with d-[6,6-h2]glucose ( 2% enriched ) according to the hot - glucose infusion protocol after overnight fasting , volunteers underwent a mrs protocol consisting of two consecutive sessions with a break of 30 min in a 3-t mr scanner ( bruker biospin , ettlingen , germany ) using a 10-cm linear polarized surface coil tuned to h and p frequencies . in session 1 , subjects were supine in the scanner with the coil positioned over the lateral aspect of the liver . hcl was measured using localized h mrs ( steam , 20  20  20 mm ; 5 scans : echo time , 15 , 20 , 30 , 50 , 70 ms ; repetition time , 2.5 s ) ( fig . fat and water peaks were integrated after phasing and baseline adjustment using xwin - nmr software ( bruker biospin , ettlingen , germany ) . a 30-mm slab within the liver was selected using 1d - isis encoding for p acquisitions ( fig . after a baseline scan , alternated spectra with saturation of the -atp resonance frequency and the frequency mirrored relative to the pi resonance were acquired ( fig . in addition , a combined saturation transfer inversion recovery experiment was performed in each individual to determine the effective longitudinal relaxation time ( t1 * ) . the peak amplitude was used for quantification because only peak ratios between consecutive scans were used as described ( 12 ) . in session 2 , lasting 50 min , the subjects were prone in the scanner on top of the surface coil with the lateral aspect of the liver over the coil center . after shimming , gradient echo - based images were acquired during one breath - hold . a 3-dimensional k - space weighted chemical shift imaging sequence ( field of view : 20  20  20 cm , repetition time : 1 s , bandwidth : 5 khz , 2,048 acquisitions in 34 min ) was applied , reconstructed , and quantified offline using custom - made software in combination with jmrui / amares , as described ( 12,14 ) . the chemical exchange constant , k of the saturation transfer experiment is assessed from the pi signal before ( m0 ) and after saturation of -atp ( ms ) . then , k is calculated as k = 1/t1 *  ( 1  ms / m0 ) , and fatp as fatp = k  pi . rates of glucose appearance ( ra ) were calculated by dividing the tracer ( d-[6,6-h2]glucose ) infusion rate multiplied by tracer enrichment by the percent of tracer enrichment in plasma and subtracting the tracer infusion rate . during the clamp , ra was calculated using steele 's nonsteady state equations . egp suppression was calculated as 100  [ ( 1  egp insulin - mediated)/(egp baseline ) ] . data are presented as means  sd and compared between the groups using the two - tailed student t test . relationships between variables were analyzed with pearson correlations for normally distributed data and with spearman correlations for the scattered hcl values . regression analyses for the dependent variables fatp and pi were done using the independent variables of age , bmi , waist circumference , hcl , alanine aminotransferase , a1c , egp , and ffa suppression , m / i , serum triglycerides , and low - density lipoprotein . after recruitment by means of public advertisement , all participants underwent complete medical history , clinical examination , and laboratory tests to exclude cardiovascular , renal , and thyroid diseases , and viral hepatitis . all volunteers were white and had already participated in our previous study on absolute quantification of liver phosphorus metabolites ( 11 ) . the study excluded patients taking insulin or thiazolidinediones , or presenting with islet cell antibodies and diabetes - related complications . controls had similar body mass and age , but normal glucose tolerance during a 75-g oral glucose tolerance test and had neither a family history of diabetes nor regular drug treatment . of 24 individuals recruited , 3 did not complete the experiment because they refused to continue the mrs session . data sets of three participants could not be used due to low quality resulting from a poor local homogeneity of the static magnetic field . data from one individual could not be used because of a technical problem with the scanner . thus , complete mrs datasets for 17 individuals were available for analyses ( table 1 ) . anthropometric and laboratory parameters data are expressed as mean  sd , unless indicated otherwise . * p < 0.001 , significant differences between groups . type 2 diabetic patients showed acceptable glycemic ( a1c : 7.06  0.98% vs. 5.53  0.27% in controls ; p = 0.001 ) and lipid control on treatment with diet only ( n = 1 ) , sulfonylurea only ( n = 3 ) , or combined with metformin ( n = 5 ) and statins ( n = 7 ) . measurements of fatp and insulin sensitivity were performed on different days under identical conditions of overnight fasting and withdrawal of medications . waist was defined as the midpoint between the highest point of the iliac crest and the lowest part of the costal margin in the midaxillary line ( 13 ) . plasma glucose was measured by the glucose oxidase method ( glucose analyzer ii , beckman instruments inc . , fullerton , ca ) and plasma insulin by double antibody radio immunoassay ( 11 ) . all other parameters were measured in the clinical chemistry laboratory with standardized quality - controlled assays . whole - body glucose disposal ( m , or m related to plasma insulin [ m / i ] ) and egp were measured in seven diabetic subjects ( three women / four men ) and in six controls ( four women / two men ) as described previously ( 7 ) , who exhibited identical demographics as the whole group . studies started at 6:30 a.m. with primed - continuous infusion ( 5 min : 3.6 mg  kg body wt  fasting glucose [ mg / dl]/90 [ mg / dl ] ; 355 min : 0.036 mg  min  kg body wt ) of d-[6,6-h2]glucose ( 98% enriched ; cil , andover , ma ) . the euglycemic ( 5.5 mmol / l)-hyperinsulinemic ( 70 mu / l ) clamp test was started at 10:00 a.m. , with a primed continuous infusion of human regular insulin ( actrapid ; novo nordisk , bagsvaerd , denmark ; 40 mu  [ m body surface area ]  min ) for 240 min . a 20% dextrose infusion labeled with d-[6,6-h2]glucose ( 2% enriched ) according to the hot - glucose infusion protocol after overnight fasting , volunteers underwent a mrs protocol consisting of two consecutive sessions with a break of 30 min in a 3-t mr scanner ( bruker biospin , ettlingen , germany ) using a 10-cm linear polarized surface coil tuned to h and p frequencies . in session 1 , subjects were supine in the scanner with the coil positioned over the lateral aspect of the liver . hcl was measured using localized h mrs ( steam , 20  20  20 mm ; 5 scans : echo time , 15 , 20 , 30 , 50 , 70 ms ; repetition time , 2.5 s ) ( fig . fat and water peaks were integrated after phasing and baseline adjustment using xwin - nmr software ( bruker biospin , ettlingen , germany ) . a 30-mm slab within the liver was selected using 1d - isis encoding for p acquisitions ( fig . after a baseline scan , alternated spectra with saturation of the -atp resonance frequency and the frequency mirrored relative to the pi resonance were acquired ( fig . in addition , a combined saturation transfer inversion recovery experiment was performed in each individual to determine the effective longitudinal relaxation time ( t1 * ) . the peak amplitude was used for quantification because only peak ratios between consecutive scans were used as described ( 12 ) . in session 2 , lasting 50 min , the subjects were prone in the scanner on top of the surface coil with the lateral aspect of the liver over the coil center . after shimming , gradient echo - based images were acquired during one breath - hold . a 3-dimensional k - space weighted chemical shift imaging sequence ( field of view : 20  20  20 cm , repetition time : 1 s , bandwidth : 5 khz , 2,048 acquisitions in 34 min ) was applied , reconstructed , and quantified offline using custom - made software in combination with jmrui / amares , as described ( 12,14 ) . the chemical exchange constant , k of the saturation transfer experiment is assessed from the pi signal before ( m0 ) and after saturation of -atp ( ms ) . then , k is calculated as k = 1/t1 *  ( 1  ms / m0 ) , and fatp as fatp = k  pi . rates of glucose appearance ( ra ) were calculated by dividing the tracer ( d-[6,6-h2]glucose ) infusion rate multiplied by tracer enrichment by the percent of tracer enrichment in plasma and subtracting the tracer infusion rate . during the clamp egp suppression was calculated as 100  [ ( 1  egp insulin - mediated)/(egp baseline ) ] . data are presented as means  sd and compared between the groups using the two - tailed student t test . relationships between variables were analyzed with pearson correlations for normally distributed data and with spearman correlations for the scattered hcl values . regression analyses for the dependent variables fatp and pi were done using the independent variables of age , bmi , waist circumference , hcl , alanine aminotransferase , a1c , egp , and ffa suppression , m / i , serum triglycerides , and low - density lipoprotein . diabetic participants had 67% higher fasting plasma glucose concentrations , 27% higher a1c , and similar body weight , but tended to have greater waist circumference ( p = 0.167 ) than controls ( table 1 ) . insulin sensitivity was 29% ( m ) and 45% ( m / i ) lower in diabetic volunteers than in controls , confirming peripheral insulin resistance ( table 2 ) . liver fat , energy metabolism , and hepatic and peripheral insulin sensitivity data are shown as mean  sd . significant differences between groups : * p = 0.011 ; mean hcl content was not different between groups ( table 2 ) . the distribution of the individual hcl values covered a broad range from < 1 to 33% across the groups , with values > 5.5% in six diabetic and five nondiabetic participants . fasting egp was similar in both groups , whereas egp was completely suppressed during insulin stimulation only in controls but not in diabetic participants , indicating hepatic insulin resistance . fasting hepatocellular fatp was 42% lower and k , the chemical exchange rate constant , tended to be lower in patients compared with controls ( table 2 ) . previous medical treatment with or without metformin did not affect fatp ( 16.5  6.0 vs. 16.0  5.0 mmol / l / min ) . fatp positively correlated with both peripheral ( m : r = 0.67 , p = 0.01 ; m / i : r = 0.61 , p = 0.03 ) and hepatic insulin resistance ( suppression of egp : r = 0.77 , p = 0.002 ) , even after adjustment for plasma insulin during the clamp ( r = 0.84 , p = 0.001 ) across all participants ( fig . also , k values related to m ( r = 0.67 , p = 0.01 ) but not to m / i ( r = 0.47 , p = 0.11 ) . furthermore , fatp correlated negatively with waist circumference ( r = 0.78 p = 0.001 ; fig . 2b ) , fasting plasma glucose ( r = 0.58 , p = 0.01 ) , bmi ( r = 0.55 , p = 0.02 ) , hcl ( r = 0.51 , p = 0.04 ; fig . 2c ) , and a1c ( r = 0.50 , p = 0.04 ) . hepatocellular pi ( r = 0.77 , p = 0.0003 ) related positively to fatp ( fig . after adjusting for hcl , the relationships of fatp with waist circumference ( r = 0.81 p = 0.001 ) , m ( r = 0.66 , p = 0.02 ) , m / i ( r = 0.64 , p = 0.03 ) , egp suppression ( r = 0.72 , p = 0.009 ) , bmi ( 0.54 , p = 0.03 ) , and fasting plasma glucose ( r = 0.52 , p = 0.04 ) remained significant . fatp did not correlate significantly with fasting ffa ( r = 0.203 , p = 0.507 ) or ffa suppression ( r = 0.51 , p = 0.07 ) . correlations with hepatocellular atp production ( fatp ) with ( a ) hepatic insulin resistance ( egp suppression ; all : r = 0.77 , p = 0.002 ; type 2 diabetic subjects : r = 0.79 , p = 0.01 ; controls : r = 0.54 , p = 0.17 ) , ( b ) waist circumference ( all : r = 0.78 , p = 0.001 ; type 2 diabetic subjects : r = 0.57 , p = 0.11 ; controls : r = 0.68 , p = 0.064 ) , ( c ) hcl contents ( all : r = 0.51 , p = 0.038 ; type 2 diabetic subjects r = 0.20 , p = 0.61 ; controls : r = 0.62 , p = 0.10 ) , ( d ) hepatic concentrations of pi ( all : r = 0.77 , p = 0.0003 ; type 2 diabetic subjects : r = 0.57 , p = 0.11 ; controls : r = 0.68 , p = 0.064 ) across all participants ( type 2 diabetic subjects :  , controls :  ) . multiple regression analysis for the dependent variable fatp identified waist circumference as the independent predictor of fatp ( r = 0.65 , adjusted r = 0.62 , p = 0.001 ) . likewise , multiple regression analysis for the dependent variable pi identified waist circumference as the independent predictor of pi ( r = 0.56 , adjusted r = 0.52 , p = 0.003 ) . this study found that patients with type 2 diabetes have markedly lower hepatic atp production than nondiabetic individuals of comparable age and body mass . of note , this alteration of fatp occurred without evidence for clinical liver disease , as indicated by the lack of a history of hepatitis , alcohol consumption , or significant elevation of liver enzymes ( 11 ) . across all subjects , hepatocellular fatp correlated negatively with hcl , as recently reported for the relationships between hepatic absolute pi and atp concentrations and hcl ( 11 ) . however , we detected a clear difference in fatp between diabetic and nondiabetic participants , despite comparable hcl values and equal distribution of steatosis ( hcl > 5.5% ) in both groups ( 67% of diabetic and 63% of nondiabetic participants ) . after adjustment for hcl , hepatic and whole - body insulin sensitivity remained tightly and negatively related to fatp . absolute hepatic concentrations of pi and atp are also associated with hepatic but not with peripheral insulin sensitivity ( 11 ) . under similar conditions of overnight fasting , myocellular fatp correlates negatively to peripheral insulin sensitivity , which is mostly accounted for by skeletal muscle , but not to hepatic insulin sensitivity ( 7 ) . this suggests that hepatocellular fatp could serve as a better indicator of overall insulin resistance than myocellular fatp . in type 2 diabetes , insulin resistance develops as a result of inherited factors as well as from acquired factors that result in glucolipotoxicity due to chronic elevation of ffas , triglycerides , or glucose ( 2,3 ) . in rats , a high - fat diet for 7 weeks induced obesity , insulin resistance , and hepatic steatosis and also reduced respiratory capacity and increased oxidative stress in liver mitochondria ( 15 ) . further , mitochondria from fatty rat liver have lower amounts of atp synthase protein ( 16 ) . a short - term high - fat diet also leads to insulin resistance in liver , but not in skeletal muscle ( 17 ) , whereas a hypocaloric diet decreases both hepatic fat content and insulin resistance within 48 h ( 18 ) . in conclusion , impaired energy metabolism in the liver could be an early defect in the pathogenesis of type 2 diabetes . the lack of a correlation of fatp with circulating plasma triglycerides or ffas suggests that increased lipid availability can not explain the abnormality of hepatic fatp . however , hepatocellular fatp related negatively to bmi and even more tightly to waist circumference , which explained most of the variation of fatp . of note , waist circumference is the best anthropometric marker of abdominal visceral fat ( 19 ) , which suggests , but does not prove , a role of visceral fat for hepatic energy metabolism . hepatocellular fatp further related to short- and long - term glycemia , as measured by fasting plasma glucose and a1c , so that defective hepatic energy metabolism could also reflect deterioration of glucose metabolism in the progression of type 2 diabetes . abnormalities in fatp have been found in skeletal muscle not only of patients with overt type 2 diabetes ( 7,20 ) but also of their nondiabetic first - degree relatives , who are at increased risk for type 2 diabetes ( 8) . impairment of atp synthesis in skeletal muscle has been further related to insulin resistance , myocellular lipid deposition , and genetic susceptibility to the onset of type 2 diabetes ( 21 ) . to our knowledge , however , no data on a similar association between lipid contents , insulin sensitivity , and atp production , which also includes atp generation from glycolytic flux , have been reported in the livers of patients with type 2 diabetes . although our results suggest that abnormal phosphorus metabolism relates to hepatic insulin resistance and hcl , we can not rule out that reduced atp turnover is rather a consequence of reduced energy demand or altered energy homeostasis that may relate to increased lipid availability and hyperglycemia . in this case , impaired fatp would result from chronic hyperglycemia and/or insulin resistance due to glucose toxicity . of note , there is evidence that insulin stimulates uptake of pi by way of a sodium - dependent transporter , at least in cultured rat hepatocytes , so that desensitization of this system could lead to lower hepatocellular pi levels ( 22 ) . our mrs protocol is time - consuming , which also explains the dropout rate in this study . alternatively , the tight relationship between hepatocellular fatp and pi , which is a direct consequence of a comparable rate constant , k , suggests that quantification of the absolute pi concentration could be sufficient for assessing hepatic energy metabolism . altered tissue morphology could affect the mr relaxation rates , which in turn could affect the absolute quantification of metabolites and fatp . this simplification should , therefore , only be considered when there is strong evidence against alterations in mr relaxation times . the tight correlation between atp and pi ( 11 ) renders errors due to altered mr relaxation times highly unlikely . participants in the current study did not show clinical signs of cholestasis or impaired liver function , as expressed by normal results on liver enzyme tests and function parameters , although we can not be completely certain because we had no histologic confirmation . thus , absolute quantification of hepatic p metabolites ( 11,12 ) , together with k from this study , appears to be a clinically applicable measure of hepatic energy metabolism . even more , it appears that waist circumference could also serve as a viable predictor of hepatic atp production . we studied elderly volunteers who had a mean age of ~60 years , which might result in lower fatp analogous to data on myocellular fatp ( 8) . the ages of diabetic and nondiabetic participants in this study were similar , however , and fatp in these elderly controls was almost identical to that previously reported for young , lean , healthy volunteers ( 12 ) . finally , the saturation transfer experiment provides a measure of total atp turnover , which includes cycling at the levels of glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase of the glycolytic and gluconeogenic pathways ( 23 ) . because fasting egp was comparable between both groups , despite different degrees of glycemia , finally , some spectra of the saturation transfer experiment contained residual phosphocreatine resulting from a minor compromise between optimizing localization and signal - to - noise ratio . of note , the phosphocreatine is dominating skeletal muscle spectra , implying the contamination in pi , which determines fatp , is lower by an order of magnitude . 1a ) ; this could , besides off - resonance effects , indicate chemical exchange such as atp used in phosphodiesterase formation ( 24 ) . in conclusion , patients with type 2 diabetes without clinical liver disease exhibit lower atp production than nondiabetic otherwise comparable individuals . the reduction in flux through atp synthesis depends primarily on the lower hepatic pi and atp concentrations . insulin resistance relates to the perturbed hepatic energy metabolism , which can be at least be partly explained by fat mass as assessed from waist circumference .
objectivesteatosis associates with insulin resistance and may even predict type 2 diabetes and cardiovascular complications . because muscular insulin resistance relates to myocellular fat deposition and disturbed energy metabolism , we hypothesized that reduced hepatic atp turnover ( fatp ) underlies insulin resistance and elevated hepatocellular lipid ( hcl ) contents.research design and methodswe measured hepatic fatp using 31p magnetic resonance spectroscopy in patients with type 2 diabetes and age- and body mass  matched controls . 
 peripheral ( m and m / i ) and hepatic ( suppression of endogenous glucose production ) insulin sensitivity were assessed with euglycemic - hyperinsulinemic clamps.resultsdiabetic individuals had 29% and 28% lower peripheral and hepatic insulin sensitivity as well as 42% reduced fatp than controls . after adjusting for hcl , fatp correlated positively with peripheral and hepatic insulin sensitivity but negatively with waist circumference , bmi , and fasting plasma glucose . 
 multiple regression analysis identified waist circumference as an independent predictor of fatp and inorganic phosphate ( pi ) concentrations , explaining 65% ( p = 0.001 ) and 56% ( p = 0.003 ) of the variations . 
 hepatocellular pi primarily determined the alterations in fatp.conclusionsin patients with type 2 diabetes , insulin resistance relates to perturbed hepatic energy metabolism , which is at least partly accounted for by fat depots .