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1.  Reduced cortical thickness associated with visceral fat and BMI 
NeuroImage : Clinical  2014;6:307-311.
Structural brain imaging studies have shown that obesity is associated with widespread reductions in gray matter (GM) volume. Although the body mass index (BMI) is an easily accessible anthropometric measure, substantial health problems are more related to specific body fat compartments, like visceral adipose tissue (VAT). We investigated cortical thickness measures in a group of 72 healthy subjects (BMI range 20–35 kg/m2, age range 19–50 years). Multiple regression analyses were performed using VAT and BMI as predictors and age, gender, total surface area and education as confounds. BMI and VAT were independently associated with reductions in cortical thickness in clusters comprising the left lateral occipital area, the left inferior temporal cortex, and the left precentral and inferior parietal area, while the right insula, the left fusiform gyrus and the right inferior temporal area showed a negative correlation with VAT only. In addition, we could show significant reductions in cortical thickness with increasing VAT adjusted for BMI in the left temporal cortex. We were able to detect widespread cortical thinning in a young to middle-aged population related to BMI and VAT; these findings show close resemblance to studies focusing on GM volume differences in diabetic patients. This may point to the influence of VAT related adverse effects, like low-grade inflammation, as a potentially harmful factor on brain integrity already in individuals at risk of developing diabetes, metabolic syndromes and arteriosclerosis.
•We investigated cortical thickness in healthy adults.•Body mass index (BMI) and visceral adipose tissue (VAT) were used as predictors.•BMI and VAT were independently associated with cortical thickness.•Cortical thinning was observed in the temporal cortex with increasing VAT.•Our findings show close resemblance to GM volume differences in diabetic patients.
PMCID: PMC4215386  PMID: 25379443
Obesity; Cortical thickness; MR imaging; Visceral adipose tissue
2.  Circulating Lysophosphatidylcholines Are Markers of a Metabolically Benign Nonalcoholic Fatty Liver 
Diabetes Care  2013;36(8):2331-2338.
Nonalcoholic fatty liver (NAFL) is thought to contribute to insulin resistance and its metabolic complications. However, some individuals with NAFL remain insulin sensitive. Mechanisms involved in the susceptibility to develop insulin resistance in humans with NAFL are largely unknown. We investigated circulating markers and mechanisms of a metabolically benign and malignant NAFL by applying a metabolomic approach.
A total of 265 metabolites were analyzed before and after a 9-month lifestyle intervention in plasma from 20 insulin-sensitive and 20 insulin-resistant subjects with NAFL. The relevant plasma metabolites were then tested for relationships with insulin sensitivity in 17 subjects without NAFL and in plasma from 29 subjects with liver tissue samples.
The best separation of the insulin-sensitive from the insulin-resistant NAFL group was achieved by a metabolite pattern including the branched-chain amino acids leucine and isoleucine, ornithine, the acylcarnitines C3:0-, C16:0-, and C18:0-carnitine, and lysophosphatidylcholine (lyso-PC) C16:0 (area under the ROC curve, 0.77 [P = 0.00023] at baseline and 0.80 [P = 0.000019] at follow-up). Among the individual metabolites, predominantly higher levels of lyso-PC C16:0, both at baseline (P = 0.0039) and at follow-up (P = 0.001), were found in the insulin-sensitive compared with the insulin-resistant subjects. In the non-NAFL groups, no differences in lyso-PC C16:0 levels were found between the insulin-sensitive and insulin-resistant subjects, and these relationships were replicated in plasma from subjects with liver tissue samples.
From a plasma metabolomic pattern, particularly lyso-PCs are able to separate metabolically benign from malignant NAFL in humans and may highlight important pathways in the pathogenesis of fatty liver–induced insulin resistance.
PMCID: PMC3714475  PMID: 23514731
3.  Common Genetic Variation in the Human CTF1 Locus, Encoding Cardiotrophin-1, Determines Insulin Sensitivity 
PLoS ONE  2014;9(7):e100391.
Recently, cardiotrophin-1, a member of the interleukin-6 family of cytokines was described to protect beta-cells from apoptosis, to improve glucose-stimulated insulin secretion and insulin resistance, and to prevent streptozotocin-induced diabetes in mice. Here, we studied whether common single nucleotide polymorphisms (SNPs) in the CTF1 locus, encoding cardiotrophin-1, influence insulin secretion and insulin sensitivity in humans.
We genotyped 1,771 German subjects for three CTF1 tagging SNPs (rs1046276, rs1458201, and rs8046707). The subjects were metabolically characterized by an oral glucose tolerance test. Subgroups underwent magnetic resonance (MR) imaging/spectroscopy and hyperinsulinaemic-euglycaemic clamps.
After appropriate adjustment, the minor allele of CTF1 SNP rs8046707 was significantly associated with decreased in vivo measures of insulin sensitivity. The other tested SNPs were not associated with OGTT-derived sensitivity parameters, nor did the three tested SNPs show any association with OGTT-derived parameters of insulin release. In the MR subgroup, SNP rs8046707 was nominally associated with lower visceral adipose tissue. Furthermore, the SNP rs1458201 showed a nominal association with increased VLDL levels.
In conclusion, this study, even though preliminary and awaiting further confirmation by independent replication, provides first evidence that common genetic variation in CTF1 could contribute to insulin sensitivity in humans. Our SNP data indicate an insulin-desensitizing effect of cardiotrophin-1 and underline that cardiotrophin-1 represents an interesting target to influence insulin sensitivity.
PMCID: PMC4099130  PMID: 25025664
4.  Impact of the Adipokine Adiponectin and the Hepatokine Fetuin-A on the Development of Type 2 Diabetes: Prospective Cohort- and Cross-Sectional Phenotyping Studies 
PLoS ONE  2014;9(3):e92238.
Among adipokines and hepatokines, adiponectin and fetuin-A were consistently found to predict the incidence of type 2 diabetes, both by regulating insulin sensitivity.
To determine to what extent circulating adiponectin and fetuin-A are independently associated with incident type 2 diabetes in humans, and the major mechanisms involved.
Relationships with incident diabetes were tested in two cohort studies: within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study (628 cases) and the Nurses' Health Study (NHS; 470 cases). Relationships with body fat compartments, insulin sensitivity and insulin secretion were studied in the Tübingen Lifestyle Intervention Program (TULIP; N = 358).
Circulating adiponectin and fetuin-A, independently of several confounders and of each other, associated with risk of diabetes in EPIC-Potsdam (RR for 1 SD: adiponectin: 0.45 [95% CI 0.37–0.54], fetuin-A: 1.18 [1.05–1.32]) and the NHS (0.51 [0.42–0.62], 1.35 [1.16–1.58]). Obesity measures considerably attenuated the association of adiponectin, but not of fetuin-A. Subjects with low adiponectin and concomitantly high fetuin-A had the highest risk. Whereas both proteins were independently (both p<1.8×10−7) associated with insulin sensitivity, circulating fetuin-A (r = −0.37, p = 0.0004), but not adiponectin, associated with insulin secretion in subjects with impaired glucose tolerance.
We provide novel information that adiponectin and fetuin-A independently of each other associate with the diabetes risk. Furthermore, we suggest that they are involved in the development of type 2 diabetes via different mechanisms, possibly by mediating effects of their source tissues, expanded adipose tissue and nonalcoholic fatty liver.
PMCID: PMC3958485  PMID: 24643166
5.  Cinnamon Extract Improves Insulin Sensitivity in the Brain and Lowers Liver Fat in Mouse Models of Obesity 
PLoS ONE  2014;9(3):e92358.
Treatment of diabetic subjects with cinnamon demonstrated an improvement in blood glucose concentrations and insulin sensitivity but the underlying mechanisms remained unclear. This work intends to elucidate the impact of cinnamon effects on the brain by using isolated astrocytes, and an obese and diabetic mouse model.
Cinnamon components (eugenol, cinnamaldehyde) were added to astrocytes and liver cells to measure insulin signaling and glycogen synthesis. Ob/ob mice were supplemented with extract from cinnamomum zeylanicum for 6 weeks and cortical brain activity, locomotion and energy expenditure were evaluated. Insulin action was determined in brain and liver tissues.
Treatment of primary astrocytes with eugenol promoted glycogen synthesis, whereas the effect of cinnamaldehyde was attenuated. In terms of brain function in vivo, cinnamon extract improved insulin sensitivity and brain activity in ob/ob mice, and the insulin-stimulated locomotor activity was improved. In addition, fasting blood glucose levels and glucose tolerance were greatly improved in ob/ob mice due to cinnamon extracts, while insulin secretion was unaltered. This corresponded with lower triglyceride and increased liver glycogen content and improved insulin action in liver tissues. In vitro, Fao cells exposed to cinnamon exhibited no change in insulin action.
Together, cinnamon extract improved insulin action in the brain as well as brain activity and locomotion. This specific effect may represent an important central feature of cinnamon in improving insulin action in the brain, and mediates metabolic alterations in the periphery to decrease liver fat and improve glucose homeostasis.
PMCID: PMC3958529  PMID: 24643026
6.  Monounsaturated Fatty Acids Prevent the Aversive Effects of Obesity on Locomotion, Brain Activity, and Sleep Behavior 
Diabetes  2012;61(7):1669-1679.
Fat and physical inactivity are the most evident factors in the pathogenesis of obesity, and fat quality seems to play a crucial role for measures of glucose homeostasis. However, the impact of dietary fat quality on brain function, behavior, and sleep is basically unknown. In this study, mice were fed a diet supplemented with either monounsaturated fatty acids (MUFAs) or saturated fatty acids (SFAs) and their impact on glucose homeostasis, locomotion, brain activity, and sleep behavior was evaluated. MUFAs and SFAs led to a significant increase in fat mass but only feeding of SFAs was accompanied by glucose intolerance in mice. Radiotelemetry revealed a significant decrease in cortical activity in SFA-mice whereas MUFAs even improved activity. SFAs decreased wakefulness and increased non–rapid eye movement sleep. An intracerebroventricular application of insulin promoted locomotor activity in MUFA-fed mice, whereas SFA-mice were resistant. In humans, SFA-enriched diet led to a decrease in hippocampal and cortical activity determined by functional magnetic resonance imaging techniques. Together, dietary intake of MUFAs promoted insulin action in the brain with its beneficial effects for cortical activity, locomotion, and sleep, whereas a comparable intake of SFAs acted as a negative modulator of brain activity in mice and humans.
PMCID: PMC3379681  PMID: 22492529
7.  Characteristics, changes and influence of body composition during a 4486 km transcontinental ultramarathon: results from the Transeurope Footrace mobile whole body MRI-project 
BMC Medicine  2013;11:122.
Almost nothing is known about the medical aspects of runners doing a transcontinental ultramarathon over several weeks. The results of differentiated measurements of changes in body composition during the Transeurope Footrace 2009 using a mobile whole body magnetic resonance (MR) imager are presented and the proposed influence of visceral and somatic adipose and lean tissue distribution on performance tested.
22 participants were randomly selected for the repeated MR measurements (intervals: 800 km) with a 1.5 Tesla MR scanner mounted on a mobile unit during the 64-stage 4,486 km ultramarathon. A standardized and validated MRI protocol was used: T1 weighted turbo spin echo sequence, echo time 12 ms, repetition time 490 ms, slice thickness 10 mm, slice distance 10 mm (breath holding examinations). For topographic tissue segmentation and mapping a modified fuzzy c-means algorithm was used. A semi-automatic post-processing of whole body MRI data sets allows reliable analysis of the following body tissue compartments: Total body volume (TV), total somatic (TSV) and total visceral volume (TVV), total adipose (TAT) and total lean tissue (TLT), somatic (SLT) and visceral lean tissue (VLT), somatic (SAT) and visceral adipose tissue (VAT) and somatic adipose soft tissue (SAST). Specific volume changes were tested on significance. Tests on difference and relationship regarding prerace and race performance and non-finishing were done using statistical software SPSS.
Total, somatic and visceral volumes showed a significant decrease throughout the race. Adipose tissue showed a significant decrease compared to the start at all measurement times for TAT, SAST and VAT. Lean adipose tissues decreased until the end of the race, but not significantly. The mean relative volume changes of the different tissue compartments at the last measurement compared to the start were: TV −9.5% (SE 1.5%), TSV −9.4% (SE 1.5%), TVV −10.0% (SE 1.4%), TAT −41.3% (SE 2.3%), SAST −48.7% (SE 2.8%), VAT −64.5% (SE 4.6%), intraabdominal adipose tissue (IAAT) −67.3% (SE 4.3%), mediastinal adopose tissue (MAT) −41.5% (SE 7.1%), TLT −1.2% (SE 1.0%), SLT −1.4% (SE 1.1%). Before the start and during the early phase of the Transeurope Footrace 2009, the non-finisher group had a significantly higher percentage volume of TVV, TAT, SAST and VAT compared to the finisher group. VAT correlates significantly with prerace training volume and intensity one year before the race and with 50 km- and 24 hour-race records. Neither prerace body composition nor specific tissue compartment volume changes showed a significant relationship to performance in the last two thirds of the Transeurope Footrace 2009.
With this mobile MRI field study the complex changes in body composition during a multistage ultramarathon could be demonstrated in detail in a new and differentiated way. Participants lost more than half of their adipose tissue. Even lean tissue volume (mainly skeletal muscle tissue) decreased due to the unpreventable chronic negative energy balance during the race. VAT has the fastest and highest decrease compared to SAST and lean tissue compartments during the race. It seems to be the most sensitive morphometric parameter regarding the risk of non-finishing a transcontinental footrace and shows a direct relationship to prerace-performance. However, body volume or body mass and, therefore, fat volume has no correlation with total race performances of ultra-athletes finishing a 4,500 km multistage race.
PMCID: PMC3668188  PMID: 23657091
Magnetic resonance imaging; MRI; Body mass; Body volume; Body composition; Running; Marathon; Ultramarathon; Performance; Adipose tissue; Body fat; Lean tissue; Visceral; Somatic; Topography; Segmentation; Mapping
8.  The Transeurope Footrace Project: longitudinal data acquisition in a cluster randomized mobile MRI observational cohort study on 44 endurance runners at a 64-stage 4,486km transcontinental ultramarathon 
BMC Medicine  2012;10:78.
The TransEurope FootRace 2009 (TEFR09) was one of the longest transcontinental ultramarathons with an extreme endurance physical load of running nearly 4,500 km in 64 days. The aim of this study was to assess the wide spectrum of adaptive responses in humans regarding the different tissues, organs and functional systems being exposed to such chronic physical endurance load with limited time for regeneration and resulting negative energy balance. A detailed description of the TEFR project and its implemented measuring methods in relation to the hypotheses are presented.
The most important research tool was a 1.5 Tesla magnetic resonance imaging (MRI) scanner mounted on a mobile unit following the ultra runners from stage to stage each day. Forty-four study volunteers (67% of the participants) were cluster randomized into two groups for MRI measurements (22 subjects each) according to the project protocol with its different research modules: musculoskeletal system, brain and pain perception, cardiovascular system, body composition, and oxidative stress and inflammation. Complementary to the diverse daily mobile MR-measurements on different topics (muscle and joint MRI, T2*-mapping of cartilage, MR-spectroscopy of muscles, functional MRI of the brain, cardiac and vascular cine MRI, whole body MRI) other methods were also used: ice-water pain test, psychometric questionnaires, bioelectrical impedance analysis (BIA), skinfold thickness and limb circumference measurements, daily urine samples, periodic blood samples and electrocardiograms (ECG).
Thirty volunteers (68%) reached the finish line at North Cape. The mean total race speed was 8.35 km/hour. Finishers invested 552 hours in total. The completion rate for planned MRI investigations was more than 95%: 741 MR-examinations with 2,637 MRI sequences (more than 200,000 picture data), 5,720 urine samples, 244 blood samples, 205 ECG, 1,018 BIA, 539 anthropological measurements and 150 psychological questionnaires.
This study demonstrates the feasibility of conducting a trial based centrally on mobile MR-measurements which were performed during ten weeks while crossing an entire continent. This article is the reference for contemporary result reports on the different scientific topics of the TEFR project, which may reveal additional new knowledge on the physiological and pathological processes of the functional systems on the organ, cellular and sub-cellular level at the limits of stress and strain of the human body.
Please see related articles: and
PMCID: PMC3409063  PMID: 22812450
9.  Quantifying the Improvement of Surrogate Indices of Hepatic Insulin Resistance Using Complex Measurement Techniques 
PLoS ONE  2012;7(6):e39029.
We evaluated the ability of simple and complex surrogate-indices to identify individuals from an overweight/obese cohort with hepatic insulin-resistance (HEP-IR). Five indices, one previously defined and four newly generated through step-wise linear regression, were created against a single-cohort sample of 77 extensively characterised participants with the metabolic syndrome (age 55.6±1.0 years, BMI 31.5±0.4 kg/m2; 30 males). HEP-IR was defined by measuring endogenous-glucose-production (EGP) with [6–62H2] glucose during fasting and euglycemic-hyperinsulinemic clamps and expressed as EGP*fasting plasma insulin. Complex measures were incorporated into the model, including various non-standard biomarkers and the measurement of body-fat distribution and liver-fat, to further improve the predictive capability of the index. Validation was performed against a data set of the same subjects after an isoenergetic dietary intervention (4 arms, diets varying in protein and fiber content versus control). All five indices produced comparable prediction of HEP-IR, explaining 39–56% of the variance, depending on regression variable combination. The validation of the regression equations showed little variation between the different proposed indices (r2 = 27–32%) on a matched dataset. New complex indices encompassing advanced measurement techniques offered an improved correlation (r = 0.75, P<0.001). However, when validated against the alternative dataset all indices performed comparably with the standard homeostasis model assessment for insulin resistance (HOMA-IR) (r = 0.54, P<0.001). Thus, simple estimates of HEP-IR performed comparable to more complex indices and could be an efficient and cost effective approach in large epidemiological investigations.
PMCID: PMC3382235  PMID: 22761721
10.  Genetic Ablation of cGMP-Dependent Protein Kinase Type I Causes Liver Inflammation and Fasting Hyperglycemia 
Diabetes  2011;60(5):1566-1576.
The nitric oxide/cGMP/cGMP-dependent protein kinase type I (cGKI) signaling pathway regulates cell functions that play a pivotal role in the pathogenesis of type 2 diabetes. However, the impact of a dysfunction of this pathway for glucose metabolism in vivo is unknown.
The expression of cGKI in tissues relevant to insulin action was analyzed by immunohistochemistry. The metabolic consequences of a genetic deletion of cGKI were studied in mice that express cGKI selectively in smooth muscle but not in other cell types (cGKI-SM mice).
In wild-type mice, cGKI protein was detected in hepatic stellate cells, but not in hepatocytes, skeletal muscle, fat cells, or pancreatic β-cells. Compared with control animals, cGKI-SM mice had higher energy expenditure in the light phase associated with lower body weight and fat mass and increased insulin sensitivity. Mutant mice also showed higher fasting glucose levels, whereas insulin levels and intraperitoneal glucose tolerance test results were similar to those in control animals. Interleukin (IL)-6 signaling was strongly activated in the liver of cGKI-SM mice as demonstrated by increased levels of IL-6, phospho-signal transducer and activator of transcription 3 (Tyr 705), suppressor of cytokine signaling-3, and serum amyloid A2. Insulin-stimulated tyrosine phosphorylation of the insulin receptor in the liver was impaired in cGKI-SM mice. The fraction of Mac-2–positive macrophages in the liver was significantly higher in cGKI-SM mice than in control mice. In contrast with cGKI-SM mice, conditional knockout mice lacking cGKI only in the nervous system were normal with respect to body weight, energy expenditure, fasting glucose, IL-6, and insulin action in the liver.
Genetic deletion of cGKI in non-neuronal cells results in a complex metabolic phenotype, including liver inflammation and fasting hyperglycemia. Loss of cGKI in hepatic stellate cells may affect liver metabolism via a paracrine mechanism that involves enhanced macrophage infiltration and IL-6 signaling.
PMCID: PMC3292332  PMID: 21464444
11.  Common Genetic Variation in the SERPINF1 Locus Determines Overall Adiposity, Obesity-Related Insulin Resistance, and Circulating Leptin Levels 
PLoS ONE  2012;7(3):e34035.
Pigment epithelium-derived factor (PEDF) belongs to the serpin family of peptidase inhibitors (serpin F1) and is among the most abundant glycoproteins secreted by adipocytes. In vitro and mouse in vivo data revealed PEDF as a candidate mediator of obesity-induced insulin resistance. Therefore, we assessed whether common genetic variation within the SERPINF1 locus contributes to adipose tissue-related prediabetic phenotypes in humans.
A population of 1,974 White European individuals at increased risk for type 2 diabetes was characterized by an oral glucose tolerance test with glucose and insulin measurements (1,409 leptin measurements) and genotyped for five tagging SNPs covering 100% of common genetic variation (minor allele frequency ≥0.05) in the SERPINF1 locus. In addition, a subgroup of 486 subjects underwent a hyperinsulinaemic-euglycaemic clamp and a subgroup of 340 magnetic resonance imaging (MRI) and spectroscopy (MRS).
After adjustment for gender and age and Bonferroni correction for the number of SNPs tested, SNP rs12603825 revealed significant association with MRI-derived total adipose tissue mass (p = 0.0094) and fasting leptin concentrations (p = 0.0035) as well as nominal associations with bioelectrical impedance-derived percentage of body fat (p = 0.0182) and clamp-derived insulin sensitivity (p = 0.0251). The association with insulin sensitivity was completely abolished by additional adjustment for body fat (p = 0.8). Moreover, the fat mass-increasing allele of SNP rs12603825 was significantly associated with elevated fasting PEDF concentrations (p = 0.0436), and the PEDF levels were robustly and positively associated with all body fat parameters measured and with fasting leptin concentrations (p<0.0001, all).
In humans at increased risk for type 2 diabetes, a functional common genetic variant in the gene locus encoding PEDF contributes to overall body adiposity, obesity-related insulin resistance, and circulating leptin levels.
PMCID: PMC3311576  PMID: 22457810
12.  Relationships of Circulating Sex Hormone–Binding Globulin With Metabolic Traits in Humans 
Diabetes  2010;59(12):3167-3173.
Recent data suggested that sex hormone–binding globulin (SHBG) levels decrease when fat accumulates in the liver and that circulating SHBG may be causally involved in the pathogenesis of type 2 diabetes in humans. In the present study, we investigated mechanisms by which high SHBG may prevent development to diabetes.
Before and during a 9-month lifestyle intervention, total body and visceral fat were precisely measured by magnetic resonance (MR) tomography and liver fat was measured by 1H-MR spectroscopy in 225 subjects. Insulin sensitivity was estimated from a 75-g oral glucose tolerance test (ISOGTT) and measured by a euglycemic hyperinsulinemic clamp (ISclamp, n = 172). Insulin secretion was measured during the OGTT and an ivGTT (n = 172).
SHBG levels correlated positively with insulin sensitivity (ISOGTT, P = 0.037; ISclamp, P = 0.057), independently of age, sex, and total body fat. In a multivariate model, these relationships were also significant after additional adjustment for levels of the adipokine adiponectin and the hepatokine fetuin-A (ISOGTT, P = 0.0096; ISclamp, P = 0.029). Adjustment of circulating SHBG for liver fat abolished the relationships of SHBG with insulin sensitivity. In contrast, circulating SHBG correlated negatively with fasting glycemia, before (r = −0.17, P = 0.009) and after (r = −0.14, P = 0.04) adjustment for liver fat. No correlation of circulating SHBG with adjusted insulin secretion was observed (OGTT, P = 0.16; ivGTT, P = 0.35). The SNP rs1799941 in SHBG was associated with circulating SHBG (P ≤ 0.025) but not with metabolic characteristics (all P > 0.18).
Possible mechanisms by which high circulating SHBG prevents the development of type 2 diabetes involve regulation of fasting glycemia but not alteration of insulin secretory function.
PMCID: PMC2992779  PMID: 20841609
13.  Gene Variants of TCF7L2 Influence Weight Loss and Body Composition During Lifestyle Intervention in a Population at Risk for Type 2 Diabetes 
Diabetes  2009;59(3):747-750.
The impact of the diabetes risk gene transcription factor 7-like 2 (TCF7L2) on body weight is unclear. As TCF7L2 is expressed in adipose tissue and involved in Wnt-dependent regulation of adipogenesis, we studied the impact of TCF7L2 variants on body composition and weight loss during lifestyle intervention.
We genotyped 309 German subjects at increased risk for type 2 diabetes for single nucleotide polymorphisms (SNPs) rs7903146, rs12255372, rs11196205, and rs7895340 in TCF7L2 and performed oral glucose tolerance tests before and after a 9-month lifestyle intervention. Fat distribution was quantified using whole-body magnetic resonance imaging/spectroscopy in a subgroup of 210 subjects.
After adjustment for confounding variables, we observed a negative impact of the type 2 diabetes allele of SNP rs7903146 on change in BMI (P = 0.0034) and on changes in nonvisceral (P = 0.0032) and visceral fat (P = 0.0165) during lifestyle intervention. An association of rs7903146 with lifestyle intervention-induced changes in insulin secretion, glucose concentrations, liver fat, or insulin sensitivity were not detected (all P > 0.2). Essentially the same results were obtained with SNP rs1255372. In contrast, we found no effects of SNPs rs11196205 and rs7895340 on change in BMI (all P ≥ 0.5).
Our data reveal that diabetes-associated alleles of TCF7L2 are associated with less weight loss in response to lifestyle intervention. Thus, diabetes-associated TCF7L2 gene variation predicts the success of lifestyle intervention in terms of weight loss and determines individual susceptibility toward environmental factors.
PMCID: PMC2828665  PMID: 20028944
14.  Circulating Palmitoleate Strongly and Independently Predicts Insulin Sensitivity in Humans 
Diabetes Care  2009;33(2):405-407.
We investigated whether palmitoleate, which prevents insulin resistance in mice, predicts insulin sensitivity in humans.
The fasting fatty acid pattern in the plasma free fatty acid (FFA) fraction was determined in 100 subjects at increased risk for type 2 diabetes. Insulin sensitivity was estimated during an oral glucose tolerance test (OGTT) at baseline and after 9 months of lifestyle intervention and measured during the euglycemic-hyperinsulinemic clamp (n = 79).
Circulating palmitoleate (OGTT:F ratio = 8.2, P = 0.005; clamp:F ratio = 7.8, P = 0.007) but not total FFAs (OGTT:F ratio = 0.6, P = 0.42; clamp:F ratio = 0.7, P = 0.40) correlated positively with insulin sensitivity, independently of age, sex, and adiposity. High baseline palmitoleate predicted a larger increase in insulin sensitivity. For 1-SD increase in palmitoleate, the odds ratio for being in the highest versus the lowest tertile of adjusted change in insulin sensitivity was 2.35 (95% CI 1.16–5.35).
Circulating palmitoleate strongly and independently predicts insulin sensitivity, suggesting that it plays an important role in the pathophysiology of insulin resistance in humans.
PMCID: PMC2809292  PMID: 19889804
15.  The D299G/T399I Toll-Like Receptor 4 Variant Associates with Body and Liver Fat: Results from the TULIP and METSIM Studies 
PLoS ONE  2010;5(11):e13980.
Toll-like-receptor 4 (TLR) is discussed to provide a molecular link between obesity, inflammation and insulin resistance. Genetic studies with replications in non-diabetic individuals in regard to their fat distribution or insulin resistance according to their carrier status of a common toll-like receptor 4 (TLR4) variant (TLR4D299G/T399I) are still lacking.
Methodology/Principal Findings
We performed a cross-sectional analysis in individuals phenotyped for prediabetic traits as body fat composition (including magnetic resonance imaging), blood glucose levels and insulin resistance (oral glucose tolerance testing, euglycemic hyperinsulinemic clamp), according to TLR4 genotype determined by candidate SNP analyses (rs4986790). We analyzed N = 1482 non-diabetic individuals from the TÜF/TULIP cohort (South Germany, aged 39±13 y, BMI 28.5±7.9, mean±SD) and N = 5327 non-diabetic participants of the METSIM study (Finland, males aged 58±6 y, BMI 26.8±3.8) for replication purposes. German TLR4D299G/T399I carriers had a significantly increased body fat (XG in rs4986790: +6.98%, p = 0.03, dominant model, adjusted for age, gender) and decreased insulin sensitivity (XG: −15.3%, Matsuda model, p = 0.04; XG: −20.6%, p = 0.016, clamp; both dominant models adjusted for age, gender, body fat). In addition, both liver fat (AG: +49.7%; p = 0.002) and visceral adipose tissue (AG: +8.2%; p = 0.047, both adjusted for age, gender, body fat) were significantly increased in rs4986790 minor allele carriers, and the effect on liver fat remained significant also after additional adjustment for visceral fat (p = 0.014). The analysis in METSIM confirmed increased body fat content in association with the rare G allele in rs4986790 (AG: +1.26%, GG: +11.0%; p = 0.010, additive model, adjusted for age) and showed a non-significant trend towards decreased insulin sensitivity (AG: −0.99%, GG: −10.62%).
TLR4D299G/T399I associates with increased total body fat, visceral fat, liver fat and decreased insulin sensitivity in non-diabetic Caucasians and may contribute to diabetes risk. This finding supports the role of TLR4 as a molecular link between obesity and insulin resistance.
PMCID: PMC2981584  PMID: 21125016
16.  Dissociation Between Fatty Liver and Insulin Resistance in Humans Carrying a Variant of the Patatin-Like Phospholipase 3 Gene 
Diabetes  2009;58(11):2616-2623.
In a genome-wide association scan, the rs738409 C>G single nucleotide polymorphism (SNP) in the patatin-like phospholipase 3 gene (PNPLA3) was strongly associated with increased liver fat but not with insulin resistance estimated from fasting values. We investigated whether the SNP determines liver fat independently of visceral adiposity and whether it may even play a role in protecting from insulin resistance.
Liver fat was measured by 1H magnetic resonance spectroscopy and total and visceral fat by magnetic resonance tomography in 330 subjects. Insulin sensitivity was estimated during an oral glucose tolerance test and the euglycemic-hyperinsulinemic clamp (n = 222). PNPLA3 and tumor necrosis factor-α mRNA and triglyceride content were measured in liver biopsies from 16 subjects.
Liver fat correlated strongly with insulin sensitivity (P < 0.0001) independently of age, sex, total fat, and visceral fat. G allele carriers of the SNP rs738409 had higher liver fat (P < 0.0001) and an odds ratio of 2.38 (95% CI 1.37–4.20) for having fatty liver compared to C allele homozygotes. Interestingly, insulin sensitivity (oral glucose tolerance test: P = 0.99; clamp: P = 0.32), serum C-reactive protein levels, lipids, or liver enzymes (all P > 0.14) were not different among the genotypes. Additional adjustment for liver fat actually revealed increased insulin sensitivity in more obese carriers of the G allele (P = 0.01). In liver biopsies triglyceride content correlated positively with expression of the proinflammatory gene tumor necrosis factor-α in C allele homozygotes (n = 6, P = 0.027) but not in G allele carriers (n = 10, P = 0.149).
PNPLA3 may be an important key to understand the mechanisms discriminating fatty liver with and without metabolic consequences.
PMCID: PMC2768178  PMID: 19651814
17.  Individual Stearoyl-CoA Desaturase 1 Expression Modulates Endoplasmic Reticulum Stress and Inflammation in Human Myotubes and Is Associated With Skeletal Muscle Lipid Storage and Insulin Sensitivity In Vivo 
Diabetes  2009;58(8):1757-1765.
Increased plasma levels of free fatty acids occur in obesity and type 2 diabetes and contribute to the development of insulin resistance. Saturated fatty acids (SFAs) such as palmitate especially have lipotoxic effects leading to endoplasmatic reticulum (ER) stress, inflammation, and insulin resistance. Stearoyl-CoA desaturase 1 (SCD1) plays a key role in preventing lipotoxic effects, as it converts SFAs to less harmful monounsaturated fatty acids. Here, we tested the hypothesis that individual differences in the regulation of SCD1 expression by palmitate exist and influence insulin sensitivity and the cellular response to palmitate.
Palmitate-induced gene expression was studied in primary human myotubes of 39 metabolically characterized individuals, as well as in an SCD1-overexpressing cell culture model.
SCD1 mRNA expression and inducibility by palmitate in cultured myotubes showed a broad interindividual variation, presumably due to inheritable characteristics of the donors. Overexpression of SCD1 prevented the inflammatory and ER stress response to palmitate exposure. In primary human myotubes, high SCD1 inducibility was associated with a low inflammatory (interleukin [IL]-6, IL-8, and chemokine [CXC motif] ligand 3 [CXCL3]) and ER stress (CCAAT/enhancer binding protein [C/EBP] homologous protein, activating transcription factor 3 [ATF3], and X-box binding protein 1 [XBP1]) response to palmitate exposure. Finally, palmitate-stimulated SCD1 mRNA expression, positively correlated with intramyocellular lipid (IMCL) content of the donors, was measured by 1H-magnetic resonance spectroscopy. After adjustment for IMCL, SCD1 expression and inducibility were positively correlated with insulin sensitivity.
We hypothesize that myocellular SCD1 inducibility by palmitate is an individual characteristic that modulates lipid storage, palmitate-induced inflammation, ER stress, and insulin resistance. This may describe individuals with increased capability of innoxious free fatty acid handling and benign triglyceride storage.
PMCID: PMC2712792  PMID: 19478146
18.  Medium Chain Acylcarnitines Dominate the Metabolite Pattern in Humans under Moderate Intensity Exercise and Support Lipid Oxidation 
PLoS ONE  2010;5(7):e11519.
Exercise is an extreme physiological challenge for skeletal muscle energy metabolism and has notable health benefits. We aimed to identify and characterize metabolites, which are components of the regulatory network mediating the beneficial metabolic adaptation to exercise.
Methodology and Principal Findings
First, we investigated plasma from healthy human subjects who completed two independent running studies under moderate, predominantly aerobic conditions. Samples obtained prior to and immediately after running and then 3 and 24 h into the recovery phase were analyzed by a non-targeted (NT-) metabolomics approach applying liquid chromatography-qTOF-mass spectrometry. Under these conditions medium and long chain acylcarnitines were found to be the most discriminant plasma biomarkers of moderately intense exercise. Immediately after a 60 min (at 93% VIAT) or a 120 min run (at 70% VIAT) a pronounced, transient increase dominated by octanoyl-, decanoyl-, and dodecanoyl-carnitine was observed. The release of acylcarnitines as intermediates of partial β-oxidation was verified in skeletal muscle cell culture experiments by probing 13C-palmitate metabolism. Further investigations in primary human myotubes and mouse muscle tissue revealed that octanoyl-, decanoyl-, and dodecanoyl-carnitine were able to support the oxidation of palmitate, proving more effective than L-carnitine.
Medium chain acylcarnitines were identified and characterized by a functional metabolomics approach as the dominating biomarkers during a moderately intense exercise bout possessing the power to support fat oxidation. This physiological production and efflux of acylcarnitines might exert beneficial biological functions in muscle tissue.
PMCID: PMC2902514  PMID: 20634953
19.  No association between variation in the NR4A1 gene locus and metabolic traits in white subjects at increased risk for type 2 diabetes 
BMC Medical Genetics  2010;11:84.
The nuclear receptor NR4A1 is implicated in metabolic regulation in insulin-sensitive tissues, such as liver, adipose tissue, and skeletal muscle. Functional loss of NR4A1 results in insulin resistance and enhanced intramuscular and hepatic lipid content. Therefore, we investigated in a cohort of white European subjects at increased risk for type 2 diabetes whether genetic variation within the NR4A1 gene locus contributes to prediabetic phenotypes, such as insulin resistance, ectopic fat distribution, or β-cell dysfunction.
We genotyped 1495 subjects (989 women, 506 men) for five single nucleotide polymorphisms (SNPs) tagging 100% of common variants (MAF = 0.05) within the NR4A1 gene locus with an r2 = 0.8. All subjects underwent an oral glucose tolerance test (OGTT), a subset additionally had a hyperinsulinemic-euglycemic clamp (n = 506). Ectopic hepatic (n = 296) and intramyocellular (n = 264) lipids were determined by magnetic resonance spectroscopy. Peak aerobic capacity, a surrogate parameter for oxidative capacity of skeletal muscle, was measured by an incremental exercise test on a motorized treadmill (n = 270).
After appropriate adjustment and Bonferroni correction for multiple comparisons, none of the five SNPs was reliably associated with insulin sensitivity, ectopic fat distribution, peak aerobic capacity, or indices of insulin secretion (all p ≥ 0.05).
Our data suggest that common genetic variation within the NR4A1 gene locus may not play a major role in the development of prediabetic phenotypes in our white European population.
PMCID: PMC2894787  PMID: 20525362
20.  Liver fat content determined by magnetic resonance imaging and spectroscopy 
Hepatic steatosis as the most prevalent liver disorder can either be related to alcoholic liver disease (ALD) or non-alcoholic fatty liver disease (NAFLD). In both conditions, hepatocytes excessively accumulate fat-containing vacuoles within their cytoplasm, which is the key histological feature. In contrast to ALD, NAFLD is commonly associated with metabolic syndrome, obesity and insulin resistance. To determine increased liver fat content, liver biopsy is currently considered the gold standard. Besides the invasive technique, various other non-invasive techniques have been developed, such as ultrasound, computed tomography (CT), magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) based methods. Among these techniques, ultrasound and CT provide only qualitative information about hepatic steatosis, whereas MRS- or MRI-based methods are able to determine even small amounts of fat accurately. These non-invasive magnetic resonance techniques have already proven their great potential, especially in longitudinal and cross-sectional studies regarding various metabolic conditions and medical treatment regimens. In this review, the most common, non-invasive MRS/MRI techniques for assessment of intrahepatic lipid content are described with their inherent advantages and limitations.
PMCID: PMC2848364  PMID: 20355234
Hepatic steatosis; Magnetic resonance imaging; Proton magnetic resonance spectroscopy; Lipids
21.  Muscle-Derived Angiopoietin-Like Protein 4 Is Induced by Fatty Acids via Peroxisome Proliferator–Activated Receptor (PPAR)-δ and Is of Metabolic Relevance in Humans 
Diabetes  2009;58(3):579-589.
OBJECTIVE— Long-chain fatty acids (LCFAs) contribute to metabolic homeostasis in part via gene regulation. This study's objective was to identify novel LCFA target genes in human skeletal muscle cells (myotubes).
RESEARCH DESIGN AND METHODS— In vitro methods included culture and treatment of human myotubes and C2C12 cells, gene array analysis, real-time RT-PCR, Western blotting, ELISA, chromatin immunoprecipitation, and RNA interference. Human subjects (two cohorts) were characterized by oral glucose tolerance test, hyperinsulinemic-euglycemic clamp, magnetic resonance imaging and spectroscopy, and standard blood analyses (glucose, insulin, C-peptide, and plasma lipids).
RESULTS— We show here that ANGPTL4 (encoding angiopoietin-like protein 4) represents a prominent LCFA-responsive gene in human myotubes. LCFA activated peroxisome proliferator-activated receptor (PPAR)-δ, but not PPAR-α or -γ, and pharmacological activation of PPAR-δ markedly induced ANGPTL4 production and secretion. In C2C12 myocytes, knockdown of PPARD, but not of PPARG, blocked LCFA-mediated ANGPTL4 induction, and LCFA treatment resulted in PPAR-δ recruitment to the ANGPTL4 gene. In addition, pharmacological PPAR-δ activation induced LIPE (encoding hormone-sensitive lipase), and this response crucially depended on ANGPTL4, as revealed by ANGPTL4 knockdown. In a human cohort of 108 thoroughly phenotyped subjects, plasma ANGPTL4 positively correlated with fasting nonesterified fatty acids (P = 0.0036) and adipose tissue lipolysis (P = 0.0012). Moreover, in 38 myotube donors, plasma ANGPTL4 levels and adipose tissue lipolysis in vivo were reflected by basal myotube ANGPTL4 expression in vitro (P = 0.02, both).
CONCLUSIONS— ANGPTL4 is produced by human myotubes in response to LCFA via PPAR-δ, and muscle-derived ANGPTL4 seems to be of systemic relevance in humans.
PMCID: PMC2646056  PMID: 19074989
22.  SIRT1 genetic variants associate with the metabolic response of Caucasians to a controlled lifestyle intervention – the TULIP Study 
BMC Medical Genetics  2008;9:100.
Sirtuin1 (SIRT1) regulates gene expression in distinct metabolic pathways and mediates beneficial effects of caloric restriction in animal models. In humans, SIRT1 genetic variants associate with fasting energy expenditure. To investigate the relevance of SIRT1 for human metabolism and caloric restriction, we analyzed SIRT1 genetic variants in respect to the outcome of a controlled lifestyle intervention in Caucasians at risk for type 2 diabetes.
A total of 1013 non-diabetic Caucasians from the Tuebingen Family Study (TUEF) were genotyped for four tagging SIRT1 SNPs (rs730821, rs12413112, rs7069102, rs2273773) for cross-sectional association analyses with prediabetic traits. SNPs that associated with basal energy expenditure in the TUEF cohort were additionally analyzed in 196 individuals who underwent a controlled lifestyle intervention (Tuebingen Lifestyle Intervention Program; TULIP). Multivariate regressions analyses with adjustment for relevant covariates were performed to detect associations of SIRT1 variants with the changes in anthropometrics, weight, body fat or metabolic characteristics (blood glucose, insulin sensitivity, insulin secretion and liver fat, measured by magnetic resonance techniques) after the 9-month follow-up test in the TULIP study.
Minor allele (X/A) carriers of rs12413112 (G/A) had a significantly lower basal energy expenditure (p = 0.04) and an increased respiratory quotient (p = 0.02). This group (rs12413112: X/A) was resistant against lifestyle-induced improvement of fasting plasma glucose (GG: -2.01%, X/A: 0.53%; p = 0.04), had less increase in insulin sensitivity (GG: 17.3%, X/A: 9.6%; p = 0.05) and an attenuated decline in liver fat (GG: -38.4%, X/A: -7.5%; p = 0.01).
SIRT1 plays a role for the individual lifestyle intervention response, possibly owing to decreased basal energy expenditure and a lower lipid-oxidation rate in rs12413112 X/A allele carriers. SIRT1 genetic variants may, therefore, represent a relevant determinant for the response rate of individuals undergoing caloric restriction and increased physical activity.
PMCID: PMC2626584  PMID: 19014491
23.  Impact of Different Fat Depots on Insulin Sensitivity: Predominant Role of Liver Fat 
Overall obesity and, as it is increasingly appreciated, body fat distribution and ectopic fat deposition in liver and skeletal muscle, determine insulin resistance in humans. However, little is known about the independence of these relationships. Therefore, we determined the impact of different fat depots as well as fat accumulation in ectopic tissues such as liver and skeletal muscle in the prediction of insulin resistance in healthy humans.
Visceral and subcutaneous abdominal fat were determined by magnetic resonance (MR) tomography and liver fat and intramyocellular fat in the tibialis anterior muscle by 1H-MR spectroscopy in 220 subjects. Insulin sensitivity was estimated from the oral glucose tolerance test (OGTT) and measured by a euglycemic hyperinsulinemic clamp in a subgroup (n = 157).
Insulin sensitivity estimated from the OGTT correlated negatively with total body fat (r = −0.27, p < 0.0001), subcutaneous abdominal fat (r = −0.35, p < 0.0001), and visceral fat (r = −0.43, p < 0.0001). Furthermore, insulin sensitivity correlated negatively with liver fat (r = −0.53, p < 0.0001) and intramyocellular fat (r = −0.26, p < 0.0001). In multivariate regression models, high liver and visceral fat emerged as the strongest predictors of low insulin sensitivity.
Among various fat compartments, high liver fat and high visceral fat are the strongest determinants of insulin sensitivity in humans.
PMCID: PMC2769646  PMID: 19885144
ectopic fat; insulin resistance; intramyocellular fat; liver fat; visceral fat

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