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Intramyocellular and intrahepatic (IHL) lipids are significantly associated with insulin resistance in adults and adolescents and may represent an early marker for developing the metabolic syndrome or type 2 diabetes.
During the pilot phase of a larger cross-sectional study, we used proton magnetic resonance spectroscopy (1H-MRS) to determine the feasibility of noninvasively evaluating IHL in 11 male (n=4) and female (n=7) prepubertal children using a standard clinical system and to determine whether IHL is correlated with adiposity, fasting insulin and glucose, and liver enzymes.
Body mass index (BMI) (range, 13.4–32.4kg/m2) and IHL stores (range, 0.07–3.2% relative to an oil phantom) were variable. IHL was correlated with body mass (r=0.66, P=0.037), BMI (r=0.73, P=0.016), percentage body fat (r=0.73, P=0.01, n=10), waist circumference (r=0.85, P=0.016), and serum lactate dehydrogenase concentration (r=0.77, P=0.03) but was not significantly correlated with other markers of liver damage, including aspartate aminotransferase activity (r=0.59, P=0.09, n=9) and alkaline phosphatase concentrations (r=0.60, P=0.087). IHL was also (P<0.01) correlated with fasting insulin concentration (r=0.85, P=0.03, n=6) and insulin resistance (r=0.94, P=0.006, n=6), but these correlations were driven by the results for one child.
These preliminary data suggest that 1H-MRS obtained in a standard pediatric clinical environment may be used to determine IHL in healthy normal and overweight prepubertal youth. This noninvasive technique may prove useful in identifying early markers of the metabolic syndrome in at-risk youth.
Lipid accumulation within skeletal muscle (intramyocellular lipid [IMCL])1–5 and liver (intrahepatic lipid [IHL])6–9 (i.e., ectopic fat) is significantly associated with insulin resistance in adults and adolescents and may represent an early marker for developing the metabolic syndrome or type 2 diabetes mellitus (T2DM). Although it is not well established whether ectopic fat accumulates independent of, or as a consequence of, increased obesity, many studies have found correlations between increasing whole-body and/or abdominal adiposity and IMCL2,5,10 and IHL11,12 accumulation. IHL in addition is correlated with visceral abdominal adiposity in some7,11 but not all6,9 previous studies and appears to be more sensitive to weight reduction via caloric restriction than is IMCL.7
Little is currently known, however, about ectopic fat accommodation and its relation to adiposity and insulin resistance before puberty. Using proton magnetic resonance spectroscopy (1H-MRS), one study found that IMCL accumulated to a greater degree in prepubertal boys with a higher body mass index (BMI) and larger waist circumference and that such accumulation was related to the fasting glucose:insulin ratio.13 Further evaluation of ectopic fat, particularly IHL, is of timely importance given the increasing prevalence of insulin resistance14,15 and its potential long-term consequences in adulthood.16 Unfortunately, many of the techniques used to assess ectopic fat, including tissue biopsies and computed tomography, are not appropriate for use in children because of their invasiveness or ionizing radiation exposure. 1H-MRS, which is noninvasive and has been used successfully to assess IMCL, may be difficult to use in liver because it requires that participants lay motionless in a semi-enclosed tube during the procedure. Nevertheless, 1H-MRS is a logical method for assessing IHL even in prepubertal children due to its safety (does not use ionizing radiation), availability at pediatric medical centers (providing purchase of the system's spectroscopy package), and proven reliability and validity.17,18 Thus, the primary purpose of this pilot study was to determine the feasibility of assessing IHL in healthy 7–9-year-old prepubertal children using a standard clinical MRS system. A secondary purpose was to determine whether IHL stores, if present, are correlated with adiposity, fasting insulin and glucose, and liver enzymes19 in such young healthy prepubertal children.
This pilot study was designed to examine the feasibility of conducting insulin sensitivity/resistance and 1H-MRS testing in healthy prepubertal children with low birth weight, referred to as the SILLY study (Study of Insulin sensitivity in Louisiana Low-birth weight Youth). Subjects were the first children (n=11) enrolled in the pilot study and were apparently healthy 7–9-year-old normal and overweight prepubertal children (Tanner stage <2) who were not taking regular prescribed medications. A screening blood sample confirmed their health status. The study was approved by the General Clinical Research Center, Children's Hospital, Louisiana State University Health Sciences Center, the Pennington Biomedical Research Center, and the University of Wyoming Institutional Review Boards, and written informed consent and assent were obtained from the child and his or her legal guardian.
Subjects were recruited from June 2005 through July 2006 and were tested at Children's Hospital in New Orleans, LA. Children were tested during a 3–4-h outpatient visit at Children's Hospital in New Orleans and a half-day visit to the General Clinical Research Center. During these visits, body mass and height were measured without shoes using standardized procedures. Waist circumference was measured at the level of the umbilicus. Insulin resistance was measured by the homeostatic model assessment (HOMA) methodology.20 IHL was assessed by 1H-MRS (as detailed below). Medical history was obtained from a family medical history questionnaire, which included information on the child's birth weight and current or past health problems. A fasting blood sample was drawn for analysis of liver function enzymes, insulin, and glucose.
Intrahepatic lipid was measured on a commercially available clinical 1.5-T whole-body imaging and spectroscopy system (Signa XL Horizon 2001 MRI Unit, General Electric, Medical Systems, Milwaukee, WI) located in the radiation department of Children's Hospital, using the PRESS (Point RESolved Spectroscopy) technique. Children were positioned in a prone, feet-first orientation (i.e., on their belly, feet-first going into the magnet). An external amplitude reference phantom was created using a test tube filled with a known volume of peanut oil and was positioned vertically across the child's tailbone and secured with surgical tape. This placement helped assure that the phantom was placed close to the homogeneous spot of the magnet. A single water-suppressed PRESS voxel (~3×3×3cm3, echo time [TE]=40ms, repetition time [TR]=1,500ms) was then collected using the commercially provided 1H-body coil using standard GE PRESS acquisitions in an area of the middle right lobe that was visually free from heavy vascularization as determined from T1-weighted axial images (25 slices, 8mm thick, 2mm separation; Fig. 1A) and not located too close to the surface subcutaneous fat. The larger voxel size allowed collection of less number of averages and a shorter scan time. Children were informed when the scan began and were asked to remain still and take shallow breaths during the procedure. An investigator practiced shallow breathing with the children before positioning in the magnet and also stressed the importance of remaining motionless during the acquisition. A single non–water-suppressed PRESS voxel was then collected from the phantom (~3×1.5×1.5cm3 voxel, TE=40ms, TR=1,500ms, 64 scans). To keep the children entertained during the 20-min procedure and to reduce the discomfort associated with the loud noises, children listened to personal music or story CDs via child-sized headphones.
Peak positions and areas of interest including the CH2 (frequency=1.4) and CH3 (frequency=1.0) fatty acid chain resonances were determined by time domain fitting using jMRUI (Java-Based Magnetic Resonance User Interface)19 by a single investigator. The residual water peak was removed using a HLSVD algorithm, and lipid resonances were fit using single lorentzian line shapes and referenced to the oil (phantom) resonance. The CH2 resonance relative to the phantom (expressed as a percentage) was used as IHL.
Statistical analysis was performed using SPSS analysis software (SPSS version 13.0 for Windows, SPSS, Inc., Chicago, IL). Data are presented as mean±SD values. Pearson's correlation procedure was applied to determine associations between variables of interest. Significance was set at α>0.05.
A usable liver spectrum was not available in one participant (patient 06) because of significant participant movement during data acquisition. This resulted in bad signal acquisition that produced a spectrum file with a low signal-to-noise ratio, which could not be analyzed. Data were also missing for several other parameters, including waist circumference and insulin resistance, because of staff shortages and blood transportation problems during post-Katrina recovery. Thus, the “n” values for all statistical analysis are reported in parentheses.
The characteristics of the 11 children, including their anthropometrics, blood measurements, and IHL content, are shown in Table 1. IHL stores were variable (range, 0.07–3.2%) and could also be detected visually in the non–water-suppressed spectra (Fig. 1). One participant (patient 02) was found to have elevated fasting glucose and insulin concentrations and was diagnosed with T2DM during the study. This participant was not dropped from the pilot analysis because our main purpose was to determine the feasibility of assessing IHL and determine if IHL accumulation occurs in prepubertal children. Correlations between IHL stores and fasting insulin and glucose and insulin resistance, however, were evaluated with and without this participant.
IHL was significantly correlated with body mass (r=0.66, P=0.037, n=10), BMI (Fig. 2, upper panel; r=0.73, P=0.016, n=10), percentage body fat (Fig. 2, middle panel; r=0.73, P=0.01, n=10), waist circumference (Fig. 2, lower panel; r=0.85, P=0.016, n=7), and serum lactate dehydrogenase concentration (r=0.77, P=0.03, n=8) but was not significantly correlated with serum aspartate aminotransferase activity (r=0.59, P=0.09, n=9) and alkaline phosphatase concentration (P=0.60, P=0.087, n=9). IHL was also (P<0.01) correlated with fasting serum insulin concentration (r=0.85, P=0.03, n=6) and insulin resistance (r=0.94, P=0.006, n=6), but these correlations were driven by subject 02 and were not significantly correlated when this subject was removed from the analysis. IHL was not correlated with fasting glucose concentration (r=0.59, P=0.09, n=9) and birth weight (r=0.46, P=0.22, n=9).
This pilot study demonstrates that noninvasive assessment of liver lipid by 1H-MRS is feasible in healthy, normal and overweight prepubertal children and highlights the methodology of 1H-MRS in a standard pediatric clinical environment. Most notably, our preliminary results are the first to demonstrate in exclusively prepubertal, healthy children who are both normal and overweight that many of the relations between IHL and components of the metabolic syndrome in adults and adolescents6–9,11,12 are also present in normal and overweight, apparently healthy prepubertal children. For instance, children with higher BMI and total adiposity and larger waist circumferences had increased IHL stores. Increased IHL stores, in turn, were significantly correlated with lactate dehydrogenase, a nonspecific marker of tissue damage (including liver).
Although the present pilot study demonstrated the feasibility of using 1H-MRS to assess IHL in healthy normal and overweight youth, it is important to note, based on our pilot experience, that these measurements should be performed by a well-trained, dedicated clinical staff who are comfortable working with children. We originally anticipated losing some subject data due to movement artifacts (i.e., from wiggly participants). However, we were pleased that only one of 11 liver acquisitions was unusable because of motion artifact. Thus, 7–9-year-old children can successfully be kept “relatively” still and entertained during IHL acquisitions by stressing the importance of being still, practicing shallow breathing, and providing music/stories via headphones.
The most exciting findings from our pilot study were that IHL accumulation is initiated (at least in some children) before puberty and that this accumulation tends to be most noteworthy in those with presumed central adiposity and higher total adiposity. Although excessive liver fat accumulation, or hepatic steatosis, has been identified in ill or obese prepubertal21,22 and adolescent8,23 children, our studies are the first to identify this phenomenon noninvasively in healthy young prepubertal children. It is particularly interesting to note that IHL stores in the current study in children were mostly in the range we have previously seen in overweight, insulin-sensitive adults (range, 0.07–3.2% vs. 0.3–7.2%) using nearly identical methodology.7 Although the highest IHL accumulation was found in the child with newly diagnosed T2DM, it is clear that significant IHL stores were also present in other apparently healthy prepubertal children (Table 1). Certainly, these noninvasive measures should prove useful in identifying early markers of the metabolic syndrome in at-risk youth and also help to elucidate whether liver lipid accumulation is a cause or consequence of tissue insulin resistance. Ongoing research from this pilot is currently measuring both IHL and intramyocellular lipids by 1H-MRS along with insulin sensitivity by the frequently sampled intravenous glucose tolerance test in a larger cohort of 400 healthy normal and overweight prepubertal African American and white children born with normal, high, and low birth weight.
In conclusion, while our study provides important preliminary results, they are limited by our small mixed sample of male and female white and African American children and our use of HOMA to assess insulin resistance. Regardless, these preliminary data suggest that 1H-MRS may be successfully used to determine IHL in exclusively prepubertal, healthy, normal and overweight youth when assessed by a well-trained, dedicated staff who are comfortable working with children. Most importantly, IHL data may be successfully obtained in a standard pediatric clinical environment. This noninvasive measure may prove useful in identifying early markers of the metabolic syndrome in at-risk youth.
This work was supported by National Institute of Child Health and Human Development grant 1 R01 HD41071-01A2 entitled “Insulin Sensitivity in African-American and Caucasian Youth with Low Birth” and grant 1 R01 HD49046-01 entitled “Mechanisms of the Metabolic Syndrome in Pre-Pubertal Youth.” Also, this study was supported in part by Tulane University and the Clinical and Translational Research Center, Louisiana State University Health Sciences Center as part of the Louisiana Board of Regents RC/EEP Fund (LSUHSC IRB 6134) and partially supported by CNRU Center grant 1P30 DK072476 entitled “Nutritional Programming: Environmental and Molecular Interactions” sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases.
All authors declare that no competing financial interests exist.