Daniel T. Stein

Measurement of intracellular triglyceride stores by 1H spectroscopy: validation in vivo

We validate the use of 1H magnetic resonance spectroscopy (MRS) to quantitatively differentiate between adipocyte and intracellular triglyceride (TG) stores by monitoring the TG methylene proton signals at 1.6 and 1.4 ppm, respectively. In two animal models of intracellular TG accumulation, intrahepatic and intramyocellular TG accumulation was confirmed histologically. Consistent with the histological changes, the methylene signal intensity at 1.4 ppm increased in both liver and muscle, whereas the signal at 1.6 ppm was unchanged. In response to induced fat accumulation, the TG concentration in liver derived from 1H MRS increased from 0 to 44.9 ± 13.2 μmol/g, and this was matched by increases measured biochemically (2.1 ± 1.1 to 46.1 ± 10.9 μmol/g). Supportive evidence that the methylene signal at 1.6 ppm in muscle is derived from investing interfascial adipose tissue was the finding that, in four subjects with generalized lipodystrophy, a disease characterized by absence of interfacial fat, no signal was detected at 1.6 ppm; however, a strong signal was seen at 1.4 ppm. An identical methylene chemical shift at 1.4 ppm was obtained in human subjects with fatty liver where the fat is located exclusively within hepatocytes. In experimental animals, there was a close correlation between hepatic TG content measured in vivo by 1H MRS and chemically by liver biopsy [ R = 0.934; P < .0001; slope 0.98, confidence interval (CI) 0.70-1.17; y-intercept 0.26, CI -0.28 to 0.70]. When applied to human calf muscle, the coefficient of variation of the technique in measuring intramyocellular TG content was 11.8% in nonobese subjects and 7.9% in obese subjects and of extramyocellular (adipocyte) fat was 22.6 and 52.5%, respectively. This study demonstrates for the first time that noninvasive in vivo 1H MRS measurement of intracellular TG, including that within myocytes, is feasible at 1.5-T field strengths and is comparable in accuracy to biochemical measurement. In addition, in mixed tissue such as muscle, the method is clearly advantageous in differentiating between TG from contaminating adipose tissue compared with intramyocellular lipids.

THE INSULINOTROPIC POTENCY OF FATTY ACIDS IS INFLUENCED PROFOUNDLY BY THEIR CHAIN LENGTH AND DEGREE OF SATURATION

Lowering of the elevated plasma FFA concentration in 18- 24-h fasted rats with nicotinic acid (NA) caused complete ablation of subsequent glucose-stimulated insulin secretion (GSIS). Although the effect of NA was reversed when the fasting level of total FFA was maintained by coinfusion of soybean oil or lard oil (plus heparin), the more saturated animal fat proved to be far more potent in enhancing GSIS. We therefore examined the influence of individual fatty acids on insulin secretion in the perfused rat pancreas. When present in the perfusion fluid at 0.5 mM (in the context of 1% albumin), the fold stimulation of insulin release from the fasted pancreas in response to 12.5 mM glucose was as follows: octanoate (C8:0), 3.4; linoleate (C18:2 cis/cis), 5.3; oleate (C18:1 cis), 9.4; palmitate (C16:0), 16. 2; and stearate (C18:0), 21.0. The equivalent value for palmitoleate (C16:1 cis) was 3.1. A cis--> trans switch of the double bond in the C16:1 and C18:1 fatty acids had only a modest, if any, impact on their potency. A similar profile emerged with regard to basal insulin secretion (3 mM glucose). When a subset of these fatty acids was tested in pancreases from fed animals, the same rank order of effectiveness at both basal and stimulatory levels of glucose was seen. The findings reaffirm the essentiality of an elevated plasma FFA concentration for GSIS in the fasted rat. They also show, however, that the insulinotropic effect of individual fatty acids spans a remarkably broad range, increasing and decreasing dramatically with chain length and degree of unsaturation, respectively. Thus, for any given level of glucose, insulin secretion will be influenced greatly not only by the combined concentration of all circulating (unbound) FFA, but also by the makeup of this FFA pool. Both factors will likely be important considerations in understanding the complex interplay between the nature of dietary fat and whole body insulin, glucose, and lipid dynamics.

Essentiality of circulating fatty acids for glucose-stimulated insulin secretion in the fasted rat

We asked whether the well known starvation-induced impairment of glucose-stimulated insulin secretion (GSIS) seen in isolated rat pancreas preparations also applies in vivo. Accordingly, fed and 18-24-h-fasted rats were subjected to an intravenous glucose challenge followed by a hyperglycemic clamp protocol, during which the plasma-insulin concentration was measured. Surprisingly, the acute (5 min) insulin response was equally robust in the two groups. However, after infusion of the antilipolytic agent, nicotinic acid, to ensure low levels of plasma FFA before the glucose load, GSIS was essentially ablated in fasted rats, but unaffected in fed animals. Maintenance of a high plasma FFA concentration by coadministration of Intralipid plus heparin to nicotinic acid-treated rats (fed or fasted), or further elevation of the endogenous FFA level in nonnicotinic acid-treated fasted animals by infusion of etomoxir (to block hepatic fatty acid oxidation), resulted in supranormal GSIS. The in vivo findings were reproduced in studies with the perfused pancreas from fed and fasted rats in which GSIS was examined in the absence and presence of palmitate. The results establish that in the rat, the high circulating concentration of FFA that accompanies food deprivation is a sine qua non for efficient GSIS when a fast is terminated. They also serve to underscore the powerful interaction between glucose and fatty acids in normal beta cell function and raise the possibility that imbalances between the two fuels in vivo could have pathological consequences.

MRI of muscular fat

An MRI technique with high selectivity and sensitivity to the signal components in the chemical shift range of methylene and methyl protons of fatty acids has been developed for noninvasive assessment of muscular fat in vivo. A spoiled gradient‐echo sequence with spatial‐spectral excitation by six equidistant pulses with 2°‐(−9°)‐17°‐(−17°)‐9°‐(−2°) and a multi‐echo train (TE = 16, 36, 56, 76, 96, and 116 ms) allowed a series of images to be recorded with a receiver bandwidth of 78 Hz per pixel. SIs from phantoms with lipid contents between 0.1% and 100% were compared to those from pure water. Thirty healthy volunteers underwent fat‐selective imaging of their lower leg, and parallel localized proton spectroscopy of the tibialis anterior and the soleus muscle by a single‐voxel stimulated echo acquisition mode (STEAM) technique (TR = 2 s, TE = 10 ms, TM = 15 ms). Results show a high correlation (r = 0.91) between fat imaging and the spectroscopic approach in the soleus muscle, considering the percentage total fat content of musculature. The correlation coefficient was clearly lower (r = 0.55) in the tibialis anterior muscle due to signal contaminations from adjacent subcutaneous fat in the images, inhomogeneous fat distribution, and generally lower lipid content in this muscle. Applications of the new imaging technique showed marked intra‐ and interindividual variability in the spatial distribution of lipids in the musculature of the lower leg. No significant correlation of the muscular fat with the thickness of the subcutaneous fat layer was found. In addition, the body mass index does not appear to determine muscular fat content, except in very obese cases. Magn Reson Med 47:720–727, 2002.

Human Brain β-Hydroxybutyrate and Lactate Increase in Fasting-Induced Ketosis:

Ketones are known to constitute an important fraction of fuel for consumption by the brain, with brain ketone content generally thought to be low. However, the recent observation of 1-mmol/L levels of brain β-hydroxybutyrate (BHB) in children on the ketogenic diet suggests otherwise. The authors report the measurement of brain BHB and lactate in the occipital lobe of healthy adults using high field (4-T) magnetic resonance spectroscopy, measured in the nonfasted state and after 2-and 3-day fasting-induced ketosis. A 9-mL voxel located in the calcarine fissure was studied, detecting the BHB and lactate upfield resonances using a 1H homonuclear editing sequence. Plasma BHB levels also were measured. The mean brain BHB concentration increased from a nonfasted level of 0.05 ± 0.05 to 0.60 ± 0.26 mmol/L (after second day of fasting), increasing further to 0.98 ± 0.16 mmol/L (after the third day of fasting). The mean nonfasted brain lactate was 0.69 ± 0.17 mmol/L, increasing to 1.47 ± 0.22 mmol/L after the third day. The plasma and brain BHB levels correlated well (r = 0.86) with a brain–plasma slope of 0.26. These data show that brain BHB rises significantly with 2-and 3-day fasting-induced ketosis. The lactate increase likely results from ketones displacing lactate oxidation without altering glucose phosphorylation and glycolysis.

Bulk magnetic susceptibility effects on the assessment of intra- and extramyocellular lipids in vivo

Localized proton spectroscopy provides a novel method for noninvasive measurement of lipid content in skeletal muscle. It has been suggested that the chemical shift difference between lipid signals from distinct compartments in skeletal muscle might be caused by bulk magnetic susceptibility (BMS) differences from lipids stored in intra‐ (IMCL) and extramyocellular (EMCL) compartments. Direct evidence is provided to confirm the theoretical prediction that compartment symmetry is responsible for discrimination between resonances of IMCL and EMCL. Phantoms imitating lipids in skeletal muscle were constructed using soybean oil to represent EMCL, and Intralipid™, an intravenous fat emulsion of fine droplets, to represent IMCL. It was found that the chemical shift of Intralipid™ is independent of the BMS effects, while the resonance of soybean oil shifts in a predictable manner determined by the geometry of the compartment. Magn Reson Med 47:607–610, 2002.

Insulin enhances glucose-stimulated insulin secretion in healthy humans

Islet β-cells express both insulin receptors and insulin-signaling proteins. Recent evidence from rodents in vivo and from islets isolated from rodents or humans suggests that the insulin signaling pathway is physiologically important for glucose sensing. We evaluated whether insulin regulates β-cell function in healthy humans in vivo. Glucose-induced insulin secretion was assessed in healthy humans following 4-h saline (low insulin/sham clamp) or isoglycemic-hyperinsulinemic (high insulin) clamps using B28-Asp insulin that could be immunologically distinguished from endogenous insulin. Insulin and C-peptide clearance were evaluated to understand the impact of hyperinsulinemia on estimates of β-cell function. Preexposure to exogenous insulin increased the endogenous insulin secretory response to glucose by ≈40%. C-peptide response also increased, although not to the level predicted by insulin. Insulin clearance was not saturated at hyperinsulinemia, but metabolic clearance of C-peptide, assessed by infusion of stable isotope–labeled C-peptide, increased modestly during hyperinsulinemic clamp. These studies demonstrate that insulin potentiates glucose-stimulated insulin secretion in vivo in healthy humans. In addition, hyperinsulinemia increases C-peptide clearance, which may lead to modest underestimation of β-cell secretory response when using these methods during prolonged dynamic testing.

Standardization of C-Peptide Measurements

BACKGROUND C-peptide is a marker of insulin secretion in diabetic patients. We assessed within- and between-laboratory imprecision of C-peptide assays and determined whether serum calibrators with values assigned by mass spectrometry could be used to harmonize C-peptide results. METHODS We sent 40 different serum samples to 15 laboratories, which used 9 different routine C-peptide assay methods. We also sent matched plasma samples to another laboratory for C-peptide analysis with a reference mass spectrometry method. Each laboratory analyzed 8 of these samples in duplicate on each of 4 days to evaluate within- and between-day imprecision. The same 8 samples were also used to normalize the results for the remaining samples to the mass spectrometry reference method. RESULTS Within- and between-run CVs ranged from <2% to >10% and from <2% to >18%, respectively. Normalizing the results with serum samples significantly improved the comparability among laboratories and methods. After normalization, the differences among laboratories in mean response were no longer statistically significant (P = 0.24), with least-squares means of 0.93-1.02. CONCLUSIONS C-peptide results generated by different methods and laboratories do not always agree, especially at higher C-peptide concentrations. Within-laboratory imprecision also varied, with some methods giving much more consistent results than others. These data show that calibrating C-peptide measurement to a reference method can increase comparability between laboratories.

Spectroscopic imaging of glutamate C4 turnover in human brain.

One‐dimensional spectroscopic imaging of 13C‐4‐glutamate turnover is performed in the human brain with a 6 cc nominal voxel resolution at 4T. Data were acquired with an indirect detection approach using a short spin echo single quantum 1H‐13C heteronuclear editing method and a 7 cm surface coil with quadrature 13C decoupling coils. To analyze the data as a function of tissue type, T1‐based image segmentation through the surface coil was performed to determine the gray and white matter contributions to each voxel. The tricarboxylic acid (TCA) cycle rate in gray and white matter was then determined using a two‐compartment model with the tissue fractionation derived from the image segmentation. The mean values for the TCA cycle rate for occipital gray and white matter from three volunteers was 0.88 ± 0.12 and 0.28 ± 0.13 respectively, in agreement with literature data. Magn Reson Med 44:673–679, 2000.

Acute elevation of NEFA causes hyperinsulinemia without effect on insulin secretion rate in healthy human subjects.

Abstract: Increased circulating levels of nonesterified free fatty acids (NEFA) have been observed in such hyperinsulinemic states as obesity, impaired glucose tolerance, diabetes, and dyslipidemia where they have been causally linked to the development of insulin resistance and hyperinsulinemia. The concentration of NEFA in plasma is believed to have direct modifying effects on insulin secretion and clearance. It remains controversial whether acute increases in NEFA potentiate insulin secretion in human subjects. We studied the effect of an acute elevation of NEFA during lipid‐heparin infusion compared to a glycerol‐only control on glucose‐stimulated insulin secretion and clearance during a 120‐min hyperglycemic (10 mM) clamp in 7 healthy normoglucose‐tolerant volunteers. The metabolic clearance rate of C‐peptide (MCRCP) was measured in each subject during the study by simultaneous infusion of C‐peptide. Insulin secretion rate (ISR) was calculated from deconvolution of C‐peptide data after correction for the rate of C‐peptide infusion. Clearance rate of insulin (MCRINS) was calculated based upon endogenous ISR. Plasma glucose (mg/dL): basal (90‐115 min) 90.2 ± 2.8 vs. 90.2 ± 2.3; clamp (150‐240 min) 180.5 ± 2.8 vs. 180.9 ± 1.3. Plasma insulin (pmol/L): prebasal (fasting) 29.6 ± 10.0 vs. 29.8 ± 10.6; basal (90‐115 min) 30.1 ± 9.2 vs. 34.5 ± 12.1; second phase clamp (210‐240 min) 127.6 ± 18.2 vs. 182.5 ± 17.3*. Plasma NEFA (mM): prebasal 0.47 ± 0.08 vs. 0.52 ± 0.09; basal 0.35 ± 0.05 vs. 0.98 ± 0.02*; clamp (122‐240 min) 0.06 ± 0.02 vs. 0.77 ± 0.06*. ISR (pmol/min): prebasal 72.7 ± 7.5 vs. 72.0 ± 7.9; second phase clamp (210‐240 min) 268.5 ± 27.2 vs. 200.2 ± 23.7. MCRINS (mL/min): prebasal 3393 ± 488 vs. 3370 ± 511; clamp 2284 ± 505 vs. 1214 ± 153* (*p < 0.05 glycerol vs. intralipid/heparin). This study demonstrates that acute NEFA elevation causes hyperinsulinemia due to a significant decrease in systemic insulin clearance without increasing rates of insulin secretion.