Lidia S. Szczepaniak

Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity.

Despite the increasing prevalence of nonalcoholic fatty liver disease (NAFLD), its pathogenesis and clinical significance remain poorly defined. In this study, we examined and compared the distribution of hepatic triglyceride content (HTGC) in 2,287 subjects from a multiethnic, population‐based sample (32.1% white, 48.3% black, and 17.5% Hispanic) using proton magnetic resonance spectroscopy. HTGC varied over a wide range (0.0%‐41.7%; median, 3.6%) in the population. Almost one third of the population had hepatic steatosis, and most subjects with hepatic steatosis had normal levels of serum alanine aminotransferase (79%). The frequency of hepatic steatosis varied significantly with ethnicity (45% in Hispanics; 33% in whites; 24% in blacks) and sex (42% in white men; 24% in white women). The higher prevalence of hepatic steatosis in Hispanics was due to the higher prevalence of obesity and insulin resistance in this ethnic group. However, the lower frequency of hepatic steatosis in blacks was not explained by ethnic differences in body mass index, insulin resistance, ethanol ingestion, or medication use. The prevalence of hepatic steatosis was greater in men than women among whites, but not in blacks or Hispanics. The ethnic differences in the frequency of hepatic steatosis in this study mirror those observed previously for NAFLD‐related cirrhosis (Hispanics > whites > blacks). In conclusion, the significant ethnic and sex differences in the prevalence of hepatic steatosis documented in this study may have a profound impact on susceptibility to steatosis‐related liver disease. (HEPATOLOGY 2004;40:1387–1395.)

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.

Cardiac Steatosis in Diabetes Mellitus A 1H-Magnetic Resonance Spectroscopy Study

Background— The risk of heart failure in type 2 diabetes mellitus is greater than can be accounted for by hypertension and coronary artery disease. Rodent studies indicate that in obesity and type 2 diabetes mellitus, lipid overstorage in cardiac myocytes produces lipotoxic intermediates that cause apoptosis, which leads to heart failure. In humans with diabetes mellitus, cardiac steatosis previously has been demonstrated in explanted hearts of patients with end-stage nonischemic cardiomyopathy. Whether cardiac steatosis precedes the onset of cardiomyopathy in individuals with impaired glucose tolerance or in patients with type 2 diabetes mellitus is unknown. Methods and Results— To represent the progressive stages in the natural history of type 2 diabetes mellitus, we stratified 134 individuals (age 45±12 years) into 1 of 4 groups: (1) lean normoglycemic (lean), (2) overweight and obese normoglycemic (obese), (3) impaired glucose tolerance, and (4) type 2 diabetes mellitus. Localized 1H magnetic resonance spectroscopy and cardiac magnetic resonance imaging were used to quantify myocardial triglyceride content and left ventricular function, respectively. Compared with lean subjects, myocardial triglyceride content was 2.3-fold higher in those with impaired glucose tolerance and 2.1-fold higher in those with type 2 diabetes mellitus (P<0.05). Left ventricular ejection fraction was normal and comparable across all groups. Conclusions— In humans, impaired glucose tolerance is accompanied by cardiac steatosis, which precedes the onset of type 2 diabetes mellitus and left ventricular systolic dysfunction. Thus, lipid overstorage in human cardiac myocytes is an early manifestation in the pathogenesis of type 2 diabetes mellitus and is evident in the absence of heart failure.

Myocardial triglycerides and systolic function in humans: in vivo evaluation by localized proton spectroscopy and cardiac imaging.

Recent experimental data suggest that adiposity directly damages the heart by promoting ectopic deposition of triglyceride, a process known as myocardial steatosis. The goal of this study was to develop and validate proton magnetic resonance spectroscopy (1H MRS) as an in vivo tool to measure myocardial lipid content. Complementary studies in rat tissue ex vivo and in 15 healthy humans in vivo provided evidence that 1H MRS constitutes a reproducible technique for the measurement of myocardial triglyceride. In myocardial tissue from Zucker rats, the 1H MRS measurement of triglyceride matched that obtained by biochemical measurement (P < 0.001). In human subjects triglyceride was evident in the hearts of even the very lean individuals and was elevated in overweight and obese subjects. Increased myocardial triglyceride content was accompanied by elevated LV mass and suppressed septal wall thickening as measured by cardiac imaging. Magn Reson Med 49:417–423, 2003.

Adiposity of the Heart*, Revisited

The unrelenting obesity epidemic is one likely explanation for the recent adverse secular trends in cardiovascular morbidity and mortality rates in the United States (1, 2). Hospitalizations for congestive heart failure have increased, and the steady decline in coronary heart diseaserelated deaths since the 1950s has leveled off (3). The recent obesity epidemic poses a major threat to human health in the United States because these persons will be predisposed to a burden of major chronic disease (1, 2). Obesity has both metabolic and cardiovascular health consequences; in particular, obese individuals are at much greater risk for type 2 diabetes and cardiovascular disease (3, 4). Obesity is traditionally considered to be an indirect cause of heart disease. Obese persons typically present with several Framingham risk factors, including hypertension, dyslipidemia, and diabetes mellitus. These risk factors predispose the patient to myocardial infarction that, in severe cases, results in ischemic cardiomyopathy (4). In addition to an elevated Framingham risk score, the hemodynamic hallmarks of obesity are increased heart rate and stroke volume (5). This hyperdynamic circulation is thought to be a compensatory adaptation to increased adipose tissue mass at the expense of eccentric left ventricular remodeling. In extreme obesity, this condition can progress to nonischemic dilated cardiomyopathy (2, 6). In contrast to these 2 rather traditional concepts, an emerging body of basic research is revisiting a previous hypothesis (7, 8): that fat is a direct cardiotoxin (9, 10). In 1933, the original autopsy studies of Smith and Willius (8) suggested that fatty degeneration of the heart is a common consequence of obesity and a possible cause of dilated cardiomyopathy in humans. After Alexander and colleagues (11) called this theory into question in the 1960s, the issue lay dormant for the next several decades (9). Now a growing body of evidence is revisiting the hypothesis that excessive deposits of lipids within myocardial tissue (that is, cardiac lipotoxicity) is an important but forgotten cause of nonischemic dilated cardiomyopathy in humans (12, 13). Under healthy conditions, most triglyceride is stored in adipocytes; the amount of triglyceride stored in nonadipocyte tissues (such as the pancreas, liver, and myocardium) is minimal and very tightly regulated. Various genetic rodent models of obesity have shown that cytosolic triglyceride accumulates excessively in these organs (termed steatosis) when this regulation is disrupted. This accumulation has been implicated in activating adverse signaling cascades that culminate in irreversible cell death (termed lipotoxicity) and lead to several well-recognized clinical syndromes (13). These include nonalcoholic hepatic steatosis; pancreatic -cell failure in type 2 diabetes; and most recently, dilated cardiomyopathy (Figure 1). Figure 1. Concept of lipotoxicity. bottom The purposes of this article are to review recent basic animal research that demonstrates direct toxic effects of lipid accumulation on the myocardium and to highlight emerging efforts to translate this work into the clinical setting by using novel cardiac magnetic resonance imaging and spectroscopy technology. The results of this research could provide insight into the pathogenesis of heart disease in obese humans and guide the development of a novel biomarker and drug target for the prevention of heart failure in these persons. Steatosis in Rodents The seminal research that showed a role for steatosis in obesity-related organ dysfunction was performed with the Zucker diabetic fatty rat, which is a genetic model of progressive type 2 diabetes (14-16). In this obese rodent, type 2 diabetes developed secondary to a loss-of-function mutation in tissue receptors for leptin, the adipocyte-derived hormone that regulates appetite and body weight (17). This model of genetic obesity is more extreme than the milder leptin resistance that commonly accompanies dietary obesity in humans (17). Initial studies demonstrated that pancreatic steatosis directly caused islet cell failure and the subsequent hyperglycemia that characterized this model (14-16). Although leptin was generally thought to act centrally to regulate caloric intake and energy expenditure (17), a series of studies provided experimental evidence that leptin also acts directly on the pancreatic islet cells to stimulate fatty acid oxidation, thereby limiting cellular triglyceride accumulation (18). These findings suggested that leptin signaling is also essential in regulating peripheral lipid stores. Furthermore, the investigators described a pathway whereby failure of the leptin receptor led to excessive cytosolic accumulation of triglyceride and its by-product, ceramide, within islet cells. This accumulation activated the inducible form of nitric oxide synthase, which accelerated cell death (apoptosis) and failure of the -cell (14, 15). Interventions that stimulated free-fatty acid oxidation, like restoration of leptin signaling or thiazolidinedione therapy, effectively attenuated triglyceride accumulation in islet cells and prevented the onset of type 2 diabetes (19). These findings provided evidence that steatosis is an integral determinant of -cell failure in the pathogenesis of obesity-associated type 2 diabetes. In addition to pancreatic -cell failure, the Zucker diabetic fatty rat experienced age-related cardiac dysfunction that was characterized by eccentric left ventricular remodeling, increased left ventricular pressure, and decreased systolic performance (9, 20). The abnormalities in cardiac structure and function are accompanied by a 2-fold increase in myocardial triglyceride content and ceramide that is similar to the accumulation seen in islet cells. Myocardial DNA laddering, which is a marker of apoptosis, is also increased (9). Of note, early administration of thiazolidinedione therapy is effective in attenuating myocardial triglyceride accumulation and normalizing left ventricular contractile performance (9, 20), as shown in Figure 2. Because reduced myocardial lipid content and improved cardiac structure and function were observed independent of changes in body weight, they strongly suggest a role for myocardial steatosis in obesity-related cardiomyopathy. Figure 2. Myocardial lipotoxicity in the Zucker diabetic fatty rat. Top panel. white bars light gray bars dark gray bars Bottom panel. The extreme obesity in the Zucker rat model makes it difficult to determine whether the cardiac maladaptations are related to excessive myocardial lipid accumulation or to increased expression of conventional risk factors for cardiovascular disease. To address this limitation, various lean genetic mouse models of cardiac-restricted steatosis have recently been developed (10, 21-29). These animals display diffuse myocardial lipid content in the absence of obesity or any other traditional cardiovascular risk factors, thereby allowing researchers to study the acute effects of myocardial steatosis on left ventricular structure and function. Overexpression of long-chain acyl-CoA synthetase, a key enzyme involved in triglyceride synthesis, produces an example of cardiac-restricted steatosis. Increased protein expression of acyl-CoA synthetase in the myocardium disrupts the balance between lipid import and export in the myocardium (Figure 3), which results in diffuse lipid accumulation and a greater than 2-fold increase in heart mass (10). The severe myocardial steatosis that is observed in this animal is associated with substantial left ventricular hypertrophy by 4 weeks of age that coincides with left ventricular dilatation and eventually progresses to heart failure. Of importance, the changes in cardiac lipid content, structure, and function develop without any change in lipid profile or body weight of the animal. This pattern of steatosis-induced heart failure has been reproduced by targeted overexpression of genes that are involved in lipid delivery (24, 26) and synthesis (10, 25) and by targeted deletion of genes that are involved in lipoprotein secretion (21) from the myocardium. Taken together, these data demonstrate that cardiac-specific steatosis, independent of systemic obesity, is a direct cause of dilated cardiomyopathy. Figure 3. Myocardial-specific lipotoxicity. Top panel. Middle panel. gray bars white bars Bottom panel. The development of cardiac-restricted transgenic murine models have also shown the therapeutic potential of several countermeasures, including adenoviral administration of leptin (Figure 3) and apolipoprotein B (26, 28), dietary replacement of long-chain triglycerides with medium-chain triglycerides (22), and blockade of production of reactive oxygen species (29). Each of these interventions has effectively ameliorated the myocardial steatosis in these mouse models and has rescued the myocardium from progression to dilated cardiomyopathy. These data reinforce the observations in the Zucker diabetic fatty rat that lipid accumulation is toxic in the myocardium. It is important to note that current thinking suggests that the cardiomyopathy is not a direct consequence of triglyceride accumulation alone, but that cardiomyopathy develops secondary to an accumulation of by-products of lipid metabolism, such as ceramide or other fatty acid derivatives that are known to interfere with intracellular signaling pathways (9, 30). This research provides convincing evidence for an acute role of steatosis in the development of left ventricular hypertrophy and dysfunction in animal models of obesity; until recently, however, few data from human research were available to support this theory. Quantification of Lipids in Human Tissues To study the role of steatosis in the clinical setting, we and others have developed a magnetic resonance imaging and spectroscopy technique that permits the precise and reproducible quantification of intracellular trig

Noninvasive Quantification of Pancreatic Fat in Humans

OBJECTIVE To validate magnetic resonance spectroscopy (MRS) as a tool for non-invasive quantification of pancreatic triglyceride (TG) content and to measure the pancreatic TG content in a diverse human population with a wide range of body mass index (BMI) and glucose control. METHODS To validate the MRS method, we measured TG content in the pancreatic tissue of 12 lean and 12 fatty ZDF rats (ages 5-14 weeks) both by MRS and the gold standard biochemical assay. We used MRS to measure pancreatic TG content in vivo in 79 human volunteers. Additionally, to assess the reproducibility of the method, in 33 volunteers we obtained duplicate MRS measurements 1-2 weeks apart. RESULTS MRS quantifies pancreatic TG content with high reproducibility and concordance to the biochemical measurement (Spearmans rank correlation coefficient = 0.91). In humans, median pancreatic TG content was as follows: (1) normal weight and normoglycemic group 0.46 f/w%, (2) overweight or obese but normoglycemic group 3.16 f/w%, (3) impaired fasting glucose or impaired glucose tolerance group (BMI matched with group 2) 5.64 f/w%, and (4) untreated type 2 diabetes group (BMI matched with group 2) 5.54 f/w% (Jonckheere-Terpstra trend test across groups p < 0.001). CONCLUSIONS Human pancreatic steatosis, as measured by MRS, increases with BMI and with impaired glycemia. MRS is a quantitative and reproducible non-invasive clinical research tool which will enable systematic studies of the relationship between ectopic fat accumulation in the pancreas and development of type 2 diabetes.

Metabolic correlates of nonalcoholic fatty liver in women and men

Nonalcoholic hepatic steatosis associates with a clustering of metabolic risk factors and steatohepatitis. One risk factor for hepatic steatosis is obesity, but other factors likely play a role. We examined metabolic concomitants of hepatic steatosis in nonobese and obese men and women. Sixty‐one obese women and 35 obese men were studied; both those with and without hepatic steatosis were compared against each other and against nonobese controls (17 women and 32 men) without hepatic steatosis. Obesity (defined as ≥25% body fat in men and ≥35% in women), was identified by x‐ray absorptiometry, whereas hepatic steatosis (≥5.5% liver fat) was detected by magnetic resonance spectroscopy. The primary endpoint was a difference in insulin sensitivity. Obese groups with and without steatosis had similar body fat percentages. Compared with obese women without hepatic steatosis, those with steatosis were more insulin resistant; the same was true for men, although differences were less striking. Obese subjects with hepatic steatosis had higher ratios of truncal‐to‐lower body fat and other indicators of adipose tissue dysfunction compared with obese subjects without steatosis. Conclusion: These results support the concept that obesity predisposes to hepatic steatosis; but in addition, insulin resistance beyond that induced by obesity alone and a relatively high ratio of truncal‐to‐lower body fat usually combined with obesity to produce an elevated liver fat content. (HEPATOLOGY 2007.)

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.

Effect of Pioglitazone Therapy on Myocardial and Hepatic Steatosis in Insulin-Treated Patients with Type 2 Diabetes

High levels of myocardial and hepatic triglyceride are common in obesity and type 2 diabetes. Monotherapy with thiazolidinedione agents reduces hepatic steatosis by up to 50% in patients with type 2 diabetes. It is not known if treatment with a thiazolidinedione added to insulin has a similar beneficial antisteatotic effect. The aim of our study was to determine whether the addition of pioglitazone to insulin treatment in patients with type 2 diabetes has antisteatotic action in the heart and the liver. Thirty-two patients were randomized to 6 months of treatment with insulin or insulin plus pioglitazone. In addition to blood tests, we evaluated myocardial and hepatic triglyceride content, as well as subcutaneous and visceral fat mass at the L2 level, by magnetic resonance spectroscopy and imaging, respectively. Despite weight and subcutaneous fat mass gain, hemoglobin A1c was significantly reduced by both treatments. Myocardial and hepatic triglyceride contents were reduced by the treatment with pioglitazone plus insulin (p = .02 and .03, respectively) but not by the treatment with insulin. Systolic and diastolic blood pressure and heart function remained unchanged in both groups. The addition of pioglitazone to insulin therapy reduced myocardial and hepatic steatosis, consistent with the reported ability of the thiazolidinedione agents to redistribute fat from nonadipose to subcutaneous adipose depots.

Comparison of efficacy and safety of leptin replacement therapy in moderately and severely hypoleptinemic patients with familial partial lipodystrophy of the Dunnigan variety.

CONTEXT Leptin replacement therapy improves metabolic complications in patients with lipodystrophy and severe hypoleptinemia (SH), but whether the response is related to the degree of hypoleptinemia remains unclear. OBJECTIVE The aim of the study was to compare efficacy of leptin therapy in familial partial lipodystrophy, Dunnigan variety (FPLD) patients with SH (serum leptin<7th percentile of normal) vs. those with moderate hypoleptinemia (MH; serum leptin in 7th to 20th percentiles). DESIGN, SETTING, AND PATIENTS We conducted an open-label, parallel group, observational study in 14 SH (mean±sd, serum leptin, 1.9±1.1 ng/ml) and 10 MH (serum leptin, 5.3±1.0 ng/ml) women with FPLD. INTERVENTION Patients received 0.08 mg/kg·d of metreleptin by twice daily sc injections for 6 months. MAIN OUTCOME MEASURES The primary outcome variable was change in fasting serum triglycerides. Other secondary variables were fasting plasma glucose and insulin, insulin sensitivity, hemoglobin A1c, and hepatic triglyceride content. RESULTS Median fasting serum triglycerides decreased from 228 to 183 mg/dl in the SH group (P=0.04) and from 423 to 339 mg/dl in the MH group (P=0.02), but with no difference between the groups (P value for interaction=0.96). Hepatic triglyceride levels similarly declined significantly from 8.8 to 4.9% in the SH group and from 23.7 to 9.2% in the MH group (P value for interaction=0.9). Loss of body weight and body fat occurred in both groups. Fasting glucose, insulin, glucose tolerance, and hemoglobin A1c levels did not change. K value on insulin tolerance test improved slightly in the SH group (0.98 to 1.24%; P=0.01), but not in the MH group (1.1 to 1.27%; P=0.4). CONCLUSION Metreleptin replacement therapy is equally effective in FPLD patients with both SH and MH in reducing serum and hepatic triglyceride levels, but did not improve hyperglycemia.