Jacob E. Friedman

Maternal high-fat diet triggers lipotoxicity in the fetal livers of nonhuman primates

Maternal obesity is thought to increase the offsprings risk of juvenile obesity and metabolic diseases; however, the mechanism(s) whereby excess maternal nutrition affects fetal development remain poorly understood. Here, we investigated in nonhuman primates the effect of chronic high-fat diet (HFD) on the development of fetal metabolic systems. We found that fetal offspring from both lean and obese mothers chronically consuming a HFD had a 3-fold increase in liver triglycerides (TGs). In addition, fetal offspring from HFD-fed mothers (O-HFD) showed increased evidence of hepatic oxidative stress early in the third trimester, consistent with the development of nonalcoholic fatty liver disease (NAFLD). O-HFD animals also exhibited elevated hepatic expression of gluconeogenic enzymes and transcription factors. Furthermore, fetal glycerol levels were 2-fold higher in O-HFD animals than in control fetal offspring and correlated with maternal levels. The increased fetal hepatic TG levels persisted at P180, concurrent with a 2-fold increase in percent body fat. Importantly, reversing the maternal HFD to a low-fat diet during a subsequent pregnancy improved fetal hepatic TG levels and partially normalized gluconeogenic enzyme expression, without changing maternal body weight. These results suggest that a developing fetus is highly vulnerable to excess lipids, independent of maternal diabetes and/or obesity, and that exposure to this may increase the risk of pediatric NAFLD.

Maternal obesity and fetal metabolic programming: a fertile epigenetic soil

The incidence of obesity and overweight has reached epidemic levels in the United States and developed countries worldwide. Even more alarming is the increasing prevalence of metabolic diseases in younger children and adolescents. Infants born to obese, overweight, and diabetic mothers (even when normal weight) have increased adiposity and are at increased risk of later metabolic disease. In addition to maternal glucose, hyperlipidemia and inflammation may contribute to the childhood obesity epidemic through fetal metabolic programming, the mechanisms of which are not well understood. Pregravid obesity, when combined with normal changes in maternal metabolism, may magnify increases in inflammation and blood lipids, which can have profound effects on the developing embryo and the fetus in utero. Fetal exposure to excess blood lipids, particularly saturated fatty acids, can activate proinflammatory pathways, which could impact substrate metabolism and mitochondrial function, as well as stem cell fate, all of which affect organ development and the response to the postnatal environment. Fetal and neonatal life are characterized by tremendous plasticity and the ability to respond to environmental factors (nutrients, oxygen, hormones) by altering gene expression levels via epigenetic modifications. Given that lipids act as both transcriptional activators and signaling molecules, excess fetal lipid exposure may regulate genes involved in lipid sensing and metabolism through epigenetic mechanisms. Epigenetic regulation of gene expression is characterized by covalent modifications to DNA and chromatin that alter gene expression independent of gene sequence. Epigenetic modifications can be maintained through positive and negative feedback loops, thereby creating stable changes in the expression of metabolic genes and their main transcriptional regulators. The purpose of this article is to review current literature on maternal-fetal lipid metabolism and maternal obesity outcomes and to suggest some potential mechanisms for fetal metabolic programming in key organ systems that regulate postnatal energy balance, with an emphasis on epigenetics and the intrauterine environment.

Longitudinal changes in maternal serum leptin concentrations, body composition, and resting metabolic rate in pregnancy

OBJECTIVE We sought to evaluate the longitudinal changes in maternal serum leptin concentrations, body composition, and resting metabolic rate during pregnancy. STUDY DESIGN Ten women were evaluated before pregnancy, in early pregnancy (12 to 14 weeks), and in late pregnancy (34 to 36 weeks). Leptin concentrations were measured by radioimmunoassay, body composition with hydrodensitometry with adjustment for total body water, and resting metabolic rate by use of indirect calorimetry. RESULTS Using analysis of variance with repeated measures from pregravid to late pregnancy, a 66% increase (mean +/- SD) was found in leptin concentrations (in nanograms per milliliter) (before pregnancy, 25.4 +/- 19.9; in early pregnancy, 37.5 +/- 26.2; and in late pregnancy, 38.4 +/- 27.3, p = 0.003); a 9% increase in body fat (in kilograms) (before pregnancy, 29.4 +/- 15.7; in early pregnancy, 28.7 +/- 14.0; in late pregnancy, 31.4 +/- 14.6; p = 0.04); a 28% increase in oxygen consumption (in milliliters of oxygen per minute) (before pregnancy, 221.2 +/- 29.5; in early pregnancy, 230.4 +/- 42.9; in late pregnancy, 285.3 +/- 51.9; p < 0.0001); and a 9% increase in oxygen consumption (milliliters of oxygen per kilogram per minute) (before pregnancy, 3.02 +/- 0.43; in early pregnancy, 3.05 +/- 0.30; in late pregnancy, 3.31 +/- 0.37, p = 0.002) with advancing gestation. A significant positive correlation was present between leptin and body fat before pregnancy (r = 0.90, p < 0.0001), in early pregnancy (r = 0.91, p < 0.0001), and in late pregnancy (r = 0.87, p = 0.0005) and between leptin and oxygen consumption before pregnancy (r = 0.80, p = 0.004), in early pregnancy (r = 0.92, p < 0.0001), and in late pregnancy (r = 0.62, p = 0.06). When oxygen consumption was adjusted for maternal and fetal tissue mass, a significant negative correlation was found between leptin and oxygen consumption before pregnancy (r = -0.96, p < 0.0001), in early pregnancy (r = -0.80, p = 0.0034), and in late pregnancy (r = -0.70, p = 0.02). CONCLUSION We conclude that leptin increases significantly during early pregnancy before any major changes in body fat and resting metabolic rate. These data suggest that pregnancy represents a leptin-resistant state.

Fatty liver is associated with reduced SIRT3 activity and mitochondrial protein hyperacetylation.

Acetylation has recently emerged as an important mechanism for controlling a broad array of proteins mediating cellular adaptation to metabolic fuels. Acetylation is governed, in part, by SIRTs (sirtuins), class III NAD(+)-dependent deacetylases that regulate lipid and glucose metabolism in liver during fasting and aging. However, the role of acetylation or SIRTs in pathogenic hepatic fuel metabolism under nutrient excess is unknown. In the present study, we isolated acetylated proteins from total liver proteome and observed 193 preferentially acetylated proteins in mice fed on an HFD (high-fat diet) compared with controls, including 11 proteins not previously identified in acetylation studies. Exposure to the HFD led to hyperacetylation of proteins involved in gluconeogenesis, mitochondrial oxidative metabolism, methionine metabolism, liver injury and the ER (endoplasmic reticulum) stress response. Livers of mice fed on the HFD had reduced SIRT3 activity, a 3-fold decrease in hepatic NAD(+) levels and increased mitochondrial protein oxidation. In contrast, neither SIRT1 nor histone acetyltransferase activities were altered, implicating SIRT3 as a dominant factor contributing to the observed phenotype. In Sirt3⁻(/)⁻ mice, exposure to the HFD further increased the acetylation status of liver proteins and reduced the activity of respiratory complexes III and IV. This is the first study to identify acetylation patterns in liver proteins of HFD-fed mice. Our results suggest that SIRT3 is an integral regulator of mitochondrial function and its depletion results in hyperacetylation of critical mitochondrial proteins that protect against hepatic lipotoxicity under conditions of nutrient excess.

Hepatitis C virus infection: Molecular pathways to metabolic syndrome

Chronic infection with hepatitis C virus (HCV) can induce insulin resistance (IR) in a genotype‐dependent fashion, thus contributing to steatosis, progression of fibrosis and resistance to interferon therapy. The molecular mechanisms in genotype 1 patients that lead to metabolic syndrome are still ambiguous. Based on our current understanding, HCV proteins associate with mitochondria and endoplasmic reticulum and promote oxidative stress. The latter mediates signals involving the p38 mitogen‐activated protein kinase and activates nuclear factor kappa B. This transcription factor plays a key role in the expression of cytokines, tumor necrosis factor alpha (TNF‐α), interleukin 6, interleukin 8, tumor growth factor beta, and Fas ligand. TNF‐α inhibits the function of insulin receptor substrates and decreases the expression of the glucose transporter and lipoprotein lipase in peripheral tissues, which is responsible for the promotion of insulin resistance. Furthermore, reduced adiponectin levels, loss of adiponectin receptors, and decreased anti‐inflammatory peroxisome proliferator‐activated receptor alpha in the liver of HCV patients may contribute to reduced fatty acid oxidation, inflammation, and eventually lipotoxicity. This chain of events may be initiated by HCV‐associated IR and provides a direction for future research in the areas of therapeutic intervention. (HEPATOLOGY 2008.)

Phosphoenolpyruvate Carboxykinase Overexpression Selectively Attenuates Insulin Signaling and Hepatic Insulin Sensitivity in Transgenic Mice

The ability of insulin to suppress gluconeogenesis in type II diabetes mellitus is impaired; however, the cellular mechanisms for this insulin resistance remain poorly understood. To address this question, we generated transgenic (TG) mice overexpressing the phosphoenolpyruvate carboxykinase (PEPCK) gene under control of its own promoter. TG mice had increased basal hepatic glucose production (HGP), but normal levels of plasma free fatty acids (FFAs) and whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp compared with wild-type controls. The steady-state levels of PEPCK and glucose-6-phosphatase mRNAs were elevated in livers of TG mice and were resistant to down-regulation by insulin. Conversely, GLUT2 and glucokinase mRNA levels were appropriately regulated by insulin, suggesting that insulin resistance is selective to gluconeogenic gene expression. Insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate (IRS)-1, and associated phosphatidylinositol 3-kinase were normal in TG mice, whereas IRS-2 protein and phosphorylation were down-regulated compared with control mice. These results establish that a modest (2-fold) increase in PEPCK gene expression in vivo is sufficient to increase HGP without affecting FFA concentrations. Furthermore, these results demonstrate that PEPCK overexpression results in a metabolic pattern that increases glucose-6-phosphatase mRNA and results in a selective decrease in IRS-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression. However, acute suppression of HGP and glycolytic gene expression remained intact, suggesting that FFA and/or IRS-1 signaling, in addition to reduced IRS-2, plays an important role in downstream insulin signal transduction pathways involved in control of gluconeogenesis and progression to type II diabetes mellitus.

Phosphoenolpyruvate Carboxykinase (GTP) Gene Transcription and Hyperglycemia Are Regulated by Glucocorticoids in Genetically Obesedb/db Transgenic Mice

The molecular mechanisms underlying increased hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene transcription and gluconeogenesis in type II diabetes are largely unknown. To examine the involvement of glucocorticoids and thecis-acting insulin response sequence (IRS, −416/−407) in the genetically obese db/db mouse model, we generated crosses between C57BL/KsJ-db/+ mice and transgenic mice that express −460 or −2000 base pairs of the rat PEPCK gene promoter containing an intact or mutated IRS, linked to a reporter gene. Transgenic mice expressing the intact PEPCK(460)-CRP (C-reactive protein) transgene bred to near homozygosity at thedb locus were obese, hyperinsulinemic, and developed fasting hyperglycemia (389 ± 26 mg/100 ml) between 4 and 10 weeks of age. Levels of CRP reporter gene expression were increased 2-fold despite severe hyperinsulinemia compared with non-diabetic non-obese transgenic mice. Reporter gene expression was also increased 2-fold in transgenic obese diabetic db/db mice bearing a mutation in the IRS, −2000(IRS)-hGx, compared with non-obese non-diabetic transgenic 2000(IRS)-hGx mice. Treatment of obese diabeticdb/db transgenic mice with the glucocorticoid receptor blocker RU 486 decreased plasma glucose by 50% and reduced PEPCK, GLUT2, glucose-6-phosphatase, tyrosine aminotransferase, CRP, and hGx reporter gene expression to levels similar to those of non-obese normoglycemic transgenic mice. Taken together, these results establish that −460 bp of 5′-flanking sequence is sufficient to mediate the induction of PEPCK gene transcription in genetically obesedb/db mice during the development of hyperglycemia. The results further demonstrate that the mechanism underlying increased expression of gluconeogenic enzymes in thedb/db mouse requires the action of glucocorticoids and occurs independently of factors acting through the PEPCK IRS (−416/−407) promoter binding site.

The I1-imidazoline receptor: from binding site to therapeutic target in cardiovascular disease

Objective To review previous work and present additional evidence characterizing the I1-imidazoline receptor and its role in cellular signaling, central cardiovascular control, and the treatment of metabolic syndromes. Second-generation centrally-acting antihypertensives inhibit sympathetic activity mainly via imidazoline receptors, whereas first-generation agents act via α2-adrenergic receptors. The I1subtype of imidazoline receptor resides in the plasma membrane and binds central antihypertensives with high affinity. Methods and results Radioligand binding assays have characterized I1-imidazoline sites in the brainstem site of action for these agents in the rostral ventrolateral medulla. Binding affinity at I1-imidazoline sites, but not at other classes of imidazoline binding sites, correlates closely with the potency of central antihypertensive agents in animals and in human clinical trials. The antihypertensive action of systemic moxonidine is eliminated by the I1α2-antagonist efaroxan, but not by selective blockade of α2-adrenergic receptors. Until now, the cell signaling pathway coupled to I1-imidazoline receptors was unknown. Using a model system lacking α2-adrenergic receptors (PC12 pheochromocytoma cells) we have found that moxonidine acts as an agonist at the cell level and I1-imidazoline receptor activation leads to the production of the second messenger diacylglycerol, most likely through direct activation of phosphatidylcholine-selective phospholipase C. The obese spontaneously hypertensive rat (SHR; SHROB strain) shows many of the abnormalities that cluster in human syndrome X, including elevations in blood pressure, serum lipids and insulin. SHROB and their lean SHR littermates were treated with moxonidine at 8 mg/kg per day. SHROB and SHR treated with moxonidine showed not only lowered blood pressure but also improved glucose tolerance and facilitated insulin secretion in response to a glucose load. Because α2-adrenergic agonists impair glucose tolerance, I1-imidazoline receptors may contribute to the multiple beneficial effects of moxonidine treatment. Conclusion The I1-imidazoline receptor is a specific high- affinity binding site corresponding to a functional cell-sur-face receptor mediating the antihypertensive actions of moxonidine and other second-generation centrally-acting agents, and may play a role in countering insulin resistance in an animal model of metabolic syndrome X.

Increased P85α Is a Potent Negative Regulator of Skeletal Muscle Insulin Signaling and Induces in Vivo Insulin Resistance Associated with Growth Hormone Excess

Insulin resistance is a cardinal feature of normal pregnancy and excess growth hormone (GH) states, but its underlying mechanism remains enigmatic. We previously found a significant increase in the p85 regulatory subunit of phosphatidylinositol kinase (PI 3-kinase) and striking decrease in IRS-1-associated PI 3-kinase activity in the skeletal muscle of transgenic animals overexpressing human placental growth hormone. Herein, using transgenic mice bearing deletions in p85α, p85β, or insulin-like growth factor-1, we provide novel evidence suggesting that overexpression of p85α is a primary mechanism for skeletal muscle insulin resistance in response to GH. We found that the excess in total p85 was entirely accounted for by an increase in the free p85α-specific isoform. In mice with a liver-specific deletion in insulin-like growth factor-1, excess GH caused insulin resistance and an increase in skeletal muscle p85α, which was completely reversible using a GH-releasing hormone antagonist. To understand the role of p85α in GH-induced insulin resistance, we used mice bearing deletions of the genes coding for p85α or p85β, respectively (p85α +/– and p85β–/–). Wild type and p85β–/– mice developed in vivo insulin resistance and demonstrated overexpression of p85α and reduced insulin-stimulated PI 3-kinase activity in skeletal muscle in response to GH. In contrast, p85α+/–mice retained global insulin sensitivity and PI 3-kinase activity associated with reduced p85α expression. These findings demonstrated the importance of increased p85α in mediating skeletal muscle insulin resistance in response to GH and suggested a potential role for reducing p85α as a therapeutic strategy for enhancing insulin sensitivity in skeletal muscle.

CCAAT/enhancing binding protein β deletion in mice attenuates inflammation, endoplasmic reticulum stress, and lipid accumulation in diet-induced nonalcoholic steatohepatitis†

Nonalcoholic steatohepatitis (NASH) is characterized by steatosis, inflammation, and oxidative stress. To investigate whether the transcription factor CCAAT/Enhancer binding protein (C/EBPβ) is involved in the development of NASH, C57BL/6J wild‐type (WT) or C/EBPβ knockout (C/EBPβ−/−) mice were fed either a methionine and choline deficient (MCD) diet or standard chow. These WT mice fed a MCD diet for 4 weeks showed a 2‐ to 3‐fold increase in liver C/EBPβ messenger RNA and protein, along with increased expression of lipogenic genes peroxisome proliferators‐activated receptor γ and Fas. WT mice also showed increased levels of the endoplasmic reticulum stress pathway proteins phosphorylated eukaryotic translation initiation factor α, phosphorylated pancreatic endoplasmic reticulum kinase, and C/EBP homologous protein, along with inflammatory markers phosphorylated nuclear factor κB and phosphorylated C‐jun N‐terminal kinase compared to chow‐fed controls. Cytochrome P450 2E1 protein and acetyl coA oxidase messenger RNA involved in hepatic lipid peroxidation were also markedly increased in WT MCD diet‐fed group. In contrast, C/EBPβ−/− mice fed a MCD diet showed a 60% reduction in hepatic triglyceride accumulation and decreased liver injury as evidenced by reduced serum alanine aminotransferase and aspartate aminotransferase levels, and by H&E staining. Immunoblots and real‐time qPCR data revealed a significant reduction in expression of stress related proteins and lipogenic genes in MCD diet‐fed C/EBPβ−/− mice. Furthermore, circulating TNFα and expression of acute phase response proteins CRP and SAP were significantly lower in C/EBPβ−/− mice compared to WT mice. Conversely, C/EBPβ over‐expression in livers of WT mice increased steatosis, nuclear factor‐κB, and endoplasmic reticulum stress, similar to MCD diet‐fed mice. Conclusion: Taken together, these data suggest a previously unappreciated molecular link between C/EBPβ, hepatic steatosis and inflammation and suggest that increased C/EBPβ expression may be an important factor underlying events leading to NASH. (HEPATOLOGY 2007;45:1108–1117.)