Reproductive and immune effects emerge at similar thresholds of PFHxS in deer mice.

Although perfluorohexane sulfonate (PFHxS) is structurally similar to perfluorooctane sulfonate (PFOS) and also widely detected in humans and the environment, comparatively fewer toxicity data exists on this 6-chain perfluoroalkyl sulfonic acid. In this study, repeated oral doses of PFHxS were administered to deer mice (Peromyscus maniculatus) to evaluate subchronic toxicity and potential effects on reproduction and development. Maternal oral exposure to PFHxS caused increased stillbirths, which is relevant for ecological risk assessment, and resulted in a benchmark dose lower limit (BMDL) of 5.72 mg/kg-d PFHxS. Decreased plaque formation, which is relevant for human health risk assessment, occurred in both sexes of adult animals (BMDL = 8.79 mg/kg-d PFHxS). These data are the first to suggest a direct link between PFHxS and decreased functional immunity in an animal model. Additionally, female animals exhibited increased liver:body weight and animals of both sexes exhibited decreased serum thyroxine (T4) levels. Notably, since reproductive effects were used to support 2016 draft health advisories and immune effects were used in 2022 drinking water health advisories released by the United States Environmental Protection Agency for PFOS and perfluorooctanoic acid (PFOA), these novel data can potentially support advisories for PFHxS because relevant points of departure emerge at similar thresholds in a wild mammal and corroborate the general understanding of per- and polyfluoroalkyl substances (PFAS).

Potential chemical risks from tattoos and their relevance to military health policy in the United States.

We summarize and consolidate disparate sources of information about the practice of tattooing and its potential implications for military population health and policy. Each branch of the United States military has policies about tattoos for service members, but these have varied over time and do not cover health protection. The number of veterans receiving disability payments and the cost of those payments has been rising over time; the broad category of skin conditions accounts for 11% of disability claims. Any additional factor, such as tattoos that may increase the occurrence of adverse skin reactions, can substantially impact veteran benefit expenses and budgets. This may be a consideration for the military as it evaluates its policies related to tattoos among service members.

Next-generation PFAS 6:2 fluorotelomer sulfonate reduces plaque formation in exposed white-footed mice.

6:2 fluorotelomer sulfonate (6:2 FTS) has been used as a replacement for legacy per- and polyfluoroalkyl substances (PFAS). We assessed reproductive and developmental effects in a human-wildlife hybrid animal model based on the association of adverse effects linked to legacy PFAS with these sensitive life stages. In this study, white-footed mice were exposed orally to 0, 0.2, 1, 5, or 25 mg/kg-day 6:2 FTS for 112 days (4 weeks premating exposure plus at least 4 weeks mating exposure). Pregnancy and fertility indices were calculated, and litter production, total litter size, live litter size, stillbirths, litter loss, average pup weight, and pinna unfolding were assessed. Sex steroid and thyroid hormone serum levels were assessed. Body weight, histopathology, and immune function were also assessed in this study. Reproductive endpoints were not significantly altered in response to 6:2 FTS. Spleen weight increased in male mice dosed with 6:2 FTS. Immune function determined via a plaque-forming cell (PFC) assay was decreased in both male and female mice in the 2 highest doses. A low benchmark dose was calculated based on PFCs as the critical effect and was found to be 2.63 and 2.26 mg/kg-day 6:2 FTS in male and female mice, respectively. This study characterizes 6:2 FTS as being potentially immunotoxic with little evidence of effect on reproduction and development; furthermore, it models acceptable levels of exposure. These 2 pieces of information together will aid regulators in setting environmental exposure limits for this PFAS currently thought to be less toxic than other PFAS.

Macrophages augment the skeletal muscle proinflammatory response through TNFα following LPS-induced acute lung injury.

Muscle may contribute to the systemic inflammatory environment during critical illness, but leukocyte interaction and cytokine influence on muscle and its response has not been fully explored in this context. Using an in vivo model of intratracheal lipopolysaccharide (IT LPS)-induced acute lung injury, we show that skeletal muscle rapidly responds with expression of proinflammatory genes, which may be explained by migration of LPS into the circulation. Treatment of mature C2C12 myotubes with LPS at a level achieved in the circulation following IT LPS elicited a proinflammatory cytokine expression profile similar to that of in vivo murine muscle following IT LPS. Stimulation with toll-like receptor (TLR) 2 and 3 agonists provoked comparable responses in C2C12 myotubes. Additionally, co-cultures of C2C12 myotubes and bone marrow-derived macrophages (BMDM) identified the capacity of macrophages to increase myotube proinflammatory gene expression, with tumor necrosis factor-α (TNFα) gene and protein expression largely attributable to BMDM. To investigate the contribution of TNFα in the synergy of the co-culture environment, C2C12 myotubes were treated with recombinant TNFα, co-cultures were established using TNF-deficient BMDM, and co-cultures were also depleted of TNFα using antibodies. To determine whether the in vitro observations were relevant in vivo, mice received intramuscular administration of LPS ± TNFα or TNFα-neutralizing antibodies and showed that TNFα is both sufficient and necessary to induce synergistic cytokine release from muscle. Taken together, these data demonstrate how skeletal muscle tissue may contribute proinflammatory cytokines following acute endotoxin injury and the potential of leukocytes to augment this response via TNFα secretion.

Effect of neuromuscular electrical stimulation on skeletal muscle size and function in patients with breast cancer receiving chemotherapy.

Exercise has numerous benefits for patients with cancer, but implementation is challenging because of practical and logistical hurdles. This study examined whether neuromuscular electrical stimulation (NMES) can serve as a surrogate for classic exercise by eliciting an exercise training response in skeletal muscle of women diagnosed with breast cancer undergoing chemotherapy. Patients (n = 22) with histologically confirmed stage I, II, or III breast cancer scheduled to receive neoadjuvant or adjuvant chemotherapy were randomized to 8 wk of bilateral neuromuscular electrical stimulation (NMES; 5 days/wk) to their quadriceps muscles or control. Biopsy of the vastus lateralis was performed at baseline and after 8 wk of intervention to assess muscle fiber size, contractility, and mitochondrial content. Seventeen patients (8 control/9 NMES) completed the trial and were included in analyses. NMES promoted muscle fiber hypertrophy (P < 0.001), particularly in fast-twitch, myosin heavy chain (MHC) IIA fibers (P < 0.05) and tended to induce fiber type shifts in MHC II fibers. The effects of NMES on single-muscle fiber contractility were modest, and it was unable to prevent declines in the function in MHC IIA fibers. NMES did not alter intermyofibrillar mitochondrial content/structure but was associated with reductions in subsarcolemmal mitochondria. Our results demonstrate that NMES induces muscle fiber hypertrophy and fiber type shifts in MHC II fibers but had minimal effects on fiber contractility and promoted reductions in subsarcolemmal mitochondria. Further studies are warranted to evaluate the utility of NMES as an exercise surrogate in cancer patients and other conditions.NEW & NOTEWORTHY This is the first study to evaluate whether neuromuscular electrical stimulation (NMES) can be used as an exercise surrogate to improve skeletal muscle fiber size or function in cancer patients receiving treatment. We show that NMES promoted muscle fiber hypertrophy and fiber type shifts but had minimal effects on single-fiber contractility and reduced subsarcolemmal mitochondria.

Effects of conditioned media from murine lung cancer cells and human tumor cells on cultured myotubes.

Factors secreted from tumors/tumor cells are hypothesized to cause skeletal muscle wasting in cancer patients. We examined whether cancer cells secrete factors to promote atrophy by evaluating the effects of conditioned media (CM) from murine lung cancer cells and primary cultures of human lung tumor cells on cultured myotubes. We evaluated murine Lewis lung carcinoma (LLC) and KRASG12D cells, and primary cell lines derived from tumor biopsies from patients with lung cancer (hTCM; n = 6). In all experiments, serum content was matched across treatment groups. We hypothesized that CM from murine and human tumor cells would reduce myotube myosin content, decrease mitochondrial content, and increase mitochondrial reactive oxygen species (ROS) production. Treatment of myotubes differentiated for 7 days with CM from LLC and KRASG12D cells did not alter any of these variables. Effects of murine tumor cell CM were observed when myotubes differentiated for 4 days were treated with tumor cell CM and compared with undiluted differentiation media. However, these effects were not apparent if tumor cell CM treatments were compared with control cell CM or dilution controls. Finally, CM from human lung tumor primary cell lines did not modify myosin content or mitochondrial content or ROS production compared with either undiluted differentiated media, control cell CM, or dilution controls. Our results do not support the hypothesis that factors released from cultured lung cancer/tumor cells promote myotube wasting or mitochondrial abnormalities, but we cannot dismiss the possibility that these cells could secrete such factors in vivo within the native tumor microenvironment.

Electrical stimulation prevents doxorubicin-induced atrophy and mitochondrial loss in cultured myotubes.

Muscle contraction may protect against the effects of chemotherapy to cause skeletal muscle atrophy, but the mechanisms underlying these benefits are unclear. To address this question, we utilized in vitro modeling of contraction and mechanotransduction in C2C12 myotubes treated with doxorubicin (DOX; 0.2 µM for 3 days). Myotubes expressed contractile proteins and organized these into functional myofilaments, as electrical field stimulation (STIM) induced intracellular calcium (Ca2+) transients and contractions, both of which were prevented by inhibition of membrane depolarization. DOX treatment reduced myotube myosin content, protein synthesis, and Akt (S308) and forkhead box O3a (FoxO3a; S253) phosphorylation and increased muscle RING finger 1 (MuRF1) expression. STIM (1 h/day) prevented DOX-induced reductions in myotube myosin content and Akt and FoxO3a phosphorylation, as well as increases in MuRF1 expression, but did not prevent DOX-induced reductions in protein synthesis. Inhibition of myosin-actin interaction during STIM prevented contraction and the antiatrophic effects of STIM without affecting Ca2+ cycling, suggesting that the beneficial effect of STIM derives from mechanotransductive pathways. Further supporting this conclusion, mechanical stretch of myotubes recapitulated the effects of STIM to prevent DOX suppression of FoxO3a phosphorylation and upregulation of MuRF1. DOX also increased reactive oxygen species (ROS) production, which led to a decrease in mitochondrial content. Although STIM did not alter DOX-induced ROS production, peroxisome proliferator-activated receptor-γ coactivator-1α and antioxidant enzyme expression were upregulated, and mitochondrial loss was prevented. Our results suggest that the activation of mechanotransductive pathways that downregulate proteolysis and preserve mitochondrial content protects against the atrophic effects of chemotherapeutics.

Skeletal muscle atrophy and dysfunction in breast cancer patients: role for chemotherapy-derived oxidant stress.

How breast cancer and its treatments affect skeletal muscle is not well defined. To address this question, we assessed skeletal muscle structure and protein expression in 13 women who were diagnosed with breast cancer and receiving adjuvant chemotherapy following tumor resection and 12 nondiseased controls. Breast cancer patients showed reduced single-muscle fiber cross-sectional area and fractional content of subsarcolemmal and intermyofibrillar mitochondria. Drugs commonly used in breast cancer patients (doxorubicin and paclitaxel) caused reductions in myosin expression, mitochondrial loss, and increased reactive oxygen species (ROS) production in C2C12 murine myotube cell cultures, supporting a role for chemotherapeutics in the atrophic and mitochondrial phenotypes. Additionally, concurrent treatment of myotubes with the mitochondrial-targeted antioxidant MitoQ prevented chemotherapy-induced myosin depletion, mitochondrial loss, and ROS production. In patients, reduced mitochondrial content and size and increased expression and oxidation of peroxiredoxin 3, a mitochondrial peroxidase, were associated with reduced muscle fiber cross-sectional area. Our results suggest that chemotherapeutics may adversely affect skeletal muscle in patients and that these effects may be driven through effects of these drugs on mitochondrial content and/or ROS production.

Saturated and unsaturated fat induce hepatic insulin resistance independently of TLR-4 signaling and ceramide synthesis in vivo.

Hepatic insulin resistance is a principal component of type 2 diabetes, but the cellular and molecular mechanisms responsible for its pathogenesis remain unknown. Recent studies have suggested that saturated fatty acids induce hepatic insulin resistance through activation of the toll-like receptor 4 (TLR-4) receptor in the liver, which in turn transcriptionally activates hepatic ceramide synthesis leading to inhibition of insulin signaling. In this study, we demonstrate that TLR-4 receptor signaling is not directly required for saturated or unsaturated fat-induced hepatic insulin resistance in both TLR-4 antisense oligonucleotide treated and TLR-4 knockout mice, and that ceramide accumulation is not dependent on TLR-4 signaling or a primary event in hepatic steatosis and impairment of insulin signaling. Further, we show that both saturated and unsaturated fats lead to hepatic accumulation of diacylglycerols, activation of PKCε, and impairment of insulin-stimulated IRS-2 signaling. These data demonstrate that saturated fat-induced insulin resistance is independent of TLR-4 activation and ceramides.

Cellular mechanisms by which FGF21 improves insulin sensitivity in male mice.

Fibroblast growth factor 21 (FGF21) is a potent regulator of glucose and lipid metabolism and is currently being pursued as a therapeutic agent for insulin resistance and type 2 diabetes. However, the cellular mechanisms by which FGF21 modifies insulin action in vivo are unclear. To address this question, we assessed insulin action in regular chow- and high-fat diet (HFD)-fed wild-type mice chronically infused with FGF21 or vehicle. Here, we show that FGF21 administration results in improvements in both hepatic and peripheral insulin sensitivity in both regular chow- and HFD-fed mice. This improvement in insulin responsiveness in FGF21-treated HFD-fed mice was associated with decreased hepatocellular and myocellular diacylglycerol content and reduced protein kinase Cε activation in liver and protein kinase Cθ in skeletal muscle. In contrast, there were no effects of FGF21 on liver or muscle ceramide content. These effects may be attributed, in part, to increased energy expenditure in the liver and white adipose tissue. Taken together, these data provide a mechanism by which FGF21 protects mice from lipid-induced liver and muscle insulin resistance and support its development as a novel therapy for the treatment of nonalcoholic fatty liver disease, insulin resistance, and type 2 diabetes.

Cellular mechanism by which estradiol protects female ovariectomized mice from high-fat diet-induced hepatic and muscle insulin resistance.

Estrogen replacement therapy reduces the incidence of type 2 diabetes in postmenopausal women; however, the mechanism is unknown. Therefore, the aim of this study was to evaluate the metabolic effects of estrogen replacement therapy in an experimental model of menopause. At 8 weeks of age, female mice were ovariectomized (OVX) or sham (SHAM) operated, and OVX mice were treated with vehicle (OVX) or estradiol (E2) (OVX+E2). After 4 weeks of high-fat diet feeding, OVX mice had increased body weight and fat mass compared with SHAM and OVX+E2 mice. OVX mice displayed reduced whole-body energy expenditure, as well as impaired glucose tolerance and whole-body insulin resistance. Differences in whole-body insulin sensitivity in OVX compared with SHAM mice were accounted for by impaired muscle insulin sensitivity, whereas both hepatic and muscle insulin sensitivity were impaired in OVX compared with OVX+E2 mice. Muscle diacylglycerol (DAG), content in OVX mice was increased relative to SHAM and OVX+E2 mice. In contrast, E2 treatment prevented the increase in hepatic DAG content observed in both SHAM and OVX mice. Increases in tissue DAG content were associated with increased protein kinase Cε activation in liver of SHAM and OVX mice compared with OVX+E2 and protein kinase Cθ activation in skeletal muscle of OVX mice compared with SHAM and OVX+E2. Taken together, these data demonstrate that E2 plays a pivotal role in the regulation of whole-body energy homeostasis, increasing O(2) consumption and energy expenditure in OVX mice, and in turn preventing diet-induced ectopic lipid (DAG) deposition and hepatic and muscle insulin resistance.

CGI-58 knockdown sequesters diacylglycerols in lipid droplets/ER-preventing diacylglycerol-mediated hepatic insulin resistance.

Comparative gene identification 58 (CGI-58) is a lipid droplet-associated protein that promotes the hydrolysis of triglyceride by activating adipose triglyceride lipase. Loss-of-function mutations in CGI-58 in humans lead to Chanarin-Dorfman syndrome, a condition in which triglyceride accumulates in various tissues, including the skin, liver, muscle, and intestines. Therefore, without adequate CGI-58 expression, lipids are stored rather than used for fuel, signaling intermediates, and membrane biosynthesis. CGI-58 knockdown in mice using antisense oligonucleotide (ASO) treatment also leads to severe hepatic steatosis as well as increased hepatocellular diacylglycerol (DAG) content, a well-documented trigger of insulin resistance. Surprisingly, CGI-58 knockdown mice remain insulin-sensitive, seemingly dissociating DAG from the development of insulin resistance. Therefore, we sought to determine the mechanism responsible for this paradox. Hyperinsulinemic-euglycemic clamp studies reveal that the maintenance of insulin sensitivity with CGI-58 ASO treatment could entirely be attributed to protection from lipid-induced hepatic insulin resistance, despite the apparent lipotoxic conditions. Analysis of the cellular compartmentation of DAG revealed that DAG increased in the membrane fraction of high fat-fed mice, leading to PKCε activation and hepatic insulin resistance. However, DAG increased in lipid droplets or lipid-associated endoplasmic reticulum rather than the membrane of CGI-58 ASO-treated mice, and thus prevented PKCε translocation to the plasma membrane and induction of insulin resistance. Taken together, these results explain the disassociation of hepatic steatosis and DAG accumulation from hepatic insulin resistance in CGI-58 ASO-treated mice, and highlight the importance of intracellular compartmentation of DAG in causing lipotoxicity and hepatic insulin resistance.

Fatty acid amide hydrolase ablation promotes ectopic lipid storage and insulin resistance due to centrally mediated hypothyroidism.

Fatty acid amide hydrolase (FAAH) knockout mice are prone to excess energy storage and adiposity, whereas mutations in FAAH are associated with obesity in humans. However, the molecular mechanism by which FAAH affects energy expenditure (EE) remains unknown. Here we show that reduced energy expenditure in FAAH(-/-) mice could be attributed to decreased circulating triiodothyronine and thyroxine concentrations secondary to reduced mRNA expression of both pituitary thyroid-stimulating hormone and hypothalamic thyrotropin-releasing hormone. These reductions in the hypothalamic-pituitary-thyroid axis were associated with activation of hypothalamic peroxisome proliferating-activated receptor γ (PPARγ), and increased hypothalamic deiodinase 2 expression. Infusion of NAEs (anandamide and palmitoylethanolamide) recapitulated increases in PPARγ-mediated decreases in EE. FAAH(-/-) mice were also prone to diet-induced hepatic insulin resistance, which could be attributed to increased hepatic diacylglycerol content and protein kinase Cε activation. Our data indicate that FAAH deletion, and the resulting increases in NAEs, predispose mice to ectopic lipid storage and hepatic insulin resistance by promoting centrally mediated hypothyroidism.

Dissociation of inositol-requiring enzyme (IRE1α)-mediated c-Jun N-terminal kinase activation from hepatic insulin resistance in conditional X-box-binding protein-1 (XBP1) knock-out mice.

Hepatic insulin resistance has been attributed to both increased endoplasmic reticulum (ER) stress and accumulation of intracellular lipids, specifically diacylglycerol (DAG). The ER stress response protein, X-box-binding protein-1 (XBP1), was recently shown to regulate hepatic lipogenesis, suggesting that hepatic insulin resistance in models of ER stress may result from defective lipid storage, as opposed to ER-specific stress signals. Studies were designed to dissociate liver lipid accumulation and activation of ER stress signaling pathways, which would allow us to delineate the individual contributions of ER stress and hepatic lipid content to the pathogenesis of hepatic insulin resistance. Conditional XBP1 knock-out (XBP1Δ) and control mice were fed fructose chow for 1 week. Determinants of whole-body energy balance, weight, and composition were determined. Hepatic lipids including triglyceride, DAGs, and ceramide were measured, alongside markers of ER stress. Whole-body and tissue-specific insulin sensitivity were determined by hyperinsulinemic-euglycemic clamp studies. Hepatic ER stress signaling was increased in fructose chow-fed XBP1Δ mice as reflected by increased phosphorylated eIF2α, HSPA5 mRNA, and a 2-fold increase in hepatic JNK activity. Despite JNK activation, XBP1Δ displayed increased hepatic insulin sensitivity during hyperinsulinemic-euglycemic clamp studies, which was associated with increased insulin-stimulated IRS2 tyrosine phosphorylation, reduced hepatic DAG content, and reduced PKCε activity. These studies demonstrate that ER stress and IRE1α-mediated JNK activation can be disassociated from hepatic insulin resistance and support the hypothesis that hepatic insulin resistance in models of ER stress may be secondary to ER stress modulation of hepatic lipogenesis.

Thyroid hormone receptor-α gene knockout mice are protected from diet-induced hepatic insulin resistance.

Nonalcoholic fatty liver disease (NAFLD) is the most frequent chronic liver disease in the United States and is strongly associated with hepatic insulin resistance. We examined whether the thyroid hormone receptor-α (Thra) would be a potential therapeutic target to prevent diet-induced NAFLD and insulin resistance. For that purpose, we assessed insulin action in high-fat diet-fed Thra gene knockout (Thra-0/0) and wild-type mice using hyperinsulinemic-euglycemic clamps combined with (3)H/(14)C-labeled glucose to assess basal and insulin-stimulated rates of glucose and fat metabolism. Body composition was assessed by (1)H magnetic resonance spectroscopy and energy expenditure by indirect calorimetry. Relative rates of hepatic glucose and fat oxidation were assessed in vivo using a novel proton-observed carbon-edited nuclear magnetic resonance technique. Thra-0/0 were lighter, leaner, and manifested greater whole-body insulin sensitivity than wild-type mice during the clamp, which could be attributed to increased insulin sensitivity both in liver and peripheral tissues. Increased hepatic insulin sensitivity could be attributed to decreased hepatic diacylglycerol content, resulting in decreased activation of protein kinase Cε and increased insulin signaling. In conclusion, loss of Thra protects mice from high-fat diet-induced hepatic steatosis and hepatic and peripheral insulin resistance. Therefore, thyroid receptor-α inhibition represents a novel pharmacologic target for the treatment of NAFLD, obesity, and type 2 diabetes.

Impact of CD1d deficiency on metabolism.

Invariant natural killer T cells (iNKTs) are innate-like T cells that are highly concentrated in the liver and recognize lipids presented on the MHC-like molecule CD1d. Although capable of a myriad of responses, few essential functions have been described for iNKTs. Among the many cell types of the immune system implicated in metabolic control and disease, iNKTs seem ideally poised for such a role, yet little has been done to elucidate such a possible function. We hypothesized that lipid presentation by CD1d could report on metabolic status and engage iNKTs to regulate cellular lipid content through their various effector mechanisms. To test this hypothesis, we examined CD1d deficient mice in a variety of metabolically stressed paradigms including high fat feeding, choline-deficient feeding, fasting, and acute inflammation. CD1d deficiency led to a mild exacerbation of steatosis during high fat or choline-deficient feeding, accompanied by impaired hepatic glucose tolerance. Surprisingly, however, this phenotype was not observed in Jα18⁻/⁻ mice, which are deficient in iNKTs but express CD1d. Thus, CD1d appears to modulate some metabolic functions through an iNKT-independent mechanism.

Hepatic insulin resistance in mice with hepatic overexpression of diacylglycerol acyltransferase 2.

Mice overexpressing acylCoA:diacylglycerol (DAG) acyltransferase 2 in the liver (Liv-DGAT2) have been shown to have normal hepatic insulin responsiveness despite severe hepatic steatosis and increased hepatic triglyceride, diacylglycerol, and ceramide content, demonstrating a dissociation between hepatic steatosis and hepatic insulin resistance. This led us to reevaluate the role of DAG in causing hepatic insulin resistance in this mouse model of severe hepatic steatosis. Using hyperinsulinemic-euglycemic clamps, we studied insulin action in Liv-DGAT2 mice and their wild-type (WT) littermate controls. Here, we show that Liv-DGAT2 mice manifest severe hepatic insulin resistance as reflected by decreased suppression of endogenous glucose production (0.8 ± 41.8 vs. 87.7 ± 34.3% in WT mice, P < 0.01) during the clamps. Hepatic insulin resistance could be attributed to an almost 12-fold increase in hepatic DAG content (P < 0.01) resulting in a 3.6-fold increase in protein kinase Cε (PKCε) activation (P < 0.01) and a subsequent 52% decrease in insulin-stimulated insulin receptor substrate 2 (IRS-2) tyrosine phosphorylation (P < 0.05), as well as a 64% decrease in fold increase pAkt/Akt ratio from basal conditions (P < 0.01). In contrast, hepatic insulin resistance in these mice was not associated with increased endoplasmic reticulum (ER) stress or inflammation. Importantly, hepatic insulin resistance in Liv-DGAT2 mice was independent of differences in body composition, energy expenditure, or food intake. In conclusion, these findings strengthen the link between hepatic steatosis and hepatic insulin resistance and support the hypothesis that DAG-induced PKCε activation plays a major role in nonalcoholic fatty liver disease (NAFLD)-associated hepatic insulin resistance.