Development Of Hepatic Insulin Resistance Research Articles

Mitochondria-associated membranes contribution to exercise-mediated alleviation of hepatic insulin resistance: Contrasting high-intensity interval training with moderate-intensity continuous training in a high-fat diet mouse model.

Mitochondria-associated membranes (MAMs) serve pivotal functions in hepatic insulin resistance (IR). Our aim was to explore the potential role of MAMs in mitigating hepatic IR through exercise and to compare the effects of different intensities of exercise on hepatic MAMs formation in high-fat diet (HFD) mice. Male C57BL/6J mice were fed an HFD and randomly assigned to undergo supervised high-intensity interval training (HIIT) or moderate-intensity continuous training (MICT). IR was evaluated using the serum triglyceride/high-density lipoprotein cholesterol ratio (TG/HDL-C), glucose tolerance test (GTT), and insulin tolerance test (ITT). Hepatic steatosis was observed using hematoxylin-eosin (H&E) and oil red O staining. The phosphatidylinositol 3-kinase/protein kinase B/glycogen synthase kinase 3 beta (PI3K-AKT-GSK3β) signaling pathway was assessed to determine hepatic IR. MAMs were evaluated through immunofluorescence (colocalization of voltage-dependent anion-selective channel 1 [VDAC1] and inositol 1,4,5-triphosphate receptor [IP3R]). After 8 weeks on an HFD, there was notable inhibition of the hepatic PI3K/Akt/GSK3β signaling pathway, accompanied by a marked reduction in hepatic IP3R-VDAC1 colocalization levels. Both 8-week HIIT and MICT significantly enhanced the hepatic PI3K/Akt/GSK3β signaling and colocalization levels of IP3R-VDAC1 in HFD mice, with MICT exhibiting a stronger effect on hepatic MAMs formation. Furthermore, the colocalization of hepatic IP3R-VDAC1 positively correlated with the expression levels of phosphorylation of protein kinase B (p-AKT) and phosphorylation of glycogen synthase kinase 3 beta (p-GSK3β), while displaying a negative correlation with serum triglyceride/high-density lipoprotein cholesterol levels. The reduction in hepatic MAMs formation induced by HFD correlates with the development of hepatic IR. Both HIIT and MICT effectively bolster hepatic MAMs formation in HFD mice, with MICT demonstrating superior efficacy. Thus, MAMs might wield a pivotal role in exercise-induced alleviation of hepatic IR.

The Role of Acyl-CoA Synthetase 1 in Bioactive Lipid Accumulation and the Development of Hepatic Insulin Resistance.

The liver plays a crucial role in glucose metabolism. Obesity and a diet rich in fats (HFD) contribute to the accumulation of intracellular lipids. The aim of the study was to explore the involvement of acyl-CoA synthetase 1 (ACSL1) in bioactive lipid accumulation and the induction of liver insulin resistance (InsR) in animals fed an HFD. The experiments were performed on male C57BL/6 mice divided into the following experimental groups: 1. Animals fed a control diet; 2. animals fed HFD; and 3. HFD-fed animals with the hepatic ACSL1 gene silenced through a hydrodynamic gene delivery technique. Long-chain acyl-CoAs, sphingolipids, and diacylglycerols were measured by LC/MS/MS. Glycogen was measured by means of a commercially available kit. The protein expression and phosphorylation state of the insulin pathway was estimated by Western blot. HFD-fed mice developed InsR, manifested as an increase in fasting blood glucose levels (202.5 mg/dL vs. 130.5 mg/dL in the control group) and inhibition of the insulin pathway, which resulted in an increase in the rate of gluconeogenesis (0.420 vs. 0.208 in the control group) and a decrease in the hepatic glycogen content (1.17 μg/mg vs. 2.32 μg/mg in the control group). Hepatic ACSL1 silencing resulted in decreased lipid content and improved insulin sensitivity, accounting for the decreased rate of gluconeogenesis (0.348 vs. 0.420 in HFD(+/+)) and the increased glycogen content (4.3 μg/mg vs. 1.17 μg/mg in HFD(+/+)). The elevation of gluconeogenesis and the decrease in glycogenesis in the hepatic tissue of HFD-fed mice resulted from cellular lipid accumulation. Inhibition of lipid synthesis through silencing ACSL1 alleviated HFD-induced hepatic InsR.

From cause to consequence: Insights into incident diabetes mellitus after hepatic steatosis

From cause to consequence: Insights into incident diabetes mellitus after hepatic steatosis

MiR-183-5p Induced by Saturated Fatty Acids Hinders Insulin Signaling by Downregulating IRS-1 in Hepatocytes.

Excessive saturated fatty acids (SFA) uptake is known to be a primary cause of obesity, a widely acknowledged risk factor of insulin resistance and type 2 diabetes. Although specific microRNAs (miRNAs) targeting insulin signaling intermediates are dysregulated by SFA, their effects on insulin signaling and sensitivity are largely unknown. Here, we investigated the role of SFA-induced miR-183-5p in the regulation of proximal insulin signaling molecules and the development of hepatic insulin resistance. HepG2 hepatocytes treated with palmitate and the livers of high-fat diet (HFD)-fed mice exhibited impaired insulin signaling resulting from dramatic reductions in the protein expressions of insulin receptor (INSR) and insulin receptor substrate-1 (IRS-1). Differential expression analysis showed the level of miR-183-5p, which tentatively targets the 3′UTR of IRS-1, was significantly elevated in palmitate-treated HepG2 hepatocytes and the livers of HFD-fed mice. Dual-luciferase analysis showed miR-183-5p bound directly to the 3′UTR of IRS-1 and reduced IRS-1 expression at the post-transcriptional stage. Moreover, transfection of HepG2 hepatocytes with miR-183-5p mimic significantly inhibited IRS-1 expression and hindered insulin signaling, consequently inhibiting insulin-stimulated glycogen synthesis. Collectively, this study reveals a novel mechanism whereby miR-183-5p induction by SFA impairs insulin signaling and suggests miR-183-5p plays a crucial role in the pathogenesis of hepatic insulin resistance in the background of obesity.

Investigation of insulin resistance through a multiorgan microfluidic organ-on-chip

The development of hepatic insulin resistance (IR) is a critical factor in developing type 2 diabetes (T2D), where insulin fails to inhibit hepatic glucose production but retains its capacity to promote hepatic de novo lipogenesis leading to hyperglycemia and hypertriglyceridemia. Improving insulin sensitivity can be effective in preventing and treating T2D. However, selective control of glucose and lipid synthesis has been difficult. It is known that excess white adipose tissue is detrimental to insulin sensitivity, whereas brown adipose tissue transplantation can restore it in diabetic mice. However, challenges remain in our understanding of liver-adipose communication because the confounding effects of hypothalamic regulation of metabolic function cannot be ruled out in previous studies. There is a lack of in vitro models that use primary cells to study cellular-crosstalk under insulin resistant conditions. Building upon our previous work on the microfluidic primary liver and adipose organ-on-chips, we report for the first time, the development of an integrated insulin resistant liver-adipose (white and brown) organ-on-chip. The design of the microfluidic device was carried out using computational fluid dynamics; the experimental studies were conducted by carrying out detailed biochemical analysis RNA-seq analysis on both cell types. Further, we tested the hypothesis that brown adipocytes (BAC) regulated both hepatic insulin sensitivity and de novo lipogenesis. Our results show that BAC effectively restored insulin sensitivity and supressed hepatic glucose production and de novo lipogenesis suggesting that the experimental platform could be useful for identifying potential therapeutics to treat IR and diabetes.

Hypothyroidism-Associated Dyslipidemia: Potential Molecular Mechanisms Leading to NAFLD.

Thyroid hormones control lipid metabolism by exhibiting specific effects on the liver and adipose tissue in a coordinated manner. Different diseases of the thyroid gland can result in hypothyroidism. Hypothyroidism is frequently associated with dyslipidemia. Hypothyroidism-associated dyslipidemia subsequently results in intrahepatic accumulation of fat, leading to nonalcoholic fatty liver disease (NAFLD), which leads to the development of hepatic insulin resistance. The prevalence of NAFLD in the western world is increasing, and evidence of its association with hypothyroidism is accumulating. Since hypothyroidism has been identified as a modifiable risk factor of NAFLD and recent data provides evidence that selective thyroid hormone receptor β (THR-β) agonists are effective in the treatment of dyslipidemia and NAFLD, interest in potential therapeutic options for NAFLD targeting these receptors is growing. In this review, we summarize current knowledge regarding clinical and molecular data exploring the association of hypothyroidism, dyslipidemia and NAFLD.

Dietary induction of obesity and insulin resistance is associated with changes in Fgf21 DNA methylation in liver of mice

DNA methylation is dynamically regulated in metabolic diseases, but it remains unclear whether the changes are causal or consequential. Therefore, we used a longitudinal approach to refine the onset of metabolic and DNA methylation changes at high temporal resolution. Male C57BL/6N mice were fed with 60 % high-fat diet (HFD) for up to 12 weeks and metabolically characterized weekly. Liver was collected after 1, 2, 4, 5, 6, 7, 8, and 12 weeks and hepatic DNA methylation and gene expression were analyzed. A subset of obese mice underwent vertical sleeve gastrectomy (VSG) or metformin treatment and livers were studied. Distinct hepatic gene expression patterns developed upon feeding HFD, with genes from the fatty acid metabolism pathway being predominantly altered. When comparing metabolic data with gene expression and DNA methylation, in particular Fgf21 DNA methylation decreased before the onset of increased Fgf21 expression and metabolic changes. Neither weight loss induced by VSG nor improved glucose tolerance by metformin treatment could revert hepatic Fgf21 DNA methylation or expression. Our data emphasize the dynamic induction of DNA methylation upon metabolic stimuli. Reduced Fgf21 DNA methylation established before massive overexpression of Fgf21, which is likely an adaptive effort of the liver to maintain glucose homeostasis despite the developing insulin resistance and steatosis. Fgf21 DNA methylation resisted reversion by intervention strategies, illustrating the long-term effects of unhealthy lifestyle. Our data provide a temporal roadmap to the development of hepatic insulin resistance, comprehensively linking DNA methylation with gene expression and metabolic data.

Denosumab improves glucose parameters in patients with impaired glucose tolerance: a systematic review and meta-analysis

Objective Receptor activator of NF-κβ ligand (RANKL) is crucial for the development of hepatic insulin resistance and poor glucose uptake; therefore, inhibiting RANKL with Denosumab could improve fasting plasma glucose (FPG) and insulin (FPI). Methods A systematic review was conducted to evaluate the effects of Denosumab on glycemic parameters. PubMed, SCOPUS, EBSCO, and LILACS databases were searched for studies that investigated the effect of Denosumab on FPG, glycated hemoglobin (HbA1c), FPI, and Homeostatic Model Assessment for Insulin Resistance (HOMA1-IR). The pooled standard difference in means (SDM) and 95% confidence intervals (95%CI) were calculated. The results were stratified into (1) Normal Glucose Tolerance (NGT) and (2) Impaired Glucose Tolerance (IGT). Results Six publications (1203 participants) were included. There was a significant association between Denosumab and FPG (SDM = −0.388, 95%CI: −0.705 to −0.070, p = .017) and with HOMA1-IR (SDM = −0.223, 95%CI: −0.388 to −0.058, p = .008), but not for HbA1c and FPI. When stratified by glucose tolerance, the association between Denosumab and FPG, HbA1c, and HOMA1-IR was present for the IGT group. Lastly, Denosumab had a time-dependent effect on HbA1c (slope = −0.037, 95%CI: −0.059 to −0.015, p < .005). Conclusions Denosumab significantly improved glycemic parameters. This outcome was more prominent for subjects with compromised glucose tolerance, positing that Denosumab can be used as a treatment to improve glucose metabolism for persons with pre-diabetes and diabetes.

ABCB10 exports mitochondrial biliverdin, driving metabolic maladaptation in obesity.

Although the role of hydrophilic antioxidants in the development of hepatic insulin resistance and nonalcoholic fatty liver disease has been well studied, the role of lipophilic antioxidants remains poorly characterized. A known lipophilic hydrogen peroxide scavenger is bilirubin, which can be oxidized to biliverdin and then reduced back to bilirubin by cytosolic biliverdin reductase. Oxidation of bilirubin to biliverdin inside mitochondria must be followed by the export of biliverdin to the cytosol, where biliverdin is reduced back to bilirubin. Thus, the putative mitochondrial exporter of biliverdin is expected to be a major determinant of bilirubin regeneration and intracellular hydrogen peroxide scavenging. Here, we identified ABCB10 as a mitochondrial biliverdin exporter. ABCB10 reconstituted into liposomes transported biliverdin, and ABCB10 deletion caused accumulation of biliverdin inside mitochondria. Obesity with insulin resistance up-regulated hepatic ABCB10 expression in mice and elevated cytosolic and mitochondrial bilirubin content in an ABCB10-dependent manner. Revealing a maladaptive role of ABCB10-driven bilirubin synthesis, hepatic ABCB10 deletion protected diet-induced obese mice from steatosis and hyperglycemia, improving insulin-mediated suppression of glucose production and decreasing lipogenic SREBP-1c expression. Protection was concurrent with enhanced mitochondrial function and increased inactivation of PTP1B, a phosphatase disrupting insulin signaling and elevating SREBP-1c expression. Restoration of cellular bilirubin content in ABCB10 KO hepatocytes reversed the improvements in mitochondrial function and PTP1B inactivation, demonstrating that bilirubin was the maladaptive effector linked to ABCB10 function. Thus, we identified a fundamental transport process that amplifies intracellular bilirubin redox actions, which can exacerbate insulin resistance and steatosis in obesity.

Role of Chitinase 3-Like 1 Protein in the Pathogenesis of Hepatic Insulin Resistance in Nonalcoholic Fatty Liver Disease.

A recently discovered human glycoprotein, chitinase 3-like 1 (Chi3L1), may play a role in inflammation, tissue remodeling, and visceral fat accumulation. We hypothesize that Chi3L1 gene expression is important in the development of hepatic insulin resistance characterized by the generation of pAKT, pGSK, and pERK in wild type and Chi3L1 knockout (KO) murine liver following insulin stimulation. The Chi3L1 gene and protein expression was evaluated by Real Time PCR and ELISA; lipid accumulation in hepatocytes was also assessed. To alter Chi3L1 function, three different anti-Chi3L1 monoclonal antibodies (mAbs) were administered in vivo and effects on the insulin signaling cascade and hepatic lipid deposition were determined. Transmission of the hepatic insulin signal was substantially improved following KO of the CHi3L1 gene and there was reduced lipid deposition produced by a HFD. The HFD-fed mice exhibited increased Chi3L1 expression in the liver and there was impaired insulin signal transduction. All three anti-Chi3L1 mAbs partially restored hepatic insulin sensitivity which was associated with reduced lipid accumulation in hepatocytes as well. A KO of the Chi3L1 gene reduced lipid accumulation and improved insulin signaling. Therefore, Chi3L1 gene upregulation may be an important factor in the generation of NAFLD/NASH phenotype.

Hepatic nNOS impaired hepatic insulin sensitivity through the activation of p38 MAPK.

Neuronal nitric oxide synthase (nNOS) interacts with its adaptor protein NOS1AP through its PZD domain in the neurons. Previously, we had reported that NOS1AP enhanced hepatic insulin sensitivity through its PZD-binding domain, which suggested that nNOS might mediate the effect of NOS1AP. This study aimed to examine the role and underlying mechanisms of nNOS in regulating hepatic insulin sensitivity. nNOS co-localized with NOS1AP in mouse liver. The overexpression of NOS1AP in mouse liver decreased the level of phosphorylated nNOS (p-nNOS (Ser1417)), the active form of nNOS. Conversely, the liver-specific deletion of NOS1AP increased the level of p-nNOS (Ser1417). The overexpression of nNOS in the liver of high-fat diet-induced obese mice exacerbated glucose intolerance, enhanced intrahepatic lipid accumulation, decreased glycogen storage, and blunted insulin-induced phosphorylation of IRbeta and Akt in the liver. Similarly, nNOS overexpression increased triglyceride production, decreased glucose utilization, and downregulated insulin-induced expression of p-IRbeta, p-Akt, and p-GSK3beta in the HepG2 cells. In contrast, treatment with Nω-propyl-L-arginine (L-NPA), a selective nNOS inhibitor, improved glucose tolerance and upregulated insulin-induced phosphorylation of IRbeta and Akt in the liver of ob/ob mice. Furthermore, overexpression of nNOS increased p38MAPK phosphorylation in the HepG2 cells. In contrast, inhibition of p38MAPK with SB203580 significantly reversed the nNOS-induced inhibition of insulin-signaling activity (all P < 0.05). This indicated that hepatic nNOS inhibited the insulin-signaling pathway through the activation of p38MAPK. These findings suggest that nNOS is involved in the development of hepatic insulin resistance and that nNOS might be a potential therapeutic target for diabetes.

MiR-27b Modulates Insulin Signaling in Hepatocytes by Regulating Insulin Receptor Expression.

Insulin resistance (IR) is one of the key contributing factors in the development of type 2 diabetes mellitus (T2DM). However, the molecular mechanisms leading to IR are still unclear. The implication of microRNAs (miRNAs) in the pathophysiology of multiple cardiometabolic pathologies, including obesity, atherosclerotic heart failure and IR, has emerged as a major focus of interest in recent years. Indeed, upregulation of several miRNAs has been associated with obesity and IR. Among them, miR-27b is overexpressed in the liver in patients with obesity, but its role in IR has not yet been thoroughly explored. In this study, we investigated the role of miR-27b in regulating insulin signaling in hepatocytes, both in vitro and in vivo. Therefore, assessment of the impact of miR-27b on insulin resistance through the hepatic tissue is of special importance due to the high expression of miR-27b in the liver together with its known role in regulating lipid metabolism. Notably, we found that miR-27b controls post-transcriptional expression of numerous components of the insulin signaling pathway including the insulin receptor (INSR) and insulin receptor substrate 1 (IRS1) in human hepatoma cells. These results were further confirmed in vivo showing that overexpression and inhibition of hepatic miR-27 enhances and suppresses hepatic INSR expression and insulin sensitivity, respectively. This study identified a novel role for miR-27 in regulating insulin signaling, and this finding suggests that elevated miR-27 levels may contribute to early development of hepatic insulin resistance.

1823-P: Bromocriptine (BC) Normalizes Elevated Expression of Liver Genes Involved in Fatty Acid Oxidation, Lipogenesis, Inflammation, and Endoplasmic Reticulum (ER) Stress and Glucose Intolerance of High-Fat/Sucrose Diet-Fed Rats

Consumption of the “westernized diet” (WD) of high fat and sucrose promotes the development of hepatic insulin resistance and metabolic syndrome (MS). This diet is well known to reduce brain dopamine activity and stimulate chronic over-activation of the sympathetic nervous system that potentiate liver insulin resistance by as yet unknown mechanisms. This study evaluated the effect of circadian-timed bromocriptine, a sympatholytic dopamine agonist, to reverse the adverse effects of the WD on glucose tolerance and liver expression of several genes involved in hepatic aspects of insulin sensitivity including lipid balance, inflammation and ER stress. Female Sprague-Dawley rats were maintained on a WD of high fat (40% by kcal/g) and sucrose (29% by kcal/g) for 4 weeks and then administered BC (7.5 mg/kg) or vehicle (N =10/group) at the onset of daily waking for an additional 10 weeks while maintained on WD to study end when a GTT (3g/kg glucose) was performed and 5 days later when animals were sacrificed for analyses of hepatic genes (via PCR) involved in insulin sensitivity. A regular chow fed group (N=10) served as positive control. BC treatment reduced the GTT glucose AUC by 39% vs. controls (P&amp;lt;0.001) without change in the insulin AUC and reduced body fat level by 35% (P= 0.01) without change in feeding vs. controls. BC also reduced hepatic gene transcripts for fatty acid oxidation - PPARa and CPT1A by 35 and 48%, respectively (P&amp;lt;0.03), for lipid synthesis - DGAT2 by 20% (P&amp;lt;0.04), and for inflammation and ER stress - monocyte chemoattractant protein 1 and C/EBP homologous protein by 40 and 47% (P&amp;lt;0.05), respectively, vs. controls, all to levels observed in regular chow fed rats. This study demonstrated that bromocriptine can reverse the adverse effects of WD on hepatic lipid, inflammation, and ER stress gene expression pathways that promote hepatic insulin resistance and MS. Disclosure Y. Zhang: Employee; Self; VeroScience LLC. M. Ezrokhi: Employee; Self; VeroScience LLC. N. Cominos: Employee; Self; VeroScience LLC. A.H. Cincotta: Employee; Self; VeroScience LLC.

1817-P: Elafibranor and Glucagon-Like Peptide-1 Receptor Agonists Synergize to Attenuate Hepatic Inflammation and Fibrosis in a Rodent Model of Obesity, Insulin Resistance, and NASH

Elafibranor and GLP-1 receptor agonists synergize to attenuate hepatic inflammation and fibrosis in a rodent model of obesity, insulin resistance and NASH. Liver steatosis contributes to the development of hepatic insulin resistance and is present in the majority of T2DM patients, who are 50 or older. Therefore, many NAFLD patients with diabetes are currently treated with the antidiabetics, including GLP-1 receptor agonists (GLP-1RA). Elafibranor (ELA), a phase 3 candidate for the treatment of NASH, ameliorates insulin sensitivity and lowers HbA1c in patients with NASH-associated T2DM. The aim of our study was to evaluate the pharmacological benefit of combining GLP-1RA and ELA in the context of NASH-associated insulin resistance. GLP-1RA administration induced body weight loss, reduced insulinemia, HOMA-IR and liver steatosis in obese mice with NASH and insulin resistance, but its action on liver fibrosis was negligible. ELA co-administration allowed for GLP-1RA dose reduction and resolved all NASH features. Food intake was also normalized at reduced GLP-1RA doses. Plasma CRP concentration was reduced by 56% in mice that received the combination treatment. Transcriptomic analyses revealed that the synergism between ELA and GLP-1RA in the liver was limited to the modulation of the inflammatory pathways with no effect on metabolic genes. Since ELA and GLP-1RA also synergize in vitro to reduce TNFa secretion in LPS-activated THP1 macrophages, we postulate that both drugs have a direct action on inflammatory cells in the liver. Further analyses to characterize ELA and GLP-1RA effects on innate and adaptive immune responses will be presented. Conclusion: ELA and GLP-1RA synergize to reduce hepatic inflammation and fibrosis in a model of NASH and obesity. These results suggest a potential therapeutic interest of combining ELA and GLP-1RA for the treatment of T2DM in NASH patients. Disclosure V. Legry: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT. E. Descamps: Employee; Self; GENFIT. Employee; Spouse/Partner; GENFIT. Stock/Shareholder; Spouse/Partner; GENFIT. Stock/Shareholder; Self; GENFIT. S. Debaecker: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT. N. Stankovic-Valentin: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT. P. Parroche: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT. N. Degallaix: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT. P. Poulain: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT. B. Staels: Consultant; Self; Genfit. R. Walczak: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT.

Hepatic Neuronal Nitric Oxide Synthase Impaired Hepatic Insulin Sensitivity Through Activation of p38 MAPK

Background: nNOS contains PZD domain and binds with NOS1AP. We reported previously that NOS1AP enhanced hepatic insulin activity on its PZD binding domain suggesting an involvement of nNOS in NOS1AP effect. In present study, we aimed to explore the role of nNOS on hepatic insulin activity and the mechanism underlined. Methods: Liver specific nNOS overexpression was achieved by tail-vein-injection of adenovirus-nNOS in C57BL/6 mice fed with a high-fat diet for 8 weeks. nNOS specific inhibitor (L-NPA) was used to inhibit its activity. Findings: nNOS co-localized with NOS1AP in liver. Its phosphorylation in Ser1417 increased in the livers of ob/ob and diet-induced obese (DIO) mice and mice with liver NOS1AP deletion. It decreased in mice with NOS1AP overexpression. Overexpression of nNOS in the livers of DIO mice aggravated glucose intolerance, increased intrahepatic lipid accumulation, decreased glycogen storage, and blunted insulin-induced phosphorylation of IRβ and Akt in livers. Similarly, overexpression of nNOS increased triglyceride production, decreased glucose utilization, and insulin-induced p-IRβ, p-Akt and p-GSK3β in HepG2 cells. In contrast, inhibition of nNOS with L-NPA in ob/ob mice improved glucose tolerance and increased insulin-induced phosphorylation of IRβ and Akt in livers. Moreover, overexpression of nNOS in HepG2 cells increased p-p38 MAPK expression, and inhibition of p38 MAPK with SB203580 significantly reversed the defect in insulin signaling activation. Interpretation: Hepatic nNOS inhibited insulin signaling activity through activation of p38 MAPK in obesity, suggesting a role of nNOS in the development of hepatic insulin resistance, and a potential therapeutic target for the prevention of diabetes. Funding: This work was supported by the National Natural Science Foundation of China (81670707 to C. Wang). Funding Statement: This study was supported by the National Natural Science Foundation of China (81670707 to C. Wang). Declaration of Interests: The authors declared that they have no conflict of interest. Ethics Approval Statement: The protocol was approved by the Ethics Committee of Shanghai Jiao Tong University (2018-KY-026 (K)). Informed consent was given by all subjects and principles of the Declaration of Helsinki were followed. Procedures involving the care and use of animals were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals of Shanghai Jiao Tong University and approved by the Animal Ethics Committee of the University Affiliated Sixth People’s Hospital (DWSY2011-096/DWLL2019-0274).

Hematopoietic Cell–Expressed Endothelial Nitric Oxide Protects the Liver From Insulin Resistance

Mice genetically deficient in endothelial nitric oxide synthase (Nos3-/-) have fasting hyperinsulinemia and hepatic insulin resistance, indicating the importance of Nos3 (nitric oxide synthase) in maintaining metabolic homeostasis. Although the current paradigm holds that these metabolic effects are derived specifically from the expression of Nos3 in the endothelium, it has been established that bone marrow-derived cells also express Nos3. The aim of this study was to investigate whether bone marrow-derived cell Nos3 is important in maintaining metabolic homeostasis. Approach and Results: To test the hypothesis that bone marrow-derived cell Nos3 contributes to metabolic homeostasis, we generated chimeric male mice deficient or competent for Nos3 expression in circulating blood cells. These mice were placed on a low-fat diet for 5 weeks, a time period which is known to induce hepatic insulin resistance in global Nos3-deficient mice but not in wild-type C57Bl/6 mice. Surprisingly, we found that the absence of Nos3 in the bone marrow-derived component is associated with hepatic insulin resistance and that restoration of Nos3 in the bone marrow-derived component in global Nos3-deficient mice is sufficient to restore hepatic insulin sensitivity. Furthermore, we found that overexpression of Nos3 in bone marrow-derived component in wild-type mice attenuates the development of hepatic insulin resistance during high-fat feeding. Finally, compared with wild-type macrophages, the loss of macrophage Nos3 is associated with increased inflammatory responses to lipopolysaccharides and reduced anti-inflammatory responses to IL-4, a macrophage phenotype associated with the development of hepatic and systemic insulin resistance. These results would suggest that the metabolic and hepatic consequences of high-fat feeding are mediated by loss of Nos3/nitric oxide actions in bone marrow-derived cells, not in endothelial cells.

Lower cardiovagal tone and baroreflex sensitivity associated with hepatic insulin resistance and promote cardiovascular disorders in Tibetan minipigs induced by a high fat and high cholesterol diet

AimsA long-term high-fat/cholesterol (HFC) diet leads to hepatic insulin resistance (IR), which is associated with autonomic dysfunction and cardiovascular diseases risk increasing. However, whether this occurs in Tibetan minipigs remains unknown. We tested that a long-term HFC diet caused hepatic IR and promote cardiovascular disorders in Tibetan minipigs, and are associated with the reduction of cardiovagal tone and baroreflex sensitivity (BRS). MethodsMale Tibetan minipigs were fed either a standard diet or a HFC diet, and were euthanized at 12 weeks. Thereafter, the minipigs were tested for biochemical blood indices, glucose tolerance, blood pressure, heart rate variability (HRV), BRS, and insulin receptor substrate (IRS)-associated gene and protein expression levels, as well as cardiac function. ResultsHFC-fed minipigs developed IR by increasing body weight, total cholesterol, fasting blood glucose and insulin levels, and nonesterified fatty acid (NEFA) and high sensitive C-reactive protein (hs-CRP) levels, glucose intolerance. Increased adipose cell size, hepatic fat deposition, malondialdehyde (MDA) content and NEFA level, down-regulation of IRS1, IRS2, PI3K, Akt, p-Akt, Glut2 and PGC1ɑ expression concomitant with up-regulation of mTOR, GSK3β, TNF-ɑ, FOXO1, p-mTOR and p-p70S6K expression in the liver tissue, as well as hypertension and left ventricular diastolic dysfunction were observed in HFC-fed minipigs. HRV parameters and BRS values were further significantly reduced. Furthermore, multiple linear regression analysis showed that the development of hepatic IR toward cardiovascular disease was associated with low HFnu, RMSSD, BRS and LV −dp/dtmax, high NEFA, high hepatic TG content. ConclusionThese data suggest that HFC-fed Tibetan minipigs develop hepatic IR and promote cardiovascular disorders, and are associated with lower cardiovagal tone and BRS.

Intestine‐Specific Overexpression of Carboxylesterase 2c Protects Mice From Diet‐Induced Liver Steatosis and Obesity

Murine hepatic carboxylesterase 2c (Ces2c) and the presumed human ortholog carboxylesterase 2 (CES2) have been implicated in the development of nonalcoholic fatty liver disease (NAFLD) in mice and obese humans. These studies demonstrated that Ces2c hydrolyzes triglycerides (TGs) in hepatocytes. Interestingly, Ces2c/CES2 is most abundantly expressed in the intestine, indicating a role of Ces2c/CES2 in intestinal TG metabolism. Here we show that Ces2c is an important enzyme in intestinal lipid metabolism in mice. Intestine‐specific Ces2c overexpression (Ces2cint) provoked increased fatty acid oxidation (FAO) in the small intestine accompanied by enhanced chylomicron clearance from the circulation. As a consequence, high‐fat diet–fed Ces2cint mice were resistant to excessive diet‐induced weight gain and adipose tissue expansion. Notably, intestinal Ces2c overexpression increased hepatic insulin sensitivity and protected mice from NAFLD development. Although lipid absorption was not affected in Ces2cint mice, fecal energy content was significantly increased. Mechanistically, we demonstrate that Ces2c is a potent neutral lipase, which efficiently hydrolyzes TGs and diglycerides (DGs) in the small intestine, thereby generating fatty acids (FAs) for FAO and monoglycerides (MGs) and DGs for potential re‐esterification. Consequently, the increased availability of MGs and DGs for re‐esterification and primordial apolipoprotein B48 particle lipidation may increase chylomicron size, ultimately mediating more efficient chylomicron clearance from the circulation. Conclusion: This study suggests a critical role for Ces2c in intestinal lipid metabolism and highlights the importance of intestinal lipolysis to protect mice from the development of hepatic insulin resistance, NAFLD, and excessive diet‐induced weight gain during metabolic stress.

PEPCK1 Antisense Oligonucleotide Prevents Adiposity and Impairs Hepatic Glycogen Synthesis in High-Fat Male Fed Rats.

The increased hepatic gluconeogenesis in type 2 diabetes mellitus has often been ascribed to increased transcription of phosphoenolpyruvate carboxykinase 1, cystolic form (PEPCK1), although recent evidence has questioned this attribution. To assess the metabolic role of PEPCK1, we treated regular chow fed and high-fat fed (HFF) male Sprague-Dawley rats with a 2'-O-methoxyethyl chimeric antisense oligonucleotide (ASO) against PEPCK1 and compared them with control ASO-treated rats. PEPCK1 ASO effectively decreased PEPCK1 expression in the liver and white adipose tissue. In chow fed rats, PEPCK1 ASO did not alter adiposity, plasma glucose, or insulin. In contrast, PEPCK1 ASO decreased the white adipose tissue mass in HFF rats but without altering basal rates of lipolysis, de novo lipogenesis, or glyceroneogenesis in vivo. Despite the protection from adiposity, hepatic insulin sensitivity was impaired in HFF PEPCK1 ASO-treated rats. PEPCK1 ASO worsened hepatic steatosis, although without additional impairments in hepatic insulin signaling or activation of inflammatory signals in the liver. Instead, the development of hepatic insulin resistance and the decrease in hepatic glycogen synthesis during a hyperglycemic clamp was attributed to a decrease in hepatic glucokinase (GCK) expression and decreased synthesis of glycogen via the direct pathway. The decrease in GCK expression was associated with increased expression of activating transcription factor 3, a negative regulator of GCK transcription. These studies have demonstrated that PEPCK1 is integral to coordinating cellular metabolism in the liver and adipose tissue, although it does not directly effect hepatic glucose production or adipose glyceroneogenesis.

Very low-carbohydrate, high-fat, weight reduction diet decreases hepatic gene response to glucose in obese rats

BackgroundVery low carbohydrate (VLC) diets are used to promote weight loss and improve insulin resistance (IR) in obesity. Since the high fat content of VLC diets may predispose to hepatic steatosis and hepatic insulin resistance, we investigated the effect of a VLC weight-reduction diet on measures of hepatic and whole body insulin resistance in obese rats.MethodsIn Phase 1, adult male Sprague-Dawley rats were made obese by ad libitum consumption of a high-fat (HF1, 60% of energy) diet; control rats ate a lower-fat (LF, 15%) diet for 10 weeks. In Phase 2, obese rats were fed energy-restricted amounts of a VLC (5%C, 65%F), LC (19%C, 55%F) or HC (55%C, 15%F) diet for 8 weeks while HF2 rats continued the HF diet ad libitum. In Phase 3, VLC rats were switched to the HC diet for 1 week. At the end of each phase, measurements of body composition and metabolic parameters were obtained. Hepatic insulin resistance was assessed by comparing expression of insulin-regulated genes following an oral glucose load,that increased plasma insulin levels, with the expression observed in the feed-deprived state.ResultsAt the end of Phase 1, body weight, percent body fat, and hepatic lipid levels were greater in HF1 than LF rats (p < 0.05). At the end of Phase 2, percent body fat and intramuscular triglyceride decreased in LC and HC (p < 0.05), but not VLC rats, despite similar weight loss. VLC and HF2 rats had higher HOMA-IR and higher insulin at similar glucose levels following an ip glucose load than HC rats (p < 0.05). HC, but not VLC or HF2 rats, showed changes in Srebf1, Scd1, and Cpt1a expression (p < 0.05) in response to an oral glucose load. At the end of Phase 3, switching from the VLC to the HC diet mitigated differences in hepatic gene expression.ConclusionWhen compared with a high-carbohydrate, low-fat diet that produced similar weight loss, a commonly used VLC diet failed to improve whole body insulin resistance; it also reduced insulin’s effect on hepatic gene expression, which may reflect the development of hepatic insulin resistance.