Hepatic Insulin Signaling Research Articles

Cinnamaldehyde supplementation acts as an insulin mimetic compound improving glucose metabolism during adolescence, but not during adulthood, in healthy male rats.

Adolescence is a critical period of increased vulnerability to nutritional modifications, and adolescents may respond differently from adults to dietary intake and nutraceuticals. Cinnamaldehyde, a major bioactive compound of cinnamon, improves energy metabolism, as has been shown in studies conducted primarily in adult animals. We hypothesized that cinnamaldehyde treatment may have a higher impact on the glycemic homeostasis of healthy adolescent rats than on healthy adult rats. Male adolescent (30 days) or adult (90 days) Wistar rats received cinnamaldehyde (40 mg/kg) for 28 days by gavage. The oral glucose tolerance test (OGTT), liver glycogen content, serum insulin concentration, serum lipid profile, and hepatic insulin signaling marker expression were evaluated. Cinnamaldehyde-treated adolescent rats showed less weight gain (P = 0.041), improved OGTT (P = 0.004), increased expression of phosphorylated IRS-1 (P = 0.015), and a trend to increase phosphorylated IRS-1 (P = 0.063) in the liver of adolescent rats in the basal state. None of these parameters was modified after treatment with cinnamaldehyde in the adult group. Cumulative food intake, visceral adiposity, liver weight, serum insulin, serum lipid profile, hepatic glycogen content, and liver protein expression of IRβ, phosphorylated IRβ, AKT, phosphorylated AKT, and PTP-1B in the basal state were similar between both age groups. In a healthy metabolic condition, cinnamaldehyde supplementation affects glycemic metabolism in adolescent rats while promoting no changes in adult rats.

Brain-derived neurotrophic factor in diabetes mellitus: A systematic review and meta-analysis.

Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor expressed in several tissues, including the brain, gut, and pancreas. Activation of the BDNF/TrkB/CREB reduces hepatic gluconeogenesis, induces hepatic insulin signal transduction, and protects against pancreatic beta-cell loss in diabetes mellitus (DM). Several studies have investigated the possible association between BDNF and DM and its complications, but the results have been conflicting. In the present study, we aimed at systematically reviewing the literature on the serum and plasma levels of BDNF in DM and its subgroups such as T2DM, DM patients with depression, and patients with retinopathy. A comprehensive search was conducted in PubMed, Scopus, and Web of Science. We identified 28 eligible studies and calculated the standardized mean difference (SMD) of outcomes as an effect measure. The meta-analysis included 2734 patients with DM and 6004 controls. Serum BDNF levels were significantly lower in patients with DM vs. controls (SMD = -1.00, P<0.001). Plasma BDNF levels were not different in patients with DM compared with controls. When conducting subgroup analysis, serum BDNF levels were lower among patients with T2DM (SMD = -1.26, P<0.001), DM and depression (SMD = -1.69, P<0.001), and patients with diabetic retinopathy (DR) vs. controls (SMD = -1.03, P = 0.01). Serum BDNF levels were lower in patients with DM, T2DM, DM with depression, and DM and DR than the controls. Our findings are in line with the hypothesis that decreased BDNF levels might impair glucose metabolism and contribute to the pathogenesis of DM and its complications.

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

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

Empagliflozin protects mice against diet-induced obesity, insulin resistance and hepatic steatosis

Aims/hypothesisSodium–glucose cotransporter 2 (SGLT2) inhibitors are widely used in the treatment of type 2 diabetes, heart failure and chronic kidney disease. Their role in the prevention of diet-induced metabolic deteriorations, such as obesity, insulin resistance and fatty liver disease, has not been defined yet. In this study we set out to test whether empagliflozin prevents weight gain and metabolic dysfunction in a mouse model of diet-induced obesity and insulin resistance.MethodsC57Bl/6 mice were fed a western-type diet supplemented with empagliflozin (WDE) or without empagliflozin (WD) for 10 weeks. A standard control diet (CD) without or with empagliflozin (CDE) was used to control for diet-specific effects. Metabolic phenotyping included assessment of body weight, food and water intake, body composition, hepatic energy metabolism, skeletal muscle mitochondria and measurement of insulin sensitivity using hyperinsulinaemic–euglycaemic clamps.ResultsMice fed the WD were overweight, hyperglycaemic, hyperinsulinaemic and insulin resistant after 10 weeks. Supplementation of the WD with empagliflozin prevented these metabolic alterations. While water intake was significantly increased by empagliflozin supplementation, food intake was similar in WDE- and WD-fed mice. Adipose tissue depots measured by MRI were significantly smaller in WDE-fed mice than in WD-fed mice. Additionally, empagliflozin supplementation prevented significant steatosis found in WD-fed mice. Accordingly, hepatic insulin signalling was deteriorated in WD-fed mice but not in WDE-fed mice. Empagliflozin supplementation positively affected size and morphology of mitochondria in skeletal muscle in both CD- and WD-fed mice.Conclusions/interpretationEmpagliflozin protects mice from diet-induced weight gain, insulin resistance and hepatic steatosis in a preventative setting and improves muscle mitochondrial morphology independent of the type of diet.Graphical abstract

Platycodon grandiflorus root extract activates hepatic PI3K/PIP3/Akt insulin signaling by enriching gut Akkermansia muciniphila in high fat diet fed mice

BackgroundIncreasing hepatic insulin signaling is found to be an important mechanism of Platycodon grandiflorus root to alleviate metabolic syndrome (MetS) symptoms such as insulin resistance, obesity, hyperlipidemia and hepatic steatosis, but the details are not yet clear. Since the main constituents of Platycodon grandiflorus root were hard to be absorbed by gastrointestinal tract, getting opportunity to interact with gut microbiota, we speculate the gut microorganisms may mediate its effect. PurposeOur work aimed to confirm the critical role of gut microbes in the intervention of Platycodon grandiflorus root extract (PRE) on MetS, and investigate the mechanism. MethodsBiochemical analyses, glucose tolerance test and hepatic lipidomics analysis were used to evaluate the anti-MetS effect of PRE on high fat diet (HFD) fed mice. Perform 16S rDNA analysis, qPCR analysis and in vitro co-incubation experiment to study its effect on gut microbes, followed by fecal microbiota transplantation (FMT) experiment and antibiotics intervention experiment. Also, the effect of Akkermansia muciniphila treatment on HFD mice was investigated. ResultsPRE alleviated lipid accumulation and insulin resistance in HFD mice and remodeled the fecal microbiome. It also increased the gene expression of colonic tight junction proteins, alleviated metabolic endotoxemia and inflammation, so that reduced TNF-α induced hepatic JNK-dependent IRS-1 serine phosphorylation and the impairment of PI3K/PIP3/Akt insulin signaling pathway. A. muciniphila was one of the most significantly enriched microbes by PRE treatment, and its administration to HFD mice showed similar effects to PRE, repairing the gut barrier and activating hepatic PI3K/PIP3/Akt pathway. Finally, anti-MetS effect of PRE could be delivered to FMT recipients, and PRE could not further attenuate MetS in gut microbiota depleted mice. ConclusionWe demonstrated for the first time that PRE alleviated MetS in a gut microbiota dependent manner, and found activation of hepatic insulin signaling mediated by gut A. muciniphila was a potential mechanism of it.

Genistein protects against hyperglycemia and fatty liver disease in diet-induced prediabetes mice via activating hepatic insulin signaling pathway.

Insulin signaling via the insulin receptor (IR) may be associated with the amelioration of diet-induced metabolic syndrome. Genistein, a soy isoflavone, has been suggested to play a role in the amelioration of high-fat diet-induced metabolic disorders. Here, we aimed to explore whether genistein regulates glucose and hepatic lipid by activating the insulin signaling pathway in diet-induced obesity mice. We showed that treatment of western-style diet-fed mice with genistein (60 mg/kg) significantly improved insulin resistance with decreased hyperglycemia and HOMA-IR index. These effects were linked to activating hepatic IRβ/PI3K/Akt signaling. Furthermore, genistein suppressed gluconeogenesis and promoted glycogen synthesis to maintain glucose homeostasis by increasing the phosphorylation of hepatic FOXO1/GSK3β in vivo and in vitro. The reduced level of insulin and upregulation of insulin signaling in genistein-treated mice also lead to an increase in hepatic energy status by inducing energy-sensing AMPK, reducing hepatic SREBP1c/ACC/FAS without affecting β-oxidation to prevent hepatic lipid accumulation. The protective effect of genistein on hepatic lipid accumulation was also validated in vitro. Besides, genistein had little effect on improvements in intestinal function and liver inflammation. Taken together, our results showed that genistein prevents insulin resistance and hyperglycemia through improvements in hepatic function. This study provides new insight into the mechanisms of genistein mediating glucose metabolism and suggests that genistein may be a promising diet ingredient for preventing prediabetes and hepatic lipid accumulation.

Parenteral Exposure of Mice to Ricin Toxin Induces Fatal Hypoglycemia by Cytokine-Mediated Suppression of Hepatic Glucose-6-Phosphatase Expression.

Ricin toxin is an agent of biodefense concern and we have been developing countermeasures for ricin threats. In doing so, we sought biomarkers of ricin toxicosis and found that in mice parenteral injection of ricin toxin causes profound hypoglycemia, in the absence of other clinical laboratory abnormalities. We now seek to identify the mechanisms underlying this hypoglycemia. Within the first hours following injection, while still normoglycemic, lymphopenia and pro-inflammatory cytokine secretion were observed, particularly tumor necrosis factor (TNF)-α. The cytokine response evolved over the next day into a complex storm of both pro- and anti-inflammatory cytokines. Evaluation of pancreatic function and histology demonstrated marked islet hypertrophy involving predominantly β-cells, but only mildly elevated levels of insulin secretion, and diminished hepatic insulin signaling. Drops in blood glucose were observed even after destruction of β-cells with streptozotocin. In the liver, we observed a rapid and persistent decrease in the expression of glucose-6-phosphatase (G6Pase) RNA and protein levels, accompanied by a drop in glucose-6-phosphate and increase in glycogen. TNF-α has previously been reported to suppress G6Pase expression. In humans, a genetic deficiency of G6Pase results in glycogen storage disease, type-I (GSD-1), a hallmark of which is potentially fatal hypoglycemia.

Sphingosine kinase 2 regulates insulin receptor trafficking in hepatocytes.

Disturbed insulin receptor (InsR) trafficking is associated with impaired insulin signaling and the development of diabetes. Sphingosine kinase (SphK), including SphK1 and SphK2, is a key enzyme of sphingolipid metabolism, which has been implicated in the regulation of membrane trafficking. More recently, we have reported that SphK2 is a key regulator of hepatic insulin signaling and glucose homeostasis. However, the role of SphK in InsR trafficking is still undefined. Huh7 cells were treated with specific SphK1 and SphK2 inhibitors or SphK1- and SphK2-specific small interfering RNA (siRNA) in the presence or absence of insulin. Flow cytometry and immunofluorescence assays were carried out to investigate the role of SphK in InsR trafficking. InsR endocytosis, recycling, and insulin signaling were analyzed. Inhibition of SphK2, but not SphK1, by either specific pharmaceutic inhibitors or siRNA, significantly suppressed InsR endocytosis and recycling following insulin stimulation. Consequently, the insulin-stimulated Akt activation was significantly attenuated by SphK2 inhibition in hepatocytes. Moreover, the effect of SphK2 on InsR trafficking was mediated via the clathrin-dependent mechanism. Thus, our results show that SphK2 is able to regulate InsR trafficking. These findings suggest that SphK2 may impinge on hepatic insulin signaling by regulating InsR trafficking, providing further mechanistic evidence that SphK2 could serve as a potential intervention target against insulin resistance and T2D (type 2 diabetes).

RF22 | PSUN318 Hepatocytes Exposed to PFOA Prior to Differentiation Leads to Epigenetic Changes in Genes Linked With NAFLD

Abstract Background Perfluorooctanoic acid (PFOA), is a persistent fluorinated compound with oil and water repelling properties found in cookware, food packaging and municipal water systems. Adult animals exposed to PFOA develop hepatomegaly, fatty liver, peroxisome proliferation, and immunotoxicity. Rodents exposed to PFOA in utero have altered hepatic lipid metabolism, increased hepatic de novo lipogenesis and susceptibility to non-alcoholic fatty liver disease (NAFLD), but underlying molecular mechanisms remain unknown. With increasing rates of obesity, diabetes, and NAFLD it is critical to examine the mechanisms by which in utero exposure to PFOA contributes to the development of metabolic syndrome in offspring. Objectives To characterize how PFOA exposure during hepatocyte differentiation leads to the development of NAFLD through alterations in DNA methylation profiles and changes in the availability of transcription factor binding sites. Methods HepaRG cells (human-derived hepatocyte progenitor cells) were treated with 0.5uM PFOA or vehicle for 48 hours followed by differentiation. Undifferentiated and differentiated hepatocytes exposed to PFOA were assessed relative to controls (n=4). RNASeq was completed; DESeq2 identified differentially expressed genes via false discovery rate (FDR) of &amp;lt;0.05 after Bonferroni correction. Genome-wide DNA methylation analysis via MethylSeq was completed to identify differentially methylated regions (DMRs) defined as minimum number of CpN =5, absolute change in percent methylation &amp;gt;10%, and FDR of &amp;lt;0.05. Enrichment analysis of transcription factor binding motifs within DMRs and single CpG sites (± 200 bp) was performed using HOMER. Results PFOA treatment resulted in decreased expression of the transcription factors early growth response protein 1 (EGR1), nuclear receptor Nur77 (NR4A1), early growth response protein 2 (EGR2), krueppel-like factor 10 (KLF10) and fos-related antigen 1 (FOSL1), which are key genes linked to impaired hepatic insulin signaling, lipid metabolism, steatosis and fibrosis (q&amp;lt;0.05) in undifferentiated hepatocytes. Differentiated hepatocytes showed decreased expression of peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α), peroxisome proliferator-activated receptor γ (PPARγ), forkhead box protein O1 (FOXO1) and increased expression of pyruvate dehydrogenase kinase isozyme 4 (PDK4), genes linked to regulation of lipid metabolism and insulin signaling (q &amp;lt;0.05). MethylSeq analysis identified changes in DMRs located in exons, introns, and intergenic regions, with 57 DMRs in undifferentiated hepatocytes (46 with gains; 11 with losses; q &amp;lt;0.05) and 75 DMRs in differentiated hepatocytes (32 with gains; 41 with losses; q&amp;lt;0.05). HOMER identified 29 known transcription factor binding motifs with changes in methylation in undifferentiated hepatocytes (p&amp;lt;0.05) and 19 in differentiated hepatocytes after PFOA treatment (p&amp;lt;0.05), with significant changes in the EGR1 consensus sequence identified in both comparisons. Conclusions We conclude hepatocyte progenitor cells exposed to low dose PFOA results in changes in DNA methylation and expression of key metabolic genes linked to NAFLD, notably EGR1, a gene previously linked to NAFLD, suggesting PFOA exposure in utero has lasting effects. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m., Sunday, June 12, 2022 12:48 p.m. - 12:53 p.m.

Chronotherapy with a glucokinase activator profoundly improves metabolism in obese Zucker rats.

Circadian rhythms play a critical role in regulating metabolism, including daily cycles of feeding/fasting. Glucokinase (GCK) is central for whole-body glucose homeostasis and oscillates according to a circadian clock. GCK activators (GKAs) effectively reduce hyperglycemia, but their use is also associated with hypoglycemia, hyperlipidemia, and hepatic steatosis. Given the circadian rhythmicity and natural postprandial activation of GCK, we hypothesized that GKA treatment would benefit from being timed specifically during feeding periods. Acute treatment of obese Zucker rats with the GKA AZD1656 robustly increased flux into all major metabolic pathways of glucose disposal, enhancing glucose elimination. Four weeks of continuous AZD1656 treatment of obese Zucker rats improved glycemic control; however, hepatic steatosis and inflammation manifested. In contrast, timing AZD1656 to feeding periods robustly reduced hepatic steatosis and inflammation in addition to improving glycemia, whereas treatment timed to fasting periods caused overall detrimental metabolic effects. Mechanistically, timing AZD1656 to feeding periods diverted newly synthesized lipid toward direct VLDL secretion rather than intrahepatic storage. In line with increased hepatic insulin signaling, timing AZD1656 to feeding resulted in robust activation of AKT, mTOR, and SREBP-1C after glucose loading, pathways known to regulate VLDL secretion and hepatic de novo lipogenesis. In conclusion, intermittent AZD1656 treatment timed to feeding periods promotes glucose disposal when needed the most, restores metabolic flexibility and hepatic insulin sensitivity, and thereby avoids hepatic steatosis. Thus, chronotherapeutic approaches may benefit the development of GKAs and other drugs acting on metabolic targets.

Hepatic interleukin-1 receptor type 1 signalling regulates insulin sensitivity in the early phases of nonalcoholic fatty liver disease.

BackgroundNonalcoholic fatty liver disease (NAFLD) is associated with hepatic as well as systemic insulin resistance even in the absence of type 2 diabetes. The extent and pathways through which hepatic inflammation modulates insulin sensitivity in NAFLD are only partially understood. We explored the contribution of hepatic interleukin (IL)‐1 signalling in a novel conditional knockout mouse model and expand the knowledge on this signalling pathway with regard to its liver‐specific functions.MethodsA high‐fat, high‐carbohydrate diet (HFD) over 12 weeks was used in male hepatocyte‐specific IL‐1 receptor type 1 (IL‐1R1) knockout mice (Il1r1 Hep−/–) and wild‐type (WT) littermates.ResultsBoth genotypes developed an obese phenotype and accompanying macrovesicular hepatic steatosis. In contrast to WT mice, microvesicular steatosis and ballooning injury was less pronounced in HFD‐fed Il1r1 Hep−/– mice, and alanine aminotransferase remained in the normal range. This was paralleled by the suppression of injurious and proinflammatory hepatic c‐Jun N‐terminal kinases and extracellular signal‐regulated kinases signalling, stable peroxisome proliferator activated receptor gamma coactivator‐1alpha and farnesoid X receptor‐alpha expression and preservation of mitochondrial function. Strikingly, despite HFD‐feeding Il1r1 Hep−/– mice remained highly insulin sensitive as indicated by lower insulin levels, homeostatic model assessment for insulin resistance, higher glucose tolerance, more stable hepatic insulin signalling cascade, and less adipose tissue inflammation compared to the WT.ConclusionsThe current data highlights that hepatocyte IL‐1R1 contributes to hepatic and extrahepatic insulin resistance. Future liver‐directed therapies in NAFLD could have effects on insulin sensitivity when improving hepatic inflammation and IL‐1R1 signalling.

The ketogenic diet prevents steatosis and insulin resistance by reducing lipogenesis, diacylglycerol accumulation and protein kinase C activity in male rat liver

Obesity-associated insulin resistance plays a major role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). The accumulation of diacylglycerol (DAG), ceramides and inflammation are key factors that cause NAFLD. In recent years, the ketogenic diet (KD) has emerged as an effective non-pharmacological intervention for the treatment of NAFLD and other obesity-related metabolic disorders. What remains undetermined is how the KD affects DAG and ceramide content and insulin sensitivity in the liver. Thus, this research was designed to assess these variables, as well as glucose and fat metabolism and markers of inflammation in livers of rats exposed for 8weeks to one of the following diets: standard chow (SC), obesogenic high-fat, sucrose-enriched diet (HFS) or a KD. Despite having a higher fat content than the HFS diet, the KD did not cause steatosis and preserved hepatic insulin signalling. The KD reduced DAG content and protein kinase C-ε activity, but markedly increased liver ceramide content. However, whereas the KD increased ceramide synthase 2 (CerS2) expression, it suppressed CerS6 expression, an effect that promoted the production of beneficial very long-chain ceramides instead of harmful long-chain ceramides. The KD also enhanced the liver expression of key genes involved in mitochondrial biogenesis and fatty acid oxidation (Pgc-1α and Fgf21), suppressed inflammatory genes (Tnfα, Nf-kb, Tlr4 and Il6), and shifted substrate away from de novo lipogenesis. Thus, through multiple mechanisms the KD exerted anti-steatogenic and insulin-sensitizing effects in the liver, which supports the use of this dietary intervention to treat NAFLD. KEY POINTS: The accumulation of diacylglycerol (DAG), ceramides and inflammation are key factors that cause insulin resistance and non-alcoholic fatty liver disease (NAFLD). This study provides evidence that a ketogenic diet (KD) rich in fat and devoid of carbohydrate reduced DAG content and preserved insulin signalling in the liver. The KD shifted metabolism away from lipogenesis by enhancing genes involved in mitochondrial biogenesis and fatty acid oxidations in the liver. The KD also promoted the production of beneficial very long-chain ceramides instead of potentially harmful long-chain ceramides. Through multiple mechanisms, the KD exerted anti-steatogenic and insulin-sensitizing effects in the liver, which supports the use of this dietary intervention to treat NAFLD.

Lycopene attenuates oxidative stress-induced hepatic dysfunction of insulin signal transduction: involvement of FGF21 and mitochondria

Lycopene (LYC) has been regarded as a nutraceutical that has powerful antioxidant and hepatoprotective bioactivities. In the present study, we aimed to investigate the beneficial effects of LYC on hepatic insulin signal transduction under oxidative stress conditions and the possible involvement of FGF21 and mitochondria pathways. Two-month-old CD-1 mice were treated by intraperitoneal injection of D-galactose (D-gal) 150 mg/kg/day for 8 weeks and received 0.03% LYC (w/w, mixed into diet). The results showed that LYC increased the expression of FGF21, alleviated mitochondrial dysfunction and improved hepatic insulin signal transduction in D-gal-treated mice. Furthermore, knockdown of FGF21 by small interfering RNA notably suppressed mitochondrial function and blunted LYC-stimulated insulin signal transduction in H2O2-treated HepG2 cells. Moreover, suppressed mitochondrial function via oligomycin also inhibited insulin signal transduction, indicating that LYC supplementation ameliorated oxidative stress-induced hepatic dysfunction of insulin signal transduction by up-regulating FGF21 and enhancing mitochondrial function.

IPMK modulates hepatic glucose production and insulin signaling.

Hepatic glucose production (HGP) is crucial for the maintenance of normal glucose homeostasis. Although hepatic insulin resistance contributes to excessive glucose production, its mechanism is not well understood. Here, we show that inositol polyphosphate multikinase (IPMK), a key enzyme in inositol polyphosphate biosynthesis, plays a role in regulating hepatic insulin signaling and gluconeogenesis both in vitro and in vivo. IPMK-deficient hepatocytes exhibit decreased insulin-induced activation of Akt-FoxO1 signaling. The expression of messenger RNA levels of phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose 6-phosphatase (G6pc), key enzymes mediating gluconeogenesis, are increased in IPMK-deficient hepatocytes compared to wild type hepatocytes. Importantly, re-expressing IPMK restores insulin sensitivity and alleviates glucose production in IPMK-deficient hepatocytes. Moreover, hepatocyte-specific IPMK deletion exacerbates hyperglycemia and insulin sensitivity in mice fed a high-fat diet, accompanied by an increase in HGP during pyruvate tolerance test and reduction in Akt phosphorylation in IPMK deficient liver. Our results demonstrate that IPMK mediates insulin signaling and gluconeogenesis and may be potentially targeted for treatment of diabetes.

Terminalia catappa aqueous leaf extract reverses insulin resistance, improves glucose transport and activates PI3K/AKT signalling in high fat/streptozotocin-induced diabetic rats

Rising prevalence of type 2 diabetes mellitus (T2DM) in sub-Saharan Africa has necessitated surveys of antidiabetic medicinal plants. This study assessed the antidiabetic mechanism of Terminalia catappa aqueous leaf extract (TCA) in high fat/low dose streptozotocin-induced type 2 diabetic rats. T2DM was induced by a combination of high-fat diet and low dose STZ (30 mg/kg bw) and the animals were administered with TCA (400 and 800 mg/kg bw) orally daily for 28 days. Biochemical parameters and indices for diabetes including renal function tests and pancreatic histology were evaluated. Relative expression of hepatic insulin resistance, signalling and glucose transport genes were also assessed. Induction of T2DM resulted in significant (p < 0.05) weight loss, dysregulated glucose level and clearance, electrolyte imbalance and disrupted diabetic biochemical parameters. Diabetes onset also perturbed β-cell function and insulin resistance indices, damaged pancreas microanatomy, while disrupting the expression of insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT) and glucose transporter isoform 4 (GLUT-4) mRNA. Oral treatment of diabetic animals with TCA significantly (p < 0.05) ameliorated alterations due to T2DM induction in a manner comparable with glibenclamide. These results suggest TCA exerts its antidiabetic action by reversing insulin resistance, improving glucose transport and activating PI3K/AKT signalling.

Abstract 527: Loss Of Hepatic Insulin Signaling Promotes Apolipoprotein Ai Retention In Liver And Alters HDL In Plasma In Mice

Background: Insulin signaling regulates a variety of features of hepatic lipid and lipoprotein metabolism. Liver-specific deletion of insulin receptor (LIRKO) reduces plasma HDL, an effect attributed to a reduction in deiodinase 2 expression and apolipoprotein (Apo) AI mRNA expression. We report a concomitant accumulation of ApoAI protein in the liver of LIRKO mice in a yet to be fully characterized compartment within hepatocytes. Methods: Mice harboring loxP sites within the insulin receptor gene (IR fl/fl ) were administered a control adenoassociated viral vector (AAV-Empty) or an AAV encoding Cre recombinase under the control of a liver-specific promoter (AAV_TBG-Cre) at 8 weeks of age to generate Control and LIRKO mice. Male and female mice were evaluated for the abundance of ApoAI in liver and total and FPLC-fractionated plasma. ApoAI secretion rates were evaluated in primary hepatocytes from both genotypes. Subcellular localization of ApoAI in liver of Control and LIRKO mice was evaluated by immunofluorescence microscopy and biochemical fractionation. Results: The loss of hepatic insulin receptor and signaling was confirmed by rtPCR, immunoblot analysis and glucose tolerance test. Whereas ApoAI was unaffected in LIRKO mice compared to controls, protein levels were reduced in plasma and elevated in liver. In contrast, no changes were observed for ApoB or ApoE in either liver or plasma. FPLC fractionation of plasma indicates a shift in HDL size and an accumulation of ApoM in the HDL fractions. Secretion rates of newly synthesized ApoAI were modestly reduced in primary hepatocytes isolated from LIRKO mice. Immunolocalization of ApoAI demonstrated accumulation in large, membrane-bound organelles located peripherally in cells. These organelles stained positively for LampI and other markers of the endosomal-lysosomal system. However, markers of the endosomal-lysosomal compartment in perinuclear regions of the cells were negative for ApoAI. Conclusions: Insulin signaling is a regulator of ApoAI secretion that alters the intracellular itinerary of ApoAI in the secretory and endocytic pathways and alters the plasma HDL proteome in mice.

Pharmacological Inhibition of Cullin Neddylation Improves Hepatic Insulin Sensitivity

Background and significanceHepatic insulin resistance is a hallmark feature of non‐alcoholic fatty liver disease (NAFLD) and type‐2 diabetes (T2D) and significantly contributes to systemic insulin resistance. Abnormal activation of nutrient and stress‐sensing kinases leads to serine/threonine phosphorylation of insulin receptor substrate (IRS) and subsequent IRS proteasome degradation, which is a key underlying cause of hepatic insulin resistance. Recently, members of the cullin‐RING E3 ligases (CRLs) have emerged as mediators of IRS protein turnover. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin‐like protein called Nedd8 to a cullin scaffold.AimTo investigate the role of CRLs in regulating hepatic insulin signaling in NAFLD.MethodsNeddylation inhibitor MLN4924 was used to study its effect on insulin sensitivity in liver cells. Hyperinsulinemic‐euglycemic clamp was used to assess MNL4924 effect on insulin sensitivity in vivo. siRNA‐mediated screening was used to identify the Cullin members that mediates IRS protein turnover in liver cells. Co‐immunoprecipitation was performed to study Cullin and IRS interaction. Glucose homeostasis and insulin sensitivity were studied in mice with AAV‐mediated liver specific knockdown of Cul1 or Cul3.ResultsNeddylation inhibition delays CRL‐mediated IRS protein turnover to prolong insulin action in cultured liver cells. Consistently, acute MLN4924 treatment rapidly decreases hepatic glucose production without causing hypoglycemia in mice, while chronic MLN4924 treatment completely normalizes hyperglycemia in obese mice independent of obesity. Hyperinsulinemic‐euglycemic clamp study revealed that MLN4924 significantly increased insulin sensitivity and improved hepatic glucose output. In vitro siRNA based screening showed that knockdown of Cul1 or Cul3, but not other cullin family members, stabilized IRS protein and increased insulin sensitivity in AML12 cells. Consistently, Co‐immunoprecipitation demonstrated IRS interaction with Cul1 and Cul3. AAV‐mediated liver specific knockdown of Cul1 in mice improved insulin sensitivity, lowered fasting glucose, and prevented Western diet‐induced obesity and NAFLD. In contrast, AAV‐mediated liver Cul3 knockdown only decreased fasting glucose in chow‐fed mice but not Western diet‐fed mice.ConclusionThese findings suggest that pharmacological inhibition of cullin neddylation is a novel therapeutic strategy for hepatic insulin sensitization in NAFLD and T2D. This effect may be at least in part mediated by hepatic Cul1 and Cul3 neddylation.

Hepatic FoxOs link insulin signaling with plasma lipoprotein metabolism through an apolipoprotein M/sphingosine-1-phosphate pathway.

Multiple beneficial cardiovascular effects of HDL depend on sphingosine-1-phosphate (S1P). S1P associates with HDL by binding to apolipoprotein M (ApoM). Insulin resistance is a major driver of dyslipidemia and cardiovascular risk. However, the mechanisms linking alterations in insulin signaling with plasma lipoprotein metabolism are incompletely understood. The insulin-repressible FoxO transcription factors mediate key effects of hepatic insulin action on glucose and lipoprotein metabolism. This work tested whether hepatic insulin signaling regulates HDL-S1P and aimed to identify the underlying molecular mechanisms. We report that insulin-resistant, nondiabetic individuals had decreased HDL-S1P levels, but no change in total plasma S1P. This also occurred in insulin-resistant db/db mice, which had low ApoM and a specific reduction of S1P in the HDL fraction, with no change in total plasma S1P levels. Using mice lacking hepatic FoxOs (L-FoxO1,3,4), we found that hepatic FoxOs were required for ApoM expression. Total plasma S1P levels were similar to those in controls, but S1P was nearly absent from HDL and was instead increased in the lipoprotein-depleted plasma fraction. This phenotype was restored to normal by rescuing ApoM in L-FoxO1,3,4 mice. Our findings show that insulin resistance in humans and mice is associated with decreased HDL-associated S1P. Our study shows that hepatic FoxO transcription factors are regulators of the ApoM/S1P pathway.

Acute Deletion of the FOXO1-dependent Hepatokine FGF21 Does not Alter Basal Glucose Homeostasis or Lipolysis in Mice.

The hepatic transcription factor forkhead box O1 (FOXO1) is a critical regulator of hepatic and systemic insulin sensitivity. Previous work by our group and others demonstrated that genetic inhibition of FOXO1 improves insulin sensitivity both in genetic and dietary mouse models of metabolic disease. Mechanistically, this is due in part to cell nonautonomous control of adipose tissue insulin sensitivity. However, the mechanisms mediating this liver-adipose tissue crosstalk remain ill defined. One candidate hepatokine controlled by hepatic FOXO1 is fibroblast growth factor 21 (FGF21). Preclinical and clinical studies have explored the potential of pharmacological FGF21 as an antiobesity and antidiabetic therapy. In this manuscript, we performed acute loss-of-function experiments to determine the role of hepatocyte-derived FGF21 in glucose homeostasis and insulin tolerance both in control and mice lacking hepatic insulin signaling. Surprisingly, acute deletion of FGF21 did not alter glucose tolerance, insulin tolerance, or adipocyte lipolysis in either liver-specific FGF21KO mice or mice lacking hepatic AKT-FOXO1-FGF21, suggesting a permissive role for endogenous FGF21 in the regulation of systemic glucose homeostasis and insulin tolerance in mice. In addition, these data indicate that liver FOXO1 controls glucose homeostasis independently of liver-derived FGF21.

Modulation of Hepatic Insulin and Glucagon Signaling by Nutritional Factors in Broiler Chicken.

Influencing the endocrine metabolic regulation of chickens by nutritional factors might provide novel possibilities for improving animal health and productivity. This study was designed to evaluate the impact of dietary cereal type (wheat-based (WB) vs. maize-based (MB) diets), crude protein level (normal (NP) vs. lowered (LP)), and sodium (n-)butyrate (1.5 g/kg diet) supplementation (vs. no butyrate) on the responsiveness of hepatic glucagon receptor (GCGR), insulin receptor beta (IRβ) and mammalian target of rapamycin (mTOR) in the phase of intensive growth of chickens. Liver samples of Ross 308 broiler chickens (Gallus gallus domesticus) were collected on day 21 for quantitative real-time polymerase chain reaction and Western blot analyses. Hepatic GCGR and mTOR gene expressions were up-regulated by WB and LP diet. GCGR and IRβ protein level decreased in groups with butyrate supplementation; however, the quantity of IRβ and mTOR protein increased in WB groups. Based on these data, the applied dietary strategies may be useful tools to modulate hepatic insulin and glucagon signaling of chickens in the period of intensive growth. The obtained results might contribute to the better understanding of glycemic control of birds and increase the opportunity of ameliorating insulin sensitivity, hence, improving the production parameters and the welfare of broilers.