Insulin-stimulated Phosphorylation Research Articles

Tyrosine-phosphorylated DNER sensitizes insulin signaling in hepatic gluconeogenesis by inducing proteasomal degradation of TRB3

ObjectiveHepatic insulin resistance, which leads to increased hepatic gluconeogenesis, is a major contributor to fasting hyperglycemia in type 2 diabetes mellitus (T2DM). However, the mechanism of impaired insulin-dependent suppression of hepatic gluconeogenesis remains elusive. Delta/Notch-like epidermal growth factor (EGF)-related receptor (DNER), firstly described as a neuron-specific Notch ligand, has been recently identified as a susceptibility gene for T2DM through genome-wide association studies. We herein investigated whether DNER regulates hepatic gluconeogenesis and whether this is mediated by enhanced insulin signaling. MethodsThe association between DNER, tribbles homolog 3 (TRB3) and Akt signaling was evaluated in C57BL/6J, ob/ob and db/db mice by western blot analysis. DNER loss-of-function and gain-of-function in hepatic gluconeogenesis were analyzed by western blot analysis, quantitative real-time PCR, glucose uptake and output assay in AML-12 cells and partially validated in primary mouse hepatocytes. Hepatic DNER knockdown mice were generated by tail vein injection of adenovirus to confirm the effects of DNER in vivo. The interaction between DNER and TRB3 was investigated by rescue experiments, cycloheximide chase analysis, co-immunoprecipitation and immunofluorescence. The potential insulin-stimulated phosphorylation sites of DNER were determined by co-immunoprecipitation, LC-MS/MS analysis and site-specific mutagenesis. ResultsHere we show that DNER enhanced hepatic insulin signaling in gluconeogenesis by inhibiting TRB3, an endogenous Akt inhibitor, through the ubiquitin-proteasome degradation pathway. In AML-12 hepatocytes, insulin-stimulated activation of Akt and suppression of gluconeogenesis are attenuated by DNER knockdown, but potentiated by DNER over-expression. In C57BL/6J mice, hepatic DNER knockdown is accompanied by impaired glucose and pyruvate tolerance. Furthermore, the in vitro effects of DNER knockdown or over-expression on both Akt activity and hepatic gluconeogenesis can be rescued by TRB3 knockdown or over-expression, respectively. In response to insulin stimulation, DNER interacted directly with insulin receptor and was phosphorylated at Tyr677. This site-specific phosphorylation is essential for DNER to upregulate Akt activity and then downregulate G6Pase and PEPCK expression, by interacting with TRB3 directly and inducing TRB3 proteasome-dependent degradation. ConclusionsTaken together, the crosstalk between insulin-Akt and DNER-TRB3 pathways represents a previously unrecognized mechanism by which insulin regulates hepatic gluconeogenesis.

Elevated levels of alcohol dehydrogenase aggravate ethanol-evoked cardiac remodeling and contractile anomalies through FKBP5-yap-mediated regulation of ferroptosis and ER stress

Alcohol intake provokes severe organ injuries including alcoholic cardiomyopathy with hallmarks of cardiac remodeling and contractile defects. This study examined the toxicity of facilitated ethanol metabolism in alcoholism-evoked changes in myocardial morphology and contractile function, insulin signaling and various cell death domains using cardiac-selective overexpression of alcohol dehydrogenase (ADH). WT and ADH mice were offered an alcohol liquid diet for 12 weeks prior to assessment of cardiac geometry, function, ER stress, apoptosis and ferroptosis. Alcohol intake provoked pronounced glucose intolerance, cardiac remodeling and contractile anomalies with apoptosis, ER stress, and ferroptosis, the effects were accentuated by ADH with the exception of global glucose intolerance. Hearts from alcohol ingesting mice displayed dampened insulin-stimulated phosphorylation of insulin receptor (tyr1146) and IRS-1 (tyrosine) along with elevated IRS-1 serine phosphorylation, the effect was augmented by ADH. Alcohol challenge dampened phosphorylation of Akt and GSK-3β, and increased phosphorylation of c-Jun and JNK, the effects were accentuated by ADH. Alcohol challenge promoted ER stress, FK506 binding protein 5 (FKBP5), YAP, apoptosis and ferroptosis, the effects were exaggerated by ADH. Using a short-term ethanol challenge model (3 g/kg, i.p., twice in three days), we found that inhibition of FKBP5-YAP signaling or facilitated ethanol detoxification by Alda-1 alleviated ethanol cardiotoxicity. In vitro study revealed that the ethanol metabolite acetaldehyde evoked cardiac contractile anomalies, lipid peroxidation, and apoptosis, the effects of which were mitigated by Alda-1, inhibition of ER stress, FKBP5 and YAP. These data suggest that facilitated ethanol metabolism via ADH exacerbates alcohol-evoked myocardial remodeling, functional defects, and insulin insensitivity possibly through a FKBP5-YAP-associated regulation of ER stress and ferroptosis.

Effect of resveratrol on insulin action in primary myotubes from lean individuals and individuals with severe obesity.

Resveratrol, a natural polyphenol compound contained in numerous plants, has been proposed as a treatment for obesity-related disease processes such as insulin resistance. However, in humans there are conflicting results concerning the efficacy of resveratrol in improving insulin action; the purpose of the present study was to determine whether obesity status (lean, severely obese) affects the response to resveratrol in human skeletal muscle. Primary skeletal muscle cells were derived from biopsies obtained from age-matched lean and insulin-resistant women with severe obesity and incubated with resveratrol (1 µM) for 24 h. Insulin-stimulated glucose oxidation and incorporation into glycogen, insulin signal transduction, and energy-sensitive protein targets [AMP-activated protein kinase (AMPK), Sirt1, and PGC1α] were analyzed. Insulin-stimulated glycogen synthesis, glucose oxidation, and AMPK phosphorylation increased with resveratrol incubation compared with the nonresveratrol conditions (main treatment effect for resveratrol). Resveratrol further increased IRS1, Akt, and TBC1D4 insulin-stimulated phosphorylation and SIRT1 content in myotubes from lean women, but not in women with severe obesity. Resveratrol improves insulin action in primary human skeletal myotubes derived from lean women and women with severe obesity. In women with obesity, these improvements may be associated with enhanced AMPK phosphorylation with resveratrol treatment.NEW & NOTEWORTHY A physiologically relevant dose of resveratrol increases insulin-stimulated glucose oxidation and glycogen synthesis in myotubes from individuals with severe obesity. Furthermore, resveratrol improved insulin signal transduction in myotubes from lean individuals but not from individuals with obesity. Activation of AMPK plays a role in resveratrol-induced improvements in glucose metabolism in individuals with severe obesity.

Tryptophanylation of insulin receptor by WARS attenuates insulin signaling.

Increased circulating amino acid levels have been linked to insulin resistance and development of type 2 diabetes (T2D), but the underlying mechanism remains largely unknown. Herein, we show that tryptophan modifies insulin receptor (IR) to attenuate insulin signaling and impair glucose uptake. Mice fed with tryptophan-rich chow developed insulin resistance. Excessive tryptophan promoted tryptophanyl-tRNA synthetase (WARS) to tryptophanylate lysine 1209 of IR (W-K1209), which induced insulin resistance by inhibiting the insulin-stimulated phosphorylation of IR, AKT, and AS160. SIRT1, but not other sirtuins, detryptophanylated IRW-K1209 to increase the insulin sensitivity. Collectively, we unveiled the mechanisms of how tryptophan impaired insulin signaling, and our data suggested that WARS might be a target to attenuate insulin resistance in T2D patients.

Akt may associate with insulin-responsive vesicles via interaction with sortilin.

Insulin-responsive vesicles (IRVs) deliver the glucose transporter Glut4 to the plasma membrane in response to activation of the insulin signaling cascade: insulin receptor-IRS-PI3 kinase-Akt-TBC1D4-Rab10. Previous studies have shown that Akt, TBC1D4, and Rab10 are compartmentalized on the IRVs. Although functionally significant, the mechanism of Akt association with the IRVs remains unknown. Using pull-down assays, immunofluorescence microscopy, and cross-linking, we have found that Akt may be recruited to the IRVs via the interaction with the juxtamembrane domain of the cytoplasmic C terminus of sortilin, a major IRV protein. Overexpression of full-length sortilin increases insulin-stimulated phosphorylation of TBC1D4 and glucose uptake in adipocytes, while overexpression of the cytoplasmic tail of sortilin has the opposite effect. Our findings demonstrate that the IRVs represent both a scaffold and a target of insulin signaling.

Sex-specific increases in myostatin and SMAD3 contribute to obesity-related insulin resistance in human skeletal muscle and primary human myotubes.

The purpose of the present study was to determine the effects of obesity and biological sex on myostatin expression in humans and to examine the direct effects of myostatin, SMAD2, and SMAD3 on insulin signaling in primary human skeletal muscle cells (HSkMCs). For cohort 1, 15 lean [body mass index (BMI): 22.1 ± 0.5 kg/m2; n = 8 males; n = 7 females] and 14 obese (BMI: 40.6 ± 1.4 kg/m2; n = 7 males; n = 7 females) individuals underwent skeletal muscle biopsies and an oral glucose tolerance test. For cohort 2, 14 young lean (BMI: 22.4 ± 1.9 kg/m2; n = 6 males; n = 8 females) and 14 obese (BMI: 39.3 ± 7.9 kg/m2; n = 6 males; n = 8 females) individuals underwent muscle biopsies for primary HSkMC experiments. Plasma mature myostatin (P = 0.041), skeletal muscle precursor myostatin (P = 0.048), and skeletal muscle SMAD3 (P = 0.029) were elevated in obese females compared to lean females, and plasma mature myostatin (r = 0.58, P = 0.029) and skeletal muscle SMAD3 (r = 0.56, P = 0.037) were associated with insulin resistance in females but not males. Twenty-four hours of myostatin treatment impaired insulin signaling in primary HSkMCs derived from females (P < 0.024) but not males. Overexpression of SMAD3, but not SMAD2, impaired insulin-stimulated AS160 phosphorylation in HSkMCs derived from lean females (-27%, P = 0.040), whereas silencing SMAD3 improved insulin-stimulated AS160 phosphorylation and insulin-stimulated glucose uptake (25%, P < 0.014) in HSkMCs derived from obese females. These results suggest for the first time that myostatin-induced impairments in skeletal muscle insulin signaling are sex specific and that increased body fat in females is associated with detrimental elevations in myostatin and SMAD3, which contribute to obesity-related insulin resistance.NEW & NOTEWORTHY Obesity is considered a main risk factor for the development of insulin resistance and type 2 diabetes. The present study utilizes in vivo and in vitro experiments in human skeletal muscle to demonstrate for the first time that females are inherently more susceptible to myostatin-induced insulin resistance, which is further enhanced with obesity due to increased myostatin and SMAD3 expression.

OR18-02 Mammary Insulin Sensitivity Increases At The Onset Of Lactation And Promotes Metabolic Pathways Supporting Breast Milk Synthesis

Abstract Disclosure: X. Meng: None. H. Rostom: None. A. Fry: None. T. Elajnaf: None. F. Hannan: None. The initiation of lactation during postpartum days 1-4 is hormonally regulated and critical for successful breastfeeding. Insulin represents a key lactogenic hormone as evidenced by women with diabetes mellitus who have delayed lactation onset. However, the role of insulin in lactation and its influence on mammary cells are unclear. We utilised clinical and cellular approaches to investigate this, and recruited n=38 healthy pregnant women (age range 26-42 years) following informed consent and measured serum insulin at 36 weeks’ gestation and during post-partum days 1-4 (1). Serum insulin progressively decreased from 36 weeks’ gestation to day 4 post-partum (244±33pmol/L vs 37±4pmol/L, p&amp;lt;0.0001). Moreover, reverse transcription-quantitative PCR (RT-qPCR) of lactating human mammary epithelial cells (HMECs) isolated from breast milk showed a &amp;gt;20-fold increase in insulin receptor expression compared to non-lactating HMECs (p&amp;lt;0.01, n=4). These findings indicated an increase in both systemic and mammary insulin sensitivity at lactation onset. We hypothesised that insulin promotes lactation by influencing mammary metabolism and used cultured HMECs to evaluate this. Stimulation of HMECs with 10nM insulin caused a &amp;gt;7-fold increase (p=0.001, n=5) in the phosphorylation of Akt, a signalling protein required for initiating lactation. Akt influences oxidative phosphorylation and glycolysis, and we assessed these processes by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively. Cultured HMECs treated with 10nM insulin for ≤18hrs showed increased OCR (22±0.9 vs. 17±0.4pmol O2/min/106 cells for control HMECs, p&amp;lt;0.05, n=4) and ECAR (0.23±0.003 vs. 0.18±0.002mpH/min/106 cells for control HMECs, p&amp;lt;0.001, n=4), consistent with increased oxidative phosphorylation and glycolysis. HMECs treated with 10nM insulin for 8hrs had significant upregulation of genes encoding glycolytic enzymes, namely hexokinase 2 (&amp;gt;4-fold increase, p&amp;lt;0.0001, n=4) and pyruvate kinase M1/2 (1.4-fold increase, p&amp;lt;0.05, n=4). However, RT-qPCR analysis of HMECs showed that insulin did not increase expression of genes mediating oxidative phosphorylation, suggesting an Akt-mediated post-transcriptional mechanism. In summary, increased mammary insulin sensitivity together with insulin-stimulated oxidative phosphorylation and glycolysis may support mammary function and milk component synthesis at the onset of lactation. Reference: (1) Rostom, H. et al. Protocol for an observational study investigating hormones triggering the onset of sustained lactation: the INSIGHT study. BMJ Open 12, e062478. Presentation: Saturday, June 17, 2023

Simulated weightlessness induces hippocampal insulin resistance and cognitive impairment

Growing evidence highlights the potential consequences of long-term spaceflight, including gray matter volume reduction and cognitive dysfunction with subclinical manifestations of diabetes mellitus among astronauts, but the underlying mechanisms remain unknown. In this study, we found that long-term simulated weightlessness induced hippocampal insulin resistance and subsequent neuronal damage and cognitive impairment in rats. Rats subjected to 4-week tail suspension exhibited peripheral insulin resistance, evidenced by increased fasting blood glucose and abnormal glucose tolerance and insulin tolerance, alongside reduced spontaneous activity and impaired recognition memory. In addition, 4 weeks of simulated weightlessness induced neuronal apoptosis and degeneration in the hippocampus, as evidenced by increased TUNEL and Fluoro-Jade B staining-positive neurons. Mechanistically, insulin-stimulated hippocampal Akt phosphorylation was decreased, while PTEN, the negative regulator of insulin signaling, was increased in the hippocampus in tail-suspended rats. Interestingly, treatment with berberine, an insulin sensitizer, partly reversed the above-mentioned effects induced by simulated weightlessness. These data suggest that long-term simulated weightlessness induces cognitive impairment as well as neuronal apoptosis and neural degeneration, partially through hippocampal insulin resistance via PTEN up-regulation. Berberine treatment attenuates hippocampal insulin resistance and improves cognitive function.

192-OR: Lipid-Induced Insulin Resistance in Brown Adipose Tissue Is Mediated by the sn-1,2 DAG-PKCe-IRKT1150 Phosphorylation Pathway

While there is substantial evidence that brown adipose tissue (BAT) is insulin-responsive, it remains unclear whether BAT becomes insulin resistant with high fat diet (HFD) feeding. Here, we examined the hypothesis that the sn-1,2-diacylglycerol (DAG)/Protein Kinase C epsilon (PKCε)/insulin receptor kinase (IRK)T1150 phosphorylation pathway may trigger HFD-induced BAT insulin resistance. To address this hypothesis we first studied two groups of male Sprague Dawley rats fed either a regular chow diet (RC) or a HFD. HFD-fed rats manifested BAT insulin resistance as reflected by reduced insulin-stimulated phosphorylation of IRKT1158 (p=0.03), AKTT308 (p=0.003), PDHS232 (p=0.0002), and PDHS300 (p=0.0004) compared to RC rats during a hyperinsulinemic-euglycemic clamp (HEC). These reductions in insulin signaling occured at the level of the IRK and were associated with increased sn-1,2 diacylglycerols (p=0.03) and increased PKCε translocation (p&amp;lt;0.004). These defects in insulin signaling were also associated with a 35% (p&amp;lt;0.009) reduction in insulin-stimulated BAT mitochondrial pyruvate dehydrogenase/citrate synthase flux (VPDH/VCS) as assessed by LC-MS/MS and GC-MS measurements of C4C5 glutamate (m+2)/pyruvate (m+3) during a HEC where [13C6]glucose was the infusate. In order to assess the role of PKCε-induced IRKT1150 phosphorylation in this process we next studied the effects of HFD feeding in: 1) wild type mice fed a regular chow diet (WT), 2) wild type mice fed a HFD (WT-HFD) and 3) IRKT1150A knockin mice fed a HFD (IRKT1150A-HFD) and found that IRKT1150A-HFD mice have preserved levels of insulin-stimulated AKTS473 phosphorylation compared to WT-HFD mice (p=0.04). Conclusion: These results demonstrate a critical role of the sn-1,2-DAG-PKCε-IRKT1150 phosphorylation pathway in mediating HFD-induced insulin resistance and mitochondrial dysfunction in BAT. Disclosure R.C.Gaspar: None. B.T.Hubbard: None. I.Sakuma: None. T.E.Lamoia: None. D.Zhang: None. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck &amp; Co., Inc., Janssen Research &amp; Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc.

236-LB: FHL2-/- Perivascular Adipose Tissue Improves Vessel Tone and Insulin-Stimulated Vasodilation by Inhibiting ERK1/2

Background: Type 2 diabetes, obesity and aging increase risk of developing cardiovascular diseases. Perivascular adipose tissue (PVAT) is a crucial regulator of vascular function. Four and Half Lim domains 2 (FHL2) is a protein upregulated in aging which targets various proteins with established links to adipose tissue growth, potentially regulating metabolic signaling. Objective: we hypothesized that FHL2 regulates adipose tissue regulation of vascular function. Methods: Femoral arteries were isolated from Wild type (WT) and FHL2-/- mice and vasoreactivity to insulin and acetylcholine was studied with and without perivascular adipose tissue. Results: FHL2-/- vessels with PVAT had increased tone compared to WT vessels with PVAT as well as improved insulin-stimulated vasodilation. Exchange experiments using vessels with PVAT from the opposite genotype showed that FHL2-PVAT enhanced tone and insulin-stimulated vasodilatation compared to WT-PVAT. Gene expression analysis showed downregulation of adiponectin in FHL2-PVAT. Western blot analysis of insulin-stimulated phosphorylation of ERK and Akt in human microvascular endothelial cells (HMECs) stimulated with PVAT-conditioned medium revealed that FHL2-PVAT but not WT-PVAT reduced insulin-stimulated phosphorylation of ERK but not activation of Akt. In line with the effect of FHL2 on insulin-induced vasodilatation and activation of ERK1/2, analysis of publicly available RNA sequencing data of peri-aortic PVAT showed that FHL2 expression in PVAT positively correlates with expression of the endothelial vasoconstrictor peptide endothelin-1 (edn1). Conclusion: FHL2 regulates vascular function through enhancement of smooth muscle tone, regulation of adipokine secretion affecting insulin-stimulated ERK1/2 activation and Edn1 expression in endothelial cells. These finding provide a new mechanism for vascular dysfunction in type 2 diabetes. Disclosure J. Habibe: None. D. van Raalte: Consultant; Boehringer Ingelheim and Eli Lilly Alliance, AstraZeneca, Merck &amp; Co., Inc. Research Support; Boehringer Ingelheim and Eli Lilly Alliance, AstraZeneca, Merck &amp; Co., Inc. M. Nieuwdorp: Advisory Panel; caelus health. C. de Vries: None. E. C. Eringa: None. Funding Amsterdam Cardiovascular Sciences (ACS201703)

Physical inactivity induces insulin resistance in plantaris muscle through protein tyrosine phosphatase 1B activation in mice.

Inactivity causes insulin resistance in skeletal muscle and exacerbates various lifestyle-related diseases. We previously found that 24-h hindlimb cast immobilization (HCI) of the predominantly slow-twitch soleus muscle increased intramyocellular diacylglycerol (IMDG) and insulin resistance by activation of lipin1, and HCI after a high-fat diet (HFD) further aggravated insulin resistance. Here, we investigated the effects of HCI on the fast-twitch-predominant plantaris muscle. HCI reduced the insulin sensitivity of plantaris muscle by approximately 30%, and HCI following HFD dramatically reduced insulin sensitivity by approximately 70% without significant changes in the amount of IMDG. Insulin-stimulated phosphorylation levels of insulin receptor (IR), IR substrate-1, and Akt were reduced in parallel with the decrease in insulin sensitivity. Furthermore, tyrosine phosphatase 1B (PTP1B), a protein known to inhibit insulin action by dephosphorylating IR, was activated, and PTP1B inhibition canceled HCI-induced insulin resistance. In conclusion, HCI causes insulin resistance in the fast-twitch-predominant plantaris muscle as well as in the slow-twitch-predominant soleus muscle, and HFD potentiates these effects in both muscle types. However, the mechanism differed between soleus and plantaris muscles, since insulin resistance was mediated by the PTP1B inhibition at IR in plantaris muscle.

Adenosine monophosphate-activated protein kinase is elevated in human cachectic muscle and prevents cancer-induced metabolic dysfunction in mice.

Metabolic dysfunction and cachexia are associated with poor cancer prognosis. With no pharmacological treatments, it is crucial to define the molecular mechanisms causing cancer-induced metabolic dysfunction and cachexia. Adenosine monophosphate-activated protein kinase (AMPK) connects metabolic and muscle mass regulation. As AMPK could be a potential treatment target, it is important to determine the function for AMPK in cancer-associated metabolic dysfunction and cachexia. We therefore established AMPK's roles in cancer-associated metabolic dysfunction, insulin resistance and cachexia. In vastus lateralis muscle biopsies from n=26 patients with non-small cell lung cancer (NSCLC), AMPK signalling and protein content were examined by immunoblotting. To determine the role of muscle AMPK, male mice overexpressing a dominant-negative AMPKα2 (kinase-dead [KiDe]) specifically in striated muscle were inoculated with Lewis lung carcinoma (LLC) cells (wild type [WT]: n=27, WT+LLC: n=34, mAMPK-KiDe: n=23, mAMPK-KiDe+LLC: n=38). Moreover, male LLC-tumour-bearing mice were treated with (n=10)/without (n=9) 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to activate AMPK for 13days. Littermate mice were used as controls. Metabolic phenotyping of mice was performed via indirect calorimetry, body composition analyses, glucose and insulin tolerance tests, tissue-specific 2-[3H]deoxy-d-glucose (2-DG) uptake and immunoblotting. Patients with NSCLC presented increased muscle protein content of AMPK subunits α1, α2, β2, γ1 and γ3 ranging from +27% to +79% compared with control subjects. In patients with NSCLC, AMPK subunit protein content correlated with weight loss (α1, α2, β2 and γ1), fat-free mass (α1, β2 and γ1) and fat mass (α1 and γ1). Tumour-bearing mAMPK-KiDe mice presented increased fat loss and glucose and insulin intolerance. LLC in mAMPK-KiDe mice displayed lower insulin-stimulated 2-DG uptake in skeletal muscle (quadriceps: -35%, soleus: -49%, extensor digitorum longus: -48%) and the heart (-29%) than that in non-tumour-bearing mice. In skeletal muscle, mAMPK-KiDe abrogated the tumour-induced increase in insulin-stimulated TBC1D4thr642 phosphorylation. The protein content of TBC1D4 (+26%), pyruvate dehydrogenase (PDH; +94%), PDH kinases (+45% to +100%) and glycogen synthase (+48%) was increased in skeletal muscle of tumour-bearing mice in an AMPK-dependent manner. Lastly, chronic AICAR treatment elevated hexokinase II protein content and normalized phosphorylation of p70S6Kthr389 (mTORC1 substrate) and ACCser212 (AMPK substrate) and rescued cancer-induced insulin intolerance. Protein contents of AMPK subunits were upregulated in skeletal muscle of patients with NSCLC. AMPK activation seemed protectively inferred by AMPK-deficient mice developing metabolic dysfunction in response to cancer, including AMPK-dependent regulation of multiple proteins crucial for glucose metabolism. These observations highlight the potential for targeting AMPK to counter cancer-associated metabolic dysfunction and possibly cachexia.

Heptadecanoic Acid Is Not a Key Mediator in the Prevention of Diet-Induced Hepatic Steatosis and Insulin Resistance in Mice.

Epidemiological studies found that the intake of dairy products is associated with an increased amount of circulating odd-chain fatty acids (OCFA, C15:0 and C17:0) in humans and further indicate that especially C17:0 is associated with a lower incidence of type 2 diabetes. However, causal relationships are not elucidated. To provide a mechanistic link, mice were fed high-fat (HF) diets supplemented with either milk fat or C17:0 for 20 weeks. Cultured primary mouse hepatocytes were used to distinguish differential effects mediated by C15:0 or C17:0. Despite an induction of OCFA after both dietary interventions, neither long-term milk fat intake nor C17:0 supplementation improved diet-induced hepatic lipid accumulation and insulin resistance in mice. HF feeding with milk fat actually deteriorates liver inflammation. Treatment of primary hepatocytes with C15:0 and C17:0 suppressed JAK2/STAT3 signaling, but only C15:0 enhanced insulin-stimulated phosphorylation of AKT. Overall, the data indicate that the intake of milk fat and C17:0 do not mediate health benefits, whereas C15:0 might be promising in further studies.

Tilorone increases glucose uptake in vivo and in skeletal muscle cells by enhancing Akt2/AS160 signaling and glucose transporter levels.

Skeletal muscle plays a major role in whole-body glucose metabolism. Insulin resistance in skeletal muscle is characterized by decreased insulin-stimulated glucose uptake resulting from impaired intracellular trafficking and decreased glucose transporter 4 (GLUT4) expression. In this study, we illustrated that tilorone, a low-molecular-weight antiviral agent, improves glucose uptake in vitro and in vivo. Tilorone increased bone morphogenetic protein (BMP) signaling in C2C12 myoblasts, the transcription of multiple BMPs (BMP2, BMP4, BMP7, and BMP14), Smad4 expression, and the phosphorylation of BMP-mediated Smad1/5/8. The activation of Akt2/AS160(TBC1D4) signaling, the critical regulator of GLUT4 translocation, was also increased, as well as the levels of GLUT4 and GLUT1, leading to enhanced uptake of the radioactively labeled glucose analog 18 F-fluoro-2-deoxyglucose (18 FDG). However, this excess glucose content did not result in increased ATP formation by mitochondrial respiration; both basal and ATP-linked respiration were diminished, thereby contributing to the induction of AMPK. In differentiated myotubes, AS160 phosphorylation and 18 FDG uptake also increased. Moreover, tilorone administration further increased insulin-stimulated phosphorylation of Akt2 and glucose uptake of myotubes indicating an insulin-sensitizing effect. Importantly, during in vivo experiments, the systemic administration of tilorone resulted in increased 18 FDG uptake of skeletal muscle, liver, and adipose tissue in C57BL/6 mice. Our results provide new perspectives for the treatment of type 2 diabetes, which has a limited number of treatments that regulate protein expression or translocation.

Insulin-resistant female rat skeletal muscles display diacylglycerol-mediated protein kinase C activation and inflammation without ceramide accumulation.

This study investigated the role of diacylglycerol (DAG)-mediated protein kinase C (PKC) activation, ceramide accumulation and inflammation in insulin-resistant female oxidative and glycolytic skeletal muscles induced by an obesogenic high-fat sucrose-enriched (HFS) diet. The HFS diet impaired insulin-stimulated AKTThr308 phosphorylation and glycogen synthesis, whereas rates of fatty acid oxidation and basal lactate production were significantly elevated in soleus (Sol), extensor digitorum longus (EDL) and epitrochlearis (Epit) muscles. Insulin resistance was accompanied by increases in triacylglycerol (TAG) and DAG contents in Sol and EDL, whereas in Epit muscles only TAG content and markers of inflammation were associated with HFS diet-induced insulin resistance. Analysis of membrane-bound/cytoplasmic PKC fractions revealed that the HFS diet promoted activation/translocation of PKCδ and θ isoforms in Sol, EDL and Epit muscles. However, none of these muscles displayed alterations in ceramide content in response to HFS feeding. This could be explained by a significant increase in Dgat2 mRNA expression in Sol, EDL and Epit muscles, which likely diverted most of the intramyocellular acyl-CoAs toward TAG synthesis instead of ceramides. Overall, this study helps elucidate the molecular mechanisms underlying insulin resistance caused by diet-induced obesity in female skeletal muscles with distinct fibre type compositions. KEY POINTS: Feeding female Wistar rats a high-fat sucrose-enriched diet (HFS) led to diacylglycerol (DAG)-induced PKC activation and insulin resistance in oxidative and glycolytic skeletal muscles. HFS diet-induced toll-like receptor 4 (Tlr4) expression did not lead to increased ceramide content in female skeletal muscles. In highly glycolytic female muscles, elevated TAG content and markers of inflammation underlay HFS diet-induced insulin resistance. The HFS diet suppressed glucose oxidation and increased lactate production in oxidative and glycolytic female muscles. Increased Dgat2 mRNA expression likely diverted most of the intramyocellular acyl-CoAs toward TAG synthesis and prevented ceramide formation in skeletal muscles of HFS-fed female rats.

Chronic cortisol elevation restricts glucose uptake but not insulin responsiveness in zebrafish skeletal muscle

Although teleosts show an elevated insulin response to hyperglycemia, the circulating glucose levels are not normalized as rapidly as in mammals. While this may suggest a lack of target tissue insulin responsiveness, the underlying mechanisms are unclear. We investigated whether changes in skeletal muscle insulin sensitivity and glucose uptake underlie the cortisol-mediated elevated blood glucose levels. Adult zebrafish (Danio rerio) were exposed to water-borne cortisol for 3 days followed by an intraperitoneal injection of glucose with or without insulin. Cortisol treatment resulted in a temporal delay in the reduction in blood glucose levels, and this corresponded with a reduced glucose uptake capacity and lower glycogen content in the skeletal muscle. The transcript abundance of slc2a1b (which encodes for GLUT1b) and a suite of genes encoding enzymes involved in muscle glycogenesis and glycolysis were upregulated in the cortisol group. Both the control and cortisol groups showed higher whole body insulin expression in response to blood glucose elevation, which also resulted in enhanced insulin-stimulated phosphorylation of AKT in the skeletal muscle. The insulin-mediated phosphorylation of S6 kinase was lower in the cortisol group. Altogether, chronic cortisol stimulation restricts glucose uptake and enhances the glycolytic capacity without affecting insulin responsiveness in zebrafish skeletal muscle.

A role for β-catenin in diet-induced skeletal muscle insulin resistance.

A central characteristic of insulin resistance is the impaired ability for insulin to stimulate glucose uptake into skeletal muscle. While insulin resistance can occur distal to the canonical insulin receptor-PI3k-Akt signaling pathway, the signaling intermediates involved in the dysfunction are yet to be fully elucidated. β-catenin is an emerging distal regulator of skeletal muscle and adipocyte insulin-stimulated GLUT4 trafficking. Here, we investigate its role in skeletal muscle insulin resistance. Short-term (5-week) high-fat diet (HFD) decreased skeletal muscle β-catenin protein expression 27% (p= 0.03), and perturbed insulin-stimulated β-cateninS552 phosphorylation 21% (p= 0.009) without affecting insulin-stimulated Akt phosphorylation relative to chow-fed controls. Under chow conditions, mice with muscle-specific β-catenin deletion had impaired insulin responsiveness, whereas under HFD, both mice exhibited similar levels of insulin resistance (interaction effect of genotype × diet p< 0.05). Treatment of L6-GLUT4-myc myocytes with palmitate lower β-catenin protein expression by 75% (p= 0.02), and attenuated insulin-stimulated β-catenin phosphorylationS552 and actin remodeling (interaction effect of insulin × palmitate p< 0.05). Finally, β-cateninS552 phosphorylation was 45% lower in muscle biopsies from men with type 2 diabetes while total β-catenin expression was unchanged. These findings suggest that β-catenin dysfunction is associated with the development of insulin resistance.

Finnish-specific AKT2 gene variant leads to impaired insulin signalling in myotubes.

Finnish-specific gene variant p.P50T/AKT2 (MAF=1.1%), is associated with insulin resistance and increased predisposition to type 2 diabetes. Here, we have investigated in vitro the impact of the gene variant on glucose metabolism and intracellular signalling in human primary skeletal muscle cells, that were established from 14 male p.P50T/AKT2 variant carriers and 14 controls. Insulin-stimulated glucose uptake and glucose incorporation into glycogen were detected with 2-[1,2-3H]-deoxy-D-glucose and D-[14C]-glucose, respectively, and the rate of glycolysis was measured with a Seahorse XFe96 analyzer. Insulin signalling was investigated with western blotting. Binding of variant and control AKT2-PH domains to phosphatidylinositol (3,4,5)-trisphosphate ((PI(3,4,5)P3)) was assayed using PIP Strips™ Membranes. Tyrosine (PTK) and serine-threonine (STK) kinase assay was performed using the PamGene® kinome profiling system. Insulin-stimulated glucose uptake and glycogen synthesis in myotubes in vitro were not significantly affected by the genotype. However, insulin-stimulated glycolytic rate was impaired in variant myotubes. Western blot analysis showed that insulin-stimulated phosphorylation of Akt-Thr308, AS160-Thr642 and GSK3β-Ser9 was reduced in variant myotubes compared to controls. Binding of variant AKT2-PH domain to PI(3,4,5)P3 was reduced as compared to control protein. PamGene® kinome profiling revealed multiple differentially phosphorylated kinase substrates, e.g. calmodulin, between the genotypes. Further in silico upstream kinase analysis predicted a large-scale impairment in activities of kinases participating for example in intracellular signal transduction, protein translation and cell cycle events. In conclusion, myotubes from p.P50T/AKT2 variant carriers show multiple signalling alterations which may contribute to predisposition to insulin resistance and T2D in the carriers of this signalling variant.

Distinct subcellular localisation of intramyocellular lipids and reduced PKCε/PKCθ activity preserve muscle insulin sensitivity in exercise-trained mice.

Athletes exhibit increased muscle insulin sensitivity, despite increased intramuscular triacylglycerol content. This phenomenon has been coined the 'athlete's paradox' and is poorly understood. Recent findings suggest that the subcellular distribution of sn-1,2-diacylglycerols (DAGs) in the plasma membrane leading to activation of novel protein kinase Cs (PKCs) is a crucial pathway to inducing insulin resistance. Here, we hypothesised that regular aerobic exercise would preserve muscle insulin sensitivity by preventing increases in plasma membrane sn-1,2-DAGs and activation of PKCε and PKCθ despite promoting increases in muscle triacylglycerol content. C57BL/6J mice were allocated to three groups (regular chow feeding [RC]; high-fat diet feeding [HFD]; RC feeding and running wheel exercise [RC-EXE]). We used a novel LC-MS/MS/cellular fractionation method to assess DAG stereoisomers in five subcellular compartments (plasma membrane [PM], endoplasmic reticulum, mitochondria, lipid droplets and cytosol) in the skeletal muscle. We found that the HFD group had a greater content of sn-DAGs and ceramides in multiple subcellular compartments compared with the RC mice, which was associated with an increase in PKCε and PKCθ translocation. However, the RC-EXE mice showed, of particular note, a reduction in PM sn-1,2-DAG and ceramide content when compared with HFD mice. Consistent with the PM sn-1,2-DAG-novel PKC hypothesis, we observed an increase in phosphorylation of threonine1150 on the insulin receptor kinase (IRKT1150), and reductions in insulin-stimulated IRKY1162 phosphorylation and IRS-1-associated phosphoinositide 3-kinase activity in HFD compared with RC and RC-EXE mice, which are sites of PKCε and PKCθ action, respectively. These results demonstrate that lower PKCθ/PKCε activity and sn-1,2-DAG content, especially in the PM compartment, can explain the preserved muscle insulin sensitivity in RC-EXE mice.

BPA exposure in L6 myotubes increased basal glucose metabolism in an estrogen receptor-dependent manner but induced insulin resistance

Exposure to bisphenol A (BPA) is associated with insulin resistance and type 2 diabetes (T2D). Since muscle insulin resistance is the primary defect in T2D, we aimed to determine whether BPA alters glucose metabolism in L6 muscle cells. L6 or L6-GLUT4-myc cells were exposed to 1-104 nM BPA or the vehicle (0.1% DMSO) for 7 days. BPA at 103-104 nM significantly decreased the levels of the muscle differentiation markers troponin- T and myosin heavy chain 3. Insulin-stimulated phosphorylation of Akt and GSK3, insulin-stimulated glucose uptake, and insulin-stimulated GLUT4 translocation were significantly decreased with 103-104 nM BPA. Basal glucose uptake and glycolysis (extracellular acidification rates measured by Seahorse XFe96) were increased with 103-104 nM BPA. Levels of ROS detoxifying enzymes were increased with BPA >10nM, while catalase activity was increased with 103-104 nM BPA. However, BPA did not induce oxidative stress (measured by protein carbonylation and lipid peroxidation) nor mitochondrial dysfunction. The effects of BPA on basal glucose uptake and catalase activity, but not on insulin sensitivity, were restored when estrogen receptors (ERs) were inhibited with ICI. These findings suggest that high concentrations of BPA increase muscle glucose uptake through the ERs but induce insulin resistance through another pathway.