Insulin-stimulated Conditions Research Articles

An E115A Missense Variant in CERS2 Is Associated With Increased Sleeping Energy Expenditure and Hepatic Insulin Resistance in American Indians.

Genetic determinants of inter-individual differences in energy expenditure (EE) are largely unknown. Sphingolipids, such as ceramides, have been implicated in the regulation of human EE via mitochondrial uncoupling. In this study, we investigated whether genetic variants within enzymes involved in sphingolipid synthesis and degradation affect EE and insulin-related traits in a cohort of American Indians informative for 24-hour EE and glucose disposal rates during a hyperinsulinemic-euglycemic clamp. Association analysis of 10,084 genetic variants within 28 genes involved in sphingolipid pathways identified a missense variant (rs267738, A>C, E115A) in exon 4 of CERS2 that was associated with higher sleeping EE (+116 kcal/day) and increased rates of endogenous glucose production during basal (+5%) and insulin-stimulated (+43%) conditions, both indicators of hepatic insulin resistance. The rs267738 variant did not affect ceramide synthesis in HepG2 cells but resulted in a 30% decrease in basal mitochondrial respiration. In conclusion, we provide evidence that the CERS2 rs267738 missense variant may influence hepatic glucose production and post-absorptive sleeping metabolic rate.

Acute effects of testosterone on whole body protein metabolism in hypogonadal and eugonadal conditions: a randomized, placebo-controlled, crossover study.

Chronic testosterone (T) substitution and short-term T administration positively affect protein metabolism, however, data on acute effects in humans are sparse. This study aimed to investigate T's acute effects on whole body protein metabolism in hypogonadal and eugonadal conditions. We designed a randomized, double-blind, placebo-controlled, crossover study, including 12 healthy young males. Whole body protein metabolism was evaluated during 1) eugonadism, and after medically induced hypogonadism, with application of a gel on each trial day containing either 2) placebo, 3) T 50 mg, or 4) T 150 mg; under basal (5-h basal period) and insulin-stimulated conditions (3-h clamp). The main outcome measure was a change in net protein balance. The net protein loss was 62% larger in the placebo-treated hypogonadal state compared with the eugonadal state during the basal period (-5.5 ± 3.5 µmol/kg/h vs. -3.4 ± 1.2 µmol/kg/h, P = 0.038), but not during the clamp (P = 0.06). Also, hypogonadism resulted in a 25% increase in whole body urea flux (P = 0.006). However, T did not result in any significant changes in protein breakdown, synthesis, or net balance during either the basal period or clamp (all P > 0.05). Protein breakdown was reduced during clamp compared with the basal period regardless of gonadal status or T exposure (all P ≤ 0.001). In conclusion, the application of transdermal T did not counteract the negative effects of hypogonadism with no effects on protein metabolism within 5 h of administration. Insulin (during clamp) mitigated the effects of hypogonadism. This study is the first to investigate acute protein metabolic effects of T in hypogonadal men.NEW & NOTEWORTHY In a model of medically induced hypogonadism in male volunteers, we found increased whole body urea flux and net protein loss as an expected consequence of hypogonadism. Our study demonstrates the novel finding that the application of transdermal testosterone had no acute effects on whole body protein metabolism under eugonadal conditions, nor could it mitigate the hypogonadism-induced changes in protein metabolism. In contrast, insulin (during clamp) mitigated the effects of hypogonadism on protein metabolism.

ACAD10 is not required for metformin's metabolic actions or for maintenance of whole-body metabolism in C57BL/6J mice.

Acyl-coenzyme A dehydrogenase family member 10 (ACAD10) is a mitochondrial protein purported to be involved in the fatty acid oxidation pathway. Metformin is the most prescribed therapy for type 2 diabetes; however, its precise mechanisms of action(s) are still being uncovered. Upregulation of ACAD10 is a requirement for metformin's ability to inhibit growth in cancer cells and extend lifespan in Caenorhabditis elegans. However, it is unknown whether ACAD10 plays a role in metformin's metabolic actions. We assessed the role for ACAD10 on whole-body metabolism and metformin action by generating ACAD10KO mice on a C57BL/6J background via CRISPR-Cas9 technology. In-depth metabolic phenotyping was conducted in both sexes on a normal chow and high fat-high sucrose diet. Compared with wildtype mice, we detected no difference in body composition, energy expenditure or glucose tolerance in male or female ACAD10KO mice, on a chow diet or high-fat, high-sucrose diet (p ≥ .05). Hepatic mitochondrial function and insulin signalling was not different between genotypes under basal or insulin-stimulated conditions (p ≥ .05). Glucose excursions following acute administration of metformin before a glucose tolerance test were not different between genotypes nor was body composition or energy expenditure altered after 4 weeks of daily metformin treatment (p ≥ .05). Despite the lack of a metabolic phenotype, liver lipidomic analysis suggests ACAD10 depletion influences the abundance of specific ceramide species containing very long chain fatty acids, while metformin treatment altered clusters of cholesterol ester, plasmalogen, phosphatidylcholine and ceramide species. Loss of ACAD10 does not alter whole-body metabolism or impact the acute or chronic metabolic actions of metformin in this model.

1632-P: Effects of MTOR Signaling in Muscle-Specific Irs1/2 Knockout Mice

Disruption of Irs1 and Irs2 expression in skeletal and cardiac muscle (MDKO mice) caused death from dilated cardiomyopathy within the first 30 days of life. MDKO showed muscle atrophy but were not glucose intolerant, despite impaired muscle glucose uptake. Beiging of white adipose fat increased glucose uptake into white adipose tissue of MDKO mice, and glucose uptake into brown adipose was also increased. Knockout of atrophy-driving genes FoxO1 and FoxO3a in MDKO mice (MQKO mice) extended life span to 70 days, when MQKO mice died with cardiomyopathy. High fat diet feeding increased life span in both MDKO and MQKO mice; however, genetic activation of the MTOR pathway in muscle of MDKO mice by Tsc1 knockout (MTKO mice) increased life span to 220 days. Regardless, MTKO mice developed cardiac hypertrophy with reduced ejection fraction by 24 weeks. Cardiac glucose uptake in MTKO mice was impaired during non-insulin as well as during insulin-stimulated conditions. Moreover, fat mass of MTKO mice decreased over time, implying a shift in consumption by MTKO hearts from glucose to fatty acids as source for cardiac energy. Tracing of radiolabeled palmitic acid did not support increased cardiac fatty acid uptake in MTKO mice; however, expression of lactate transporters was significantly elevated in MTKO hearts and circulating lactate generated by skeletal muscle was significantly reduced, indicating a potential use of lactate in MTKO hearts. These results show more robust effects of activating the MTOR pathway in insulin resistant hearts of MDKO mice rather than manipulating atrophy-related genes FoxO1/3a to counter early death from cardiomyopathy. Furthermore, activated MTOR signaling potentiated a metabolic switch in MDKO hearts. Disclosure O.Stoehr: None. K.D.Copps: None. R.Tao: None. M.F.White: Board Member; Housey Pharma.

Plasma concentrations of apolipoproteins C3 and C2 do not explain differences in insulin-stimulated endothelial-bound lipoprotein lipase activity in insulin resistant humans

Elevated circulating triglycerides (i.e., dyslipidemia) is a hallmark of insulin resistance and is generally caused by decrease in lipoprotein lipase (LPL) activity. LPL hydrolyzes triglycerides (TG) into free fatty acids in plasma for use and/or storage in tissues (i.e., adipose, skeletal muscle). Plasma apolipoproteins (Apos) C3 and C2 interact with LPL to modulate its function by activating or inhibiting LPL. Previous research shows, in the metabolic environment of insulin resistance, the concentration of ApoC3 is increased in plasma while that of ApoC2 is decreased. Thus, Apos are considered key factors in regulating plasma lipid metabolism via modulation of LPL function. The purpose of this study was to investigate the concentrations of ApoC3 and ApoC2 in plasma and along endothelial-bound LPL activity in insulin resistant and healthy/non-insulin resistant individuals. We evaluated insulin resistance (IR)/insulin sensitivity (IS) using an oral glucose tolerance test (i.e., Matsuda index calculation) in subjects between ages 19 and 45 years old. Subjects were placed in the following two groups: 1) IR, if Matsuda index <4.0 (N=6; 4 males, 2 females; BMI: 23-45 kg/m2), or 2) IS, if Matsuda index >7.0 (N=11, 9 males, 2 females; BMI: 18-26 kg/m2). Subjects received an intravenous insulin infusion (0.5 mU/kg/min) for 30 minutes to resemble increased postprandial plasma insulin response. To release whole-body endothelial-bound LPL, subjects received an injection of intravenous heparin (75 UI/kg). Plasma samples were collected 10 minutes after the heparin infusion and analyzed for LPL concentration and activity, and ApoC3 and ApoC2 concentrations using ELISA. Differences between IS and IR subjects were determined using a t-test, and the P value was set at P < 0.05. Plasma LPL concentrations were not different between groups (IR = 457 ± 17 ng/ml, IS = 453 ± 27 ng/ml, P > 0.05), but plasma LPL activity was higher in the IR subjects (IR = 665 ± 113 nmol/min/ml, IS = 365 ± 59 nmol/min/ml, P = 0.02). IR subjects had higher concentrations of plasma ApoC3 (IR = 3.6 ± 0.5 mg/dl, IS = 2.7 ± 0.2 mg/dl, P=0.03) but ApoC2 was not different between groups (IR = 0.15 ± 0.03 mg/dl, IS = 0.11 ± 0.01 mg/dl, P = 0.11). The ratio of circulating LPL to circulating ApoC3 (IR = 145 ± 22, IS = 176 ± 15) or circulating ApocC2 (IR = 4104 ± 844, IS = 4391 ± 483) were not different between groups (P > 0.05). We conclude that interactions of LPL with APOC3 and ApoC2 may not be main determinants regulating differential whole-body plasma LPL activity under plasma insulin-stimulated conditions in insulin resistant humans. American Diabetes Association Grant #7-12-CT-40 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Luteolin-7-O-rutinoside Protects RIN-5F Cells from High-Glucose-Induced Toxicity, Improves Glucose Homeostasis in L6 Myotubes, and Prevents Onset of Type 2 Diabetes.

Luteolin-7-O-rutinoside (lut-7-O-rutin), a flavonoid commonly present in Mentha longifolia L. and Olea europaea L. leaves has been used as a flavoring agent with some biological activity. The present study is the first attempt to analyze the protective effect of lut-7-O-rutin on high-glucose-induced toxicity to RIN-5F cells in vitro. We found that lut-7-O-rutin improved insulin secretion in both normal and high-glucose conditions in a dose-dependent manner, without toxicity observed. In addition, 20 µmol of lut-7-O-rutin improves insulin sensitization and glucose uptake significantly (p ≤ 0.01) in L6 myotubes cultured in a high-glucose medium. Lut-7-O-rutin has shown a significant (p ≤ 0.05) effect on glucose uptake in L6 myotubes compared to the reference drug, rosiglitazone (20 µmol). Gene expression analysis confirmed significantly lowered CYP1A, TNF-α, and NF-κb expressions in RIN-5F cells, and increased mitochondrial thermogenesis-related LPL, Ucp-1 and PPARγC1A mRNA expressions in L6 myotubes after 24 h of lut-7-O-rutin treatment. The levels of signaling proteins associated with intracellular glucose uptakes, such as cAMP, ChREBP-1, and AMPK, were significantly increased in L6 myotubes. In addition, the levels of the conversion rate of glucose to lactate and fatty acids were raised in insulin-stimulated conditions; the rate of glycerol conversion was found to be higher at the basal level in L6 myotubes. In conclusion, lut-7-O-rutin protects RIN-5F cells from high-glucose-induced toxicity, stimulates insulin secretion, and promotes glucose absorption and homeostasis via molecular mechanisms.

Efficacy of Eisenia bicyclis phlorotannins in the treatment of diabetes and reducing inflammation

Crude phlorotannins from Eisenia bicyclis (CPEb), a common perennial brown seaweed were screened for pharmacological activities such as anti-inflammatory, antioxidant, and antidiabetic effects. Crude Phlorotannins (CPEb) studied by FT-IR, LC-MS, UHPLC, and UV chromatography showed the occurrence of dieckol, 8, 8'-bieckol, fucofuroeckol, and the carotenoid, zeaxanthin (0.5 mg/kg). LPS, an inflammatory mediator, enhanced the production of cytokines such IL-1 (interleukin-1), IL-6 (interleukin-6), IL-8 (interleukin-8), IL-10 (interleukin-10), TNF-α (tumour necrosis factor-α), chemokine CXCL10, and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in differentiated THP1 macrophage cells. On differentiated human monocytic cell line LPS-induced THP1 cells, CPEb was found to have anti-inflammatory effects by reducing the expression of IL-1, IL-6, IL-8 (1.2 fold), CXCL10, NF-κB, and TNF-α. CPEb promoted 2-NBDG absorption in differentiated C2C12 myotubes under both basal (16.08%) and insulin-stimulated (51.09%) conditions. CPEb increased IRS/AKT-dependent glucose absorption and also activates the AMPK pathway. Anti-α-glucosidase, anti-inflammatory and antioxidant activity of CPEb indicate their role in treating hyperglycemia by scavenging ROS and inflammatory responses.

Mechanistic Investigation of GHS-R Mediated Glucose-Stimulated Insulin Secretion in Pancreatic Islets.

Ghrelin receptor, a growth hormone secretagogue receptor (GHS-R), is expressed in the pancreas. Emerging evidence indicates that GHS-R is involved in the regulation of glucose-stimulated insulin secretion (GSIS), but the mechanism by which GHS-R regulates GSIS in the pancreas is unclear. In this study, we investigated the role of GHS-R on GSIS in detail using global Ghsr−/− mice (in vivo) and Ghsr-ablated pancreatic islets (ex vivo). GSIS was attenuated in both Ghsr−/− mice and Ghsr-ablated islets, while the islet morphology was similar between WT and Ghsr−/− mice. To elucidate the mechanism underpinning Ghsr-mediated GSIS, we investigated the key steps of the GSIS signaling cascade. The gene expression of glucose transporter 2 (Glut2) and the glucose-metabolic intermediate—glucose-6-phosphate (G6P) were reduced in Ghsr-ablated islets, supporting decreased glucose uptake. There was no difference in mitochondrial DNA content in the islets of WT and Ghsr−/− mice, but the ATP/ADP ratio in Ghsr−/− islets was significantly lower than that of WT islets. Moreover, the expression of pancreatic and duodenal homeobox 1 (Pdx1), as well as insulin signaling genes of insulin receptor (IR) and insulin receptor substrates 1 and 2 (IRS1/IRS2), was downregulated in Ghsr−/− islets. Akt is the key mediator of the insulin signaling cascade. Concurrently, Akt phosphorylation was reduced in the pancreas of Ghsr−/− mice under both insulin-stimulated and homeostatic conditions. These findings demonstrate that GHS-R ablation affects key components of the insulin signaling pathway in the pancreas, suggesting the existence of a cross-talk between GHS-R and the insulin signaling pathway in pancreatic islets, and GHS-R likely regulates GSIS via the Akt-Pdx1-GLUT2 pathway.

Acute exposure to environmentally relevant levels of DDT alters muscle mitochondrial function in vivo in rats but not in vitro in L6 myotubes: A pilot study

Under insulin-stimulated conditions, skeletal muscle is the largest glucose consumer in the body. Mitochondrial dysfunction and damage to this tissue from oxidative stress are linked to the pathogenesis of type 2 diabetes. Environmental exposure to dichlorodiphenyltrichloroethane (DDT) and its metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), has been associated with the incidence of type 2 diabetes as well as altered oxidative stress and mitochondrial dysfunction in non-muscle tissues. We hypothesized that energy metabolism and insulin sensitivity in skeletal muscle will be altered with exposure to DDT and DDE. In this pilot study, mitochondrial function was measured in permeabilized muscle fibers from Sprague-Dawley rats after one week of exposure to a single injection of DDT (40 μg/kg), a dose comparable to DDT levels in the diets of the Inuit of Northern Canada. The levels of oxidative phosphorylation chain complexes and ROS detoxification enzymes were measured in muscle tissue from these specimens. This acute in vivo exposure to DDT decreased muscle mitochondrial function by 45% without affecting the levels of mitochondrial oxidative phosphorylation chain complexes nor levels of ROS detoxification enzymes. To isolate the effects of DDT and DDE exposure on muscle, L6 myotubes were exposed to DDT or DDE (0, 10, 100, 1000, 10 000 nM) for 24 h. Only very high concentrations of DDT and DDE (1 000 – 10 000 nM) altered maximal respiration with only DDT altering basal glucose uptake in L6 myotubes. This did not alter levels of ROS detoxification enzymes or malondialdehyde (MDA) in L6 myotubes. Altogether, acute exposure to environmentally relevant doses of DDT resulted in muscle mitochondrial dysfunction in vivo in rats, but not when muscle cells were directly exposed to the pollutant or its metabolite.

Role of TRPV4 Channel in Human White Adipocytes Metabolic Activity.

BackgroundIntracellular calcium (Ca2+) homeostasis plays an essential role in adipocyte metabolism and its alteration is associated with obesity and related disorders. Transient receptor potential vanilloid 4 (TRPV4) channels are an important Ca2+ pathway in adipocytes and their activity is regulated by metabolic mediators such as insulin. In this study, we evaluated the role of TRPV4 channels in metabolic activity and adipokine secretion in human white adipocytes.MethodsHuman white adipocytes were freshly cultured and the effects of the activation and inhibition of TRPV4 channels on lipolysis, glucose uptake, lactate production, and leptin and adiponectin secretion were evaluated.ResultsUnder basal and isoproterenol-stimulated conditions, TRPV4 activation by GSK1016709A decreased lipolysis whereas HC067047, an antagonist, increased lipolysis. The activation of TRPV4 resulted in increased glucose uptake and lactate production under both basal conditions and insulin-stimulated conditions; in contrast HC067047 decreased both parameters. Leptin production was increased, and adiponectin production was diminished by TRPV4 activation and its inhibition had the opposite effect.ConclusionOur results suggested that TRPV4 channels are metabolic mediators involved in proadipogenic processes and glucose metabolism in adipocyte biology. TRPV4 channels could be a potential pharmacological target to treat metabolic disorders.

Arsenic disrupts neuronal insulin signaling through increasing free PI3K-p85 and decreasing PI3K activity

Previously, we reported that prolonged arsenic exposure impaired neuronal insulin signaling. Here we have further identified novel molecular mechanisms underlying neuronal insulin signaling impairment by arsenic. Arsenic treatment altered insulin dose-response curve and reduced maximum insulin response in differentiated human neuroblastoma SH-SY5Y cells, suggesting that arsenic hindered neuronal insulin signaling in a non-competitive like manner. Mechanistically, arsenic suppressed insulin receptor (IR) kinase activity, as witnessed by a decreased insulin-activated autophosphorylation of IR at Y1150/1151. Arsenic decreased the level of insulin receptor substrate 1 (IRS1) but increased the protein ratio between PI3K regulatory subunit, p85, and PI3K catalytic subunit, p110. Interestingly, co-immunoprecipitation demonstrated that arsenic did not alter a level of PI3K-p110/PI3K-p85 complex while increased PI3K-p85 levels in a PI3K-p110 depletion supernatant resulted from PI3K-p110 immunoprecipitation. These results indicated that arsenic increased PI3K-p85 which was free from PI3K-p110 binding. In addition, arsenic significantly increased interaction between IRS1 and PI3K-p85 but not PI3K-p110, suggesting that there may be a fraction of free PI3K-p85 interacting with IRS1. In vitro PI3K activity demonstrated that arsenic lowered PI3K activity in both basal and insulin-stimulated conditions. These results suggested that the increase in free PI3K-p85 by arsenic might compete with PI3K heterodimer for the same IRS1 binding site, in turn blocking the activation of its catalytic subunit, PI3K-p110. Taken together, our results provide additional insights into mechanisms underlying the impairment of neuronal insulin signaling by arsenic through the reduction of IR autophosphorylation, the increase in free PI3K-p85, and the impeding of PI3K activity.

Insulin Infusion Is Linked to Increased NPPC Expression in Muscle and Plasma C-type Natriuretic Peptide in Male Dogs.

The purpose of this study was to assess insulin-stimulated gene expression in canine skeletal muscle with a particular focus on NPPC, the gene that encodes C-type natriuretic peptide, a key hormonal regulator of cardiometabolic function. Four conscious canines underwent hyperinsulinemic, euglycemic clamp studies. Skeletal muscle biopsy and arterial plasma samples were collected under basal and insulin-stimulated conditions. Bulk RNA sequencing of muscle tissue was performed to identify differentially expressed genes between these 2 steady-state conditions. Our results showed that NPPC was the most highly expressed gene in skeletal muscle in response to insulin infusion, rising 4-fold between basal and insulin-stimulated conditions. In support of our RNA sequencing data, we found that raising the plasma insulin concentration 15-fold above basal elicited a 2-fold (P = 0.0001) increase in arterial plasma concentrations of N-terminal prohormone C-type natriuretic peptide. Our data suggest that insulin may play a role in stimulating secretion of C-type natriuretic peptide by skeletal muscle. In this context, C-type natriuretic peptide may act in a paracrine manner to facilitate muscle–vascular bed crosstalk and potentiate insulin-mediated vasodilation. This could serve to enhance insulin and glucose delivery, particularly in the postprandial absorptive state.

Metabolic flexibility across the spectrum of glycemic regulation in youth.

BACKGROUNDMetabolic flexibility (MF) refers to the relative ability to utilize lipid and carbohydrate substrates and to transition between them. It is not clear whether MF is impaired in obese youth and what the determining factors are.METHODSWe investigated the determinants of MF (increased respiratory exchange ratio [ΔRER] under insulin-stimulated conditions) in pubertal youth (n = 104; 15.6 ± 1.8 years) with obesity across the spectrum of glucose tolerance compared with normal weight (NW) controls, including body composition (fat-free mass [FFM], %body fat), visceral adipose fat (VAT) (MRI), glycemia, and insulin sensitivity (IS) [3-hour hyperinsulinemic-euglycemic clamp with measurement of lipolysis ([2H5] glycerol), free fatty acids (FFAs), and RER (indirect calorimetry)].RESULTSYouth with prediabetes and type 2 diabetes had lower ΔRER and oxidative and nonoxidative glucose disposal compared with NW, with no significant difference in ΔRER between NW and obese with normal glucose tolerance. In multiple regression analysis, ISFFM (β = 0.4, P = 0.004), percentage suppression of FFAs (r = 0.26, P = 0.007), and race/ethnicity (β = –0.23, P = 0.02) contributed to the variance in ΔRER (R2 = 0.30, P < 0.001) independent of percentage body fat (or VAT), sex, Tanner stage, and hemoglobin A1c.ConclusionMF is defective at the extreme of the metabolic phenotype in obese youth with dysglycemia related to a defect in IS limiting substrate utilization.FUNDINGUSDA/ARS Project Number 3092-51000-057.

Effects of Physiological Doses of Resveratrol and Quercetin on Glucose Metabolism in Primary Myotubes.

Phenolic compounds have emerged in recent years as an option to face insulin resistance and diabetes. The central aim of this study was: (1) to demonstrate that physiological doses of resveratrol (RSV) or quercetin (Q) can influence glucose metabolism in human myotubes, (2) to establish whether AMP-activated protein kinase (AMPK) and protein kinase B –PKB- (Akt) pathways are involved in this effect. In addition, the effects of these polyphenols on mitochondrial biogenesis and fatty acid oxidation were analysed. Myotubes from healthy donors were cultured for 24 h with either 0.1 μM of RSV or with 10 μM of Q. Glucose metabolism, such as glycogen synthesis, glucose oxidation, and lactate production, were measured with D[U-14C]glucose. β-oxidation using [1–14C]palmitate as well as the expression of key metabolic genes and proteins by Real Time PCR and Western blot were also assessed. Although RSV and Q increased pgc1α expression, they did not significantly change either glucose oxidation or β-oxidation. Q increased AMPK, insulin receptor substrate 1 (IRS-1), and AS160 phosphorylation in basal conditions and glycogen synthase kinase 3 (GSK3β) in insulin-stimulated conditions. RSV tended to increase the phosphorylation rates of AMPK and GSK3β. Both of the polyphenols increased insulin-stimulated glycogen synthesis and reduced lactate production in human myotubes. Thus, physiological doses of RSV or Q may exhibit anti-diabetic actions in human myotubes.

SENP2 is vital for optimal insulin signaling and insulin-stimulated glycogen synthesis in human skeletal muscle cells

Sentrin-specific protease (SENP) 2 has been suggested as a possible novel drug target for the treatment of obesity and type 2 diabetes mellitus after observations of a palmitate-induced increase in SENP2 that lead to increased fatty acid oxidation and improved insulin sensitivity in skeletal muscle cells from mice. However, no precedent research has examined the role of SENP2 in human skeletal muscle cells. In the present work, we have investigated the impact of SENP2 on fatty acid and glucose metabolism as well as insulin sensitivity in human skeletal muscle using cultured primary human myotubes. Acute (4 ​h) oleic acid oxidation was reduced in SENP2-knockdown (SENP2-KD) cells compared to control cells, with no difference in uptake. After prelabeling (24 ​h) with oleic acid, total lipid content and incorporation into triacylglycerol was decreased, while incorporation into other lipids, as well as complete oxidation and β-oxidation was increased in SENP2-KD cells. Basal glucose uptake (i.e., not under insulin-stimulated conditions) was higher in SENP2-KD cells, whereas oxidation was similar to control myotubes. Further, basal glycogen synthesis was not different in SENP2-KD myotubes, but both insulin-stimulated glycogen synthesis and AktSer473 phosphorylation was completely blunted in SENP2-KD cells. In conclusion, SENP2 plays an important role in fatty acid and glucose metabolism in human myotubes. Interestingly, it also appears to have a pivotal role in regulating myotube insulin sensitivity. Future studies should examine the role of SENP2 in regulation of insulin sensitivity in other tissues and in vivo, defining the potential for SENP2 as a drug target.

Improved Glucose Intolerance through a Distinct Mouse Olfactory Receptor 23-Induced Signaling Pathway Mediating Glucose Uptake in Myotubes and Adipocytes.

It is aimed to determine the role of mouse olfactory receptor 23 (MOR23) in regulation of glucose uptake in myotubes and adipocytes and investigate whether administration of a possible MOR23 ligand, α-cedrene, attenuates the high fat diet (HFD)-induced glucose intolerance by enhancing the OR-mediated signaling pathway in mice. MOR23 is genetically inactivated by specific small interfering RNA in C2C12 myotubes and 3T3-L1 adipocytes and stimulated with α-cedrene under both basal and insulin-stimulated conditions. In addition, Male C57BL/6N mice are fed a normal diet, HFD, or HFD supplemented with 0.2% α-cedrene. In C2C12 myotubes and 3T3-L1 adipocytes, genetic inactivation of MOR23 significantly decrease glucose uptake and MOR23 downstream signaling under both basal and insulin-stimulated conditions. On the other hand, α-cedrene-mediated MOR23 stimulation results in increased glucose uptake and upregulation of MOR23 signaling molecules, absent in MOR23-depleted myotubes and adipocytes. Moreover, in mice, α-cedrene administration ameliorates HFD-induced glucose intolerance. Activation of MOR23 signaling cascade is also confirmed in basal and insulin stimulated skeletal muscles and adipose tissues of α-cedrene-treated mice. These findings suggest that MOR23 is a novel factor for the regulation of glucose uptake and whole-body glucose homeostasis and has therapeutic potential for diabetes treatment.

Impaired glucose partitioning in primary myotubes from severely obese women with type 2 diabetes.

The purpose of this study was to determine whether intramyocellular glucose partitioning was altered in primary human myotubes derived from severely obese women with type 2 diabetes. Human skeletal muscle cells were obtained from lean nondiabetic and severely obese Caucasian females with type 2 diabetes [body mass index (BMI): 23.6 ± 2.6 vs. 48.8 ± 1.9 kg/m2, fasting glucose: 86.9 ± 1.6 vs. 135.6 ± 12.0 mg/dL, n = 9/group]. 1-[14C]-Glucose metabolism (glycogen synthesis, glucose oxidation, and nonoxidized glycolysis) and 1- and 2-[14C]-pyruvate oxidation were examined in fully differentiated myotubes under basal and insulin-stimulated conditions. Tricarboxylic acid cycle intermediates were determined via targeted metabolomics. Myotubes derived from severely obese individuals with type 2 diabetes exhibited impaired insulin-mediated glucose partitioning with reduced rates of glycogen synthesis and glucose oxidation and increased rates of nonoxidized glycolytic products, when compared with myotubes derived from the nondiabetic individuals (P < 0.05). Both 1- and 2-[14C]-pyruvate oxidation rates were significantly blunted in myotubes from severely obese women with type 2 diabetes compared with myotubes from the nondiabetic controls. Lastly, concentrations of tricarboxylic acid cycle intermediates, namely, citrate (P < 0.05), cis-aconitic acid (P = 0.07), and α-ketoglutarate (P < 0.05), were lower in myotubes from severely obese women with type 2 diabetes. These data suggest that intramyocellular insulin-mediated glucose partitioning is intrinsically altered in the skeletal muscle of severely obese women with type 2 diabetes in a manner that favors the production of glycolytic end products. Defects in pyruvate dehydrogenase and tricarboxylic acid cycle may be responsible for this metabolic derangement associated with type 2 diabetes.

1262-P: Metabolic Flexibility across the Spectrum of Glycemic Regulation in Youth

Metabolic flexibility (MF) refers to the ability to suppress lipid and increase glucose oxidation (as reflected by increase in the respiratory quotient [ΔRQ]) under insulin-stimulated conditions. It is not clear if youth with type 2 diabetes (T2D) and prediabetes have a defect in MF compared with controls. We investigated the determinants of MF in youth with normal weight (NW) and obesity (OB) across the spectrum of glucose tolerance. Youth (n=104; 15.6±1.8 yrs, Tanner stage (TS) II-V) had evaluation of body composition (DXA), visceral fat (VAT) in a subset (MRI), RQ (indirect calorimetry) fasting and during a hyperinsulinemic-euglycemic clamp for measurement of glucose disposal rate (GDR) and insulin sensitivity (IS). Youth with prediabetes and T2D had lower ∆RQ, and lower oxidative and non-oxidative GDR compared to NW with no significant difference in ΔRQ between NW and OB-NGT (Table). ∆RQ correlated with GDR, IS (r=0.3, p=0.02), VAT (r=-.23, p=0.04), and HbA1c (r=-.26, p=0.03) after adjusting for sex, race and TS, but not with % body fat. In multiple regression, Rd (or IS) (β=0.26, p=0.04) and race-ethnicity (β= -0.3, p=0.01) contributed to the variance in ∆RQ (R2=0.25, p=0.005) independent of % body fat, VAT, sex, TS and HbA1c. MF is impaired in youth with prediabetes/T2D compared with NGT-OB and NW in relation to a defect in glucose disposal and appears to be modulated by race-ethnicity (lower in Hispanics). Disclosure F. Bacha: Research Support; Self; AstraZeneca, Takeda Development Center Americas, Inc. S.K. Bartz: None. I. Jindal: None. M.R. Puyau: None. A. Adolph: None. S. Sharma: None. Funding U.S. Department of Agriculture

Acute Voluntary Wheel Running Attenuates Olanzapine‐Induced Hyperglycemia in Male Mice

BackgroundOlanzapine is a second‐generation antipsychotic often used in the treatment of schizophrenia and other “off‐label” conditions. Though effective in reducing psychoses, the use of olanzapine is associated with adverse metabolic side effects. Acutely, olanzapine increases liver glucose output and the development of insulin resistance. Chronic olanzapine treatment leads to weight gain and predisposes individuals to the development of cardiovascular disease and type 2 diabetes. Exercise is an intervention which helps to maintain glucose homeostasis, and with long‐term adherence, can decrease glucose intolerance and insulin resistance. We have previously demonstrated that a single bout of exhaustive exercise mitigates olanzapine‐induced hyperglycemia. While these findings are encouraging, the clinical relevance of exhaustive exercise in individuals taking olanzapine is limited.Purpose &amp; HypothesesThe purpose of this study was to investigate if increases in short‐term, habitual physical activity in the form of voluntary wheel running (VWR) would have a protective effect against olanzapine‐induced hyperglycemia.Methods10‐week old male C57BL/6J mice remained sedentary or were provided running wheels at the start of the dark cycle (~20:00 h), until the following morning (~8:00 h; start of light cycle). Olanzapine (5 mg/kg body weight (BW)) was administered intraperitoneally (IP) immediately following overnight VWR, or 6 hours and 24 hours post wheel‐lock. Blood glucose was measured at baseline, 30, 60, 90, and 120 mins post‐olanzapine. In parallel experiments following the same overnight VWR protocol, an insulin tolerance test (ITT) (0.5 IU/kg BW; IP) was conducted 60 mins post‐olanzapine treatment to assess olanzapine‐induced insulin resistance among groups.ResultsA single overnight session of VWR protected against acute olanzapine‐induced hyperglycemia immediately post‐wheel lock. This protective effect was still present 6 hours post‐wheel lock, and had reversed by 24 hours after the cessation of exercise. Under insulin‐stimulated conditions, the sedentary olanzapine‐treated mice had significantly higher blood glucose area‐under‐the‐curve compared to the other three groups, providing evidence that a prior bout of VWR protects against olanzapine‐induced insulin resistance. Under insulin‐stimulated conditions, serum free fatty acid concentrations were increased with olanzapine and reduced with VWR.ConclusionsOur findings demonstrate that with a single session of overnight habitual physical activity, olanzapine‐induced hyperglycemia is mitigated for at least 6 hours post‐exercise and insulin‐stimulated glucose disposal is recovered under olanzapine‐treated conditions.Support or Funding InformationThis study was supported by the Canadian Institutes of Health Research.

Influences of Hypoxia Exercise on Whole-Body Insulin Sensitivity and Oxidative Metabolism in Older Individuals.

Aging is a primary risk factor for most chronic diseases, including type 2 diabetes. Both exercise and hypoxia regulate pathways that ameliorate age-associated metabolic muscle dysfunction. We hypothesized that the combination of hypoxia and exercise would be more effective in improving glucose metabolism than normoxia exercise. We randomized 29 older sedentary individuals (62 ± 6 years; 14 women, 15 men) to bicycle exercise under normobaric hypoxia (fraction of inspired oxygen = 15%) or normoxia (fraction of inspired oxygen = 21%). Participants trained thrice weekly for 30 to 40 minutes over 8 weeks at a heart rate corresponding to 60% to 70% of peak oxygen update. Insulin sensitivity measured by hyperinsulinemic-euglycemic glucose clamp and muscle protein expression before and after hyperinsulinemic-euglycemic glucose clamp. Heart rate and perceived exertion during training were similar between groups, with lower oxygen saturation when exercising under hypoxia (88.7 ± 1.5 vs 96.2 ± 1.2%, P < 0.01). Glucose infusion rate after 8 weeks increased in both the hypoxia (5.7 ± 1.1 to 6.7 ± 1.3 mg/min/kg; P < 0.01) and the normoxia group (6.2 ± 2.1 to 6.8 ± 2.1 mg/min/kg; P = 0.04), with a mean difference between groups of -0.44 mg/min/kg; 95% CI, -1.22 to 0.34; (P = 0.25). Markers of mitochondrial content and oxidative capacity in skeletal muscle were similar after training in both groups. Changes in Akt phosphorylation and glucose transporter 4 under fasting and insulin-stimulated conditions were not different between groups over time. Eight weeks of hypoxia endurance training led to similar changes in insulin sensitivity and markers of oxidative metabolism compared with normoxia training. Normobaric hypoxia exercise did not enhance metabolic effects in sedentary older women and men beyond exercise alone.