Measurement of Insulin Sensitivity in Skeletal Muscle In Vitro

Introduction/Aim: Circadian rhythm disruption is emerging as a risk factor for metabolic disorders and particularly, alterations in clock genes circadian expression have been shown to influence insulin sensitivity. Recently, the reciprocal interplay between the circadian clock machinery and HPA axis has been largely demonstrated: the circadian clock may control the physiological circadian endogenous glucocorticoids secretion and action; glucocorticoids, in turn, are potent regulator of the circadian clock and their inappropriate replacement has been associated with metabolic impairment. The aim of the current study was to investigate in vitro the interaction between the timing-of-the-day exposure to different hydrocortisone (HC) concentrations on muscle insulin sensitivity. Methods: Serum-shock synchronized mouse skeletal muscle C2C12 cells were exposed to different HC concentrations recapitulating the circulating daily physiological cortisol profile (standard cortisol profile), the circulating daily cortisol profile that reached in adrenal insufficient (AI) patients treated with once-daily MR-HC (flat cortisol profile) and treated with thrice-daily of conventional IR-HC (steep cortisol profile). The 24 hrs spontaneous oscillation of the clock genes in synchronized C2C12 cells was used to align the timing for in vitro HC exposure (Bmal1 acrophase, midphase and bathyphase) with the reference times of cortisol peaks in AI treated with IR-HC (8 am, 1 pm, 6 pm). A panel of 84 insulin sensitivity related genes and intracellular insulin signaling proteins were analyzed by RT-qPCR and western blot, respectively. Results: Only the steep profile, characterized by a higher HC exposure during Bmal1 bathyphase, produced significant downregulation in 21 insulin sensitivity-related genes. Among these, Insr, Irs1, Irs2, Pi3kca and Adipor2 were downregulated when compared the flat to the standard or steep profile. Reduced intracellular IRS1 Tyr608, AKT Ser473, AMPK Thr172 and ACC Ser79 phosphorylations were also observed. Conclusions: The current study demonstrated that is late-in-the-day cortisol exposure that modulates insulin sensitivity-related genes expression and intracellular insulin signaling in skeletal muscle cells.

Download Full-text

Reversal of enhanced muscle glucose transport after exercise: roles of insulin and glucose

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1990.259.5.e685 ◽

1990 ◽

Vol 259 (5) ◽

pp. E685-E691 ◽

Cited By ~ 22

Author(s):

E. A. Gulve ◽

G. D. Cartee ◽

J. R. Zierath ◽

V. M. Corpus ◽

J. O. Holloszy

Keyword(s):

Skeletal Muscle ◽

Insulin Sensitivity ◽

Glucose Transport ◽

Transport Process ◽

Sugar Transport ◽

Transport Activity ◽

High Concentration ◽

Exercise Induced ◽

Muscle Glucose Transport

Exercise stimulates insulin-independent glucose transport in skeletal muscle and also increases the sensitivity of the glucose transport process in muscle to insulin. A previous study [D. A. Young, H. Wallberg-Henriksson, M. D. Sleeper, and J. O. Holloszy. Am. J. Physiol. 253 (Endocrinol. Metab. 16): E331–E335, 1987] showed that the exercise-induced increase in glucose transport activity disappears rapidly when rat epitrochlearis muscles are incubated for 3 h in vitro in the absence of insulin and that 7.5 microU/ml insulin in the incubation medium apparently slowed the loss of enhanced sugar transport. We examined whether addition of insulin several hours after exercise increases glucose transport to the same extent as continuous insulin exposure. Addition of 7.5 microU/ml insulin 2.5 h after exercise (when glucose transport has returned to basal levels) increased sugar transport to the same level as that which resulted from continuous insulin exposure. This finding provides evidence for an increase in insulin sensitivity rather than a slowing of reversal of the exercise-induced increase in insulin-independent glucose transport activity. Glucose transport was enhanced only at submaximal, not at maximal, insulin concentrations. Exposure to a high concentration of glucose and a low insulin concentration reduced the exercise-induced increase in insulin-sensitive glucose transport. Incubation with a high concentration of 2-deoxy-D-glucose (2-DG) did not alter the increase in insulin sensitivity, even though a large amount of 2-DG entered the muscle and was phosphorylated.(ABSTRACT TRUNCATED AT 250 WORDS)

Download Full-text

The perifused isolated left atrium: a preparation for studies on sugar transport in the myocardium

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y76-098 ◽

1976 ◽

Vol 54 (5) ◽

pp. 708-713 ◽

Cited By ~ 6

Author(s):

P. C. Sawh ◽

I. Bihler

Keyword(s):

Skeletal Muscle ◽

Insulin Sensitivity ◽

Left Atrium ◽

Concentration Dependence ◽

Time Course ◽

Sugar Transport ◽

Transport Processes ◽

Surgical Methods ◽

Direct Data

A procedure is described for the in vitro perifusion of the rat left atrium by recirculating medium through the atrial cavity and around the tissue, and for making the left atrium completely quiescent using surgical methods. The main advantage of this preparation is that it provides direct data on metabolic and transport processes in resting myocardium. Its use is illustrated by data on the time course, concentration dependence, insulin sensitivity and chemical specificity of the sugar transport system; in all these respects the resting left atrium resembles resting skeletal muscle.

Download Full-text

β-Hydroxybutyrate is reduced in humans with obesity-related NAFLD and displays a dose-dependent effect on skeletal muscle mitochondrial respiration in vitro

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00058.2020 ◽

2020 ◽

Vol 319 (1) ◽

pp. E187-E195 ◽

Cited By ~ 3

Author(s):

Jacob T. Mey ◽

Melissa L. Erickson ◽

Christopher L. Axelrod ◽

William T. King ◽

Chris A. Flask ◽

...

Keyword(s):

Skeletal Muscle ◽

Oxygen Consumption ◽

Insulin Sensitivity ◽

Mitochondrial Respiration ◽

Liver Fat ◽

Whole Body ◽

Fat Accumulation ◽

Impaired Insulin ◽

Hepatic Fat

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic fat accumulation and impaired insulin sensitivity. Reduced hepatic ketogenesis may promote these pathologies, but data are inconclusive in humans and the link between NAFLD and reduced insulin sensitivity remains obscure. We investigated individuals with obesity-related NAFLD and hypothesized that β-hydroxybutyrate (βOHB; the predominant ketone species) would be reduced and related to hepatic fat accumulation and insulin sensitivity. Furthermore, we hypothesized that ketones would impact skeletal muscle mitochondrial respiration in vitro. Hepatic fat was assessed by 1H-MRS in 22 participants in a parallel design, case control study [Control: n = 7, age 50 ± 6 yr, body mass index (BMI) 30 ± 1 kg/m2; NAFLD: n = 15, age 57 ± 3 yr, BMI 35 ± 1 kg/m2]. Plasma assessments were conducted in the fasted state. Whole body insulin sensitivity was determined by the gold-standard hyperinsulinemic-euglycemic clamp. The effect of ketone dose (0.5–5.0 mM) on mitochondrial respiration was conducted in human skeletal muscle cell culture. Fasting βOHB, a surrogate measure of hepatic ketogenesis, was reduced in NAFLD (−15.6%, P < 0.01) and correlated negatively with liver fat ( r2 = 0.21, P = 0.03) and positively with insulin sensitivity ( r2 = 0.30, P = 0.01). Skeletal muscle mitochondrial oxygen consumption increased with low-dose ketones, attributable to increases in basal respiration (135%, P < 0.05) and ATP-linked oxygen consumption (136%, P < 0.05). NAFLD pathophysiology includes impaired hepatic ketogenesis, which is associated with hepatic fat accumulation and impaired insulin sensitivity. This reduced capacity to produce ketones may be a potential link between NAFLD and NAFLD-associated reductions in whole body insulin sensitivity, whereby ketone concentrations impact skeletal muscle mitochondrial respiration.

Download Full-text

Effects of prolonged elevation of plasma adrenaline concentration in vivo on insulin-sensitivity in soleus muscle of the rat

Biochemical Journal ◽

10.1042/bj2440655 ◽

1987 ◽

Vol 244 (3) ◽

pp. 655-660 ◽

Cited By ~ 20

Author(s):

L Budohoski ◽

R A Challiss ◽

A Dubaniewicz ◽

H Kaciuba-Usciłko ◽

B Leighton ◽

...

Keyword(s):

Skeletal Muscle ◽

Insulin Sensitivity ◽

Glucose Transport ◽

Soleus Muscle ◽

Glycogen Synthesis ◽

Biochemical Basis ◽

Plasma Adrenaline ◽

Skeletal Muscle Insulin Sensitivity

1. Prolonged elevation of the plasma adrenaline concentration was produced in rats by implantation of adrenaline-releasing retard-tablets. With this technique, a hyperadrenalinaemic state is maintained for at least 5 days. 2. At 6 h after implantation of the retard-tablet it was found that plasma glucose and fatty acid concentrations increased and insulin concentration decreased compared with values obtained from placebo-tablet-implanted rats. Administration of a subcutaneous glucose load demonstrated an impaired glucose tolerance in vivo, and incubation of soleus muscle strips from 6 h-hyperadrenalinaemic rats in vitro demonstrated a decreased sensitivity of the rates of glycolysis and glucose transport to insulin. 3. The sensitivities of the rates of glycolysis, glucose transport and glycogen synthesis to insulin were determined for the incubated soleus muscle preparation isolated from animals after 48 h, 72 h and 120 h duration of hyperadrenalinaemia. At 48 h after retard-tablet implantation, the sensitivity of the processes of glucose transport and glycolysis was decreased; at 72 h, the insulin-sensitivities of the rates of glycolysis and glucose transport in skeletal muscle were similar to those determined for control animals; at 120 h, however, the sensitivities of the processes of glucose transport and glycolysis were both statistically significantly increased. In contrast, no changes in the sensitivity of the process of glycogen synthesis were observed at any of the time intervals studied. 4. The possible biochemical basis for the observed changes in skeletal-muscle insulin-sensitivity with prolonged hyperadrenalinaemia is discussed.

Download Full-text

Jianpi Qinghua Formula Reduced Intramyocellular Lipids (IMCLs) by Inhibiting Excessive Activation of mTORC1

10.21203/rs.3.rs-136215/v1 ◽

2021 ◽

Author(s):

Xv Han ◽

Qingguang Chen ◽

Yahua Liu ◽

Junfei Xv ◽

Hao Lu

Keyword(s):

Skeletal Muscle ◽

Insulin Sensitivity ◽

Palmitic Acid ◽

High Fat Diet ◽

C2c12 Cells ◽

Akt Pathway ◽

High Fat ◽

Akt Phosphorylation

Abstract Background:IMCLs are an important factor in skeletal muscle insulin resistance. This study aimed to explore the effect of Jianpi Qinghua formula (JPQHF) on IMCLs and its mechanism, as well as the relationship between IMCLs and other skeletal muscle insulin sensitivity factors, thereby elucidating the mechanism by which JPQHF improves insulin sensitivity.Methods: In an in vivo experiment, JPQHF and pioglitazone (PIO) were individually used to treat C57 mice with high-fat diet-induced obesity. In an in vitro experiment, JPQHF and rapamycin in serum were individually used to treat C2C12 cells induced with palmitic acid. The IMCLs of tissue and cells were subjected to oil red O staining. The RNA and protein expression of PPARγ, myogenin, mTORC1 and members of the PI3K/AKT pathway in skeletal muscle tissue and C2C12 cells was examined. Differences between the different intervention groups were determined.Results: IMCLs were significantly increased in mice with obesity induced by a high-fat diet and the C2C12 cell line treated with palmitic acid compared to the corresponding controls. mTORC1 phosphorylation and PPARγ levels were also increased, and AKT phosphorylation and myogenin levels were decreased. Intervention with JPQHF reversed the above changes. In addition, the PPARγ level in C2C12 cells was reduced after intervention with rapamycin, an inhibitor of mTORC1. However, AKT phosphorylation and myogenin levels did not recover after rapamycin intervention.Conclusion: IMCLs were significantly increased in obese C57 mice and palmitic acid-treated C2C12 cells. JPQHF reduced IMCLs both in vivo and in vitro. Mechanistically, this effect likely occurred through JPQHF-mediated inhibition of the overactivation of mTORC1 and a subsequent reduction in the expression of PPARγ. However, the function of JPQHF in elevating myogenin levels and the PI3K/AKT pathway may not be entirely dependent on mTORC1.

Download Full-text

Astragalus Polysaccharide Suppresses Skeletal Muscle Myostatin Expression in Diabetes: Involvement of ROS-ERK and NF-κB Pathways

Oxidative Medicine and Cellular Longevity ◽

10.1155/2013/782497 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 19

Author(s):

Min Liu ◽

Jian Qin ◽

Yarong Hao ◽

Min Liu ◽

Jun Luo ◽

...

Keyword(s):

Insulin Resistance ◽

Skeletal Muscle ◽

Insulin Sensitivity ◽

Saturated Acid ◽

C2c12 Cells ◽

Whole Body ◽

Protective Effects ◽

Kkay Mice ◽

Astragalus Polysaccharide

Objective. The antidiabetes drug astragalus polysaccharide (APS) is capable of increasing insulin sensitivity in skeletal muscle and improving whole-body glucose homeostasis. Recent studies suggest that skeletal muscle secreted growth factor myostatin plays an important role in regulating insulin signaling and insulin resistance. We hypothesized that regulation of skeletal muscle myostatin expression may be involved in the improvement of insulin sensitivity by APS.Methods. APS was administered to 13-week-old diabetic KKAy and nondiabetic C57BL/6J mice for 8 weeks. Complementary studies examined APS effects on the saturated acid palmitate-induced insulin resistance and myostatin expression in C2C12 cells.Results. APS treatment ameliorated hyperglycemia, hyperlipidemia, and insulin resistance and decreased the elevation of myostatin expression and malondialdehyde production in skeletal muscle of noninsulin-dependent diabetic KKAy mice. In C2C12 cells in vitro, saturated acid palmitate-induced impaired glucose uptake, overproduction of ROS, activation of extracellular regulated protein kinases (ERK), and NF-κB were partially restored by APS treatment. The protective effects of APS were mimicked by ERK and NF-κB inhibitors, respectively.Conclusion. Our study demonstrates elevated myostatin expression in skeletal muscle of type 2 diabetic KKAy mice and in cultured C2C12 cells exposed to palmitate. APS is capable of improving insulin sensitivity and decreasing myostatin expression in skeletal muscle through downregulating ROS-ERK-NF-κB pathway.

Download Full-text

Inducible Deletion of Protein Kinase Map4k4 in Obese Mice Improves Insulin Sensitivity in Liver and Adipose Tissues

Molecular and Cellular Biology ◽

10.1128/mcb.00150-15 ◽

2015 ◽

Vol 35 (13) ◽

pp. 2356-2365 ◽

Cited By ~ 15

Author(s):

Laura V. Danai ◽

Rachel J. Roth Flach ◽

Joseph V. Virbasius ◽

Lorena Garcia Menendez ◽

Dae Young Jung ◽

...

Keyword(s):

Skeletal Muscle ◽

Adipose Tissue ◽

Insulin Sensitivity ◽

Protein Kinase ◽

Insulin Signaling ◽

Fasting Blood Glucose ◽

Mitogen Activated Protein Kinase ◽

Whole Body ◽

Metabolic Phenotypes

Studiesin vitrosuggest that mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) attenuates insulin signaling, but confirmationin vivois lacking since Map4k4 knockout is lethal during embryogenesis. We thus generated mice with floxed Map4k4 alleles and a tamoxifen-inducible Cre/ERT2 recombinase under the control of the ubiquitin C promoter to induce whole-body Map4k4 deletion after these animals reached maturity. Tamoxifen administration to these mice induced Map4k4 deletion in all tissues examined, causing decreased fasting blood glucose concentrations and enhanced insulin signaling to AKT in adipose tissue and liver but not in skeletal muscle. Surprisingly, however, mice generated with a conditional Map4k4 deletion in adiponectin-positive adipocytes or in albumin-positive hepatocytes displayed no detectable metabolic phenotypes. Instead, mice with Map4k4 deleted in Myf5-positive tissues, including all skeletal muscles tested, were protected from obesity-induced glucose intolerance and insulin resistance. Remarkably, these mice also showed increased insulin sensitivity in adipose tissue but not skeletal muscle, similar to the metabolic phenotypes observed in inducible whole-body knockout mice. Taken together, these results indicate that (i) Map4k4 controls a pathway in Myf5-positive cells that suppresses whole-body insulin sensitivity and (ii) Map4k4 is a potential therapeutic target for improving glucose tolerance and insulin sensitivity in type 2 diabetes.

Download Full-text