Calorie Restriction-Induced Increase in Skeletal Muscle Insulin Sensitivity Is Not Prevented by Overexpression of the p55α Subunit of Phosphoinositide 3-Kinase

Abstract

Introduction: The Phosphoinositide 3-kinase (PI3K) signaling pathway plays an important role in skeletal muscle insulin-stimulated glucose uptake. While whole-body and tissue specific knockout (KO) of individual or combinations of the regulatory subunits of PI3K (p85α, p55α, and p50α or p85β); increase insulin sensitivity, no study has examined whether increasing the expression of the individual regulatory subunits would inhibit insulin action in vivo. Therefore, the objective of this study was to determine whether skeletal muscle-specific overexpression of the p55α regulatory subunit of PI3K impairs skeletal muscle insulin sensitivity, or prevents its enhancement by caloric restriction.Methods: We developed a novel “floxed” mouse that, through the Cre-LoxP approach, allows for tamoxifen (TMX)-inducible and skeletal muscle-specific overexpression of the p55α subunit of PI3K (referred to as, ‘p55α-mOX’). Beginning at 10 weeks of age, p55α-mOX mice and their floxed littermates (referred to as wildtype [WT]) either continued with free access to food (ad libitum; AL), or were switched to a calorie restricted diet (CR; 60% of AL intake) for 20 days. We measured body composition, whole-body energy expenditure, oral glucose tolerance and ex vivo skeletal muscle insulin sensitivity in isolated soleus and extensor digitorum longus muscles using the 2-deoxy-glucose (2DOG) uptake method.Results: p55α mRNA and protein expression was increased ∼2 fold in muscle from p55α-mOX versus WT mice. There were no differences in energy expenditure, total activity, or food intake of AL-fed mice between genotypes. Body weight, fat and lean mass, tissue weights, and fasting glucose and insulin were comparable between p55α-mOX and WT mice on AL, and were decreased equally by CR. Interestingly, overexpression of p55α did not impair oral glucose tolerance or skeletal muscle insulin signaling or sensitivity, nor did it impact the ability of CR to enhance these parameters.Conclusion: Skeletal muscle-specific overexpression of p55α does not impact skeletal muscle insulin action, suggesting that p85α and/or p50α may be more important regulators of skeletal muscle insulin signaling and sensitivity.

Increased 1,25(OH)2-Vitamin D Concentrations after Energy Restriction Are Associated with Changes in Skeletal Muscle Phenotype

“…However, other reports have shown that ER may have benefits on skeletal muscle, particularly in preventing age-related loss of muscle mass [ 4 , 5 , 6 , 7 ]. A variety of mechanisms have been proposed to explain the beneficial effects of ER on muscle, including: retarding deterioration of neuromuscular function [ 8 ], diminishing the dysfunction of slow muscles [ 9 ], modifying mitochondrial ultrastructure [ 10 ], decreasing mitochondrial oxidative damage [ 11 ], and increasing skeletal muscle insulin sensitivity [ 12 ].…”

Surgical site peptidylarginine deaminase 4 (PAD4), a biomarker of NETosis, correlates with insulin resistance in total joint arthroplasty patients: A preliminary report

“…The supernatant was then frozen in liquid nitrogen and stored at -80°C for subsequent analysis. SDS-PAGE was performed using standard methods, as previously described [ 27 ]. Briefly, 20μg of total tissue protein extract was boiled in 1X Laemmeli sample buffer and loaded on to 12% Criterion XT Bis-Tris gels (Bio-Rad, Hercules, CA, USA).…”

Germline or inducible knockout of p300 or CBP in skeletal muscle does not alter insulin sensitivity

“…p300 and CBP protein abundance were normalized against eEF2 or GAPDH and are presented relative to average WT values. Antibodies used in this study have been used in previous published studies by our laboratory: p300 and CBP (38); Akt, pAkt T308 , pAkt S473 , GSK3␣/␤, pGSK3␣/␤ S21/9 , eEF2, and GAPDH (47,67); they were validated by determining if bands were at the correct molecular weight by using recombinant proteins and/or assessing changes in band intensity according to appropriate interventions (e.g., knockout, insulin stimulation). Gels used for silver staining were assayed according to manufacturer's instructions (Pierce Silver Stain Kit 24612; Thermo Scientific, Waltham, MA).…”

“…Quantitative PCR (qPCR). Total RNA was isolated, cDNA was synthesized, and qPCR was performed as previously described (47,67). Relative expression levels were calculated by the ⌬⌬C t method, using TATA-box binding protein (tbp) as the normalization control.…”

“…2DOG uptake. Basal and insulin-stimulated 2DOG uptake was measured in isolated and paired soleus and extensor digitorum longus (EDL) muscles, as previously described (47,62,68). A physiological insulin concentration of 0.36 nM was used for muscles treated with insulin.…”