Matching Items (21)

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Muscle IGF-1 Regulation in Humans with Obesity

Description

Objective: Isoforms of insulin-like growth factor-1 (IGF-1) gene encodes different IGF-1 isoforms by alternative splicing, and which are known to play distinct roles in muscle growth and repair. These isoforms in humans exist as IGF-1Ea, IGF-1Eb and IGF-1Ec (the latter

Objective: Isoforms of insulin-like growth factor-1 (IGF-1) gene encodes different IGF-1 isoforms by alternative splicing, and which are known to play distinct roles in muscle growth and repair. These isoforms in humans exist as IGF-1Ea, IGF-1Eb and IGF-1Ec (the latter is also known as mechano-growth factor). We sought to determine whether mRNA expression of any of these isoforms is impaired in skeletal muscle of humans with obesity, and given that humans with obesity display reduced protein synthesis in muscle. Methods: We studied 10 subjects (3 females/7 males) with obesity (body mass index: 34 ± 1 kg/m2) and 14 subjects (6 females/8 males) that were lean (body mass index: 24 ± 1 kg/m2) and served as controls. The groups represented typical populations of individuals that differed (P < 0.05) in body fat (obese: 32 ± 2; lean: 22 ± 2) and insulin sensitivity (Matsuda insulin sensitivity index, obese: 5 ± 1; lean 11 ± 2). Total RNA was extracted from 20-30 mg of tissue from muscle biopsies performed after an overnight fast. Isolated RNA was used to perform cDNA synthesis. Real-time PCR was performed using predesigned TaqMan® gene expression assays (Thermo Fisher Scientific Inc) for IGF-1Ea (assay ID: Hs01547657_m1), IGF-1Eb (assay ID: Hs00153126_m1) and IGF-1Ec (assay ID: Hs03986524_m1), as well as ACTB (assay ID: Hs01060665_g1), which was used to adjust the IGF-1 isoform mRNA expression. Responses for mRNA expression were calculated using the comparative CT method (2-ΔΔCT). Results: IGF-1Eb mRNA expression was lower in the subjects with obesity compared to the lean controls (0.67 ± 0.09 vs 1.00 ± 0.13; P < 0.05) but that of IGF-1Ea (0.99 ± 0.16 vs 1.00 ± 0.33) or IGF-1Ec (0.83 ± 0.14 vs 1.00 ± 0.21) were not different between groups (P > 0.05). Conclusions: Among the IGF-1 mRNA isoforms, IGF-1Eb mRNA is uniquely decreased in humans with obesity. Lower IGF-1Eb mRNA expression in muscle of humans with obesity may explain the lower protein synthesis observed in these individuals. Furthermore, these findings may have direct implications for muscle growth and repair in humans with obesity.

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2019-05

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The Role of UCP-1 in Human Omental Tissue

Description

The prevalence of obesity continues to increase in the United States, along with its risk for other associated cardiovascular and metabolic diseases. Several therapeutic methods are aimed at targeting and reducing obesity, now defined as a state of chronic, low-grade

The prevalence of obesity continues to increase in the United States, along with its risk for other associated cardiovascular and metabolic diseases. Several therapeutic methods are aimed at targeting and reducing obesity, now defined as a state of chronic, low-grade inflammation (in addition to BMI > 30 kg/m2). In an attempt to expand on these therapeutic methods, research on the concept of browning in white adipose tissue (WAT) and brown adipose tissue (BAT) is being conducted. Brown adipose tissue (BAT), and a newly discovered type of adipocyte, beige adipocytes, are heavily involved in thermogenesis with the use of uncoupling protein-1 (UCP-1). This paper focuses on the analysis of common browning genes, ATP-related genes, and metabolic genes in varying biological groups in mice (Chow/High-Fat Diet and Inguinal FAT and Perigonadal Fat) and in humans (Lean/Obese and Subcutaneous WAT (SC) and Omental WAT (OM)) using methods such as RT-PCR and immunohistochemistry. The data obtained shows an increase in browning in the leaner group, specifically in the subcutaneous fat. Further, browning is significantly reduced in the obese groups of subjects and mice tested, in addition to omental/perigonadal versus subcutaneous/inguinal fat depots. Interestingly, two key ATP genes, UCP-1 and COX4I1 are vastly elevated in the OM WAT, indicating that browning may not be as important in the OM, but rather may have a potential role in SC. This is contrary to prior research findings that attempt to exclude mice surrogates in future experimentation of the browning phenomenon. Further experimentation is needed to expand on the findings of this paper.

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Date Created
2019-12

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Effects of Acute Exposure to Increased Plasma Branched-Chain Amino Acid Concentrations on Insulin-Mediated Plasma Glucose Turnover in Healthy Young Subjects

Description

Background: Plasma branched-chain amino acids (BCAA) are inversely related to insulin sensitivity of glucose metabolism in humans. However, currently, it is not known whether there is a cause-and-effect relationship between increased plasma BCAA concentrations and decreased insulin sensitivity.

Objective: To determine the effects

Background: Plasma branched-chain amino acids (BCAA) are inversely related to insulin sensitivity of glucose metabolism in humans. However, currently, it is not known whether there is a cause-and-effect relationship between increased plasma BCAA concentrations and decreased insulin sensitivity.

Objective: To determine the effects of acute exposure to increased plasma BCAA concentrations on insulin-mediated plasma glucose turnover in humans.

Methods: Ten healthy subjects were randomly assigned to an experiment where insulin was infused at 40 mU/m2/min (40U) during the second half of a 6-hour intravenous infusion of a BCAA mixture (i.e., BCAA; N = 5) to stimulate plasma glucose turnover or under the same conditions without BCAA infusion (Control; N = 5). In a separate experiment, seven healthy subjects were randomly assigned to receive insulin infusion at 80 mU/m2/min (80U) in association with the above BCAA infusion (N = 4) or under the same conditions without BCAA infusion (N = 3). Plasma glucose turnover was measured prior to and during insulin infusion.

Results: Insulin infusion completely suppressed the endogenous glucose production (EGP) across all groups. The percent suppression of EGP was not different between Control and BCAA in either the 40U or 80U experiments (P > 0.05). Insulin infusion stimulated whole-body glucose disposal rate (GDR) across all groups. However, the increase (%) in GDR was not different [median (1st quartile – 3rd quartile)] between Control and BCAA in either the 40U ([199 (167–278) vs. 186 (94–308)] or 80 U ([491 (414–548) vs. 478 (409–857)] experiments (P > 0.05). Likewise, insulin stimulated the glucose metabolic clearance in all experiments (P < 0.05) with no differences between Control and BCAA in either of the experiments (P > 0.05).

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Date Created
2015-03-17

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Reproducibility of an HPLC-ESI-MS/MS Method for the Measurement of Stable-Isotope Enrichment of in Vivo-Labeled Muscle ATP Synthase Beta Subunit

Description

We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase β subunit (β-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is

We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase β subunit (β-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, β-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple β-F1-ATPase peptides. There were three β-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to β-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R[superscript 2] = 0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the β-F1-ATPase[subscript 134-143] peptide. Measured stable-isotope enrichment was highly reproducible for the β-F1-ATPase[subscript 134-143] peptide (CV = 2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R[superscript 2] = 0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the β-F1-ATPase[subscript 134-143] peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle β-F1-ATPase based on the determination of the enrichment of the β-F1-ATPase[subscript 134-143] peptide.

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2011-10-12

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Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated With Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit

Description

Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The

Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = – 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish impaired β-F1-ATPase translation as an important consequence of obesity.

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Date Created
2016-08-17

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Gene Expression Profiling in Skeletal Muscle of Patients at Risk for NODAT

Description

New-onset diabetes after kidney transplantation (NODAT) occurs in 20% of kidney transplant patients. In 5 patients who are at risk for new-onset diabetes after kidney transplantation, skeletal muscle gene expression profiling was performed both before and after kidney transplant. The

New-onset diabetes after kidney transplantation (NODAT) occurs in 20% of kidney transplant patients. In 5 patients who are at risk for new-onset diabetes after kidney transplantation, skeletal muscle gene expression profiling was performed both before and after kidney transplant. The differences in gene expression before and after transplant were compared in order to identify specific genes that could be linked to developing NODAT. These findings could open new avenues for future research.

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Date Created
2014-05

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Evaluation of Anthropometric, Biochemical, and Dietary Factors as Determinants of Insulin Resistance in Healthy Humans

Description

Type 2 diabetes mellitus (T2DM) is a life-long disease that affects over 27 million individuals in the United States alone. There are many different risk factors and pre-indicators of T2DM. One of them is insulin resistance. Insulin resistance occurs when

Type 2 diabetes mellitus (T2DM) is a life-long disease that affects over 27 million individuals in the United States alone. There are many different risk factors and pre-indicators of T2DM. One of them is insulin resistance. Insulin resistance occurs when the body is unable to appropriately respond to insulin. This in turn leads to increased levels of glucose and insulin in the bloodstream. Unlike T2DM, insulin resistance is a reversible diagnosis. The purpose of this project was to identify the most influential genetic and dietary factors of insulin resistance and to see if individuals have some extent of control to possibly avoid the diagnosis of insulin resistance and possibly T2DM entirely.
A total of 26 human subjects were used in this study. Each subject was classified as either lean or obese, according to their BMI measurement. First, the subjects underwent an oral glucose tolerance test. Blood samples were taken to measure glucose levels in the blood. After the test subject characteristics for each subject was obtained. These included age, BMI, body fat percentage, fat free mass (FFM), height, total mass, waist circumference, hip circumference, and waist to hip ratio. After the subject characteristics and blood glucose were measured the blood samples taken previously were then centrifuged, and the blood plasma was extracted. The blood plasma was then used to undergo an Insulin ELISA test. After extensive analysis, the Matsuda Index of each subject was obtained. Subjects with a Matsuda value of 6.0 or under were considered insulin resistant while subjects with a Matsuda value higher than 6.0 were considered insulin sensitive. Subjects were also required to submit a dietary record over the course of three days. The food intake was then put into a food processing software which gave a daily average of the macro and micro nutrients for each subject. Both the subject and dietary values were put into a multiple regression with a significance factor of p < 0.5 to see which factors contributed most to the Matsuda value.
It was found that BMI, height, total mass, insulin and fat free mass, all of which were subject characteristics, were considered to be significant. Some of these factors an individual has no control over, such as height and insulin. However other factors such as BMI, total mass and fat free mass can be affected by both a healthy diet and frequent exercise. This study validated that diet and physical activity can greatly influence an individual’s susceptibility to insulin resistance and ultimately T2DM.

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Date Created
2019-05

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Next-Generation Sequencing for DNA Methylation Profiling in Blood and Skeletal Muscle

Description

DNA methylation, a subset of epigenetics, has been found to be a significant marker associated with variations in gene expression and activity across the entire human genome. As of now, however, there is little to no information about how DNA

DNA methylation, a subset of epigenetics, has been found to be a significant marker associated with variations in gene expression and activity across the entire human genome. As of now, however, there is little to no information about how DNA methylation varies between different tissues inside a singular person's body. By using research data from a preliminary study of lean and obese clinical subjects, this study attempts to put together a profile of the differences in DNA methylation that can be observed between two particular body tissues from this subject group: blood and skeletal muscle. This study allows us to start describing the changes that occur at the epigenetic level that influence how differently these two tissues operate, along with seeing how these tissues change between individuals of different weight classes, especially in the context of the development of symptoms of Type 2 Diabetes.

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2013-12

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Analyzing Myosin Heavy Chain Isoform Distribution in Skeletal Muscle: a methodological approach

Description

Over 40% of adults in the United States are considered obese. Obesity is known to cause abnormal metabolic effects and lead to other negative health consequences. Interestingly, differences in metabolism and contractile performance between obese and healthy weight individuals are

Over 40% of adults in the United States are considered obese. Obesity is known to cause abnormal metabolic effects and lead to other negative health consequences. Interestingly, differences in metabolism and contractile performance between obese and healthy weight individuals are associated with differences in skeletal muscle fiber type composition between these groups. Each fiber type is characterized by unique metabolic and contractile properties, which are largely determined by the myosin heavy chain isoform (MHC) or isoform combination that the fiber expresses. In previous studies, SDS-PAGE single fiber analysis has been utilized as a method to determine MHC isoform distribution and single fiber type distribution in skeletal muscle. Herein, a methodological approach to analyze MHC isoform and fiber type distribution in skeletal muscle was fine-tuned for use in human and rodent studies. In the future, this revised methodology will be implemented to evaluate the effects of obesity and exercise on the phenotypic fiber type composition of skeletal muscle.

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2021-05

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Investigating the Response of the Dopamine Metabolite Homovanillic Acid (HVA) to Acute Physical Exercise in Lean Adults and Adults with Obesity

Description

Seven human subjects with body mass indices (BMIs) ranging from 19.4 kg/ m2 to 26.7 kg/ m2 and six human subjects with BMIs ranging from 32.1 kg/ m2 to 37.6 kg/ m2 were recruited and subjected to 45-minute bouts of

Seven human subjects with body mass indices (BMIs) ranging from 19.4 kg/ m2 to 26.7 kg/ m2 and six human subjects with BMIs ranging from 32.1 kg/ m2 to 37.6 kg/ m2 were recruited and subjected to 45-minute bouts of acute exercise to look at the changes in the plasma concentration of the dopamine metabolite homovanillic acid (HVA) in response to acute physical activity. Plasma HVA concentration was measured before exercise and during the last 10 minutes of the exercise bout via competitive ELISA. On average the optical density (OD) of the samples taken from lean subjects decreased from 0.203 before exercise to 0.192 during exercise, indicating increased plasma HVA concentration. In subjects with obesity OD increased from 0.210 before exercise to 0.219 during exercise, indicating reduced plasma HVA concentration. These differences in OD were not statistically significant. Between the lean group and the group with obesity no significant difference was observed between the OD of the plasma samples taken before exercise, but a significant difference (p = 0.0209) was observed between the ODs of the samples taken after exercise. This indicated that there was a significant difference between the percent changes in OD between the lean group and the group with obesity, which suggested that there may be a body weight-dependent difference in the amount of dopamine released in response to exercise. Because of the lack of significance in the changes in OD within the lean group and the group with obesity the results of this study were insufficient to conclude that this difference is not due to chance, but further investigation is warranted.

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2021-05