Intramyocellular Lipid Content Research Articles

Inflammation-associated intramyocellular lipid alterations in human pancreatic cancer cachexia.

Cancer cachexia is a multifactorial metabolic syndrome characterized by systemic inflammation and ongoing skeletal muscle loss resulting in weakness, poor quality of life, and decreased survival. Whereas lipid accumulation in skeletal muscle is associated with cancer cachexia as well as the prognosis of cancer patients, surprisingly little is known about the nature of the lipids that accumulate in the muscle during cachexia, and whether this is related to inflammation. We aimed to identify the types and distributions of intramyocellular lipids in patients with and without cancer cachexia. Rectus abdominis muscle biopsies were collected during surgery of patients with pancreatic ductal adenocarcinoma (n=10 without cachexia, n=20 cachectic without inflammation (CRP<10mg/L), n=10 cachectic with inflammation (CRP≥10mg/L). L3-CT scans were analysed to assess body composition based on validated thresholds in Hounsfield units (HU). Muscle sections were stained with Oil-Red O and H&E to assess general lipid accumulation and atrophy. Untargeted lipidomic analyses were performed on laser-microdissected myotubes using LC-MS/MS. The spatial distribution of intramyocellular lipids with differential abundance between groups was visualized by mass-spectrometry imaging. Genes coding for inflammation markers and enzymes involved in de novo ceramide synthesis were studied by qPCR. Muscle radiation attenuation was lower in cachectic patients with inflammation (median 24.3 [18.6-30.8] HU) as compared with those without inflammation (34.2 [29.3-38.7] HU, P=0.033) or no cachexia (37.4 [33.9-42.9] HU, P=0.012). Accordingly, intramyocellular lipid content was lower in non-cachectic patients (1.9 [1.6-2.1]%) as compared with those with cachexia with inflammation (5.5 [4.5-7.3]%, P=0.002) or without inflammation (4.8 [2.6-6.0]%, P=0.017). Intramyocellular lipid accumulation was associated with both local IL-6 mRNA levels (rs=0.57, P=0.015) and systemic CRP levels (rs=0.49, P=0.024). Compared with non-cachectic subjects, cachectic patients had a higher relative abundance of intramyocellular glycerophospholipids and a lower relative abundance of glycerolipids. Furthermore, increases in several intramyocellular lipids such as SM(d36:1), PC(34:1), and TG(48:1) were found in cachectic patients with inflammation and correlated with specific cachexia features. Altered intramyocellular lipid species such as PC(34:1), LPC(18:2), and TG(48:1) showed an uneven distribution in muscle sections of cachectic and non-cachectic patients, with areas featuring abundance of these lipids next to areas almost devoid of them. Intramyocellular lipid accumulation in patients with cachexia is associated with both local and systemic inflammation, and characterized by changes in defined lipid species such as glycerolipids and glycerophospholipids.

Excess Intramyocellular Lipid Does Not Affect Muscle Fiber Biophysical Properties in Mice or People with Metabolically Abnormal Obesity.

Observational studies show correlations between intramyocellular lipid (IMCL) content and muscle strength and contractile function in people with "metabolically abnormal" obesity. However, a clear physiologic mechanism for this association is lacking and causation is debated. We combined immunofluorescent confocal imaging with force measurements on permeabilized muscle fibers from metabolically normal and metabolically abnormal mice and metabolically normal (defined as normal fasting plasma glucose and glucose tolerance) and metabolically abnormal (defined as pre-diabetes and type 2 diabetes) people with overweight/obesity to evaluate relationships among myocellular lipid droplet characteristics (droplet size and density) and biophysical (active contractile and passive viscoelastic) properties. The fiber type specificity of lipid droplet parameters varied between metabolically abnormal and normal mice and among metabolically normal and metabolically abnormal people. However, despite considerable quantities of IMCL in the metabolically abnormal groups, there were no significant differences in peak active tension or passive viscoelasticity between the metabolically abnormal groups and the control group in mice or people. Additionally, there were no significant relationships among IMCL parameters and biophysical variables. Thus, we conclude that IMCL accumulation per se does not impact muscle fiber biophysical properties or physically impede contraction.

Association of insulin resistance with the accumulation of saturated intramyocellular lipid: A comparison with other fat stores.

It has been shown using proton magnetic resonance spectroscopy (1 H MRS) that, in a group of females, whole-body insulin resistance was more closely related to accumulation of saturated intramyocellular lipid (IMCL) than to IMCL concentration alone. This has not been investigated in males. We investigated whether age- and body mass index-matched healthy males differ from the previously reported females in IMCL composition (measured as CH2 :CH3 ) and IMCL concentration (measured as CH3 ), and in their associations with insulin resistance. We ask whether saturated IMCL accumulation is more strongly associated with insulin resistance than other ectopic and adipose tissue lipid pools and remains a significant predictor when these other pools are taken into account. In this group of males, who had similar overall insulin sensitivity to the females, IMCL was similar between sexes. The males demonstrated similar and even stronger associations of IMCL with insulin resistance, supporting the idea that a marker reflecting the accumulation of saturated IMCL is more strongly associated with whole-body insulin resistance than IMCL concentration alone. However, this marker ceased to be a significant predictor of whole-body insulin resistance after consideration of other lipid pools, which implies that this measure carries no more information in practice than the other predictors we found, such as intrahepatic lipid and visceral adipose tissue. As the marker of saturated IMCL accumulation appears to be related to these two predictors and has a much smaller dynamic range, this finding does not rule out a role for it in the pathogenesis of insulin resistance.

OR22-03 Enhanced Endothelial Mineralocorticoid Receptor Signaling Mediates Diet - Induced Soleus Fat Infiltration And Insulin Resistance

Abstract Disclosure: C. Homan: None. J. Habibi: None. D. Chen: None. A. Whaley-Connell: None. J.R. Sowers: None. G. Jia: None. Skeletal muscle is an important organ in the storage and release of lipids in response to physiological changes in free fatty acid (FFA) supply and demand. Once fat tissue mass expansion exceeds its blood supply excess lipids are stored ectopically as small lipid droplets in non-adipose tissues such as skeletal muscle. Recently, our data found that mineralocorticoid receptors (MRs) mediate Western diet (WD)-induced lipid infiltration of skeletal muscle and insulin resistance. Related to this, MRs, the principal receptors for the hormone aldosterone in the kidney, also exist in vascular cells, including endothelial cells (ECs). Moreover, enhanced MR signaling in ECs (ECMR) induces arterial stiffening and impairs microcirculation. However, our understanding of the precise mechanisms by which enhanced ECMR activation promotes skeletal muscle insulin resistance remains unclear. In this study we investigated the roles of ECMR in soleus lipid accumulation and corresponding insulin resistance in diet-induced obesity. Six-week-old ECMR wild type (ECMR+/+) and knockout (ECMR-/-) mice were fed either a mouse chow diet or WD for 16 weeks. 16 weeks of WD induced increases in glucose intolerance, fasting plasma insulin levels, HOMA-IR and insulin resistance index were found to be blunted in ECMR-/- mice. ECMR-/- prevented WD-induced increase in soleus FFA levels without changing serum FFA levels. Meanwhile, WD also increased soleus intramyocellular lipid content, plasma EC derived exosomal CD36 and soleus CD36 protein levels. These abnormalities were related to reduced soleus insulin metabolic signaling in PI3K/Akt pathways, as these pathways were blunted in ECMR-/- mice. These findings indicate that EC specific MR activation contributes to diet-induced increases in CD36 expression, soleus fat infiltration, as well as systemic and skeletal muscle insulin resistance. Presentation: Saturday, June 17, 2023

Both moderate- and high-intensity exercise training increase intramyocellular lipid droplet abundance and modify myocellular distribution in adults with obesity.

Exercise training modifies lipid metabolism in skeletal muscle, but the effect of exercise training on intramyocellular lipid droplet (LD) abundance, size, and intracellular distribution in adults with obesity remains elusive. This study compared high-intensity interval training (HIIT) with more conventional moderate-intensity continuous training (MICT) on intramyocellular lipid content, as well as LD characteristics (size and number) and abundance within the intramyofibrillar (IMF) and subsarcolemmal (SS) regions of type I and type II skeletal muscle fibers in adults with obesity. Thirty-six adults with obesity [body mass index (BMI) = 33 ± 3 kg/m2] completed 12 wk (4 days/wk) of either HIIT (10 × 1 min, 90% HRmax + 1-min active recovery; n = 19) or MICT (45-min steady-state exercise, 70% HRmax; n = 17), while on a weight-maintaining diet throughout training. Skeletal muscle biopsies were collected from the vastus lateralis before and after training, and intramyocellular lipid content and intracellular LD distribution were measured by immunofluorescence microscopy. Both MICT and HIIT increased total intramyocellular lipid content by more than 50% (P < 0.01), which was attributed to a greater LD number per µm2 in the IMF region of both type I and type II muscle fibers (P < 0.01). Our findings also suggest that LD lipophagy (autophagy-mediated LD degradation) may be transiently upregulated the day after the last exercise training session (P < 0.02 for both MICT and HIIT). In summary, exercise programs for adults with obesity involving either MICT or HIIT increased skeletal muscle LD abundance via a greater number of LDs in the IMF region of the myocyte, thereby providing more lipid in close proximity to the site of energy production during exercise.NEW & NOTEWORTHY In this study, 12 wk of either moderate-intensity continuous training (MICT) or high-intensity interval training (HIIT) enhanced skeletal muscle lipid abundance by increasing lipid droplet number within the intramyofibrillar (IMF) region of muscle. Because the IMF associates with high energy production during muscle contraction, this adaptation may enhance lipid oxidation during exercise. Despite differences in training intensity and energy expenditure between MICT and HIIT, their effects on muscle lipid abundance and metabolism were remarkably similar.

Intramyocellular Triglyceride Content During the Early Course of Type 1 and Type 2 Diabetes Mellitus.

Intramyocellular lipid content (IMCL) is elevated in insulin-resistant humans, but its changes over time and relationships with comorbidities remain unclear. We examined IMCL during the initial course of diabetes and its associations with complications. Participants of the German Diabetes Study (GDS) with recent-onset type 1 (n=132) or type 2 diabetes (n=139) and glucosetolerant controls (n=128) underwent 1H-magnetic resonance spectroscopy to measure IMCL and muscle volume, whole-body insulin sensitivity (hyperinsulinemic-euglycemic clamps; Mvalue) and cycling spiroergometry (VO2max). Subgroups underwent the same measurements after 5 years. At baseline, IMCL was ∼30% higher in type 2 diabetes than in other groups independently of age, sex, BMI and muscle volume. In type 2 diabetes, M-value was ∼36% and ∼62% lower compared to type 1 diabetes and controls, respectively. After 5 years, M-value decreased by ∼29% in type 1 and ∼13% in type 2 diabetes, whereas IMCL remained unchanged. The correlation of IMCL and M-value in type 2 diabetes at baseline was modulated by VO2max. IMCL also associated with microalbuminuria, Framingham risk score for cardiovascular disease and cardiac autonomic neuropathy. Changes in IMCL within 5 years after diagnosis do not mirror progression of insulin resistance in type 2 diabetes but associate with early diabetesrelated complications.

Quantitative magnetic resonance spectroscopy and imaging analysis of the lipid content in the psoas major and its association with intervertebral disc degeneration: a cross-sectional study

Aim: It is shown that the diminished function of the psoas major is mainly associated with increased lipid content; nonetheless, whether the fat content of the psoas major is associated with intervertebral disc degeneration (IVDD) is still under study. It is hypothesized that IVDD is correlated with the lipid content of the psoas major and that IVDD can be comprehensively investigated using quantitative magnetic resonance imaging (MRI). Consequently, this study aimed to analyse the association between the psoas major extramyocellular lipid (EMCL) and intramyocellular lipid (IMCL) content and disc degeneration quantified using MRI T2 mapping in patients with chronic low back pain (CLBP). Methods: In this study, 20 men and 23 women (mean age, 63.1 years ± 2.6 years) underwent magnetic resonance spectroscopy (MRS). The psoas major IMCL and EMCL content of patients was analysed. T2 values of the anterior annulus fibrosus (AF), nucleus pulposus (NP), and posterior AF were evaluated using MRI T2 mapping. Multiple linear regression analysis was used to determine the possible correlations of the psoas major EMCL and IMCL content with the T2 values of the anterior AF, NP, and posterior AF. Results: The EMCL content and T2 values of the anterior AF were significantly and negatively correlated [standardised partial regression coefficient (β) = −0.60, P &lt; 0.01]. There were no significant correlations between the EMCL content and T2 values of the NP (β = −0.16, P = 0.30) or posterior AF (β = −0.14, P = 0.49) or between the IMCL content and T2 values of the anterior AF (β = −0.04, P = 0.82), NP (β = 0.05, P = 0.73), or posterior AF (β = −0.11, P = 0.58). Conclusions: The EMCL content and T2 values of the anterior AF were correlated. Conversely, the IMCL content and T2 values were not correlated.

Effect of an acute long-duration exercise bout on skeletal muscle lipid droplet morphology, GLUT 4 protein, and perilipin protein expression.

Smaller lipid droplet morphology and GLUT 4 protein expression have been associated with greater muscle oxidative capacity and glucose uptake, respectively. The main purpose of this study was to determine the effect of an acute long-duration exercise bout on skeletal muscle lipid droplet morphology, GLUT4, perilipin 3, and perilipin 5 expressions. Twenty healthy men (age 24.0 ± 1.0 years, BMI 23.6 ± 0.4 kg/m2) were recruited for the study. The participants were subjected to an acute bout of exercise on a cycle ergometer at 50% VO2max until they reached a total energy expenditure of 650kcal. The study was conducted after an overnight fast. Vastus lateralis muscle biopsies were obtained before and immediately after exercise for immunohistochemical analysis to determine lipid, perilipin 3, perilipin 5, and GLUT4 protein contents while GLUT 4 mRNA was quantified using RT-qPCR. Lipid droplet size decreased whereas total intramyocellular lipid content tended to reduce (p=0.07) after an acute bout of endurance exercise. The density of smaller lipid droplets in the peripheral sarcoplasmic region significantly increased (0.584 ± 0.04 to 0.638 ± 0.08 AU; p=0.01) while larger lipid droplets significantly decreased (p<0.05). GLUT4 mRNA tended to increase (p=0.05). There were no significant changes in GLUT 4, perilipin 3, and perilipin 5 protein levels. The study demonstrates that exercise may impact metabolism by enhancing the quantity of smaller lipid droplets over larger lipid droplets.

849-P: Antidiabetic Effects of TLC-3595, a Selective ACC2 Inhibitor, in ZDF Rats

Background: TLC-3595 is a potent (IC50=14nM), selective, small molecule, allosteric inhibitor of acetyl-CoA carboxylase 2 (ACC2). TLC-3595 provides a novel therapeutic approach for type 2 diabetes (T2D), via reduction in intramyocellular lipid (IMCL) content, that is complementary to approved therapies. Reduced IMCL strongly correlates with insulin sensitivity (IS). Here, we demonstrate antidiabetic effects of TLC-3595 in Zucker Diabetic Fatty (ZDF) rats. Methods: IMCL was measured by 1H-MRS. T2D progression and IS by hyperinsulinemic-euglycemic clamp were assessed in 30% diet-restricted ZDF rats treated with TLC-3595 (1.67, 5, or 15mg/kg) or pioglitazone (Pio, 0.75mg/kg) daily for 5-12 weeks. Results: TLC-3595 or an analog caused rapid and sustained IMCL reduction (~50%) in lean and dysmetabolic rodents. TLC-3595 enhanced systemic fatty acid oxidation (increased plasma beta-hydroxybutyrate) and reduced cardiac lipid in ZDF rats. TLC-3595 dose-dependently delayed β-cell failure in ZDF rats and was non-inferior to Pio for reductions in hyperglycemia (15-25% lower glucose AUC) and HbA1c (0.6-1.6% vs. vehicle) (Fig A, B). TLC-3595 improved IS and increased clamp glucose infusion rate by 29%, (Fig C). Conclusion: TLC-3595 reduced IMCL, improved IS, and delayed T2D progression comparable to Pio in ZDF rats. These data provide strong rationale for evaluation of TLC-3595 to treat insulin resistance and T2D. Disclosure A. Vijayakumar: Employee; OrsoBio Inc, Gilead Sciences, Inc. E. Murakami: Employee; OrsoBio. R.S. Huss: Employee; OrsoBio, Inc. Stock/Shareholder; OrsoBio, Inc. N. Sroda: None. A. Shimazaki: Employee; Shionogi &amp; Co. Ltd. Y. Kashiwagi: Employee; Shionogi &amp; Co. Ltd. R.P. Myers: Employee; OrsoBio, Inc. Stock/Shareholder; OrsoBio, Inc. M. Subramanian: Other Relationship; OrsoBio Inc. G.I. Shulman: Other Relationship; AstraZeneca, Merck &amp; Co., Inc., Janssen Research &amp; Development, LLC. Consultant; Novo Nordisk. Other Relationship; iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc. Consultant; DiCerna Pharmaceuticals, Inc. Other Relationship; Equator Therapeutics, Inc., Generian Pharmaceuticals. Stock/Shareholder; Levels Health, Inc. Other Relationship; Fortress Biotech, Inc. Consultant; Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc. Other Relationship; OrsoBio, Aro Biotherapeutics Company.

1630-P: Reduced Fatty Acid Oxidation Does Not Prevent Intralipid-Induced Peripheral Insulin Resistance in Humans

Mitochondrial handling of excess lipid substrate is thought to be an important determinant of insulin resistance. Evidence from animal models support insulin signaling defects resulting from either intra-cellular lipid accumulation or by mitochondrial dysfunction and altered fatty acid oxidation (FAO) flux. To explore the relationship between FAO and insulin sensitivity, we measured the change in peripheral glucose disposal (Rd) during a 5-hour hyperinsulinemic euglycemic clamp with co-infusion of a control glycerol solution versus a 20% intralipid solution in 18 subjects with an inherited defect in FAO (FAOD; including Carnitine Palmitoyltransferase 2, Very long-chain, medium-chain or long-chain 3-hydroxy AcylcoA Dehydrogenase Deficiencies) and 18 age, sex, and weight-matched normal healthy controls. Rd was similar between groups during the glycerol control clamp. Intramyocellular lipid (IMCL) content as measured by MRS increased after the 5 hour intralipid infusion in controls but not in participants with FAOD. Plasma free fatty acids during intralipid infusion were higher in FAOD compared to controls suggesting infused lipid remained in circulation. Muscle fiber type and expression of lipid transporters CD36 and FATP1 were similar in biopsy samples between groups and cannot explain decreased lipid uptake into muscle among FAOD subjects. However, intralipid suppressed Rd equally in subjects with FAOD and controls inducing peripheral insulin resistance in both groups. Impaired FAO decreased lipid accumulation in the muscle during intralipid infusion, but despite lower IMCL, subjects with FAOD developed peripheral insulin resistance similar to controls. Increased IMCL is not essential for, nor is reduced FAO flux protective against, the development of peripheral insulin resistance during intralipid infusion. Disclosure M.B.Gillingham: None. J.Q.Purnell: Advisory Panel; Boehringer Ingelheim International GmbH, Novo Nordisk. A.N.Gregor: None. O.Varlamov: None. J.J.Robino: None. C.E.Mccurdy: None. K.T.Greyslak: None. D.Choi: None. E.Baetscher: Consultant; Biogen. W.Rooney: Consultant; Ultragenix, Research Support; Revalesio. Funding National Institutes of Health (DK102813)

Endothelial MRs mediate Western diet-induced lipid disorders and skeletal muscle insulin resistance in females.

Consumption of a Western diet (WD) consisting of excess fat and carbohydrates activates the renin-angiotensin-aldosterone system, which has emerged as an important risk factor for systemic and tissue insulin resistance. We recently discovered that activated mineralocorticoid receptors (MRs) in diet-induced obesity induces CD36 expression, increases ectopic lipid accumulation, and results in systemic and tissue insulin resistance. Here, we further investigated whether endothelial cell (EC) specific MR (ECMR) activation participates in WD-induced ectopic skeletal muscle lipid accumulation, insulin resistance and dysfunction. Six-week-old female ECMR knockout (ECMR-/-) and wild type (ECMR+/+) mice were fed either a WD or a chow diet for 16 weeks. ECMR-/- mice were found to have decreased WD-induced in vivo glucose intolerance and insulin resistance at 16-weeks. Improved insulin sensitivity was accompanied by increased glucose transporter type 4 (Glut4) expression in conjunction with improved soleus insulin metabolic signaling in phosphoinositide 3-kinases/protein kinase B and endothelial nitric oxide synthase activation. Additionally, ECMR-/- also blunted WD-induced increases in CD36 expression and associated elevations in soleus free fatty acid, total intramyocellular lipid content, oxidative stress, and soleus fibrosis. Moreover, in vitro and in vivo activation of ECMR increased EC derived exosomal CD36 that was further taken up by skeletal muscle cells, leading to increased skeletal muscle CD36 levels. These findings indicate that in the context of an obesogenic WD, enhanced ECMR signaling increases EC derived exosomal CD36 resulting in increased uptake and elevated concentrations of CD36 in skeletal muscle cells, contributing to increased lipid metabolic disorders and soleus insulin resistance.

Association Between Multifidus Intramyocellular Lipids and Pain-Related Disorders, Lumbar Spine Dysfunction, Gait Disturbance in Patients with Chronic Low Back Pain: A Cross-Sectional Study Using the Japanese Orthopaedic Association Back Pain Evaluation Questionnaire

Background and aims: Fat degeneration in the multifidus muscle (Mm) was more common than in other trunk muscles. We hypothesised that the lipid contents of the Mm and patient-reported quality of life (QOL) are related. However, there are no papers examining the association between fatty degeneration of the Mm and patient-reported QOL using magnetic resonance spectroscopy (MRS). This cross-sectional study aimed to investigate the association between patient-reported QOL, evaluated with the Japanese Orthopaedic Association Back Pain Evaluation Questionnaire (JOABPEQ), and the intramyocellular lipid (IMCL) and extramyocellular lipid (EMCL) contents of the Mm and psoas major (PM) by MRS in patients with chronic low back pain (CLBP). Materials and Methods: Forty-eight patients (mean age, 64.2 ± 13.2 years; age range, 41–79 years) with nonspecific CLBP underwent MRS for the quantification of IMCL and EMCL of the right Mm and PM in a volume of interest at L4/L5. All subjects underwent MRS and completed the JOABPEQ in the same day. We performed multiple linear regression analysis of the IMCL and EMCL contents of the Mm and PM with the five domains of the JOABPEQ adjusted for age, sex, and body mass index. Results: The IMCL content of the Mm was correlated with pain-related disorders (standardised partial regression coefficient (β)=−0.59, p&lt;0.01), lumbar spine dysfunction (β=−0.64, p&lt;0.01), and social life dysfunction (β=−0.31, p&lt;0.01) which showed moderate negative correlation, but not with psychological disorders. The EMCL content of the Mm was not correlated with the five domains of the JOABPEQ. The IMCL and EMCL contents of the PM was not correlated with the five domains of the JOABPEQ. Conclusions: IMCL content of the Mm was significantly correlated with the pain-related disorders, lumbar spine dysfunction, gait disturbance, and social life dysfunction domain scores of the JOABPEQ. Future studies using magnetic resonance spectroscopy of the Mm in patients with CLBP may help optimize exercise strategies using IMCL as an index to enhance patient-reported quality of life.

Mutation c.-379 C>T in DGAT1 affects intramyocellular lipid content by altering MYOD1 binding affinity.

Intramuscular fat (IMF) is one of the most important indexes of pork taste quality. Diacylglycerol acyltransferase 1 (DGAT1), belonging to the acyl-coenzyme A: DGAT enzymes family, is a rate-limiting enzyme responsible for the final step of triglyceride (TG) synthesis. It is involved in TG storage in skeletal muscle; however, the underlying mechanism is not well understood. This study aimed to uncover functional mutations that can influence DGAT1 expression and consequently affect IMF deposition in pork. Two experimental groups containing individuals with high and low IMF content (6.23 ± 0.20 vs. 1.25 ± 0.05, p < 0.01) were formed from 260 Duroc × Large White × Yorkshire (D × L × Y) cross-bred pigs. A novel SNP c.-379 C>T was uncovered in the DGAT1 gene using comparative sequencing with pool DNA of high- and low-IMF groups. The IMF content of CT genotype individuals (3.19 ± 0.11%) was higher than that of CC genotype individuals (2.86 ± 0.11%) when analyzing 260 D × L × Y pigs (p < 0.05). The DGAT1 expression levels revealed a significant positive correlation with IMF content (r=0.33, p < 0.01). Luciferase assay revealed that the DGAT1 promoter with the c.-379 T allele has a higher transcription activity than that bearing the C allele in C2C12 myoblast cells, but not in 3T3-L1 pre-adipocytes. Online prediction followed by chromatin immunoprecipitation-polymerase chain reaction assay confirmed that myogenic determination factor 1 (MYOD1) binds to the DGAT1 promoter with the c.-379 C allele but not the T allele. In vitro experiments demonstrated that MYOD1 represses DGAT1 transcription and lipogenesis. As a muscle-specific transcription factor, MYOD1 can inhibit the transcription of DGAT1 with the c.-379 C allele in muscle cells. However, in the absence of MYOD1 binding to the mutated DGAT1 promoter with the c.-379 T allele, DGAT1 expresses at a higher level in the muscle cells of the c.-379 T genotype, leading to more intramyocellular lipid accumulation than in the muscle cells of the c.-379 C genotype. The SNP c.-379 C>T in the promoter region of the DGAT1 gene provides a promising molecular marker for improving pork IMF content without affecting other fat depots.

ASSOCIATION BETWEEN CALF MUSCLE FATTY INFILTRATION AND PHYSICAL PERFORMANCE AMONG HEALTHY OLDER ADULTS

Abstract Muscle fatty infiltration is associated with impaired physical performance. However, the association between ectopic fat, such as lower extremity fat distribution, and sex-specific differences among older adults remains to be determined. This study aimed to examine sex-specific differences in calf muscle fat infiltration and their association with physical performance among healthy older adults aged 76 to 80 years. The pilot study comprised 40 subjects (20 men; 20 women) matched by age and body mass index. The participants underwent dual-energy X-ray absorptiometry, magnetic resonance imaging (MRI), and proton magnetic resonance spectroscopy (1H-MRS) to assess body composition and lower extremity fat distribution. The tibialis anterior intramyocellular lipid (IMCL) as assessed by 1H-MRS was negatively associated with the five-times sit-to-stand test scores (rs=0.518, p=0.023) in men, while the soleus IMCL content was negatively associated with the timed up-and-go test scores (rs=0.472, p=0.048) in women. However, the soleus extramyocellular lipid (EMCL) content was positively associated with the five-times sit-to-stand test scores (rs=−0.488, p=0.040) in women, but this association was not statistically significant in men. Regarding the calf cross-sectional area (CSA) parameters assessed by MRI, calf subcutaneous fat CSA was positively associated with the usual gait speed (rs=0.447, p=0.048) in women only. To conclude, this study showed an inverse correlation between IMCL content and physical performance in healthy older individuals and calf muscle-specific IMCL based on sex differences. Furthermore, our results suggest that greater EMCL content in the soleus and calf subcutaneous fat might affect physical performance positively in older women but not older men.

The influence of exercise training versus intensive insulin therapy on insulin resistance development in type 1 diabetes

The etiology of insulin resistance (IR) in Type 1 Diabetes (T1D) is unclear; however, intramyocellular lipids (IMCL) are likely contributors. While exercise lessens IR and IMCL content; T1D patients elevate glycemia to offset exercise-induced hypoglycemic risk. The preferred treatment for T1D patients is tight glucose management through intensive insulin therapy (IIT); however, IIT is accompanied with a sedentary lifestyle. The purpose of this study was to examine IR development and IMCL in combined exercise (DARE; aerobic/resistance) and IIT-treated T1D animals. 76 rats were divided into control sedentary (C), diabetic sedentary (CD), diabetes sedentary intensive insulin therapy (DIT) and DARE groups. Following streptozotocin (STZ), glycemia was maintained at either 9-15 mM (CD, DARE) or 5-9 mM (DIT) using insulin. DARE alternated between running and weighted climbing for 12 weeks. Results demonstrate that DARE exhibited reduced onset of IR compared with C, DIT and CD, indicated by increased glucose infusion rate (hyperinsulinemic-euglycemic-clamp). A shift in lipid metabolism was evident whereby diacylglycerol was elevated in DIT compared to DARE, while triacylglycerol was elevated in DARE. These findings indicate enhanced IMCL metabolism and the sequestration of fat as neutral triacylglycerol leads to reduced IR in DARE. In contrast, IIT and sedentary behavior leads to diacylglycerol accumulation and IR.

RF04 | PSUN334 Mineralocorticoid receptors mediate diet - induced lipid infiltration of skeletal muscle and insulin resistance

Abstract Excess blood lipids increase the total intramyocellular (IMC) lipid content and ectopic fat storage resulting in lipotoxicity and insulin resistance in skeletal muscle, which is one of the main targets of insulin whose action is central for the maintenance of glucose homeostasis. Consumption of a diet high in fat and refined sugars, a Western Diet (WD), has been shown to activate mineralocorticoid receptors (MRs) to promote insulin resistance. However, our understanding of the precise mechanisms by which enhanced MR activation promotes skeletal muscle insulin resistance remains unclear. In this study we investigated the roles and mechanisms by which enhanced MR signaling in soleus muscle promotes ectopic lipid accumulation and related insulin resistance in diet-induced obesity. Six week-old C57BL6J mice were fed either a mouse chow diet or WD with or without spironolactone (1 mg/kg/day) for 16 weeks. Spironolactone attenuated 16 weeks of WD - induced in vivo glucose intolerance and improved soleus insulin metabolic signaling (protein kinase B and AMP kinase α pathways). Improved insulin sensitivity was accompanied by increased Glut-4 expression in conjunction with decreased IMC lipid content and reduced free fatty acid (FFA) levels and CD36 expression in soleus skeletal muscle tissue. Related to this, miR-99a was identified to negatively target CD36(www.targetscan.org/vert_72/) and elevated CD36 induced excessive FFA uptake, ectopic lipid accumulation, as well as systemic and tissue insulin resistance. Furthermore, in skeletal muscle cells spironolactone prevented enhanced MR signaling mediated reduction of miR-99a and related increased CD36. These data indicate that inhibition of MR activation with spironolactone reversed diet - induced reduction of miR-99a, thereby reducing CD36 expression, leading to reduced IMC lipid content and improved soleus insulin sensitivity. Presentation: Saturday, June 11, 2022 1:48 p.m. - 1:53 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Effects of SGLT2 inhibitor dapagliflozin in patients with type 2 diabetes on skeletal muscle cellular metabolism

ObjectiveSGLT2 inhibitors increase urinary glucose excretion and have beneficial effects on cardiovascular and renal outcomes; the underlying mechanism may be metabolic adaptations due to urinary glucose loss. Here, we investigated the cellular and molecular effects of 5 weeks of dapagliflozin treatment on skeletal muscle metabolism in type 2 diabetes patients. MethodsTwenty-six type 2 diabetes mellitus patients were randomized to a 5-week double-blind, cross-over study with 6-8-week wash-out. Skeletal muscle acetylcarnitine levels, intramyocellular lipid (IMCL) content and phosphocreatine (PCr) recovery rate were measured by magnetic resonance spectroscopy (MRS). Ex vivo mitochondrial respiration was measured in skeletal muscle fibers using high resolution respirometry. Intramyocellular lipid droplet and mitochondrial network dynamics were investigated using confocal microscopy. Skeletal muscle levels of acylcarnitines, amino acids and TCA cycle intermediates were measured. Expression of genes involved in fatty acid metabolism were investigated. ResultsMitochondrial function, mitochondrial network integrity and citrate synthase and carnitine acetyltransferase activities in skeletal muscle were unaltered after dapagliflozin treatment. Dapagliflozin treatment increased intramyocellular lipid content (0.060 (0.011, 0.110) %, p = 0.019). Myocellular lipid droplets increased in size (0.03 μm2 (0.01–0.06), p < 0.05) and number (0.003 μm−2 (−0.001–0.007), p = 0.09) upon dapagliflozin treatment. CPT1A, CPT1B and malonyl CoA-decarboxylase mRNA expression was increased by dapagliflozin. Fasting acylcarnitine species and C4–OH carnitine levels (0.4704 (0.1246, 0.8162) pmoles∗mg tissue−1, p < 0.001) in skeletal muscle were higher after dapagliflozin treatment, while acetylcarnitine levels were lower (−40.0774 (−64.4766, −15.6782) pmoles∗mg tissue−1, p < 0.001). Fasting levels of several amino acids, succinate, alpha-ketoglutarate and lactate in skeletal muscle were significantly lower after dapagliflozin treatment. ConclusionDapagliflozin treatment for 5 weeks leads to adaptive changes in skeletal muscle substrate metabolism favoring metabolism of fatty acid and ketone bodies and reduced glycolytic flux.The trial is registered with ClinicalTrials.gov, number NCT03338855.

Human adaptation to immobilization: Novel insights of impacts on glucose disposal and fuel utilization.

Bed rest (BR) reduces whole-body insulin-stimulated glucose disposal (GD) and alters muscle fuel metabolism, but little is known about metabolic adaptation from acute to chronic BR nor the mechanisms involved, particularly when volunteers are maintained in energy balance. Healthy males (n=10, 24.0±1.3years), maintained in energy balance, underwent 3-day BR (acute BR). A second cohort matched for sex and body mass index (n=20, 34.2±1.8years) underwent 56-day BR (chronic BR). A hyperinsulinaemic euglycaemic clamp (60mU/m2 /min) was performed to determine rates of whole-body insulin-stimulated GD before and after BR (normalized to lean body mass). Indirect calorimetry was performed before and during steady state of each clamp to calculate rates of whole-body fuel oxidation. Muscle biopsies were taken to determine muscle glycogen, metabolite and intramyocellular lipid (IMCL) contents, and the expression of 191 mRNA targets before and after BR. Two-way repeated measures analysis of variance was used to detect differences in endpoint measures. Acute BR reduced insulin-mediated GD (Pre 11.5±0.7 vs. Post 9.3±0.6mg/kg/min, P<0.001), which was unchanged in magnitude following chronic BR (Pre 10.2±0.4 vs. Post 7.9±0.3mg/kg/min, P<0.05). This reduction in GD was paralleled by the elimination of the 35% increase in insulin-stimulated muscle glycogen storage following both acute and chronic BR. Acute BR had no impact on insulin-stimulated carbohydrate (CHO; Pre 3.69±0.39 vs. Post 4.34±0.22mg/kg/min) and lipid (Pre 1.13±0.14 vs. Post 0.59±0.11mg/kg/min) oxidation, but chronic BR reduced CHO oxidation (Pre 3.34±0.18 vs. Post 2.72±0.13mg/kg/min, P<0.05) and blunted the magnitude of insulin-mediated inhibition of lipid oxidation (Pre 0.60±0.07 vs. Post 0.85±0.06mg/kg/min, P<0.05). Neither acute nor chronic BR increased muscle IMCL content. Plentiful mRNA abundance changes were detected following acute BR, which waned following chronic BR and reflected changes in fuel oxidation and muscle glycogen storage at this time point. Acute BR suppressed insulin-stimulated GD and storage, but the extent of this suppression increased no further in chronic BR. However, insulin-mediated inhibition of fat oxidation after chronic BR was less than acute BR and was accompanied by blunted CHO oxidation. The juxtaposition of these responses shows that the regulation of GD and storage can be dissociated from substrate oxidation. Additionally, the shift in substrate oxidation after chronic BR was not explained by IMCL accumulation but reflected by muscle mRNA and pyruvate dehydrogenase kinase 4 protein abundance changes, pointing to lack of muscle contraction per se as the primary signal for muscle adaptation.

Mineralocorticoid Receptors Mediate Diet-Induced Lipid Infiltration of Skeletal Muscle and Insulin Resistance.

Excess circulating lipids increase total intramyocellular (IMC) lipid content and ectopic fat storage, resulting in lipotoxicity and insulin resistance in skeletal muscle. Consumption of a diet high in fat and refined sugars-a Western diet (WD)-has been shown to activate mineralocorticoid receptors (MRs) and promote insulin resistance. However, our understanding of the precise mechanisms by which enhanced MR activation promotes skeletal muscle insulin resistance remains unclear. In this study, we investigated the mechanisms by which enhanced MR signaling in soleus muscle promotes ectopic skeletal muscle lipid accumulation and related insulin resistance. Six-week-old C57BL/6J mice were fed either a mouse chow diet or a WD with or without spironolactone (1 mg/kg/day) for 16 weeks. Spironolactone attenuated 16 weeks of WD-induced in vivo glucose intolerance and insulin resistance, and improved soleus insulin metabolic signaling. Improved insulin sensitivity was accompanied by increased glucose transporter 4 (Glut4) expression in conjunction with decreased soleus free fatty acid and IMC lipid content, as well as CD36 expression. Additionally, spironolactone prevented WD-induced soleus mitochondria dysfunction. Furthermore, MR signaling also mediated WD/aldosterone-induced reductions in soleus microRNA (miR)-99a, which was identified to negatively target CD36 and prevented palmitic acid-induced increases in CD36 expression, lipid droplet formation, mitochondria dysfunction, and insulin resistance in C2C12 cells. These data indicate that inhibition of MR activation with spironolactone prevented diet-induced abnormal expression of miR-99a, which had the capacity to reduce CD36, leading to reduced IMC lipid content and improved soleus mitochondria function and insulin sensitivity.

O105 Human adaptation to immobilisation: novel insights of impacts on glucose disposal and fuel utilization

Abstract Introduction Bed-rest (BR) reduces whole-body insulin-stimulated glucose disposal (GD) and alters muscle fuel metabolism. However, little is known about metabolic adaptation from acute to chronic BR, particularly when volunteers are maintained in energy balance. Methods Healthy males (n=10, 24±1.25 years) maintained in energy balance underwent 3 days of BR (acute BR; ABR). A second cohort matched for gender and body mass index (n=20, 34±1.8 years) underwent 56 days of BR (chronic BR; CBR). A hyperinsulinaemic euglycaemic clamp (60 mU/kg lean mass/min) was performed before and after BR. Indirect calorimetry was performed before and during the clamp steady-state to calculate rates of whole-body fuel oxidation. Vastus Lateralis muscle biopsies were taken before and after each clamp. Two-way repeated measures ANOVA was used to detect differences in end-point measures. Results ABR reduced insulin-mediated glucose disposal (GD; p&amp;lt;0.001), which was unchanged in magnitude following CBR (p&amp;lt;0.05). This reduction in GD following both acute and CBR was paralleled by the elimination of a 35% increase in insulin-stimulated muscle glycogen storage seen Pre BR. ABR had no impact on insulin-stimulated carbohydrate (CHO) and lipid oxidation, but CBR reduced CHO oxidation (p&amp;lt;0.05) and blunted the magnitude of insulin-mediated inhibition of lipid oxidation (p&amp;lt;0.05). Neither acute nor CBR increased muscle intramyocellular lipid content. Conclusion ABR suppressed insulin-stimulated GD and glycogen storage, and the extent of suppression increased no further after CBR. However, GD and storage were dissociated from substrate oxidation during CBR. Moreover, the shift in substrate oxidation after CBR was not explained by IMCL accumulation. Take home message Acute bed rest impairs insulin-stimulated glucose disposal and glycogen storage which is the same magnitude as that seen in chronic bed rest.