Intramyocellular Lipid Research Articles

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.

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle.

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted. Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

1347-P: High Prevalence of NAFLD and Lower Lean Mass in South Asian Youth

Background: South Asians (SA) have high rates of NAFLD. SA adults have NAFLD at lower BMI. We examined differences in ectopic fat between SA and White (W) youth, and its relationship to BMI z-score (BMIZ) , fat depots, and metabolic labs. Methods: SA and W youth, 12-21y, BMI ≥ 80%ile (or ≥23kg/m2 if ≥18y) underwent MRS to measure liver fat and intramyocellular lipid (IMCL) (soleus) , and DXA to quantify total % body fat (%BF) and lean mass (LM) . Abdominal MRI was used to measure abdominal fat (AF) , visceral fat (VF) , SQ fat, deep SQ, and superficial SQ. HbA1c, fasting glucose (FBG) , and lipids were obtained. Univariate analyses used parametric or non-parametric methods as appropriate. Linear regression adjusted for potential confounding variables. NAFLD defined as &amp;gt;5% liver fat. Results: Participants (28 SA, 9 W) had similar age, sex, and BMIZ. Liver fat was higher in SA [2.0% (1.1, 7.4) ] compared to W [0.8% (0.7, 1.4) ], but not statistically different. NAFLD was present in 29% of SA youth, but in no W youth. There was no difference in IMCL (p=0.9) or VF between groups (p=0.5) . Adjusting for age, sex, puberty and BMIZ, SA had lower LM [β= -8.1, p=0.003] and higher %BF [β=5.8, p=0.01], AF [β=151.6, p=0.006], total SQ fat [β=65.9, p=0.001], deep SQ fat [β=1.9, p=0.005] and HbA1c [β=3.8, p=0.01] than W. FBG and lipids were similar between groups. In SA, liver fat correlated with HDL-C (p=0.02) , LDL-C (p=0.03) , non-HDL-C (p=0.03) and borderline with FBG (p=0.05) , HbA1c (p=0.06) , but not with BMIZ, LM, %BF, or triglycerides. In SA, VF was correlated with triglycerides (p=0.002) and non-HDL-C (p=0.04) and borderline with LM (p=0.05) and HDL-C (p=0.06) . Conclusions: These preliminary results of an ongoing study demonstrate that 29% of SA youth had NAFLD compared to no W youth. Liver fat in SA youth was not related to typical metabolic risk factors. Notably SA youth had lower LM, and increased AF, deep SQ fat, and HbA1c compared to W youth, after adjustment. Further study is needed to clarify the mechanisms underlying excess liver fat in SA youth. Disclosure T.A.Hitt: None. S.C.N.Hui: None. I.R.Kamel: None. P.B.Barker: None. B.S.Zemel: None. A.Kelly: Research Support; vTv Therapeutics. S.N.Magge: None. Funding National Institutes of Health (R01DK115648)

Importance of skeletal muscle lipid levels for muscle function and physical function in older individuals

The skeletal muscle contains lipids inside (intramyocellular lipids, IMCL) or outside (extramyocellular lipids, EMCL) its cells. The muscle lipid content increases with age; however, the characteristics of IMCL and EMCL in older individuals are not well known. We aimed to examine the characteristics of skeletal muscle lipids by investigating their relationship with muscle function and physical functions. Seven elderly men and 16 elderly women participated. The skeletal muscle lipid content, including IMCL and EMCL, was measured in the vastus lateralis by proton magnetic resonance spectroscopy. Isometric knee extension with maximal voluntary contraction (MVC) and time-to-task failure for knee extension with 50% MVC were measured as muscle functions. The participants performed six physical function tests: preferred gait speed, maximal gait speed, Timed Up and Go, chair sit-to-stand, handgrip strength, and stand from the floor. The time to knee extension task failure had a significant relationship with the IMCL ( rs = −0.43, P &lt; 0.05), but not with the EMCL content. Significant relationships were confirmed in the EMCL content with the sit-to-stand ( rs = −0.48, P &lt; 0.05) and stand-from-the-floor ( rs = 0.53, P &lt; 0.05) tests. These findings indicated that muscle lipids are associated with muscle and physical functional performances in older individuals. Novelty: No relationship was confirmed between IMCL and EMCL in older individuals. Muscle endurance performance had a relationship with IMCL, but not with EMCL. Relationships between EMCL and physical functional tests (e.g., sit-to-stand and stand from the floor) were confirmed.

Use of the Lipolysis Inhibitor Acipimox Fails to Modulate Chronic Muscle Protein Synthesis Rates or Attenuate the Loss of Muscle Mass in a Rodent Model of Severe Burns

ObjectiveMassive burns result in chronic lipolysis of adipose stores resulting in ectopic lipid accumulation. Given reports that hyperlipidemia and/or increased intramyocellular lipid accumulation is associated with impaired synthesis of muscle proteins, we hypothesized that administering the lipolysis inhibitor acipimox could protect muscle protein synthesis capacity in a rodent model of burns, thereby blunting the loss of muscle mass.MethodsSprague Dawley rats (4 groups, 16 animals per group, n = 64) were randomized to receive a scald burn (~ 60 % total body surface area; n=32) or sham procedure. Animals in each group were assigned to receive either vehicle (saline) or acipimox (50 mg/kg/day) daily by i.p. injection for 7 days. All animals received a bolus of deuterated water (10 ml/kg, i.p.) followed by supplementation in drinking water (2 %) to monitor habitual muscle protein synthesis rates (MPS). On day 7, animals were sacrificed, and tissues collected. MPS was calculated by measuring intracellular and bound deuterium enrichment of alanine via Gas Chromatography Isotope Ratio Mass Spectrometry.ResultsSevere burns resulted in significant body weight loss compared to the sham injury group (7 % vs 10 %; p &lt; 0.001). However, acipimox administration did not prevent the loss of body weight. There was a trend for gastrocnemius and tibialis anterior muscle weights to be lower in burn animals (P = 0.058 and P=0.057, respectively). However, acipimox did not prevent the loss of muscle weight with burn injury. Neither burn injury nor acipimox administration impacted habitual MPS rates.ConclusionIn sharp contrast to what is observed in patients, severe burns do not alter habitual MPS rates in rats. Given evidence that rats still experience a loss of muscle mass in response to thermal injury, it suggests that the dominant mechanism by which muscle protein content declines in these animals is via enhanced muscle proteolysis. Acipimox appears to exert no beneficial impact on muscle protein homeostasis in burn injured rodents.

Sex-Specific Differences in Lower Body Fat Distribution and Association with Physical Performance among Healthy Community-Dwelling Older Adults: A Pilot Study.

This study aims to examine sex-specific differences in body composition and lower extremity fat distribution and their association with physical performance among healthy older adults. The pilot study comprises 40 subjects (20 men and 20 women) matched by age and body mass index. The participants undergo dual-energy X-ray absorptiometry, magnetic resonance imaging, and proton magnetic resonance spectroscopy (1H-MRS) to assess body composition and lower extremity fat distribution. 1H-MRS is used to measure the extramyocellular lipid (EMCL) and intramyocellular lipid (IMCL) contents of the lower leg muscles (soleus and tibialis anterior) at the maximum circumference of the calf after overnight fasting. The tibialis anterior IMCL, as assessed by 1H-MRS, is negatively associated with the five-times sit-to-stand test scores (rs = 0.518, p = 0.023) in men, while the soleus IMCL content is negatively associated with the timed up-and-go test scores (rs = 0.472, p = 0.048) in women. However, the soleus EMCL content is positively associated with the five-times sit-to-stand test scores (rs = −0.488, p = 0.040) in women, but this association is not statistically significant in men. This study shows an inverse correlation between IMCL content and physical performance in healthy older individuals and lower leg 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 women but not men.

Changes in Visceral and Ectopic Adipose Tissue Stores Across Pregnancy and Their Relationship to Gestational Weight Gain

BackgroundExcessive gestational weight gain has been associated with increased total body fat (TBF), metabolic syndrome, and abdominal obesity. However, little is known about the relationship of gestational weight gain with changes in metabolically active visceral or ectopic (hepatic and skeletal muscle) lipid stores. ObjectivesIn a prospective study of 50 healthy, pregnant women, we assessed whether changes in weight were associated with changes in total, visceral, and ectopic lipid stores. MethodsParticipants (ages 19–39) were primarily White (84%). The mean preconception BMI was 25.8 kg/m2 (SD, 4.5 kg/m2; min-max, 17.1–35.9 kg/m2). Measurements were completed at visits 1 and 2 at means of 16 and 34 weeks gestation, respectively, and included TBF using BOD POD; abdominal subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) using MRI; and intrahepatic lipids (IHL), intramyocellular lipids (IMCL), and extramyocellular lipids (EMCL) using magnetic resonance spectroscopy. We used paired t-tests to examine changes in adipose tissue and Pearson's correlation to examine associations of adipose tissue changes and weight changes. We also examined whether changes in adipose tissue stores differed by preconception BMI (normal, overweight, and obese), using 1-way ANOVA. ResultsThe TBF (mean change, +3.5 kg; 95% CI: 2.4–4.6 kg), SAT (mean change, +701 cm3; 95% CI: 421–981 cm3), VAT (mean change, +275 cm3; 95% CI: 170–379 cm3), and IHL (percentage water peak; median, +0.15; IQR = −0.01 to 0.32) values increased significantly; the IMCL and EMCL values did not change. Changes varied by BMI strata, with the least increase (or, for SAT, net loss) among women with obesity. Weight change was positively correlated with changes in TBF (r = 0.83; P < 0.001), SAT (r = 0.74; P < 0.001), and VAT (r = 0.63; P < 0.001) but not significantly correlated with changes in ectopic lipids (IHL, IMCL, and EMCL; −0.14 < r < 0.26). ConclusionsPreferential deposition of adipose tissue to the viscera in pregnancy, as seen in our sample, could serve an important metabolic function; however, excessive deposition in this region could negatively affect maternal health.

A Single Bout of Ultra-Endurance Exercise Reveals Early Signs of Muscle Aging in Master Athletes.

Middle-aged and master endurance athletes exhibit similar physical performance and long-term muscle adaptation to aerobic exercise. Nevertheless, we hypothesized that the short-term plasticity of the skeletal muscle might be distinctly altered for master athletes when they are challenged by a single bout of prolonged moderate-intensity exercise. Six middle-aged (37Y) and five older (50Y) master highly-trained athletes performed a 24-h treadmill run (24TR). Vastus lateralis muscle biopsies were collected before and after the run and assessed for proteomics, fiber morphometry, intramyocellular lipid droplets (LD), mitochondrial oxidative activity, extracellular matrix (ECM), and micro-vascularisation. Before 24TR, muscle fiber type morphometry, intramyocellular LD, oxidative activity, ECM and micro-vascularisation were similar between master and middle-aged runners. For 37Y runners, 24TR was associated with ECM thickening, increased capillary-to-fiber interface, and an 89% depletion of LD in type-I fibers. In contrast, for 50Y runners, 24TR did not alter ECM and capillarization and poorly depleted LDs. Moreover, an impaired succinate dehydrogenase activity and functional class scoring of proteomes suggested reduced oxidative phosphorylation post-24TR exclusively in 50Y muscle. Collectively, our data support that middle-aged and master endurance athletes exhibit distinct transient plasticity in response to a single bout of ultra-endurance exercise, which may constitute early signs of muscle aging for master athletes.

Muscular carnosine is a marker for cardiorespiratory fitness and cardiometabolic risk factors in men with type 1 diabetes.

Muscle is an essential organ for glucose metabolism and can be influenced by metabolic disorders and physical activity. Elevated muscle carnosine levels have been associated with insulin resistance and cardiometabolic risk factors. Little is known about muscle carnosine in type 1 diabetes (T1D) and how it is influenced by physical activity. The aim of this study was to characterize muscle carnosine in vivo by proton magnetic resonance spectroscopy (1H MRS) and evaluate the relationship with physical activity, clinical characteristics and lipoprotein subfractions. 16 men with T1D (10 athletes/6 sedentary) and 14 controls without diabetes (9/5) were included. Body composition by DXA, cardiorespiratory capacity (VO2peak) and serum lipoprotein profile by proton nuclear magnetic resonance (1H NMR) were obtained. Muscle carnosine scaled to water (carnosineW) and to creatine (carnosineCR), creatine and intramyocellular lipids (IMCL) were quantified in vivo using 1H MRS in a 3T MR scanner in soleus muscle. Subjects with T1D presented higher carnosine CR levels compared to controls. T1D patients with a lower VO2peak presented higher carnosineCR levels compared to sedentary controls, but both T1D and control groups presented similar levels of carnosineCR at high VO2peak levels. CarnosineW followed the same trend. Integrated correlation networks in T1D demonstrated that carnosineW and carnosineCR were associated with cardiometabolic risk factors including total and abdominal fat, pro-atherogenic lipoproteins (very low-density lipoprotein subfractions), low VO2peak, and IMCL. Elevated muscle carnosine levels in persons with T1D and their effect on atherogenic lipoproteins can be modulated by physical activity.

Contribution of skeletal muscle and serum lipids to muscle contraction induced by neuromuscular electrical stimulation in older individuals.

Intramyocellular lipids (IMCL) stored in droplets in muscle cells and free fatty acids (FFA) from fat cells in the blood are the main substrates of adenosine triphosphate during continuous muscle contractions of relatively lower intensity. Although it is known that the lipid oxidative capacity decreases with aging, the effect of IMCL and FFA on muscle contraction in older individuals remains unclear. The purpose of this study was to investigate the contribution of skeletal muscle lipids and blood lipids as energy sources for muscle contraction in older individuals. Eighteen older individuals (mean age: 70.4 ± 3.5 years) underwent muscle contraction intervention induced by intermittent neuromuscular electrical stimulation (NMES) to the vastus lateralis for 30 min. Fasting blood samples were obtained and proton magnetic resonance spectroscopy (1H‐MRS) was performed before and after NMES, and the parameters (including IMCL and extramyocellular lipid [EMCL]) from 1H‐MRS, along with FFA and adiponectin levels, were analyzed using the blood samples of all participants. Levels of IMCL and EMCL did not change (p > 0.05); however, FFA and adiponectin levels decreased from 1.1 ± 0.5 mEq/L to 0.8 ± 0.2 mEq/L and 12.0 ± 5.3 μg/ml to 11.4 ± 5.0 μg/ml, after NMES (p < 0.05), respectively. These findings indicate that serum lipids, but not skeletal muscle lipids, are the energy substrate utilized during involuntary muscle contraction in older individuals.

Enlarged PLIN5-uncoated lipid droplets in inner regions of skeletal muscle type II fibers associate with type 2 diabetes

Skeletal muscle physiology remains of paramount importance in understanding insulin resistance. Due to its high lipid turnover rates, regulation of intramyocellular lipid droplets (LDs) is a key factor. Perilipin 5 (PLIN5) is one of the most critical agents in such regulation, being often referred as a protector against lipotoxicity and consequent skeletal muscle insulin resistance. We examined area fraction, size, subcellular localization and PLIN5 association of LDs in two fiber types of type 2 diabetic (T2D), obese (OB) and healthy (HC) individuals by means of fluorescence microscopy and image analysis. We found that T2D type II fibers have a significant sub-population of large and internalized LDs, uncoated by PLIN5. Based on this novel result, additional hypotheses for the pathophysiology of skeletal muscle insulin resistance are formulated, together with future research directions.

Breastfeeding may benefit cardiometabolic health of children exposed to increased gestational glycemia in utero.

There is altered breastmilk composition among mothers with gestational diabetes and conflicting evidence on whether breastfeeding is beneficial or detrimental to their offspring's cardiometabolic health. We aimed to investigate associations between breastfeeding and offspring's cardiometabolic health across the range of gestational glycemia. We included 827 naturally conceived, term singletons from a prospective mother-child cohort. We measured gestational (26-28weeks) fasting plasma glucose (FPG) and 2-h plasma glucose (2hPG) after an oral glucose tolerance test as continuous variables. Participants were classified into 2 breastfeeding categories (high/intermediate vs. low) according to their breastfeeding duration and exclusivity. Main outcome measures included magnetic resonance imaging (MRI)-measured abdominal fat, intramyocellular lipids (IMCL), and liver fat, quantitative magnetic resonance (QMR)-measured body fat mass, blood pressure, blood lipids, and insulin resistance at 6years old (all continuous variables). We evaluated if gestational glycemia (FPG and 2hPG) modified the association of breastfeeding with offspring outcomes after adjusting for confounders using a multiple linear regression model that included a 'gestational glycemia × breastfeeding' interaction term. With increasing gestational FPG, high/intermediate (vs. low) breastfeeding was associated with lower levels of IMCL (p-interaction = 0.047), liver fat (p-interaction = 0.033), and triglycerides (p-interaction = 0.007), after adjusting for confounders. Specifically, at 2 standard deviations above the mean gestational FPG level, high/intermediate (vs. low) breastfeeding was linked to lower adjusted mean IMCL [0.39% of water signal (0.29, 0.50) vs. 0.54% of water signal (0.46, 0.62)], liver fat [0.39% by weight (0.20, 0.58) vs. 0.72% by weight (0.59, 0.85)], and triglycerides [0.62mmol/L (0.51, 0.72) vs. 0.86mmol/L (0.75, 0.97)]. 2hPG did not significantly modify the association between breastfeeding and childhood cardiometabolic risk. Our findings suggest breastfeeding may confer protection against adverse fat partitioning and higher triglyceride concentration among children exposed to increased glycemia in utero.

Function outperforms morphology in the assessment of muscular contribution to insulin sensitivity in premenopausal women.

IntroductionSkeletal muscle contributes significantly to insulin sensitivity in humans. However, which non-invasive measurement best reflects this contribution remains unknown. Consequently, this paper compares morphologic and functional measurements.Research methods and designWe conducted a cross-sectional analysis of 144 premenopausal women enrolled in the “Prediction, Prevention, and Sub-classification of Type 2 Diabetes” (PPSDiab) cohort study. For the analysis, we quantified insulin sensitivity by oral glucose tolerance testing and, in a subgroup of 30 women, euglycemic clamp. To assess skeletal muscle, we measured volume by magnetic resonance imaging, intramyocellular lipid content by magnetic resonance spectroscopy, and physical fitness by cardiopulmonary exercise testing.ResultsThe mean age of the cohort was 35.7 ± 4.1 years and 94 participants (65%) had a history of gestational diabetes mellitus. Of the morphologic and functional muscle parameters, the maximum workload achieved during cardiopulmonary exercise testing associated most closely with insulin sensitivity (standardized beta = 0.39; p < .001). Peak oxygen uptake also demonstrated significant associations, whereas muscle volume and intramyocellular lipid content displayed none.ConclusionFunctional measurements provided a better assessment of the muscular contribution to insulin sensitivity than morphologic measurements in premenopausal women. In particular, exercise testing rendered an easy and cost-effective method applicable in clinical settings and other human studies.

In vivo response to an oral fat load of ectopic lipids and acetylcarnitine in skeletal muscle in subjects with obesity

Rationale: Acetylcarnitine plays a pivotal role in the regulation of mitochondrial and cellular lipid trafficking. The aim of the present study was to investigate the postprandial response to an oral fat load in terms of changes in skeletal muscle acetylcarnitine and intramyocellular lipid content (IMCL) in subjects with obesity through proton magnetic resonance spectroscopy (1H-MRS), in association with changes in postprandial lipid metabolism.

High-Fructose, High-Fat Diet Alters Muscle Composition and Fuel Utilization in a Juvenile Iberian Pig Model of Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a serious metabolic condition affecting millions of people worldwide. A “Western-style diet” has been shown to induce pediatric NAFLD with the potential disruption of skeletal muscle composition and metabolism. To determine the in vivo effect of a “Western-style diet” on pediatric skeletal muscle fiber type and fuel utilization, 28 juvenile Iberian pigs were fed either a control diet (CON) or a high-fructose, high-fat diet (HFF), with or without probiotic supplementation, for 10 weeks. The HFF diets increased the total triacylglycerol content of muscle tissue but decreased intramyocellular lipid (IMCL) content and the number of type I (slow oxidative) muscle fibers. HFF diets induced autophagy as assessed by LC3I and LC3II, and inflammation, as assessed by IL-1α. No differences in body composition were observed, and there was no change in insulin sensitivity, but HFF diets increased several plasma acylcarnitines and decreased expression of lipid oxidation regulators PGC1α and CPT1, suggesting disruption of skeletal muscle metabolism. Our results show that an HFF diet fed to juvenile Iberian pigs produces a less oxidative skeletal muscle phenotype, similar to a detraining effect, and reduces the capacity to use lipid as fuel, even in the absence of insulin resistance and obesity.

Enhanced Muscle Strength in Dyslipidemic Mice and Its Relation to Increased Capacity for Fatty Acid Oxidation.

Dyslipidemia is commonly linked to skeletal muscle dysfunction, accumulation of intramyocellular lipids, and insulin resistance. However, our previous research indicated that dyslipidemia in apolipoprotein E and low-density lipoprotein receptor double knock-out mice (ApoE/LDLR -/-) leads to improvement of exercise capacity. This study aimed to investigate in detail skeletal muscle function and metabolism in these dyslipidemic mice. We found that ApoE/LDLR -/- mice showed an increased grip strength as well as increased troponins, and Mhc2 levels in skeletal muscle. It was accompanied by the increased skeletal muscle mitochondria numbers (judged by increased citrate synthase activity) and elevated total adenine nucleotides pool. We noted increased triglycerides contents in skeletal muscles and increased serum free fatty acids (FFA) levels in ApoE/LDLR -/- mice. Importantly, Ranolazine mediated inhibition of FFA oxidation in ApoE/LDLR -/- mice led to the reduction of exercise capacity and total adenine nucleotides pool. Thus, this study demonstrated that increased capacity for fatty acid oxidation, an adaptive response to dyslipidemia leads to improved cellular energetics that translates to increased skeletal muscle strength and contributes to increased exercise capacity in ApoE/LDLR -/- mice.

Short-term physical inactivity induces diacylglycerol accumulation and insulin resistance in muscle via lipin1 activation.

Physical inactivity impairs muscle insulin sensitivity. However, its mechanism is unclear. To model physical inactivity, we applied 24-h hind-limb cast immobilization (HCI) to mice with normal or high-fat diet (HFD) and evaluated intramyocellular lipids and the insulin signaling pathway in the soleus muscle. Although 2-wk HFD alone did not alter intramyocellular diacylglycerol (IMDG) accumulation, HCI alone increased it by 1.9-fold and HCI after HFD further increased it by 3.3-fold. Parallel to this, we found increased protein kinase C ε (PKCε) activity, reduced insulin-induced 2-deoxyglucose (2-DOG) uptake, and reduced phosphorylation of insulin receptor β (IRβ) and Akt, key molecules for insulin signaling pathway. Lipin1, which converts phosphatidic acid to diacylglycerol, showed increase of its activity by HCI, and dominant-negative lipin1 expression in muscle prevented HCI-induced IMDG accumulation and impaired insulin-induced 2-DOG uptake. Furthermore, 24-h leg cast immobilization in human increased lipin1 expression. Thus, even short-term immobilization increases IMDG and impairs insulin sensitivity in muscle via enhanced lipin1 activity.NEW & NOTEWORTHY Physical inactivity impairs muscle insulin sensitivity. However, its mechanism is unclear. To model physical inactivity, we applied 24-h hind-limb cast immobilization to mice with normal or high-fat diet and evaluated intramyocellular lipids and the insulin signaling pathway in the soleus muscle. We found that even short-term immobilization increases intramyocellular diacylglycerol and impairs insulin sensitivity in muscle via enhanced lipin1 activity.