Investigating the effects of 5.5 mmoL vs 25 mmoL glucose concentration in culture media on LHCN-M2 cell viability, proliferation, metabolism and differentiation

Introduction: In vitro skeletal muscle cell models are vitally important for investigating the molecular mechanisms of skeletal muscle in metabolic and endocrine diseases, such as obesity and type 2 diabetes. Culture media for skeletal muscle cells can often contain glucose concentrations (GC) five times higher than what’s considered normal in fasting human plasma, thus is not representative of the in vivo environment. Hyperglycaemia in culture media may negatively impact metabolic function, by creating a model of cell toxicity that’s representative of diseases such as diabetes mellitus. The aim of these experiments was to determine the impact of media containing GC of 5.5 mmol (physiological) vs 25 mmol (supraphysiological) on cell viability, proliferation, ATP production and differentiation in human LHCN-m2 myoblasts.Methods: LHCN-m2 myoblasts were cultured in 5.5 mmol or 25 mmol glucose growth media and cell viability, ATP production, and proliferation were determined. Differentiation of LHCN-m2 myoblasts into multinucleated myotubes was induced by reducing levels of human serum within the culture media and analysed by immunofluorescence following 10 days of differentiation.Results: We observed no differences in the viability, proliferation or basal ATP production rates of LHCN-m2 cells grown in 5.5 mmol compared to 25 mmol glucose (P> 0.05 for all). However cells had a trend of higher ATP production rates and faster proliferation in 5.5 mmol compared to 25 mmol. Fluorescence microscopy revealed the formation of multinucleated myotubes differentiated in 5.5 mmol glucose media containing various concentrations of human serum (0.5%, 1% and 2%).Conclusions: Our data demonstrates the ability to differentiate LHCN-m2 cells in 5.5 mmol GC, which allows our in vitro model to be more physiologically-relevant and more comparable to what is observed in vivo in humans. Further work is required to determine the implications of GC on the wider metabolic function in LHCN-m2 myoblasts

Dietary carbohydrates and fats in nonalcoholic fatty liver disease

This Review discusses the role of dietary fats and carbohydrates in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Studies on the dietary habits of patients with NAFLD, and the effect on liver fat accumulation of altering dietary macronutrients, are also reviewed. The global prevalence of nonalcoholic fatty liver disease (NAFLD) has dramatically increased in parallel with the epidemic of obesity. Controversy has emerged around dietary guidelines recommending low-fat-high-carbohydrate diets and the roles of dietary macronutrients in the pathogenesis of metabolic disease. In this Review, the topical questions of whether and how dietary fats and carbohydrates, including free sugars, differentially influence the accumulation of liver fat (specifically, intrahepatic triglyceride (IHTG) content) are addressed. Focusing on evidence from humans, we examine data from stable isotope studies elucidating how macronutrients regulate IHTG synthesis and disposal, alter pools of bioactive lipids and influence insulin sensitivity. In addition, we review cross-sectional studies on dietary habits of patients with NAFLD and randomized controlled trials on the effects of altering dietary macronutrients on IHTG. Perhaps surprisingly, evidence to date shows no differential effects between free sugars, with both glucose and fructose increasing IHTG in the context of excess energy. Moreover, saturated fat raises IHTG more than polyunsaturated or monounsaturated fats, with adverse effects on insulin sensitivity, which are probably mediated in part by increased ceramide synthesis. Taken together, the data support the use of diets that have a reduced content of free sugars, refined carbohydrates and saturated fat in the treatment of NAFLD.Peer reviewe

A single day of excessive dietary fat intake reduces whole-body insulin sensitivity: the metabolic consequence of binge eating

Consuming excessive amounts of energy as dietary fat for several days or weeks can impair glycemic control and reduce insulin sensitivity in healthy adults. However, individuals who demonstrate binge eating behavior overconsume for much shorter periods of time; the metabolic consequences of such behavior remain unknown. The aim of this study was to determine the effect of a single day of high-fat overfeeding on whole-body insulin sensitivity. Fifteen young, healthy adults underwent an oral glucose tolerance test before and after consuming a high-fat (68% of total energy), high-energy (78% greater than daily requirements) diet for one day. Fasting and postprandial plasma concentrations of glucose, insulin, non-esterified fatty acids, and triglyceride were measured and the Matsuda insulin sensitivity index was calculated. One day of high-fat overfeeding increased postprandial glucose area under the curve (AUC) by 17.1% (p < 0.0001) and insulin AUC by 16.4% (p = 0.007). Whole-body insulin sensitivity decreased by 28% (p = 0.001). In conclusion, a single day of high-fat, overfeeding impaired whole-body insulin sensitivity in young, healthy adults. This highlights the rapidity with which excessive consumption of calories through high-fat food can impair glucose metabolism, and suggests that acute binge eating may have immediate metabolic health consequences for the individual

Substrate Utilization by the Failing Human Heart by Direct Quantification Using Arterio-Venous Blood Sampling

Metabolic substrate utilization of the human failing heart is an area of controversy. The purpose of this study is to directly quantify myocardial substrate utilization in moderately severe heart failure, type 2 diabetes and healthy controls using simultaneous coronary sinus and arterial blood sampling. Patients with heart failure (n = 9, mean NYHA 2.7±0.5), with type 2 diabetes (n = 5) and with normal heart function (n = 10) were studied after an overnight fast in connection with electrophysiological investigations/treatments

Physiological and pathophysiological concentrations of fatty acids induce lipid droplet accumulation and impair functional performance of tissue engineered skeletal muscle

Fatty acids (FA) exert physiological and pathophysiological effects leading to changes in skeletal muscle metabolism and function, however, in vitro models to investigate these changes are limited. These experiments sought to establish the effects of physiological and pathophysiological concentrations of exogenous FA upon the function of tissue engineered skeletal muscle (TESkM). Cultured initially for 14 days, C2C12 TESkM was exposed to FA‐free bovine serum albumin alone or conjugated to a FA mixture (oleic, palmitic, linoleic, and α‐linoleic acids [OPLA] [ratio 45:30:24:1%]) at different concentrations (200 or 800 µM) for an additional 4 days. Subsequently, TESkM morphology, functional capacity, gene expression and insulin signaling were analyzed. There was a dose response increase in the number and size of lipid droplets within the TESkM (p < .05). Exposure to exogenous FA increased the messenger RNA expression of genes involved in lipid storage (perilipin 2 [p < .05]) and metabolism (pyruvate dehydrogenase lipoamide kinase isozyme 4 [p < .01]) in a dose dependent manner. TESkM force production was reduced (tetanic and single twitch) (p < .05) and increases in transcription of type I slow twitch fiber isoform, myosin heavy chain 7, were observed when cultured with 200 µM OPLA compared to control (p < .01). Four days of OPLA exposure results in lipid accumulation in TESkM which in turn results in changes in muscle function and metabolism; thus, providing insight ito the functional and mechanistic changes of TESkM in response to exogenous FA.</div

Compositional marker in vivo reveals intramyocellular lipid turnover during fasting-induced lipolysis

Intramyocellular lipid (IMCL) is of particular metabolic interest, but despite many proton magnetic resonance spectroscopy (¹H MRS) studies reporting IMCL content measured by the methylene (CH₂) resonance signal, little is known about its composition. Here we validated IMCL CH₃:CH₂ ratio as a compositional marker using ¹H MRS at short echo time, and investigated IMCL content and composition during a 28-hour fast in 24 healthy males. Increases in IMCL CH₂ relative to the creatine and phosphocreatine resonance (Cr) at 3.0 ppm (an internal standard) correlated with circulating free fatty acid (FA) concentrations, supporting the concept of increased FA influx into IMCL. Significant decreases in IMCL CH₃:CH₂ ratio indicated a less unsaturated IMCL pool after fasting, and this compositional change related inversely to IMCL baseline composition, suggesting a selective efflux of unsaturated shorter-chain FA from the IMCL pool. This novel in vivo evidence reveals IMCL turnover during extended fasting, consistent with the concept of a flexible, responsive myocellular lipid store. There were also differences between soleus and tibialis anterior in basal IMCL composition and in response to fasting. We discuss the potential of this marker for providing insights into normal physiology and mechanisms of disease.We thank the participants, staff at the Cambridge NIHR/Wellcome Trust Clinical Research Facility and the Wolfson Brain Imaging Centre, Sarah Nutland (NIHR Cambridge BioResource, Cambridge, UK) for facilitating participant recruitment and Edwina French (MRC Laboratory of Molecular Biology, Cambridge, UK) for help with phantoms. We acknowledge grants from Addenbrooke’s Charitable Trust and the British Society for Pediatric Endocrinology and Diabetes. LH is a British Heart Foundation Senior Fellow in Basic Science. DBS is supported by the Wellcome Trust (107064). AT, AK and DBD are funded by the UK NIHR Cambridge Biomedical Research Centre and Medical Research Council (UD99999906), and AS by the NIHR via the NIHR Cambridge Clinical Research Facility

Accumulation of saturated intramyocellular lipid is associated with insulin resistance.

Intramyocellular lipid (IMCL) accumulation has been linked to both insulin-resistant and insulin-sensitive (athletes) states. Biochemical analysis of intramuscular triglyceride composition is confounded by extramyocellular triglycerides in biopsy samples, and hence the specific composition of IMCLs is unknown in these states. 1H magnetic resonance spectroscopy (MRS) can be used to overcome this problem. Thus, we used a recently validated 1H MRS method to compare the compositional saturation index (CH2:CH3) and concentration independent of the composition (CH3) of IMCLs in the soleus and tibialis anterior muscles of 16 female insulin-resistant lipodystrophic subjects with that of age- and gender-matched athletes (n = 14) and healthy controls (n = 41). The IMCL CH2:CH3 ratio was significantly higher in both muscles of the lipodystrophic subjects compared with controls but was similar in athletes and controls. IMCL CH2:CH3 was dependent on the IMCL concentration in the controls and, after adjusting the compositional index for quantity (CH2:CH3adj), could distinguish lipodystrophics from athletes. This CH2:CH3adj marker had a stronger relationship with insulin resistance than IMCL concentration alone and was inversely related to VO2max The association of insulin resistance with the accumulation of saturated IMCLs is consistent with a potential pathogenic role for saturated fat and the reported benefits of exercise and diet in insulin-resistant states

Prolyl-4-hydroxylase 3 maintains β-cell glucose metabolism during fatty acid excess in mice

The α-ketoglutarate–dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is an HIF target that uses molecular oxygen to hydroxylate peptidyl prolyl residues. Although PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about the effects of this highly conserved enzyme in insulin-secreting β cells in vivo. Here, we show that the deletion of PHD3 specifically in β cells (βPHD3KO) was associated with impaired glucose homeostasis in mice fed a high-fat diet. In the early stages of dietary fat excess, βPHD3KO islets energetically rewired, leading to defects in the management of pyruvate fate and a shift from glycolysis to increased fatty acid oxidation (FAO). However, under more prolonged metabolic stress, this switch to preferential FAO in βPHD3KO islets was associated with impaired glucose-stimulated ATP/ADP rises, Ca(2+) fluxes, and insulin secretion. Thus, PHD3 might be a pivotal component of the β cell glucose metabolism machinery in mice by suppressing the use of fatty acids as a primary fuel source during the early phases of metabolic stress

Femoral Adipose Tissue May Accumulate the Fat That Has Been Recycled as VLDL and Nonesterified Fatty Acids

OBJECTIVE: Gluteo-femoral, in contrast to abdominal, fat accumulation appears protective against diabetes and cardiovascular disease. Our objective was to test the hypothesis that this reflects differences in the ability of the two depots to sequester fatty acids, with gluteo-femoral fat acting as a longer-term "sink." RESEARCH DESIGN AND METHODS: A total of 12 healthy volunteers were studied after an overnight fast and after ingestion of a mixed meal. Blood samples were taken from veins draining subcutaneous femoral and abdominal fat and compared with arterialized blood samples. Stable isotope-labeled fatty acids were used to trace specific lipid fractions. In 36 subjects, adipose tissue blood flow in the two depots was monitored with (133)Xe. RESULTS: Blood flow increased in response to the meal in both depots, and these responses were correlated (r(s) = 0.44, P &lt; 0.01). Nonesterified fatty acid (NEFA) release was suppressed after the meal in both depots; it was lower in femoral fat than in abdominal fat (P &lt; 0.01). Plasma triacylglycerol (TG) extraction by femoral fat was also lower than that by abdominal fat (P = 0.05). Isotopic tracers showed that the difference was in chylomicron-TG extraction. VLDL-TG extraction and direct NEFA uptake were similar in the two depots. CONCLUSIONS: Femoral fat shows lower metabolic fluxes than subcutaneous abdominal fat, but differs in its relative preference for extracting fatty acids directly from the plasma NEFA and VLDL-TG pools compared with chylomicron-TG

Magnetic resonance spectroscopy analysis of intramyocellular lipid composition in lipodystrophic patients and athletes

Abstract Context Paradoxically, intramyocellular lipid (IMCL) accumulation has been linked to both insulin-resistant and to insulin-sensitive (athletes) states. The composition of this lipid store is unknown in these states. Design and Methods We used a recently validated and potentially widely applicable 1 H magnetic resonance spectroscopy method to compare the compositional saturation index (CH 2 :CH 3 ratio) and concentration independent of composition (CH 3 ) of intramyocellular lipid in the soleus and tibialis anterior muscles of 16 female insulin-resistant lipodystrophic patients with that of age- and gender-matched athletes (n=14) and healthy controls (n = 41). Main Outcome IMCL compositional saturation index (CH 2 :CH 3 ratio). Results The IMCL CH 2 :CH 3 ratio was significantly higher in both muscles of the lipodystrophic patients compared with age- and gender-matched controls but not compared to athletes. IMCL CH 2 :CH 3 was dependent on IMCL concentration in the controls and after adjusting the composition index for quantity (CH 2 :CH 3adj ) was able to distinguish patients from athletes. With groups pooled, this CH 2 :CH 3adj marker had the strongest relation to insulin resistance (HOMA-IR) compared to other measures of lipid concentration and composition, especially in the soleus muscle. Contrary to the ‘athlete’s paradox’, IMCL in athletes was similar in tibialis anterior (p>0.05) and significantly lower in the soleus (p < 0.004) compared to both controls and patients. Conclusions The IMCL saturation index adjusted for quantity, which likely reflects accumulation of saturated IMCL, is more closely associated with insulin resistance than concentration alone