Intramuscular triglyceride synthesis: importance in muscle lipid partitioning in humans.

Intramuscular triglyceride (IMTG) concentration is elevated in insulin-resistant individuals and was once thought to promote insulin resistance. However, endurance-trained athletes have equivalent concentration of IMTG compared with individuals with type 2 diabetes, and have very low risk of diabetes, termed the "athlete's paradox." We now know that IMTG synthesis is positively related to insulin sensitivity, but the exact mechanisms for this are unclear. To understand the relationship between IMTG synthesis and insulin sensitivity, we measured IMTG synthesis in obese control subjects, endurance-trained athletes, and individuals with type 2 diabetes during rest, exercise, and recovery. IMTG synthesis rates were positively related to insulin sensitivity, cytosolic accumulation of DAG, and decreased accumulation of C18:0 ceramide and glucosylceramide. Greater rates of IMTG synthesis in athletes were not explained by alterations in FFA concentration, DGAT1 mRNA expression, or protein content. IMTG synthesis during exercise in Ob and T2D indicate utilization as a fuel despite unchanged content, whereas IMTG concentration decreased during exercise in athletes. mRNA expression for genes involved in lipid desaturation and IMTG synthesis were increased after exercise and recovery. Further, in a subset of individuals, exercise decreased cytosolic and membrane di-saturated DAG content, which may help explain insulin sensitization after acute exercise. These data suggest IMTG synthesis rates may influence insulin sensitivity by altering intracellular lipid localization, and decreasing specific ceramide species that promote insulin resistance.

A Transcriptional Circuit Filters Oscillating Circadian Hormonal Inputs to Regulate Fat Cell Differentiation.

Glucocorticoid and other adipogenic hormones are secreted in mammals in circadian oscillations. Loss of this circadian oscillation pattern correlates with obesity in humans, raising the intriguing question of how hormone secretion dynamics affect adipocyte differentiation. Using live, single-cell imaging of the key adipogenic transcription factors CEBPB and PPARG, endogenously tagged with fluorescent proteins, we show that pulsatile circadian hormone stimuli are rejected by the adipocyte differentiation control system. In striking contrast, equally strong persistent signals trigger maximal differentiation. We identify the mechanism of how hormone oscillations are filtered as a combination of slow and fast positive feedback centered on PPARG. Furthermore, we confirm in mice that flattening of daily glucocorticoid oscillations significantly increases the mass of subcutaneous and visceral fat pads. Together, our study provides a molecular mechanism for why stress, Cushing's disease, and other conditions for which glucocorticoid secretion loses its pulsatility may lead to obesity.

Human Carboxylesterase 2 Reverses Obesity-Induced Diacylglycerol Accumulation and Glucose Intolerance.

Serine hydrolases are a large family of multifunctional enzymes known to influence obesity. Here, we performed activity-based protein profiling to assess the functional level of serine hydrolases in liver biopsies from lean and obese humans in order to gain mechanistic insight into the pathophysiology of metabolic disease. We identified reduced hepatic activity of carboxylesterase 2 (CES2) and arylacetamide deacetylase (AADAC) in human obesity. In primary human hepatocytes, CES2 knockdown impaired glucose storage and lipid oxidation. In mice, obesity reduced CES2, whereas adenoviral delivery of human CES2 reversed hepatic steatosis, improved glucose tolerance, and decreased inflammation. Lipidomic analysis identified a network of CES2-regulated lipids altered in human and mouse obesity. CES2 possesses triglyceride and diacylglycerol lipase activities and displayed an inverse correlation with HOMA-IR and hepatic diacylglycerol concentrations in humans. Thus, decreased CES2 is a conserved feature of obesity and plays a causative role in the pathogenesis of obesity-related metabolic disturbances.

Activity-based proteomic and metabolomic approaches for understanding metabolism.

There are an increasing number of human pathologies that have been associated with altered metabolism, including obesity, diabetes, atherosclerosis, cancer, and neurodegenerative diseases. Most attention on metabolism has been focused on well-understood metabolic pathways and has largely ignored most of the biochemical pathways that operate in (patho)physiological settings, in part because of the vast landscape of uncharacterized and undiscovered metabolic pathways. One technology that has arisen to meet this challenge is activity-based protein profiling (ABPP) that uses activity-based chemical probes to broadly assess the functional states of both characterized and uncharacterized enzymes. This review will focus on how ABPP, coupled with inhibitor discovery platforms and functional metabolomic technologies, have led to discoveries that have expanded our knowledge of metabolism in health and disease.

Chemical genetics screening reveals KIAA1363 as a cytokine-lowering target.

Inflammation is a hallmark of many human diseases, including pain, arthritis, atherosclerosis, obesity and diabetes, cancer, and neurodegenerative diseases. Although there are several successfully marketed small molecules anti-inflammatory drugs such as cyclooxygenase inhibitors and glucocorticoids, many of these compounds are also associated with various adverse cardiovascular or immunosuppressive side effects. Thus, identifying novel anti-inflammatory small molecules and their targets is critical for developing safer and more effective next-generation treatment strategies for inflammatory diseases. Here, we have conducted a chemical genetics screen to identify small molecules that suppress the release of the inflammatory cytokine TNFα from stimulated macrophages. We have used an enzyme class-directed chemical library for our screening efforts to facilitate subsequent target identification using activity-based protein profiling (ABPP). Using this strategy, we have found that KIAA1363 is a novel target for lowering key pro-inflammatory cytokines through affecting key ether lipid metabolism pathways. Our study highlights the application of combining chemical genetics with chemoproteomic and metabolomic approaches toward identifying and characterizing anti-inflammatory smal molecules and their targets.

Novel and reversible mechanisms of smoking-induced insulin resistance in humans.

Smoking is the most common cause of preventable morbidity and mortality in the United States, in part because it is an independent risk factor for the development of insulin resistance and type 2 diabetes. However, mechanisms responsible for smoking-induced insulin resistance are unclear. In this study, we found smokers were less insulin sensitive compared with controls, which increased after either 1 or 2 weeks of smoking cessation. Improvements in insulin sensitivity after smoking cessation occurred with normalization of IRS-1(ser636) phosphorylation. In muscle cell culture, nicotine exposure significantly increased IRS-1(ser636) phosphorylation and decreased insulin sensitivity, recapitulating the phenotype of smoking-induced insulin resistance in humans. The two pathways known to stimulate IRS-1(ser636) phosphorylation (p44/42 mitogen-activated protein kinase [MAPK] and mammalian target of rapamycin [mTOR]) were both stimulated by nicotine in culture. Inhibition of mTOR, but not p44/42 MAPK, during nicotine exposure prevented IRS-1(ser636) phosphorylation and normalized insulin sensitivity. These data indicate nicotine induces insulin resistance in skeletal muscle by activating mTOR. Therapeutic agents designed to oppose skeletal muscle mTOR activation may prevent insulin resistance in humans who are unable to stop smoking or are chronically exposed to secondhand smoke.

Fenofibrate administration does not affect muscle triglyceride concentration or insulin sensitivity in humans.

Animal data suggest that males, in particular, rely on peroxisome proliferator activated receptor-α activity to maintain normal muscle triglyceride metabolism. We sought to examine whether this was also true in men vs women and its relationship to insulin sensitivity (Si). Normolipidemic obese men (n = 9) and women (n = 9) underwent an assessment of Si (intravenous glucose tolerance test) and intramuscular triglyceride (IMTG) metabolism (gas chromatography/mass spectrometry and gas chromatography-combustion isotope ratio mass spectrometry from plasma and muscle biopsies taken after infusion of [U-(13)C]palmitate) before and after 12 weeks of fenofibrate treatment. Women were more insulin sensitive (Si: 5.2 ± 0.7 vs 2.4 ± 0.4 ×10(-4)/ µU/mL, W vs M, P < .01) at baseline despite similar IMTG concentration (41.9 ± 15.5 vs 30.8 ± 5.1 µg/mg dry weight, W vs M, P = .43) and IMTG fractional synthesis rate (FSR) (0.27%/h ± 0.07%/h vs 0.35%/h ± 0.06%/h, W vs M, P = .41) as men. Fenofibrate enhanced FSR in men (0.35 ± 0.06 to 0.54 ± 0.06, P = .05), with no such change seen in women (0.27 ± 0.07 to 0.32 ± 0.13, P = .73) and no change in IMTG concentration in either group (23.0 ± 3.9 in M, P = .26 vs baseline; 36.3 ± 12.0 in W, P = .79 vs baseline). Insulin sensitivity was unaffected by fenofibrate (P ≥ .68). Lower percentage saturation of IMTG in women vs men before (29.1% ± 2.3% vs 35.2% ± 1.7%, P = .06) and after (27.3% ± 2.8% vs 35.1% ± 1.9%, P = .04) fenofibrate most closely related to their greater Si (R(2) = 0.34, P = .10) and was largely unchanged by the drug. Peroxisome proliferator activated receptor-α agonist therapy had little effect on IMTG metabolism in men or women. Intramuscular triglyceride saturation, rather than IMTG concentration or FSR, most closely (but not significantly) related to Si and was unchanged by fenofibrate administration.

Altered intramuscular lipid metabolism relates to diminished insulin action in men, but not women, in progression to diabetes.

Whether sex differences in intramuscular triglyceride (IMTG) metabolism underlie sex differences in the progression to diabetes are unknown. Therefore, the current study examined IMTG concentration and fractional synthesis rate (FSR) in obese men and women with normal glucose tolerance (NGT) vs. those with prediabetes (PD). PD (n = 13 men and 7 women) and NGT (n = 7 men and 12 women) groups were matched for age and anthropometry. Insulin action was quantified using a hyperinsulinemic-euglycemic clamp with infusion of [6,6-(2)H(2)]-glucose. IMTG concentration was measured by gas chromatography/mass spectrometry (GC/MS) and FSR by GC/combustion isotope ratio MS (C-IRMS), from muscle biopsies taken after infusion of [U-(13)C]palmitate during 4 h of rest. In PD men, the metabolic clearance rate (MCR) of glucose was lower during the clamp (4.71 ± 0.77 vs. 8.62 ± 1.26 ml/kg fat-free mass (FFM)/min, P = 0.04; with a trend for lower glucose rate of disappearance (Rd), P = 0.07), in addition to higher IMTG concentration (41.2 ± 5.0 vs. 21.2 ± 3.4 µg/mg dry weight, P ≤ 0.01), lower FSR (0.21 ± 0.03 vs. 0.42 ± 0.06 %/h, P ≤ 0.01), and lower oxidative capacity (P = 0.03) compared to NGT men. In contrast, no difference in Rd, IMTG concentration, or FSR was seen in PD vs. NGT women. Surprisingly, glucose Rd during the clamp was not different between NGT men and women (P = 0.25) despite IMTG concentration being higher (42.6 ± 6.1 vs. 21.2 ± 3.4 µg/mg dry weight, P = 0.03) and FSR being lower (0.23 ± 0.04 vs. 0.42 ± 0.06 %/h, P = 0.02) in women. Alterations in IMTG metabolism relate to diminished insulin action in men, but not women, in the progression toward diabetes.

Inflexibility in intramuscular triglyceride fractional synthesis distinguishes prediabetes from obesity in humans.

Whether intramuscular triglyceride (IMTG) concentration or flux is more important in the progression to type 2 diabetes is controversial. Therefore, this study examined IMTG concentration, as well as its fractional synthesis rate (FSR), in obese people with normal glucose tolerance (NGT; n = 20) vs. obese people with prediabetes (PD; n = 19), at rest and during exercise. Insulin action and secretion were assessed using an intravenous glucose tolerance test. [U-(13)C]palmitate was infused for 4 h before and throughout 1.5 h of treadmill walking at 50% VO(2(max)). IMTG concentration was measured by gas chromatograph/mass spectrometer, and FSR by gas chromatography-combustion isotope ratio mass spectrometer, from muscle biopsies taken immediately before and after exercise. Basal IMTG concentration was higher (43 +/- 5.7 vs. 27 +/- 3.9 mg/mg dry weight, P = 0.03) and FSR trended lower (0.23 +/- 0.04 vs. 0.32 +/- 0.05/h, P = 0.075), as did insulin action (S(i); 2.9 +/- 0.43 vs. 3.3 +/- 0.35 x 10(-4)/mU/ml, P = 0.07), in PD vs. NGT. IMTG concentration did not change significantly during exercise, but was no longer different in PD vs. NGT (45 +/- 7.7 vs. 37 +/- 5.8 mg/mg dry weight, P = 0.41). IMTG FSR suppressed during exercise in NGT (-81% to 0.06 +/- 0.13/h, P = 0.02), but not PD (+4% to 0.24 +/- 0.13%/h, P = 0.95). Palmitate oxidation was similar during rest (P = 0.92) and exercise (P = 0.94) between groups, but its source appeared different with more coming from muscle at rest and plasma during exercise in NGT, whereas the converse was true in PD. Altogether, higher basal IMTG concentration that is metabolically inflexible distinguishes obese people with PD from those with NGT.

Incretin action maintains insulin secretion, but not hepatic insulin action, in people with impaired fasting glucose.

AIMS: To determine whether altered GLP-1 activity contributes to the abnormal endogenous glucose production (EGP) and insulin secretion characteristic of people with impaired fasting glucose (IFG). METHODS: People with IFG (n=10) and normal glucose tolerance (NGT; n=13) underwent assessment of EGP (via [6,6-(2)H(2)]-glucose infusion). Parameters of whole body insulin action and secretion were estimated by IVGTT and OGTT. Measures of EGP and insulin secretion were made before and after sitagliptin administration. RESULTS: EGP was not different at baseline (glucose R(a); 1.47+/-0.08 vs. 1.46+/-0.05mg/kg/min, IFG vs. NGT, p=0.93). However, when differences in circulating insulin were accounted for (EGPXSSPI; 20.2+/-2.1 vs. 14.4+/-1.0AU, vs. NGT, p=0.03) the hepatic insulin resistance index was significantly higher in IFG. Baseline insulin action (S(i); 2.3+/-0.1x10(-4)/microU/ml vs. 3.5+/-0.4x10(-4)/microU/ml, p=0.01, IFG vs. NGT) and secretion (DI; 587+/-81x10(-4)/min vs. 1171+/-226x10(-4)/min, p=0.04, IFG vs. NGT) were impaired in IFG when evaluated by the IVGTT, but not by OGTT (insulin sensitivity 4.52+/-1.08x10(-4)dl/kg/min vs. 6.73+/-1.16x10(-4)dl/kg/min, IFG vs. NGT, p=0.16; indices of basal (Phi(b)), static (Phi(s)), dynamic (Phi(d)), and total (Phi(t)) insulin secretion, p>0.07). Sitagliptin did not change EGP or insulin secretion in either group. CONCLUSIONS: Incretin action maintained insulin secretion, but not hepatic insulin action, in people with IFG.

Increased intramuscular lipid synthesis and low saturation relate to insulin sensitivity in endurance-trained athletes.

Intramuscular triglyceride (IMTG) has received considerable attention as a potential mechanism promoting insulin resistance. Endurance-trained athletes have high amounts of IMTG but are insulin sensitive, suggesting IMTG content alone does not change insulin action. Recent data suggest increased muscle lipid synthesis protects against fat-induced insulin resistance. We hypothesized that rates of IMTG synthesis at rest would be increased in athletes compared with controls. Eleven sedentary men and 11 endurance-trained male cyclists participated in this study. An intravenous glucose tolerance test was performed to assess insulin action. After 3 days of dietary control and an overnight fast, [13C16]palmitate was infused at 0.0174 micromol.kg(-1).min(-1) for 4 h, followed by a muscle biopsy to measure isotope incorporation into IMTG and diacylglycerol. Compared with controls, athletes were twice as insulin sensitive (P=0.004) and had a significantly greater resting IMTG concentration (athletes: 20.4+/-1.6 microg IMTG/mg dry wt, controls: 14.5+/-1.8 microg IMTG/mg dry wt, P=0.04) and IMTG fractional synthesis rate (athletes: 1.56+/-0.37%/h, controls: 0.61+/-0.15%/h, P=0.03). Stearoyl-CoA desaturase 1 mRNA expression (P=0.02) and protein content (P=0.03) were also significantly greater in athletes. Diacylglycerol, but not IMTG, saturation was significantly less in athletes compared with controls (P=0.002). These data indicate endurance-trained athletes have increased synthesis rates of skeletal muscle IMTG and decreased saturation of skeletal muscle diacylglycerol. Increased synthesis rates are not due to recovery from exercise and are likely adaptations to chronic endurance exercise training.

Intramuscular lipid metabolism in the insulin resistance of smoking.

OBJECTIVE: Smoking decreases insulin action and increases the risk of type 2 diabetes in humans. Mechanisms responsible for smoking-induced insulin resistance are unclear. We hypothesized smokers would have increased intramuscular triglyceride (IMTG) and diacylglycerol (DAG) concentration and decreased fractional synthesis rate (FSR) compared with nonsmokers. RESEARCH DESIGN AND METHODS: Nonsmokers (n = 18, aged 20 +/- 0.5 years, BMI 22 +/- 0.4 kg/m(2), body fat 20 +/- 2%, 0 cigarettes per day) and smokers (n = 14, aged 21 +/- 0.7 years, BMI 23 +/- 0.4 kg/m(2), body fat 20 +/- 3%, 18 +/- 0.7 cigarettes per day) were studied in a fasted condition after a standardized diet. [U-(13)C]palmitate was infused during 4 h of rest followed by a skeletal muscle biopsy and intravenous glucose tolerance test. RESULTS: Smokers were less insulin sensitive (S(i)) compared with nonsmokers (S(i) 5.28 +/- 0.5 nonsmokers vs. 3.74 +/- 0.3 smokers 10(-4) x microU(-1) x ml(-1), P = 0.03). There were no differences in IMTG or DAG concentration (IMTG 24.2 +/- 3.4 nonsmokers vs. 27.2 +/- 5.9 smokers microg/mg dry wt, DAG 0.34 +/- 0.02 nonsmokers vs. 0.35 +/- 0.02 smokers microg/mg dry wt) or IMTG FSR between groups (0.66 +/- 0.1 nonsmokers vs. 0.55 +/- 0.09 smokers %/hr). Intramuscular lipid composition was different, with increased percent saturation of IMTG (32.1 +/- 1.2 nonsmokers vs. 35.2 +/- 1.0 smokers %, P = 0.05) and DAG (52.8 +/- 1.7 nonsmokers vs. 58.8 +/- 2.2 smokers %, P = 0.04) in smokers. Smokers had significantly decreased peroxisome proliferator-activated receptor-gamma (1.76 +/- 0.1 nonsmokers vs. 1.42 +/- 0.11 smokers arbitrary units [AU], P = 0.03) and increased monocyte chemotactic protein-1 (3.11 +/- 0.41 nonsmokers vs. 4.83 +/- 0.54 smokers AU, P = 0.02) mRNA expression compared with nonsmokers. We also found increased insulin receptor substrate-1 Ser(636) phosphorylation in smokers compared with nonsmokers (0.73 +/- 0.08 nonsmokers vs. 1.14 +/- 0.09 smokers AU, P = 0.002). CONCLUSIONS: These data suggest: 1) IMTG concentration and turnover are not related to alterations in insulin action in smokers compared to nonsmokers, 2) increased saturation of IMTG and DAG in skeletal muscle may be related to insulin action, and 3) basal inhibition of insulin receptor substrate-1 may decrease insulin action in smokers.