Takhar Kasumov

Plasma Ceramides Are Elevated in Obese Subjects With Type 2 Diabetes and Correlate With the Severity of Insulin Resistance

OBJECTIVE—To quantitate plasma ceramide subspecies concentrations in obese subjects with type 2 diabetes and relate these plasma levels to the severity of insulin resistance. Ceramides are a putative mediator of insulin resistance and lipotoxicity, and accumulation of ceramides within tissues in obese and diabetic subjects has been well described. RESEARCH DESIGN AND METHODS—We analyzed fasting plasma ceramide subspecies by quantitative tandem mass spectrometry in 13 obese type 2 diabetic patients and 14 lean healthy control subjects. Results were related to insulin sensitivity measured with the hyperinsulinemic-euglycemic clamp technique and with plasma tumor necrosis factor-α (TNF-α) levels, a marker of inflammation. Ceramide species (C18:1, 18:0, 20:0, 24:1, and 24:0) were quantified using electrospray ionization tandem mass spectrometry after separation with high-performance liquid chromatography. RESULTS—Insulin sensitivity (mg · kg−1 · min−1) was lower in type 2 diabetic patients (4.90 ± 0.3) versus control subjects (9.6 ± 0.4) (P < 0.0001). Type 2 diabetic subjects had higher (P < 0.05) concentrations of C18:0, C20:0, C24:1, and total ceramide. Insulin sensitivity was inversely correlated with C18:0, C20:0, C24:1, C24:0, and total ceramide (all P < 0.01). Plasma TNF-α concentration was increased (P < 0.05) in type 2 diabetic subjects and correlated with increased C18:1 and C18:0 ceramide subspecies. CONCLUSIONS—Plasma ceramide levels are elevated in type 2 diabetic subjects and may contribute to insulin resistance through activation of inflammatory mediators, such as TNF-α.

Role of ceramides in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a chronic disease with a histological spectrum ranging from steatosis alone, to nonalcoholic steatohepatitis (NASH). The latter is associated with an increased risk for progression to cirrhosis. Ceramides are a lipid species that exert biological effects through cellular proliferation, differentiation, and cell death, and interact with several pathways involved in insulin resistance, oxidative stress, inflammation, and apoptosis, all of which are linked to NAFLD. We propose a mechanism through which ceramides contribute to the development of NAFLD and progression to NASH, due in part to second messenger effects via tumor necrosis factor (TNF)-α. A better understanding of the role of ceramides in steatohepatitis has both diagnostic and therapeutic implications for the treatment of fatty liver disease.

Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry

We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.

Gastric Bypass Surgery Reduces Plasma Ceramide Subspecies and Improves Insulin Sensitivity in Severely Obese Patients

Bariatric surgery is associated with near immediate remission of type 2 diabetes and hyperlipidemia. The mechanisms underlying restoration of normal glucose tolerance postoperatively are poorly understood. Herein, we examined the effect of Roux‐en‐Y gastric bypass surgery (RYGB) on weight loss, insulin sensitivity, plasma ceramides, proinflammatory markers, and cardiovascular risk factors before and at 3 and 6 months after surgery. Thirteen patients (10 female; age 48.5 ± 2.7 years; BMI, 47.4 ± 1.5 kg/m2) were included in the study, all of whom had undergone laparoscopic RYGB surgery. Insulin sensitivity, inflammatory mediators and fasting lipid profiles were measured at baseline, 3 and 6 months postoperatively, using enzymatic analysis. Plasma ceramide subspecies (C14:0, C16:0, C18:0, C18:1, C20:0, C24:0, and C24:1) were quantified using electrospray ionization tandem mass spectrometry after separation with HPLC. At 3 months postsurgery, body weight was reduced by 25%, fasting total cholesterol, triglycerides, low‐density lipoproteins, and free fatty acids were decreased, and insulin sensitivity was increased compared to presurgery values. These changes were all sustained at 6 months. In addition, total plasma ceramide levels decreased significantly postoperatively (9.3 ± 0.5 nmol/ml at baseline vs. 7.6 ± 0.4 at 3 months, and 7.3 ± 0.3 at 6 months, P < 0.05). At 6 months, the improvement in insulin sensitivity correlated with the change in total ceramide levels (r = −0.68, P = 0.02), and with plasma tumor necrosis factor‐α (TNF‐α) (r = −0.62, P = 0.04). We conclude that there is a potential role for ceramide lipids as mediators of the proinflammatory state and improved insulin sensitivity after gastric bypass surgery.

Ceramide as a Mediator of Non-Alcoholic Fatty Liver Disease and Associated Atherosclerosis

Cardiovascular disease (CVD) is a serious comorbidity in nonalcoholic fatty liver disease (NAFLD). Since plasma ceramides are increased in NAFLD and sphingomyelin, a ceramide metabolite, is an independent risk factor for CVD, the role of ceramides in dyslipidemia was assessed using LDLR-/- mice, a diet-induced model of NAFLD and atherosclerosis. Mice were fed a standard or Western diet (WD), with or without myriocin, an inhibitor of ceramide synthesis. Hepatic and plasma ceramides were profiled and lipid and lipoprotein kinetics were quantified. Hepatic and intestinal expression of genes and proteins involved in insulin, lipid and lipoprotein metabolism were also determined. WD caused hepatic oxidative stress, inflammation, apoptosis, increased hepatic long-chain ceramides associated with apoptosis (C16 and C18) and decreased very-long-chain ceramide C24 involved in insulin signaling. The plasma ratio of ApoB/ApoA1 (proteins of VLDL/LDL and HDL) was increased 2-fold due to increased ApoB production. Myriocin reduced hepatic and plasma ceramides and sphingomyelin, and decreased atherosclerosis, hepatic steatosis, fibrosis, and apoptosis without any effect on oxidative stress. These changes were associated with decreased lipogenesis, ApoB production and increased HDL turnover. Thus, modulation of ceramide synthesis may lead to the development of novel strategies for the treatment of both NAFLD and its associated atherosclerosis.

Measuring protein synthesis using metabolic 2H labeling, high-resolution mass spectrometry, and an algorithm

We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O.

Assessment of cardiac proteome dynamics with heavy water: Slower protein synthesis rates in interfibrillar than subsarcolemmal mitochondria

Traditional proteomics provides static assessment of protein content, but not synthetic rates. Recently, proteome dynamics with heavy water ((2)H2O) was introduced, where (2)H labels amino acids that are incorporated into proteins, and the synthesis rate of individual proteins is calculated using mass isotopomer distribution analysis. We refine this approach with a novel algorithm and rigorous selection criteria that improve the accuracy and precision of the calculation of synthesis rates and use it to measure protein kinetics in spatially distinct cardiac mitochondrial subpopulations. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated from adult rats, which were given (2)H2O in the drinking water for up to 60 days. Plasma (2)H2O and myocardial (2)H-enrichment of amino acids were stable throughout the experimental protocol. Multiple tryptic peptides were identified from 28 proteins in both SSM and IFM and showed a time-dependent increase in heavy mass isotopomers that was consistent within a given protein. Mitochondrial protein synthesis was relatively slow (average half-life of 30 days, 2.4% per day). Although the synthesis rates for individual proteins were correlated between IFM and SSM (R(2) = 0.84; P < 0.0001), values in IFM were 15% less than SSM (P < 0.001). In conclusion, administration of (2)H2O results in stable enrichment of the cardiac precursor amino acid pool, with the use of refined analytical and computational methods coupled with cell fractionation one can measure synthesis rates for cardiac proteins in subcellular compartments in vivo, and protein synthesis is slower in mitochondria located among the myofibrils than in the subsarcolemmal region.

Plasma levels of asymmetric dimethylarginine in patients with biopsy-proven nonalcoholic fatty liver disease

Asymmetric (ADMA) and symmetric dimethylarginine (SDMA) are produced by breakdown of proteins that have been methylated posttranslationally at an arginine residue. Plasma levels of ADMA are elevated in insulin resistance states. Nonalcoholic fatty liver disease (NAFLD) is associated with insulin resistance and varying degrees of hepatic dysfunction. Because ADMA is metabolized in the liver, we hypothesized that ADMA levels will be high in patients with NAFLD as a consequence of hepatic dysfunction and insulin resistance. Plasma levels of ADMA, SDMA, total homocysteine, glucose, and insulin were measured in nondiabetic patients with biopsy-proven NAFLD (11 steatosis and 24 nonalcoholic steatohepatitis) and 25 healthy subjects. Plasma ADMA levels were significantly higher (P = .029) in patients with biopsy-proven NAFLD (0.43 ± 0.21 μmol/L) compared with controls (0.34 ± 0.10 μmol/L). However, when adjusted for insulin resistance (homeostasis model assessment), the difference between 2 groups was not evident. Plasma SDMA levels were similar in all 3 groups. Plasma levels of ADMA were positively correlated with plasma total homocysteine levels (P = .003). Plasma levels of SDMA were negatively correlated with estimated glomerular filtration rate (P = .016) and positively correlated with plasma total homocysteine levels (P = .003). The ratio of ADMA/SDMA was positively correlated with body mass index (P = .027). Elevated plasma concentrations of ADMA in biopsy-proven NAFLD were primarily related to insulin resistance. Hepatic dysfunction in NAFLD does not appear to make significant contribution to changes in plasma methylarginine levels.

Improved insulin sensitivity after exercise training is linked to reduced plasma C14:0 ceramide in obesity and type 2 diabetes.

To assess the effect of exercise training on insulin sensitivity and plasma ceramides in obesity and type 2 diabetes (T2D).

Sarcopenia and a physiologically low respiratory quotient in patients with cirrhosis: a prospective controlled study

Patients with cirrhosis have increased gluconeogenesis and fatty acid oxidation that may contribute to a low respiratory quotient (RQ), and this may be linked to sarcopenia and metabolic decompensation when these patients are hospitalized. Therefore, we conducted a prospective study to measure RQ and its impact on skeletal muscle mass, survival, and related complications in hospitalized cirrhotic patients. Fasting RQ and resting energy expenditure (REE) were determined by indirect calorimetry in cirrhotic patients (n = 25), and age, sex, and weight-matched healthy controls (n = 25). Abdominal muscle area was quantified by computed tomography scanning. In cirrhotic patients we also examined the impact of RQ on mortality, repeat hospitalizations, and liver transplantation. Mean RQ in patients with cirrhosis (0.63 ± 0.05) was significantly lower (P < 0.0001) than healthy matched controls (0.84 ± 0.06). Psoas muscle area in cirrhosis (24.0 ± 6.6 cm(2)) was significantly (P < 0.001) lower than in controls (35.9 ± 9.5 cm(2)). RQ correlated with the reduction in psoas muscle area (r(2) = 0.41; P = 0.01). However, in patients with cirrhosis a reduced RQ did not predict short-term survival or risk of developing complications. When REE was normalized to psoas area, energy expenditure was significantly higher (P < 0.001) in patients with cirrhosis (66.7 ± 17.8 kcal/cm(2)) compared with controls (47.7 ± 7.9 kcal/cm(2)). We conclude that hospitalized patients with cirrhosis have RQs well below the traditional lowest physiological value of 0.69, and this metabolic state is accompanied by reduced skeletal muscle area. Although low RQ does not predict short-term mortality in these patients, it may reflect a decompensated metabolic state that requires careful nutritional management with appropriate consideration for preservation of skeletal muscle mass.