OBJECTIVEThis study sought to identify the profile of circulating microRNAs (miRNAs) in type 2 diabetes (T2D) and its response to changes in insulin sensitivity. RESEARCH DESIGN AND METHODSThe circulating miRNA profile was assessed in a pilot study of 12 men: 6 with normal glucose tolerance (NGT) and 6 T2D patients. The association of 10 circulating miRNAs with T2D was cross-sectionally validated in an extended sample of 45 NGT vs. 48 T2D subjects (65 nonobese and 28 obese men) and longitudinally in 35 T2D patients who were recruited in a randomized, double-blinded, and placebo-controlled 3-month trial of metformin treatment. Circulating miRNAs were also measured in seven healthy volunteers before and after a 6-h hyperinsulinemiceuglycemic clamp and insulin plus intralipid/heparin infusion. RESULTSCross-sectional studies disclosed a marked increase of miR-140-5p, miR-142-3p, and miR-222 and decreased miR- 423-5p, miR-125b, miR-192, miR-195, miR-130b, miR-532-5p, and miR-126 in T2D patients. Multiple linear regression analyses revealed that miR-140-5p and miR-423-5p contributed independently to explain 49.5% (P < 0.0001) of fasting glucose variance after controlling for confounders. A discriminant function of four miRNAs (miR-140-5p, miR-423-5p, miR-195, and miR-126) was specific for T2D with an accuracy of 89.2% (P < 0.0001). Metformin (but not placebo) led to significant changes in circulating miR-192 (49.5%; P = 0.022), miR-140-5p (215.8%; P = 0.004), and miR-222 (247.2%; P = 0.03), in parallel to decreased fasting glucose and HbA 1c . Furthermore, while insulin infusion during clamp decreased miR-222 (262%; P = 0.002), the intralipid/heparin mixture increased circulating miR-222 (163%; P = 0.015) and miR-140-5p (67.5%; P = 0.05). CONCLUSIONSThis study depicts the close association between variations in circulating miRNAs and T2D and their potential relevance in insulin sensitivity.

In vitro studies revealed that insulin resistance might be associated with the intracellular formation of ceramide, the second messenger in the sphingomyelin signaling pathway. The aim of the present study was to examine the content and composition of fatty acids in ceramide and sphingomyelin in human muscle and to evaluate their relationships with insulin sensitivity. The study was conducted on 27 male subjects with normal glucose tolerance. Euglycemic-hyperinsulinemic clamps and biopsies of vastus lateralis muscle were performed. In 10 subjects, additional biopsies were taken after a 4-h clamp and after a clamp with concurrent Intralipid/ heparin infusion. We identified 13 ceramides and sphingomyelins according to fatty acid residues. Insulin sensitivity was related to total ceramide content (r ‫؍‬ ؊0.49, P ‫؍‬ 0.01) and to ceramide consisting of palmitic (r ‫؍‬ ؊0.48, P ‫؍‬ 0.011), palmitoleic (r ‫؍‬ ؊0.45, P ‫؍‬ 0.019), mirystic (r ‫؍‬ ؊0.42, P ‫؍‬ 0.028), and nervonic acid (r ‫؍‬ ؊0.39, P ‫؍‬ 0.047). Hyperinsulinemia did not affect estimated muscle parameters. Intralipid/heparin infusion resulted in a 24.73% decrease in insulin sensitivity (P ‫؍‬ 0.007) and a 47.81% increase in ceramide content (P ‫؍‬ 0.005). These changes were significantly related to each other (r ‫؍‬ ؊0.64, P ‫؍‬ 0.046). A relationship with the decrease in insulin sensitivity was also observed for ceramides consisting of palmitic (r ‫؍‬ ؊0.68, P ‫؍‬ 0.03) and linoleic (r ‫؍‬ ؊0.66, P ‫؍‬ 0.038) acid. Our data indicate that the sphingomyelin signaling pathway in muscle might be an important factor determining the development of insulin resistance in humans.

Aims/hypothesis Intramyocellular lipids, including ceramide, a second messenger in the sphingomyelin signalling pathway, might contribute to the development of insulin resistance. The aim of our study was to assess parameters of the skeletal muscle sphingomyelin signalling pathway in men at risk of developing type 2 diabetes. Methods We studied 12 lean (BMI<25 kg/m 2 ) men without a family history of diabetes (control group), 12 lean male offspring of type 2 diabetic patients, and 21 men with overweight or obesity comprising 12 with NGT (obese-NGT) and nine with IGT (obese-IGT). A euglycaemic-hyperinsulinaemic clamp and a biopsy of vastus lateralis muscle were performed. Ceramide, sphingomyelin, sphinganine and sphingosine levels and sphingomyelinase and ceramidase activities were measured in muscle. Muscle diacylglycerol and triacylglycerol levels were estimated in a subgroup of 27 men (comprising men from all the above groups). Results Compared with the control group, the lean offspring of diabetic patients and the men with overweight or obesity showed lower insulin sensitivity (all p<0.005) and a greater muscle ceramide level (all p<0.01). The obese-IGT group had lower insulin sensitivity (p=0.0018) and higher muscle ceramide (p=0.0022) than the obese-NGT group. There was lower muscle sphingosine level and alkaline ceramidase activity in offspring of diabetic patients (p=0.038 and p=0.031, respectively) and higher sphinganine level in the obese-NGT (p=0.049) and obese-IGT (p=0.002) groups than in the control group. Muscle sphingomyelin was lower (p=0.0028) and neutral sphingomyelinase activity was higher (p=0.00079) in the obese-IGT than in the obese-NGT group. Muscle ceramide was related to insulin sensitivity independently of other muscle lipid fractions. Conclusions/interpretations Ceramide accumulates in muscle of men at risk of developing type 2 diabetes.