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The ADAMTS9 rs4607103 C allele is one of the few gene variants proposed to increase the risk of type 2 diabetes through an impairment of insulin sensitivity. We show that the variant is associated with increased expression of the secreted ADAMTS9 and decreased insulin sensitivity and signaling in human skeletal muscle. In line with this, mice lacking Adamts9 selectively in skeletal muscle have improved insulin sensitivity. The molecular link between ADAMTS9 and insulin signaling was characterized further in a model where ADAMTS9 was overexpressed in skeletal muscle. This selective overexpression resulted in decreased insulin signaling presumably mediated through alterations of the integrin b1 signaling pathway and disruption of the intracellular cytoskeletal organization. Furthermore, this led to impaired mitochondrial function in mouse muscle, which was observed to be of translational character because humans carrying the ADAMTS9 risk allele have decreased expression of mitochondrial markers. Finally, we found that the link between ADAMTS9 overexpression and impaired insulin signaling could be due to accumulation of harmful lipid intermediates. Our findings contribute to the understanding of the molecular mechanisms underlying insulin resistance and type 2 diabetes and point to inhibition of ADAMTS9 as a potential novel mode of treating insulin resistance.

OBJECTIVE: The positive metabolic outcome of Roux-en-Y Gastric Bypass (RYGB) surgery may involve Fibroblast Growth Factor 21 (FGF21), both in the fasting state and postprandially. We measured the fasting levels of FGF21 before and after bariatric surgery as well as the postprandial FGF21 responses after a glucose load and after a mixed meal.DESIGN: Observational intervention trial.PATIENTS AND MEASUREMENTS: Eight obese, non-diabetics patients underwent RYGB. Plasma FGF21 was measured both before and after surgery on three different days during oral glucose loads (25 g or 50 g glucose) or a mixed meal. Blood samples were taken right before the meal and at 15 min intervals until 90 min and at 150 min and 210 min relative to the start of the meal.RESULTS: Overall, fasting plasma FGF21 did not change significantly before and after surgery (262±71 vs 411±119 pg/ml), but for three subjects fasting plasma FGF21 increased significantly after surgery. Furthermore, FGF21 levels increased significantly at t=90 and t=150 min in response to 50 g glucose, but not after a mixed meal.CONCLUSIONS: In conclusion, the observed increase in postprandial plasma FGF21 in response to glucose and the lack of FGF21 response to a mixed meal may have important implications for the physiologic role of FGF21. The increase in postprandial FGF21 in response to glucose in the early post-operative period may contribute to the metabolic improvements observed after gastric bypass. This article is protected by copyright. All rights reserved.

Deacetylases such as sirtuins convert nicotinamide adenine dinucleotide (NAD) to nicotinamide (NAM). Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme in the NAD salvage pathway responsible for converting NAM to NAD to maintain cellular redox state. Activation of AMP-activated protein kinase (AMPK) increases sirtuin activity by elevating NAD levels. As NAM directly inhibits sirtuins, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependant. One-legged knee-extensor exercise training in humans increased Nampt protein by 16% (p

OBJECTIVE: Fibroblast growth factor 21 (FGF21) is a metabolic factor involved in glucose and lipid metabolism. However, little is known about the physiological role of FGF21 during a dietary challenge in humans.RESEARCH DESIGN AND METHODS: Twenty healthy low birth weight (LBW) with known risk of type 2 diabetes and 26 control (normal birth weight (NBW)) young men were subjected to 5 days of high-fat (HF) overfeeding (+50%). Basal and clamp insulin-stimulated serum FGF21 levels were examined before and after the diet, and FGF21 mRNA expression was measured in muscle and fat biopsies respectively.RESULTS: Five days of HF overfeeding diet significantly (P<0.001) increased fasting serum FGF21 levels in both the groups (P<0.001). Furthermore, insulin infusion additionally increased serum FGF21 levels to a similar extent in both the groups. Basal mRNA expression of FGF21 in muscle was near the detection limit and not present in fat in both the groups before and after the dietary challenge. However, insulin significantly (P<0.001) increased FGF21 mRNA in both muscle and fat in both the groups during both diets.CONCLUSION: Short-term HF overfeeding markedly increased serum FGF21 levels in healthy young men with and without LBW but failed to increase muscle or fat FGF21 mRNA levels. This suggests that the liver may be responsible for the rise of serum FGF21 levels during overfeeding. In contrast, the increase in serum FGF21 levels during insulin infusion may arise from increased transcription in muscle and fat. We speculate that increased serum FGF21 levels during HF overfeeding may be a compensatory response to increase fatty acid oxidation and energy expenditure.

AIMS/HYPOTHESIS: In type 2 diabetes, reduced insulin-stimulated glucose disposal, primarily glycogen synthesis, is associated with defective insulin activation of glycogen synthase (GS) in skeletal muscle. Hyperglycaemia may compensate for these defects, but to what extent it involves improved insulin signalling to glycogen synthesis remains to be clarified. METHODS: Whole-body glucose metabolism was studied in 12 patients with type 2 diabetes, and 10 lean and 10 obese non-diabetic controls by means of indirect calorimetry and tracers during a euglycaemic-hyperinsulinaemic clamp. The diabetic patients underwent a second isoglycaemic-hyperinsulinaemic clamp maintaining fasting hyperglycaemia. Muscle biopsies from m. vastus lateralis were obtained before and after the clamp for examination of GS and relevant insulin signalling components. RESULTS: During euglycaemia, insulin-stimulated glucose disposal, glucose oxidation and non-oxidative glucose metabolism were reduced in the diabetic group compared with both control groups (p¿

Supra-pharmacological doses of the insulin analog X10 (AspB10) increased the incidence of mammary tumors in female Sprague-Dawley rats in chronic toxicity studies, most likely via receptor-mediated mechanisms. However, little is known about the expression of the insulin receptor family in the rat mammary gland. Using laser micro-dissection, quantitative RT-PCR and immunohistochemistry, we examined the expression of IR (insulin receptor), IGF-1R (IGF-1 receptor), IRR (insulin receptor-related receptor), ERα (estrogen receptor alpha), ERβ (estrogen receptor beta) and PR (progesteron receptor) in young, virgin, female Sprague-Dawley rats and compared to expression in reference organs. The mammary gland displayed the highest expression of IRR and IGF-1R. In contrast, low expression of IR transcripts was observed in the mammary gland tissue with expression of the IR-A isoform being 5-fold higher than the expression of the IR-B. By immunohistochemistry, expression of IR and IGF-1R was detected in all mammary gland epithelial cells. Expression of ERα and PR was comparable between mammary gland and ovary, whereas expression of ERβ was lower in mammary gland than in the ovary. Finally, expression of IGF-1R and PR in the mammary gland varied during the estrous cycle. These findings are important for the understanding of carcinogenic effects of insulin analogs in the rat mammary gland, and relevant for development of refined short-term models for preclinical safety assessment of insulin analogs.

The function and development of the rat mammary gland is dependent on the oestrus cycle. Normalization of gene expression in mammary gland samples assessed by quantitative RT-PCR therefore requires housekeeping genes (HKGs) which are stably expressed during the oestrus cycle. mRNA expression of 10 HKGs was measured in the rat mammary gland at different phases of the oestrus cycle. In addition, mRNA expression of the HKGs was measured in a panel of other rat tissues comprising laser microdissected mammary gland alveolar lobules and interlobular connective tissue and macrodissected mammary gland, liver, skeletal muscle, colon and ovary samples. Expression and ranking of HKGs varied between tissues and oestrus cycle phases and several HKGs were necessary for normalization between samples. In the mammary gland samples, three HKGs (Sdha, Tbp, and Atp5b) were identified as the optimal combination of stably expressed genes across oestrus cycle phases. For normalization between samples from the entire panel of rat tissues, eight HKGs (Rps18, Eef1a1, B2m, Actb, Tbp, Hprt, Pgk1, and Sdha) were identified as the optimal combination. These HKGs are of general relevance for studies comparing gene expression between different rat tissues.

Antibodies to receptors can block or mimic hormone action. Taking advantage of receptor isoforms, co-receptors, and other receptor modulating proteins, antibodies and other designer ligands can enhance tissue specificity and provide new approaches to the therapy of diabetes and other diseases.

The relative expression patterns of the two IR (insulin receptor) isoforms, +/- exon 11 (IR-B/IR-A respectively), are tissue-dependent. Therefore we have developed insulin analogues with different binding affinities for the two isoforms to test whether tissue-preferential biological effects can be attained. In rats and mice, IR-B is the most prominent isoform in the liver (> 95%) and fat (> 90%), whereas in muscles IR-A is the dominant isoform (> 95%). As a consequence, the insulin analogue INS-A, which has a higher relative affinity for human IR-A, had a higher relative potency [compared with HI (human insulin)] for glycogen synthesis in rat muscle strips (26%) than for glycogen accumulation in rat hepatocytes (5%) and for lipogenesis in rat adipocytes (4%). In contrast, the INS-B analogue, which has an increased affinity for human IR-B, had higher relative potencies (compared with HI) for inducing glycogen accumulation (75%) and lipogenesis (130%) than for affecting muscle (45%). For the same blood-glucose-lowering effect upon acute intravenous dosing of mice, INS-B gave a significantly higher degree of IR phosphorylation in liver than HI. These in vitro and in vivo results indicate that insulin analogues with IR-isoform-preferential binding affinity are able to elicit tissue-selective biological responses, depending on IR-A/IR-B expression.