Andreas James Thestrup Pedersen

Human muscle fiber type specific insulin signaling – Impact of obesity and type 2 diabetes

Skeletal muscle is a heterogeneous tissue composed of different fiber types. Studies suggest that insulin-mediated glucose metabolism is different between muscle fiber types. We hypothesized that differences are due to fiber type–specific expression/regulation of insulin signaling elements and/or metabolic enzymes. Pools of type I and II fibers were prepared from biopsies of the vastus lateralis muscles from lean, obese, and type 2 diabetic subjects before and after a hyperinsulinemic-euglycemic clamp. Type I fibers compared with type II fibers have higher protein levels of the insulin receptor, GLUT4, hexokinase II, glycogen synthase (GS), and pyruvate dehydrogenase-E1α (PDH-E1α) and a lower protein content of Akt2, TBC1 domain family member 4 (TBC1D4), and TBC1D1. In type I fibers compared with type II fibers, the phosphorylation response to insulin was similar (TBC1D4, TBC1D1, and GS) or decreased (Akt and PDH-E1α). Phosphorylation responses to insulin adjusted for protein level were not different between fiber types. Independently of fiber type, insulin signaling was similar (TBC1D1, GS, and PDH-E1α) or decreased (Akt and TBC1D4) in muscle from patients with type 2 diabetes compared with lean and obese subjects. We conclude that human type I muscle fibers compared with type II fibers have a higher glucose-handling capacity but a similar sensitivity for phosphoregulation by insulin.

Intact regulation of the AMPK signaling network in response to exercise and insulin in skeletal muscle of male patients with type 2 diabetes - Illumination of AMPK activation in recovery from exercise

Current evidence on exercise-mediated AMPK regulation in skeletal muscle of patients with type 2 diabetes (T2D) is inconclusive. This may relate to inadequate segregation of trimeric complexes in the investigation of AMPK activity. We examined the regulation of AMPK and downstream targets ACC-β, TBC1D1, and TBC1D4 in muscle biopsy specimens obtained from 13 overweight/obese patients with T2D and 14 weight-matched male control subjects before, immediately after, and 3 h after exercise. Exercise increased AMPK α2β2γ3 activity and phosphorylation of ACCβ Ser221, TBC1D1 Ser237/Thr596, and TBC1D4 Ser704. Conversely, exercise decreased AMPK α1β2γ1 activity and TBC1D4 Ser318/Thr642 phosphorylation. Interestingly, compared with preexercise, 3 h into exercise recovery, AMPK α2β2γ1 and α1β2γ1 activity were increased concomitant with increased TBC1D4 Ser318/Ser341/Ser704 phosphorylation. No differences in these responses were observed between patients with T2D and control subjects. Subjects were also studied by euglycemic-hyperinsulinemic clamps performed at rest and 3 h after exercise. We found no evidence for insulin to regulate AMPK activity. Thus, AMPK signaling is not compromised in muscle of patients with T2D during exercise and insulin stimulation. Our results reveal a hitherto unrecognized activation of specific AMPK complexes in exercise recovery. We hypothesize that the differential regulation of AMPK complexes plays an important role for muscle metabolism and adaptations to exercise.

Insulin increases phosphorylation of mitochondrial proteins in human skeletal muscle in vivo.

There is increasing evidence that multiple proteins involved in key regulatory processes in mitochondria are phosphorylated in mammalian tissues. Insulin regulates glucose metabolism by phosphorylation-dependent signaling and has been shown to stimulate ATP synthesis in human skeletal muscle. Here, we investigated the effect of insulin on the phosphorylation of mitochondrial proteins in human skeletal muscle in vivo. Using a combination of TiO(2) phosphopeptide-enrichment, HILIC fractionation, and LC-MS/MS, we compared the phosphoproteomes of isolated mitochondria from skeletal muscle samples obtained from healthy individuals before and after 4 h of insulin infusion. In total, we identified 207 phosphorylation sites in 95 mitochondrial proteins. Of these phosphorylation sites, 45% were identified in both basal and insulin-stimulated samples. Insulin caused a 2-fold increase in the number of different mitochondrial phosphopeptides (87 ± 7 vs 40 ± 7, p = 0.015) and phosphoproteins (46 ± 2 vs 26 ± 3, p = 0.005) identified in each mitochondrial preparation. Almost half of the mitochondrial phosphorylation sites (n = 94) were exclusively identified in the insulin-stimulated state and included the majority of novel sites. Phosphorylation sites detected more often or exclusively in insulin-stimulated samples include multiple sites in mitochondrial proteins involved in oxidative phosphorylation, tricarboxylic acid cycle, and fatty acid metabolism, as well as several components of the newly defined mitochondrial inner membrane organizing system (MINOS). In conclusion, the present study demonstrates that insulin increases the phosphorylation of several mitochondrial proteins in human skeletal muscle in vivo and provides a first step in the understanding of how insulin potentially regulates mitochondrial processes by phosphorylation-dependent mechanisms.

Topical Antimycotics for Oral Candidiasis in Warfarin Users

Treatment for oral candidiasis in warfarin users may be complicated by drug–drug interactions (DDIs) between warfarin and topically applied antimycotics. However, current knowledge of these putative DDIs is merely based on case series. We therefore performed a cohort cross‐over study with the objective to evaluate the potential DDIs between warfarin and miconazole oral gel or nystatin oral solution. The cohort consisted of individuals using warfarin in the period of 1998–2012 (n ≈ 7400). We collected data on cohort members’ measurements of the international normalized ratio (INR) from a clinical database, and obtained information on their use of topically applied miconazole and nystatin from a regional prescription register. Potential DDIs were assessed by comparing INR values before and after initiation of an antimycotic drug. Among 17 warfarin users exposed to miconazole oral gel, the mean INR increased from 2.5 (95% CI: 2.1–2.8) to 3.8 (95% CI: 2.8–4.8) after exposure, corresponding to a mean INR increase of 1.4 (95% CI: 0.3–2.4). Among 30 warfarin users exposed to nystatin oral solution, the mean INR was 2.7 (95% CI: 2.3–3.1) before and 2.5 (95% CI: 2.2–2.9) after exposure. In conclusion, we found evidence supporting a clinically relevant drug–drug interaction between warfarin and miconazole oral gel. In contrast, we did not find any indication of an interaction between warfarin and nystatin oral solution. Nystatin rather than miconazole should be preferred when treating warfarin users for oral candidiasis.

The Pharmacogenetics of Metformin in Women with PCOS: a Randomized Trial

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder affecting women of reproductive age. PCOS is associated with obesity, dyslipidaemia and insulin resistance, and metformin treatment may improve such metabolic features. The effect of genetic variants in key metformin transporters, their transcriptional regulators or in metformin target genes on metformin response in women with PCOS is unclear. Associations between pharmacodynamic responses to metformin (changes in weight, lipid profile, insulin sensitivity evaluated by oral glucose tolerance testing) and polymorphisms in OCT1 (rs12208357 and rs72552763), HNF1A (rs1169288 and rs2464196), MATE1 (rs2289669 and rs2252281), MATE2‐K (rs12943590) and ATM (rs11212617) were studied in 40 women with PCOS randomized to 12 months of treatment with metformin 1000 mg twice daily ± oral contraceptive pills (150 μg desogestrel + 30 μg ethinylestradiol). In the entire study population, treatment was associated with reduced weight (median weight change −2.4 kg, 25th–75th percentile −5.2 to 0.3 kg, p < 0.001) and increased triglycerides (0.2 mmol/L (0.0–0.6 mmol/L), p < 0.01) without significant changes in other lipid parameters or insulin sensitivity (insulinAUC, glucoseAUC during OGTT). None of the evaluated polymorphisms significantly affected any treatment outcome. In conclusion, the genetic variants investigated were not crucial for the clinical response to metformin in PCOS.

Intact regulation of muscle expression and circulating levels of myokines in response to exercise in patients with type 2 diabetes

Regular exercise plays an important role in the prevention and treatment of type 2 diabetes (T2D). The synthesis and secretion of myokines in response to contraction may contribute to the beneficial metabolic effects of exercise. However, some exercise‐induced responses may be attenuated in T2D. Here, we investigated whether the effect of acute exercise on selected myokines are impaired in T2D. Skeletal muscle biopsies and blood samples were obtained from 13 men with T2D and 14 weight‐matched, glucose‐tolerant men before, immediately after and 3‐h after acute exercise (60 min cycling) to examine muscle expression and plasma/serum levels of selected myokines. One‐hour of exercise increased muscle expression of IL6, FGF21, ANGPTL4, CHI3L1, CTGF and CYR61, of which FGF21, ANGPTL4 and CHI3L1 increased further 3‐h into recovery, whereas expression of IL6, CYR61, and CTGF returned to baseline levels. There was no immediate effect of exercise on IL15 expression, but it decreased 3‐h into recovery. Plasma IL‐6 increased robustly, whereas circulating levels of FGF21, ANGPTL4, IL‐15, and CHI3L1 increased only modestly in response to exercise. All returned toward baseline levels 3‐h into recovery except for plasma ANGPTL4, which increased further. No significant differences in these responses to exercise were observed between the groups. Our results demonstrate that muscle expression and circulating levels of selected known and putative myokines were equally regulated by acute exercise in patients with T2D and weight‐matched controls. This suggests that the potential beneficial metabolic effects of these myokines are not impaired in patients with T2D.

Changing of the guards: EMA warning on paternal use of mycophenolate mofetil: An unnecessary and insufficiently substantiated precaution

On October 23, 2015, the European Medicines Agency (EMA) issued a press release and subsequently recommended a change to the Summary of Product Characteristics (SmPC) for mycophenolate mofetil (MMF) (EMA, 2015a,b). This specifically addressed pregnancy related issues and the wording in SmPC sections 4.4 (Special warnings and precautions for use) and 4.6 (Pregnancy and lactation) (EMA, 2015b). A Direct Healthcare Professional Communication from the manufacturer followed the EMA press release (Roche, 2015). The new warnings and precautions now for the first time included a specific statement on paternal exposure before conception, stating that: “Sexually active men are recommended to use condoms during treatment and for at least 90 days after cessation of treatment” (EMA, 2015b). The rationale or supporting evidence behind these recommendations is not presented. The FDA SmPC does not hold a similar warning (FDA, 2016). These are very strong measures called upon by a regulatory authority that in effect mean that renal transplant recipients receiving MMF de facto cannot (or at the very least are strongly advised not to) father a child. Complying with EMA precautions, planned fatherhood would require substituting MMF with a different immunosuppressant drug such as azathioprine; this would not be without risk of organ rejection or serious adverse reactions. We believe these precautionary measures are unsubstantiated by any meaningful level of evidence, and we believe they introduce unnecessary concerns to clinicians and organ transplant recipients planning fatherhood as well as parents-to-be who conceived during paternal use of MMF. In our respective Drug and Teratology Information Services across three European countries, we have received many calls from confused clinicians and worried male renal transplant recipients planning fatherhood. These include questions about termination of pregnancy in case of paternal exposure. MMF is a well-documented human teratogen following first trimester in utero exposure, and appropriate precautions are suggested in the SmPC (Anderka et al., 2009; Hoeltzenbein et al., 2012; EMA, 2015b). The amount of human data relating to paternal exposure is moderate but quite reassuring, and does not suggest a level of risk that justifies the EMAwarnings and precautions. The United States National Transplantation Pregnancy Registry (NTPR) identified 205 pregnancies fathered by 152 transplant recipients who received MMF at the estimated time of conception (Jones et al., 2013). Among 194 live births, the rates of malformations, miscarriages and prematurity were 3.1% (no specific pattern), 6.8% and 11%, respectively. All of these observations are well with the expected range. The NTPR has since collected 70 additional cases with no signs of adverse fetal outcome (personal communication, Michael J. Moritz, NTPR, December 2015). A Norwegian study, reported 2463 male organ transplant recipients who fathered 4614 children before transplantation and 474 children after transplantation (Morken et al., 2015). Following organ transplantation, no increased risk was found for any adverse pregnancy outcomes compared with outcomes before organ transplantation or to general population estimates. While specific drug exposure data including MMF is not available from the paper, the majority of the transplant recipients will have received MMF. In vitro and animal data from the SmPC does not suggest a reproductive toxicity profile of MMF that justifies the current precautions (EMA, 2015b). Two genotoxicity assays (in vitro mouse lymphoma assay and in vivo mouse bone marrow micronucleus test) showed a weak potential of MMF to cause chromosomal aberrations at extremely high doses (300 mg/kg/day) in vivo in mice. Other in vitro tests for detection of gene mutation did not demonstrate genotoxic activity. MMF had no effect on fertility of male rats at oral doses up to 20 mg/kg/day. The systemic exposure at this dose represents 2 to 3 times the clinical exposure at the recommended clinical dose of 2 gram/day in renal transplant recipients (EMA, 2006; EMA, 2015b). There are no specific data on transfer of MMF in seminal fluid, but such transfer is unlikely to be of clinical relevance (Scialli et al., 2015). We conclude that the available data do not justify the new precautions to male transplant recipients issued by EMA, which are inconsistent with FDA recommendations. We believe that EMA must reconsider this particular change to the SmPC. We believe that EMA should present evidence that contradicts the available human data presented above rather than relying on speculative theoretical concerns of potential chromosomal damage or transfer of infinitesimal amounts of MMF through seminal fluid. In any case, it should be recommended that such suggested risk should be discussed on an individual level with a Published online 0 Month 2016 in Wiley Online Library (wileyonlinelibrary.com). Doi: 10.1002/bdra.23556