Barbara Brunmair

The Mammalian Target of Rapamycin Pathway Regulates Nutrient-Sensitive Glucose Uptake in Man

The nutrient-sensitive kinase mammalian target of rapamycin (mTOR) and its downstream target S6 kinase (S6K) are involved in amino acid–induced insulin resistance. Whether the mTOR/S6K pathway directly modulates glucose metabolism in humans is unknown. We studied 11 healthy men (29 years old, BMI 23 kg/m2) twice in random order after oral administration of 6 mg rapamycin, a specific mTOR inhibitor, or placebo. An amino acid mixture was infused to activate mTOR, and somatostatin-insulin-glucose clamps created conditions of low peripheral hyperinsulinemia (∼100 pmol/l, 0–180 min) and prandial-like peripheral hyperinsulinemia (∼450 pmol/l, 180–360 min). Glucose turnover was assessed using d-[6,6-2H2]glucose infusion (n = 8). Skeletal muscle biopsies were performed at baseline and during prandial-like peripheral hyperinsulinemia (n = 3). At low peripheral hyperinsulinemia, whole-body glucose uptake was not affected by rapamycin. During prandial-like peripheral hyperinsulinemia, rapamycin increased glucose uptake compared with placebo by 17% (Rd|300–360 min, 75 ± 5 vs. 64 ± 5 μmol · kg−1 · min−1, P = 0.0008). Rapamycin affected endogenous glucose production neither at baseline nor during low or prandial-like peripheral hyperinsulinemia. Combined hyperaminoacidemia and prandial-like hyperinsulinemia increased S6K phosphorylation and inhibitory insulin receptor substrate-1 (IRS-1) phosphorylation at Ser312 and Ser636 in the placebo group. Rapamycin partially inhibited this increase in mTOR-mediated S6K phosphorylation and IRS-1 Ser312 and Ser636 phosphorylation. In conclusion, rapamycin stimulates insulin-mediated glucose uptake in man under conditions known to activate the mTOR/S6K pathway.

Chronic and acute effects of thiazolidinediones BM13.1258 and BM15.2054 on rat skeletal muscle glucose metabolism

New thiazolidinediones BM13.1258 and BM15.2054 were studied with regard to their PPARγ‐agonistic activities and to their acute and chronic effects on glucose metabolism in soleus muscle strips from lean and genetically obese rats. Both BM13.1258 and BM15.2054 revealed to be potent PPARγ‐activators in transient transfection assays in vitro. In insulin‐resistant obese rats, but not in lean rats, 10 days of oral treatment with either compound increased the stimulatory effect of insulin on muscle glycogen synthesis to a similar extent (insulin‐induced increment in μmol glucose incorporated into glycogen g−1 h−1: control, +1.19±0.28; BM13.1258, +2.50±0.20; BM15.2054, +2.55±0.46; P<0.05 vs control each). In parallel to insulin sensitization, mean glucose oxidation increased insulin‐independently in response to BM13.1258 (to 191 and 183% of control in the absence and presence of insulin, respectively; P<0.01 each), which was hardly seen in response to BM15.2054 (to 137 and 124% of control, respectively; ns). Comparable effects on PPARγ activation and on amelioration of insulin resistance by BM13.1258 and BM15.2054 were therefore opposed by different effects on glucose oxidation. In contrast to chronic oral treatment, acute exposure of muscles to BM13.1258 or BM15.2054 in vitro elicited a distinct catabolic response of glucose metabolism in specimens from both lean and obese rats. The results provide evidence that BM13.1258 and BM15.2054 can affect muscle glucose metabolism via more than one mechanism of action. Further efforts are required to clarify, to what extent other mechanisms besides insulin sensitization via the activation of PPARγ are involved in the antidiabetic actions of thiazolidinediones.

Age-Dependent Development of Metabolic Derangement and Effects of Intervention with Pioglitazone in Zucker Diabetic Fatty Rats

Zucker diabetic fatty (ZDF) rats are a standard animal model for the study of type 2 diabetes and for pharmacological characterization of insulin-sensitizing drugs. To analyze the age-dependent development of their metabolic derangements and the associated changes in their responses to treatment with the insulin sensitizer pioglitazone, groups of 7, 10.5, or 15.5-week-old ZDF rats were treated orally with vehicle or pioglitazone (12 mg/kg/day). Metabolic parameters including circulating concentrations of glucose, insulin, lipids, and adiponectin as well as body weight, tissue glycogen content, and the activity of p70S6 kinase in skeletal muscle were determined. Blood glucose of ZDF rats rose steeply from 5.9 ± 0.4 to 23.7 ± 0.5 mM between 7 and 13 weeks of age and then reached a new steady state, which was associated with increased tissue glycogen content (in 15-week-old ZDF rats versus lean littermates: skeletal muscle, 18.0 ± 0.9 versus 10.5 ± 1.4 μmol/g; liver, 181 ± 6 versus 109 ± 14 μmol/g; both p < 0.001). Early intervention with pioglitazone at 7 weeks of age fully prevented the development of hyperglycemia (blood glucose, 6.4 ± 0.4 versus 18.7 ± 1.5 mM after 5.5 weeks of treatment), which was accompanied by a 40% (p = 0.01) reduction of the activity of p70S6 kinase in skeletal muscles. These beneficial effects of pioglitazone were progressively lost, if treatment was initiated at later stages of disease development. Thus, ZDF rats are suitable for preclinical characterization of insulin-sensitizing thiazolidinediones in many aspects, but several important differences versus human type 2 diabetes exist and are to be considered in the use of this animal model.

Thiazolidinediones influence plasma steroids of male obese Zucker rats.

Insulin sensitizing thiazolidinediones (TZDs) inhibit steroidogenic enzyme activities in vitro and affect plasma steroids in women with polycystic ovary syndrome. This study was to examine TZD action on circulating steroids in male genetically obese Zucker rats (fa/fa), which were treated with troglitazone or rosiglitazone (0.3% and 0.01% food admixture, respectively) and were compared to untreated obese and lean littermates. After 36 days of TZD administration, obesity- associated derangement of carbohydrate metabolism was ameliorated (e.g., insulin-stimulated glucose oxidation by isolated soleus muscle, nmol/g/h: lean controls, 1049 ± 100; obese controls, 518 ± 30; troglitazone-treated obese, 672 ± 43; rosiglitazone-treated obese, 761 ± 77; p < 0.01 each vs. obese controls). While plasma pregnenolone and testosterone were neither affected by obesity nor by TZDs, a marked reduction of 17-hydroxyprogesterone in obese vs. lean controls (27 ± 3 vs. 58 ± 10 ng/dl; p < 0.01) was partially reversed by TZD trea...

Lipophilicity as a determinant of thiazolidinedione action in vitro: findings from BLX-1002, a novel compound without affinity to PPARs

The pharmacology of thiazolidinediones (TZDs) seems to be driven not only by activation of peroxisome proliferator-activated receptor-γ (PPARγ), but also by PPARγ-independent effects on mitochondrial function and cellular fuel handling. This study portrayed such actions of the novel hydrophilic TZD compound BLX-1002 and compared them to those of conventional TZDs. Mitochondrial function and fuel handling were examined in disrupted rat muscle mitochondria, intact rat liver mitochondria, and specimens of rat skeletal muscle. BLX-1002 was superior to most other TZDs as an inhibitor of respiratory complex 1 in disrupted mitochondria, but had less effect than any other TZD on oxygen consumption by intact mitochondria and on fuel metabolism by intact tissue. The latter finding was obviously related to the hydrophilic properties of BLX-1002, because high potentials of individual TZDs to shift muscle fuel metabolism from the aerobic into the anaerobic pathway were associated with high ClogP values indicative of high lipophilicity and low hydrophilicity (e.g., % increase in lactate release induced by 10 μmol/l of respective compound: BLX-1002, ClogP 0.39, +10 ± 8%, not significant; pioglitazone, ClogP 3.53, +68 ± 12%, P < 0.001; troglitazone, ClogP 5.58, +157 ± 14%, P < 0.001). The observed specific properties of BLX-1002 could result from relatively strong direct affinity to an unknown mitochondrial target, but limited access to this target. Results suggest 1) that impairment of mitochondrial function and increased anaerobic fuel metabolism are unlikely to account for PPARγ-independent glucose lowering by BLX-1002, and 2) that higher lipophilicity of an individual TZD is associated with stronger acceleration of anaerobic glycolysis.

The effects of amino acids on glucose metabolism of isolated rat skeletal muscle are independent of insulin and the mTOR/S6K pathway

Two mechanisms have been proposed for the modulation of skeletal muscle glucose metabolism by amino acids. Whereas studies on humans and cultured cells suggested acute insulin desensitization via mammalian target of rapamycin (mTOR) and its downstream target p70 S6 kinase (S6K), investigations using native specimens of rat muscle hinted at impairment of glucose oxidation by competition for mitochondrial oxidation. To better understand these seemingly contradictory findings, we explored the effects of high concentrations of mixed amino acids on fuel metabolism and S6K activity in freshly isolated specimens of rat skeletal muscle. In this setting, increasing concentrations of amino acids dose-dependently reduced the insulin-stimulated rates of CO(2) production from glucose and palmitate (decrease in glucose oxidation induced by addition of 5.5, 11, 22, and 44 mmol/l amino acids:--16 +/- 3, -25 +/- 7, -44 +/- 4, -62 +/- 4%; P < 0.02 each). This effect could not be attributed to insulin desensitization, because it was not accompanied by any reduction of insulin-stimulated glucose transport [+12 +/- 16, +17 +/- 22, +21 +/- 33, +13 +/- 12%; all nonsignificant (NS)] or glycogen synthesis (+1 +/- 6, -5 +/- 6, -9 +/- 8, +6 +/- 5%; all NS) and because it persisted without insulin stimulation. Abrogation of S6K activity by the mTOR blocker rapamycin failed to counteract amino acid-induced inhibition of glucose and palmitate oxidation, which therefore was obviously independent of mTOR/S6K signaling (decrease in glucose oxidation by addition of 44 mmol/l amino acids: without rapamycin, -60 +/- 4%; with rapamycin, -50 +/- 13%; NS). We conclude that amino acids can directly affect muscle glucose metabolism via two mechanisms, mTOR/S6K-mediated insulin desensitization and mitochondrial substrate competition, with the latter predominating in isolated rat muscle.

Differences in troglitazone action on glucose metabolism in freshly isolated vs long-term incubated rat skeletal muscle

Exposure of isolated skeletal muscle to troglitazone has resulted in inconsistent findings ranging from inhibition to stimulation of fuel oxidation and the glycogenic pathway. To better understand such variation in outcome, the present study used isolated rat soleus muscle strips to examine the interdependent influences of prolonged maintenance in vitro and of troglitazone exposure. If freshly isolated muscle strips were exposed to troglitazone (1 μmol l−1) for 24 h, glucose oxidation was markedly reduced (−26±1%, P<0.0001), whereas glycogen synthesis remained unaffected (+9±7%, n.s.). In contrast, extended exposure to troglitazone for 72 h increased both glucose oxidation (+65±28%, P<0.05) and glycogen synthesis (+46±11%, P<0.005), and a similar stimulatory effect was also observed in muscles exposed to troglitazone only during the last 24 h of their 72 h preincubation period (glucose oxidation: +61±15%, P<0.001; glycogen synthesis: +43±15%, P<0.01). Troglitazone thus stimulated glucose utilization in long‐term incubated muscle independent of the duration of exposure (24 or 72 h), whereas it inhibited glucose utilization in freshly isolated muscle. The observed differences in troglitazone action on freshly isolated vs long‐term incubated muscle suggest that findings on muscle tissue subject to prolonged maintenance in vitro cannot be extrapolated to native muscle in vivo.

Usability and Fun of the INTERACCT Client

We present the INTERACCT system, a smartphone app and a Web page for fostering communication between young patients in aftercare after a stem cell transplantation. In this phase of rehabilitation, daily communication between the patients and their clinicians is key for detecting upcoming possibly deadly crises as early as possible. The app consists of a communication part, a gaming part, a module for daily medicine, and a daily story for further motivation. We describe the system as well as an evaluation of the app with several healthy children.