Dana Galuska

Evidence that oestrogen receptor-α plays an important role in the regulation of glucose homeostasis in mice: insulin sensitivity in the liver

Aims/hypothesisWe used oestrogen receptor-α (ERα) knockout (ERKO) and receptor-β (ERβ) knockout (BERKO) mice to investigate the mechanism(s) behind the effects of oestrogens on glucose homeostasis.MethodsEndogenous glucose production (EGP) was measured in ERKO mice using a euglycaemic–hyperinsulinaemic clamp. Insulin secretion was determined from isolated islets. In isolated muscles, glucose uptake was assayed by using radiolabelled isotopes. Genome-wide expression profiles were analysed by high-density oligonucleotide microarray assay, and the expression of the genes encoding steroyl-CoA desaturase and the Leptin receptor (Scd1 and Lepr, respectively) was confirmed by RT-PCR.ResultsERKO mice had higher fasting blood glucose, plasma insulin levels and IGT. The plasma leptin level was increased, while the adiponectin concentration was decreased in ERKO mice. Levels of both glucose- and arginine-induced insulin secretion from isolated islets were similar in ERKO and wild-type mice. The euglycaemic–hyperinsulinaemic clamp revealed that suppression of EGP by increased insulin levels was blunted in ERKO mice, which suggests a pronounced hepatic insulin resistance. Microarray analysis revealed that in ERKO mice, the genes involved in hepatic lipid biosynthesis were upregulated, while genes involved in lipid transport were downregulated. Notably, hepatic Lepr expression was decreased in ERKO mice. In vitro studies showed a modest decrease in insulin-mediated glucose uptake in soleus and extensor digitorum longus (EDL) muscles of ERKO mice. BERKO mice demonstrated normal glucose tolerance and insulin release.Conclusions/interpretationWe conclude that oestrogens, acting via ERα, regulate glucose homeostasis mainly by modulating hepatic insulin sensitivity, which can be due to the upregulation of lipogenic genes via the suppression of Lepr expression.

Exercise-induced overexpression of key regulatory proteins involved in glucose uptake and metabolism in tetraplegic persons: molecular mechanism for improved glucose homeostasis

Complete spinal cord lesion leads to profound metabolic abnormalities and striking changes in muscle morphology. Here we assess the effects of electrically stimulated leg cycling (ESLC) on whole body insulin sensitivity, skeletal muscle glucose metabolism, and muscle fiber morphology in five tetraplegic subjects with complete C5‐C7 lesions. Physical training (seven ESLC sessions/wk for 8 wk) increased whole body insulin‐stimulated glucose uptake by 33±13%, concomitant with a 2.1‐fold increase in insulin‐stimulated (100 µU/ml) 3‐O‐methylglucose transport in isolated vastus lateralis muscle. Physical training led to a marked increase in protein expression of GLUT4 (378±85%), glycogen synthase (526±146%), and hexokinase II (204±47%) in vastus lateralis muscle, whereas phosphofructokinase expression (282±97%) was not significantly changed. Hexokinase II activity was significantly increased, whereas activity of phosphofructokinase, glycogen synthase, and citrate synthase was not changed after training. Muscle fiber type distribution and fiber area were markedly altered compared to able‐bodied subjects before ESLC training, with no change noted in either parameter after ECSL training. In conclusion, muscle contraction improves insulin action on whole body and cellular glucose uptake in cervical cord‐injured persons through a major increase in protein expression of key genes involved in the regulation of glucose metabolism. Furthermore, improvements in insulin action on glucose metabolism are independent of changes in muscle fiber type distribution.—Hjeltnes, N., Galuska, D., Björnholm, M., Aksnes, A.‐K., Lannem, A., Zierath, J. R., Wallberg‐Henriksson, H. Exercise‐induced overexpression of key regulatory proteins involved in glucose uptake and metabolism in tetraplegic persons: molecular mechanism for improved glucose homeostasis. FASEB J. 12, 1701‐1712 (1998)

Low‐intensity exercise increases skeletal muscle protein expression of PPARδ and UCP3 in type 2 diabetic patients

Physical exercise provides health benefits for people with type 2 diabetes mellitus, partly by enhancing skeletal muscle insulin action. We tested the hypothesis that changes in expression of key genes in skeletal muscles relate to exercise‐induced improvements in type 2 diabetic patients.

Effects of glycaemia on glucose transport in isolated skeletal muscle from patients with NIDDM: in vitro reversal of muscular insulin resistance.

SummaryWe investigated the influence of altered glucose levels on insulin-stimulated 3-0-methylglucose transport in isolated skeletal muscle obtained from NIDDM patients (n=13) and non-diabetic subjects (n=23). Whole body insulin sensitivity was 71% lower in the NIDDM patients compared to the non-diabetic subjects (p <0.05), whereas, insulin-mediated peripheral glucose utilization in the NIDDM patients under hyperglycaemic conditions was comparable to that of the non-diabetic subjects at euglycaemia. Following a 30-min in vitro exposure to 4 mmol/l glucose, insulin-stimulated 3-0-methylglucose transport (600 pmol/l insulin) was 40% lower in isolated skeletal muscle strips from the NIDDM patients when compared to muscle strips from the non-diabetic subjects. The impaired capacity for insulin-stimulated 3-0-methylglucose transport in the NIDDM skeletal muscle was normalized following prolonged (2 h) exposure to 4 mmol/l, but not to 8 mmol/l glucose. Insulin-stimulated 3-0-methylglucose transport in the NIDDM skeletal muscle exposed to 8 mmol/l glucose was similar to that of the non-diabetic muscle exposed to 5 mmol/l glucose, but was decreased by 43% (p <0.01) when compared to non-diabetic muscle exposed to 8 mmol/l glucose. Despite the impaired insulin-stimulated 3-0-methylglucose transport capacity demonstrated by skeletal muscle from the NIDDM patients, skeletal muscle glycogen content was similar to that of the non-diabetic subjects. Kinetic studies revel a Km for 3-0-methylglucose transport of 9.7 and 8.8 mmol/l glucose for basal and insulin-stimulated conditions, respectively. In conclusion, the impaired capacity for insulinstimulated glucose transport in skeletal muscle from patients with NIDDM appears to protect the cell from excessive glucose uptake under hyperglycaemic conditions. Furthermore, the in vitro normalization of the decreased insulin-stimulated glucose transport in NIDDM skeletal muscle following exposure to 4 mmol/l glucose suggests that glycaemia per se has a profound effect on the regulation of muscular glucose transport.

Divergent cell signaling after short-term intensified endurance training in human skeletal muscle

Endurance training represents one extreme in the continuum of skeletal muscle plasticity. The molecular signals elicited in response to acute and chronic exercise and the integration of multiple intracellular pathways are incompletely understood. We determined the effect of 10 days of intensified cycle training on signal transduction in nine inactive males in response to a 1-h acute bout of cycling at the same absolute workload (164 +/- 9 W). Muscle biopsies were taken at rest and immediately and 3 h after the acute exercise. The metabolic signaling pathways, including AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), demonstrated divergent regulation by exercise after training. AMPK phosphorylation increased in response to exercise ( approximately 16-fold; P < 0.05), which was abrogated posttraining (P < 0.01). In contrast, mTOR phosphorylation increased in response to exercise ( approximately 2-fold; P < 0.01), which was augmented posttraining (P < 0.01) in the presence of increased mTOR expression (P < 0.05). Exercise elicited divergent effects on mitogen-activated protein kinase (MAPK) pathways after training, with exercise-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation being abolished (P < 0.01) and p38 MAPK maintained. Finally, calmodulin kinase II (CaMKII) exercise-induced phosphorylation and activity were maintained (P < 0.01), despite increased expression ( approximately 2-fold; P < 0.05). In conclusion, 10 days of intensified endurance training attenuated AMPK, ERK1/2, and mTOR, but not CaMKII and p38 MAPK signaling, highlighting molecular pathways important for rapid functional adaptations and maintenance in response to intensified endurance exercise and training.

Effect of metformin on insulin-stimulated glucose transport in isolated skeletal muscle obtained from patients with NIDDM.

SummaryMetformin has been demonstrated to lower blood glucose in vivo by a mechanism which increases peripheral glucose uptake. Furthermore, the therapeutic concentration of metformin has been estimated to be in the order of 0.01 mmol/l. We investigated the effect of metformin on insulin-stimulated 3-0-methylglucose transport in isolated skeletal muscle obtained from seven patients with non-insulin-dependent diabetes mellitus (NIDDM) and from eight healthy subjects. Whole body insulin-mediated glucose utilization was decreased by 45% (p<0.05) in the diabetic subjects when studied at 8 mmol/l glucose, compared to the healthy subjects studied at 5 mmol/l glucose. Metformin, at concentrations of 0.1 and 0.01 mmol/l, had no effect on basal or insulin-stimulated (100 ΜU/ml) glucose transport in muscle strips from either of the groups. However, the two control subjects and three patients with NIDDM which displayed a low rate of insulin-mediated glucose utilization (<20 Μmol·kg−1·min−1), as well as in vitro insulin resistance, demonstrated increased insulin-stimulated glucose transport in the presence of metformin at 0.1 mmol/l (p<0.05). In conclusion, the concentration of metformin resulting in a potentiating effect on insulin-stimulated glucose transport in insulin-resistant human skeletal muscle is 10-fold higher than the therapeutic concentrations administered to patients with NIDDM. Thus, it is conceivable that the hypoglycaemic effect of metformin in vivo may be due to an accumulation of the drug in the extracellular space of skeletal muscle, or to an effect of the drug distal to the glucose transport step.

Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice

To determine the role of GLUT4 on postexercise glucose transport and glycogen resynthesis in skeletal muscle, GLUT4‐deficient and wild‐type mice were studied aftera3h swim exercise. In wild‐type mice, insulin and swimming each increased 2‐deoxyglucose uptake by twofold in extensor digitorum longus muscle. In contrast, insulin did not increase 2‐deoxyglucose glucose uptake in muscle from GLUT4‐null mice. Swimming increased glucose transport twofold in muscle from fed GLUT4‐null mice, with no effect noted in fasted GLUT4‐null mice. This exercise‐associated 2‐deoxyglucose glucose uptake was not accompanied by increased cell surface GLUT1 content. Glucose transport in GLUT4‐null muscle was increased 1.6‐fold over basal levels after electrical stimulation. Contraction‐induced glucose transport activity was fourfold greater in wild‐type vs. GLUT4‐null muscle. Glycogen content in gastrocnemius muscle was similar between wild‐type and GLUT4‐null mice and was reduced ~50% after exercise. After 5 h carbohydrate refeeding, muscle glycogen content was fully restored in wild‐type, with no change in GLUT4‐null mice. After 24 h carbohydrate refeeding, muscle glycogen in GLUT4‐null mice was restored to fed levels. In conclusion, GLUT4 is the major transporter responsible for exercise‐induced glucose transport. Also, postexercise glycogen resynthesis in muscle was greatly delayed; unlike wild‐type mice, glycogen supercompensation was not found. GLUT4 it is not essential for glycogen repletion since muscle glycogen levels in previously exercised GLUT4‐null mice were totally restored after 24 h carbohydrate refeeding.—Ryder, J. W., Kawano, Y., Galuska, D., Fahlman, R., Wallberg‐Henriksson, H., Charron, M. J., Zierath, J. R. Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4‐deficient mice. FASEB J. 13, 2246–2256 (1999)

The estrogen receptor α-selective agonist propyl pyrazole triol improves glucose tolerance in ob/ob mice; potential molecular mechanisms

The aim of this study was to validate the role of estrogen receptor alpha (ERalpha) signaling in the regulation of glucose metabolism, and to compare the molecular events upon treatment with the ERalpha-selective agonist propyl pyrazole triol (PPT) or 17beta-estradiol (E(2)) in ob/ob mice. Female ob/ob mice were treated with PPT, E(2) or vehicle for 7 or 30 days. Intraperitoneal glucose and insulin tolerance tests were performed, and insulin secretion was determined from isolated islets. Glucose uptake was assayed in isolated skeletal muscle and adipocytes. Gene expression profiling in the liver was performed using Affymetrix microarrays, and the expression of selected genes was studied by real-time PCR analysis. PPT and E(2) treatment improved glucose tolerance and insulin sensitivity. Fasting blood glucose levels decreased after 30 days of PPT and E(2) treatment. However, PPT and E(2) had no effect on insulin secretion from isolated islets. Basal and insulin-stimulated glucose uptake in skeletal muscle and adipose tissue were similar in PPT and vehicle-treated ob/ob mice. Hepatic lipid content was decreased after E(2) treatment. In the liver, treatment with E(2) and PPT increased and decreased the respective expression levels of the transcription factor signal transducer and activator of transcription 3, and of glucose-6-phosphatase. In summary, our data demonstrate that PPT exerts anti-diabetic effects, and these effects are mediated via ERalpha.

Human islet amyloid polypeptide at pharmacological levels inhibits insulin and phorbol ester-stimulated glucose transport in in vitro incubated human muscle strips

SummaryHuman islet amyloid polypeptide, at concentrations of 1–100 nmol/l, has been demonstrated to inhibit the insulin-stimulated increase in rat muscle glycogen content. However, at physiological concentrations (1–10 pmol/l) of islet amyloid polypeptide, no effects have been reported. We tested the effect of a wide range of concentrations of human islet amyloid polypeptide on insulin- and phorbol ester-stimulated 3-0-methylglucose transport in in vitro incubated human skeletal muscle. Muscle specimens from the quadriceps femoris muscle were obtained from 23 healthy subjects with the use of a newly-developed open muscle biopsy technique. Human islet amyloid polypeptide at a concentration of 100 nmol/l had no effect on basal glucose transport, but inhibited the stimulatory effect of a maximal insulin concentration (1000 μU/ml) by 69% (p<0.001). The presence of human islet amyloid polypeptide at 1, 10 and 100 nmol/l decreased the effect of 100 μU/ml of insulin on glucose transport by 61% (p<0.05), 78% (p<0.05) and 76% (p<0.05), respectively. Similarly, human islet amyloid polypeptide at a concentration of 10 nmol/l inhibited phorbol ester-stimulated glucose transport by 100% (p<0.05). The inhibitory effects of human islet amyloid polypeptide on glucose transport were present in the muscle strips despite no net changes in glycogen content. Human islet amyloid polypeptide at 10 and 100 pmol/l had no effect on the rate of insulin-stimulated glucose transport. In conclusion, pharmacological concentrations of human islet amyloid polypeptide inhibit insulin as well as phorbol ester-stimulated glucose transport in human skeletal muscle, while physiological concentrations do not exert an inhibitory effect. Furthermore, these results suggest that the inhibitory effect of human islet amyloid polypeptide on glucose transport is located at a point distal to the insulin binding process.

Differences in the Ratio of RNA Encoding Two Isoforms of the Insulin Receptor Between Control and NIDDM Patients: The RNA Variant Without Exon 11 Predominates in Both Groups

Two alternative forms of the insulin receptor with different affinities for insulin are expressed as a result of alternative splicing of RNA corresponding to exon 11 of the IR gene. The percentage of IR-RNA molecules without exon 11, encoding the high-affinity isoform, was determined by cDNA-mediated PCR amplification of RNA extracts from the quadriceps femoris muscle of healthy control subjects (n = 9) and NIDDM patients (n = 7). In both patients and control individuals, a majority of the IR-RNA molecules contained exon 11. In addition, the proportion of IR-RNA molecules without exon 11 was decreased in patients (21 ± 1%) compared with control subjects (31 ± 3%) (P = 0.018). Careful investigation of the kinetics of the PCR-based assay system, as well as the conditions for separation of the PCR products, allowed us to suggest a possible explanation of the discrepant results concerning the alternative splicing presented in previous reports. The diabetic subjects as a group had higher fasting insulin levels and lower insulin-mediated glucose uptake during a euglycemic-hyperinsulinemic clamp (P = 0.042). However, identification of the regulatory pathways leading to the splicing alteration in NIDDM patients requires further investigation.