Multi-target neuroprotective effects of herbal medicines for Alzheimer's disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Alzheimer's disease is the most common form of dementia, but its treatment options remain few and ineffective. To find new therapeutic strategies, natural products have gained interest due to their neuroprotective potential, being able to target different pathological hallmarks associated with this disorder. Several plant species are traditionally used due to their empirical neuroprotective effects and it is worth to explore their mechanism of action. AIM OF THE STUDY: This study intended to explore the neuroprotective potential of seven traditional medicinal plants, namely Scutellaria baicalensis, Ginkgo biloba, Hypericum perforatum, Curcuma longa, Lavandula angustifolia, Trigonella foenum-graecum and Rosmarinus officinalis. The safety assessment with reference to pesticides residues was also aimed. MATERIALS AND METHODS: Decoctions prepared from these species were chemically characterized by HPLC-DAD and screened for their ability to scavenge four different free radicals (DPPH•, ABTS•+, O2•‒ and •NO) and to inhibit enzymes related to neurodegeneration (cholinesterases and glycogen synthase kinase-3ß). Cell viability through MTT assay was also evaluated in two different brain cell lines, namely non-tumorigenic D3 human brain endothelial cells (hCMEC/D3) and NSC-34 motor neurons. Furthermore, and using GC, 21 pesticides residues were screened. RESULTS: Regarding chemical composition, chromatographic analysis revealed the presence of several flavonoids, phenolic acids, curcuminoids, phenolic diterpenoids, one alkaloid and one naphthodianthrone in the seven decoctions. All extracts were able to scavenge free radicals and were moderate glycogen synthase kinase-3ß inhibitors; however, they displayed weak to moderate acetylcholinesterase and butyrylcholinesterase inhibition. G. biloba and L. angustifolia decoctions were the less cytotoxic to hCMEC/D3 and NSC-34 cell lines. No pesticides residues were detected. CONCLUSIONS: The results extend the knowledge on the potential use of plant extracts to combat multifactorial disorders, giving new insights into therapeutic avenues for Alzheimer's disease.

Guaiacol as a drug candidate for treating adult polyglucosan body disease.

Adult polyglucosan body disease (APBD) is a late-onset disease caused by intracellular accumulation of polyglucosan bodies, formed due to glycogen-branching enzyme (GBE) deficiency. To find a treatment for APBD, we screened 1,700 FDA-approved compounds in fibroblasts derived from APBD-modeling GBE1-knockin mice. Capitalizing on fluorescent periodic acid-Schiff reagent, which interacts with polyglucosans in the cell, this screen discovered that the flavoring agent guaiacol can lower polyglucosans, a result also confirmed in APBD patient fibroblasts. Biochemical assays showed that guaiacol lowers basal and glucose 6-phosphate-stimulated glycogen synthase (GYS) activity. Guaiacol also increased inactivating GYS1 phosphorylation and phosphorylation of the master activator of catabolism, AMP-dependent protein kinase. Guaiacol treatment in the APBD mouse model rescued grip strength and shorter lifespan. These treatments had no adverse effects except making the mice slightly hyperglycemic, possibly due to the reduced liver glycogen levels. In addition, treatment corrected penile prolapse in aged GBE1-knockin mice. Guaiacol's curative effects can be explained by its reduction of polyglucosans in peripheral nerve, liver, and heart, despite a short half-life of up to 60 minutes in most tissues. Our results form the basis to use guaiacol as a treatment and prepare for the clinical trials in APBD.

Priming Effect of a Morning Meal on Hepatic Glucose Disposition Later in the Day.

We used hepatic balance and tracer ([3H]glucose) techniques to examine the impact of "breakfast" on hepatic glucose metabolism later in the same day. From 0-240 min, 2 groups of conscious dogs (n = 9 dogs/group) received a duodenal infusion of glucose (GLC) or saline (SAL), then were fasted from 240-360 min. Three dogs from each group were euthanized and tissue collected at 360 min. From 360-600 min, the remaining dogs underwent a hyperinsulinemic (4× basal) hyperglycemic clamp (arterial blood glucose 146 ± 2 mg/dL) with portal GLC infusion. The total GLC infusion rate was 14% greater in dogs infused with GLC than in those receiving SAL (AUC360-600min 2,979 ± 296 vs. 2,597 ± 277 mg/kg, respectively). The rates of hepatic glucose uptake (5.8 ± 0.8 vs. 3.2 ± 0.3 mg ⋅ kg-1 ⋅ min-1) and glycogen storage (4.7 ± 0.6 vs. 2.9 ± 0.3 mg ⋅ kg-1 ⋅ min-1) during the clamp were markedly greater in dogs receiving GLC compared with those receiving SAL. Hepatic glycogen content was ∼50% greater, glycogen synthase activity was ∼50% greater, glycogen phosphorylase activity was ∼50% lower, and the amount of phosphorylated glycogen synthase was 34% lower, indicating activation of the enzyme, in dogs receiving GLC compared with those receiving SAL. Thus, morning GLC primed the liver to extract and store more glucose in the presence of hyperinsulinemic hyperglycemia later in the same day, indicating that breakfast enhances the liver's role in glucose disposal in subsequent same-day meals.

Molecular mechanisms of anti-hyperglycemic effects of Costus speciosus extract in streptozotocin-induced diabetic rats.

OBJECTIVES: To investigate the mechanisms of the anti-hyperglycemic effect of Costus speciosus (C. speciosus) root ethanolic extracts (CSREt) by assessing its action on insulin synthesis and glucose catabolic enzyme gene expression and activities in streptozotocin (STZ) diabetic rats. METHODS: This study was carried out at the Biochemical Laboratory, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt between July and August 2013. Sixty male albino rats (120 +/- 20 g weight, and 6 months old) were used and divided into 6 groups (n=10). Two groups served as diabetic and nondiabetic controls. Four groups of STZ diabetic animals were given oral C. speciosus (CSREt) in doses of 200, 400, and 600 mg/kg body weight, and 600 µg/kg body weight of the standard drug glibenclamide for 4 weeks. RESULTS: The CSREt 400 and 600 mg/kg body weight induced a decrease in blood glucose and an increase in serum insulin level, glucokinase (GK), aldolase, pyruvate kinase (PK), succinate dehydrogenase (SDH), and glycogen synthase activities  in addition to a higher expression level of insulin, insulin receptor A (IRA), GK, PK, SDH, and glucose transporting protein. CONCLUSION: The C. speciosus has anti-hyperglycemic activity. It induces insulin secretion and release from cells, as well as stimulates the tissue's insulin sensitivity leading to an increase of the tissues' glucose uptake, storage, and oxidation.

The role of glycogen synthase in the development of hyperglycemia in type 2 diabetes: 'To store or not to store glucose, that's the question'.

This review deals with the role of glycogen storage in skeletal muscle for the development of insulin resistance and type 2 diabetes. Specifically, the role of the enzyme glycogen synthase, which seems to be locked in its hyperphosphorylated and inactivated state, is discussed. This defect seems to be secondary to ectopic lipid disposition in the muscle cells. These molecular defects are discussed in the context of the overall pathophysiology of hyperglycemia in type 2 diabetic subjects.

Piperidine and pyrrolidine analogs as glycogen synthase activators; a patent evaluation (WO2011058122).

A small series of piperidine analogs that effectively activate glycogen synthase (GS) was prepared in WO2011058122. The treatment or prophylaxis of metabolic disease and disorders by these novel GS activators is claimed. These compounds represent further variations around a structural motif explored in the prior patent publications by Roche.

Control of glycogen synthase through ADIPOR1-AMPK pathway in renal distal tubules of normal and diabetic rats.

Diabetic nephropathies are characterized by glycogen accumulation in distal tubular cells, which eventually leads to their apoptosis. The present study aims to determine whether adiponectin and AMPK are involved in the regulation of glycogen synthase (GS) in these structures. Western blots of isolated distal tubules revealed the presence of adiponectin receptor ADIPOR1, catalytic AMPK subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding AMPK subunit beta(2). ADIPOR2 was not detected. Expression levels of ADIPOR1, AMPKalpha(1), AMPKalpha(2), and AMPKbeta(2) were increased in streptozotocin-treated diabetic rats, whereas phosphorylated active AMPK levels were strongly decreased. Immunohistochemistry revealed the presence of ADIPOR1 on the luminal portion of distal tubules and thick ascending limb cells. Catalytic subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding subunit beta(2) were also found in the same cells, confirming immunoblot results. In vitro, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR; 2 mM) and globular adiponectin (10 mug/ml) activated catalytic AMPK in distal tubules isolated from kidneys of normal rats but much more weakly in those from diabetic rats. GS inhibition paralleled AMPK activation in both groups of animals: active GS levels were low in control animals and elevated in diabetic ones. Finally, glucose-6-phosphate, an allosteric activator of GS, was also increased in diabetic rats. These results demonstrate that in distal tubular cells, adiponectin through luminal ADIPOR1 activates AMPK, leading to the inhibition of GS. During hyperglycemia, this regulation is altered, which may explain, at least in part, the accumulation of large glycogen deposits.

An assessment of the in vivo efficacy of the glycogen phosphorylase inhibitor GPi688 in rat models of hyperglycaemia.

BACKGROUND AND PURPOSE: Studies in cultured hepatocytes demonstrate glycogen synthase (GS) activation with glycogen phosphorylase (GP) inhibitors. The current study investigated whether these phenomena occurred in vivo using a novel GP inhibitor. EXPERIMENTAL APPROACH: An allosteric GP inhibitor, GPi688, was evaluated against both glucagon-mediated hyperglycaemia and oral glucose challenge-mediated hyperglycaemia to determine the relative effects against GP and GS in vivo. KEY RESULTS: In rat primary hepatocytes, GPi688 inhibited glucagons-mediated glucose output in a concentration dependent manner. Additionally GP activity was reduced and GS activity increased seven-fold. GPi688 inhibited glucagon-mediated hyperglycaemia in both Wistar (65%) & obese Zucker (100%) rats and demonstrated a long duration of action in the Zucker rat. The in vivo efficacy in the glucagon challenge model could be predicted by the equation; % glucagon inhibition=56.9+34.3[log ([free plasma]/rat IC50)], r=0.921). GPi688 also reduced the blood glucose of obese Zucker rats after a 7 h fast by 23%. In an oral glucose tolerance test in Zucker rats, however, GPi688 was less efficacious (7% reduction) than a glycogen synthase kinase-3 (GSK-3) inhibitor (22% reduction), despite also observing activation (by 45%) of GS in vivo. CONCLUSIONS AND IMPLICATIONS: Although GP inhibition can inhibit hyperglycaemia mediated by increased glucose production, the degree of GS activation induced by allosteric GP inhibitors in vivo, although discernible, is insufficient to increase glucose disposal. The data suggests that GP inhibitors might be more effective clinically against fasting rather than prandial hyperglycaemic control.

Role of glycogen synthase kinase-3 alpha in insulin action in cultured human skeletal muscle cells.

An association between glycogen synthase kinase-3 (GSK3) in skeletal muscle and insulin resistance has been demonstrated in type 2 diabetic patients. In addition, inhibition of GSK3 improves insulin action. The aim of the present study was to elucidate the role of the alpha-isoform of GSK3 in insulin resistance in human skeletal muscle cells from nondiabetic subjects maintained in culture. Transfection of muscle cells with specific antisense oligonucleotides resulted in a 30-50% decrease of GSK3alpha protein expression (P < 0.05). Whereas neither the basal fractional velocity of glycogen synthase (GS FV) (an indicator of the activation state of the enzyme) nor glucose uptake (GU) were altered, reducing GSK3alpha expression resulted in increases in insulin stimulation of both GS FV and GU. GSK3alpha overexpression (60-100% increase over control) did not alter basal GS FV or GU but impaired insulin stimulation of both responses. Knockdown of GSK alpha also led to an increase in insulin receptor substrate-1 protein expression but did not alter insulin stimulation of pS473-Akt phosphorylation. However, GSK3alpha overexpression impaired insulin action on pS473-Akt. In summary, we concluded the following: 1) modulation of GSK3alpha expression has no effect on basal GU and glycogen synthase activities; 2) reduction of GSK3alpha expression results in improvements in insulin action; and 3) elevation of GSK3alpha in human skeletal muscle cells can induce insulin resistance for several responses. We conclude that GSK3alpha is an important regulator of muscle insulin action.

Reduced plasma adiponectin concentrations may contribute to impaired insulin activation of glycogen synthase in skeletal muscle of patients with type 2 diabetes.

AIMS/HYPOTHESIS: Circulating levels of adiponectin are negatively associated with multiple indices of insulin resistance, and the concentration is reduced in humans with insulin resistance and type 2 diabetes. However, the mechanisms by which adiponectin improves insulin sensitivity remain unclear. SUBJECTS AND METHODS: Combining euglycaemic-hyperinsulinaemic clamp studies with indirect calorimetry and skeletal muscle biopsies, we examined the relationship between plasma adiponectin and parameters of whole-body glucose and lipid metabolism, and muscle glycogen synthase (GS) activity in 51 Caucasians (ten lean, 21 obese and 20 with type 2 diabetes). RESULTS: Plasma adiponectin was significantly reduced in type 2 diabetic compared with obese and lean subjects. In lean and obese subjects, insulin significantly reduced plasma adiponectin, but this response was blunted in patients with type 2 diabetes. Plasma adiponectin was positively associated with insulin-stimulated glucose disposal (r = 0.48), glucose oxidation (r = 0.54), respiratory quotient (r = 0.58) and non-oxidative glucose metabolism (r = 0.38), and negatively associated with lipid oxidation during insulin stimulation (r = -0.60) after adjustment for body fat (all p < 0.01). Most notably, we found a positive association between plasma adiponectin and insulin stimulation of GS activity in skeletal muscle (r = 0.44, p < 0.01). CONCLUSIONS/INTERPRETATION: Our results indicate that plasma adiponectin may enhance insulin sensitivity by improving the capacity to switch from lipid to glucose oxidation and to store glucose as glycogen in response to insulin, and that low adiponectin may contribute to impaired insulin activation of GS in skeletal muscle of patients with type 2 diabetes.

The vanadyl (VO2+) chelate bis(acetylacetonato)oxovanadium(IV) potentiates tyrosine phosphorylation of the insulin receptor.

We have compared the insulin-like activity of bis(acetylacetonato)oxovanadium(IV) [VO(acac)2], bis(maltolato)oxovanadium(IV) [VO(malto)2], and bis(1-N-oxide-pyridine-2-thiolato)oxovanadium(IV) [VO(OPT)2] in differentiated 3T3-L1 adipocytes. The insulin-like influence of VO(malto)2 and VO(OPT)2 was decreased compared with that of VO(acac)2. Also, serum albumin enhanced the insulin-like activity of all three chelates more than serum transferrin. Each of the three VO2+ chelates increased the tyrosine phosphorylation of proteins in response to insulin, including the beta-subunit of the insulin receptor (IRbeta) and the insulin receptor substrate-1 (IRS1). However, VO(acac)2 exhibited the greatest synergism with insulin and was therefore further investigated. Treatment of 3T3-L1 adipocytes with 0.25 mM VO(acac)2 in the presence of 0.25 mM serum albumin synergistically increased glycogen accumulation stimulated by 0.1 and 1 nM insulin, and increased the phosphorylation of IRbeta, IRS1, protein kinase B, and glycogen synthase kinase-3beta. Wortmannin suppressed all of these classical insulin-signaling activities exerted by VO(acac)2 or insulin, except for tyrosine phosphorylation of IRbeta and IRS1. Additionally, VO(acac)2 enhanced insulin signaling and metabolic action in insulin-resistant 3T3-L1 adipocytes. Cumulatively, these results provide evidence that VO(acac)2 exerts its insulin-enhancing properties by directly potentiating the tyrosine phosphorylation of the insulin receptor, resulting in the initiation of insulin metabolic signaling cascades in 3T3-L1 adipocytes.

Glycogen synthesis correlates with androgen-dependent growth arrest in prostate cancer.

BACKGROUND: Androgen withdrawal in normal prostate or androgen-dependent prostate cancer is associated with the downregulation of several glycolytic enzymes and with reduced glucose uptake. Although glycogen metabolism is known to regulate the intracellular glucose level its involvement in androgen response has not been studied. METHODS: We investigated the effects of androgen on glycogen phosphorylase (GP), glycogen synthase (GS) and on glycogen accumulation in the androgen-receptor (AR) reconstituted PC3 cell line containing either an empty vector (PC3-AR-V) or vector with HPV-E7 (PC3-AR-E7) and the LNCaP cell line. RESULTS: Androgen addition in PC3 cells expressing the AR mimics androgen ablation in androgen-dependent prostate cells. Incubation of PC3-AR-V or PC3-AR-E7 cells with the androgen R1881 induced G1 cell cycle arrest within 24 hours and resulted in a gradual cell number reduction over 5 days thereafter, which was accompanied by a 2 to 5 fold increase in glycogen content. 24 hours after androgen-treatment the level of Glucose-6-P (G-6-P) had increased threefold and after 48 hours the GS and GP activities increased twofold. Under this condition inhibition of glycogenolysis with the selective GP inhibitor CP-91149 enhanced the increase in glycogen content and further reduced the cell number. The androgen-dependent LNCaP cells that endogenously express AR responded to androgen withdrawal with growth arrest and increased glycogen content. CP-91149 further increased glycogen content and caused a reduction of cell number. CONCLUSION: Increased glycogenesis is part of the androgen receptor-mediated cellular response and blockage of glycogenolysis by the GP inhibitor CP-91149 further increased glycogenesis. The combined use of a GP inhibitor with hormone therapy may increase the efficacy of hormone treatment by decreasing the survival of prostate cancer cells and thereby reducing the chance of cancer recurrence.

A high carbohydrate diet induces insulin resistance through decreased glucose utilization in ovariectomized rats.

BACKGROUND: Recent research has reported that high sugar diets increase insulin resistance, without abdominal obesity, in male, but not female Wistar rats. Whether a high sucrose (SU) diet increased insulin resistance in ovariectomized (OVX) rats was determined. METHODS: Female Sprague Dawley rats, weighing 273 +/- 20 g, had either an ovariectomy or a sham operation (sham). OVX and sham rats were divided into two groups: one group had a 68 En% SU diet and the other a 68 En% starch (ST) diet for 8 weeks. RESULTS: The body weight was higher in the OVX than the sham rats, regardless of dietary carbohydrate subtype. The fasting serum glucose levels did not differ according to diet and ovariectomy. However, the fasting serum insulin levels were higher in the OVX than the sham rats, and in the OVX rats, a high SU diet increased the serum insulin levels more than a high ST diet. The whole body glucose disposal rates, which referred to the state of insulin sensitivity, were lower in the OVX rats fed both the high SU and ST diets, compared to sham rats. Glycogen deposits in the soleus and quadriceps muscles were lower in the OVX rats fed high SU and ST diets than in sham rats. The glucose transporter 4 content and fraction velocity of glycogen synthase in muscles showed similar glucose disposal rates. However, the triacylglycerol content in the muscles were higher in the OVX rats with a high SU diet than those with a high ST diet. CONCLUSION: These results suggested that an OVX increased the weight gain due to higher food intakes, regardless of dietary carbohydrate subtypes. OVX-induced obesity may be involved in the induction of insulin resistance from an increased triacylglycerol content, decreased glucose uptake and glycogen synthesis in skeletal muscles, regardless of dietary carbohydrate subtypes.

Hyperglycemia and inhibition of glycogen synthase in streptozotocin-treated mice: role of O-linked N-acetylglucosamine.

Glycogen synthase is post-translationally modified by both phosphate and O-linked N-acetylglucosamine (O-GlcNAc). In 3T3-L1 adipocytes exposed to high concentrations of glucose, O-GlcNAc contributes to insulin resistance of glycogen synthase. We sought to determine whether O-GlcNAc also regulates glycogen synthase in vivo. Glycogen synthase activity in fat pad extracts was inhibited in streptozotocin (STZ)-treated diabetic mice. The half-maximal activation concentration for glucose 6-phosphate (A(0.5)) was increased to 830 +/- 120 microm compared with 240 +/- 20 microm in control mice (C, p < 0.01), while the basal glycogen synthase activity (%I-form) was decreased to 2.4 +/- 1.4% compared with 10.1 +/- 1.8% in controls (p < 0.01). Glycogen synthase activity remained inhibited after compensatory insulin treatment. After insulin treatment kinetic parameters of glycogen synthase were more closely correlated with blood glucose (A(0.5), r(2) = 0.70; %I-form, r(2) = 0.59) than insulin levels (A(0.5), r(2) = 0.04; %I-form, r(2) = 0.09). Hyperglycemia also resulted in an increase in the level of O-GlcNAc on glycogen synthase (16.1 +/- 1.8 compared with 7.0 +/- 0.9 arbitrary intensity units for controls, p < 0.01), even though the level of phosphorylation was identical in diabetic and control mice either with (STZ: 2.9 +/- 1.0 and C: 3.2 +/- 0.8) or without (STZ: 12.2 +/- 2.8 and C: 13.8 +/- 3.0 arbitrary intensity units) insulin treatment. In all mice the percent activation of glycogen synthase that could be achieved in vitro by recombinant protein phosphatase 1 (230 +/- 30%) was significantly greater in the presence of beta-d-N-acetylglucosaminidase (410 +/- 60%, p < 0.01). This synergistic stimulation of glycogen synthase due to codigestion by protein phosphatase 1 and beta-d-N-acetylglucosaminidase was more pronounced in STZ-diabetic mice (310 +/- 70%) compared with control mice (100 +/- 10%, p < 0.05). The findings demonstrate that O-GlcNAc has a role in the regulation of glycogen synthase both in normoglycemia and diabetes.

A High Carbohydrate Diet Induces Insulin Resistance Through Decreased Glucose Utilization in Ovariectomized Rats

BACKGROUND: Recent research has reported that high sugar diets increase insulin resistance, without abdominal obesity, in male, but not female Wister rats. Whether a high sucrose (SU) diet increased insulin resistance in ovariectomized (OVX) rats was determined. METHODS: Female Sprague Dawley rats, weighing 273 +/- 20 g, had either an ovariectomy or a sham operation (sham). OVX and sham rats were divided into two groups: one group had a 68 En% SU diet and the other a 68 En% starch (ST) diet for 8 weeks. RESULTS: The body weight was higher in the OVX than the sham rats, regardless of dietary carbohydrate subtype. The fasting serum glucose levels did not differ according to diet and ovariectomy. However, the fasting serum insulin levels were higher in the OVX than the sham rats, and in the OVX rats, a high SU diet increased the serum insulin levels more than a high ST diet. The whole body glucose disposal rates, which referred to the state of insulin sensitivity, were lower in the OVX rats fed both the high SU and ST diets, compared to sham rats. Glycogen deposits in the soleus and quadriceps muscles were lower in the OVX rats fed high SU and ST diets than in sham rats. The glucose transporter 4 content and fraction velocity of glycogen synthase in muscles showed similar glucose disposal rates. However, the triacylglycerol content in the muscles were higher in the OVX rats with a high SU diet than those with a high ST diet. CONCLUSION: These results suggested that an OVX increased the weight gain due to higher food intakes, regardless of dietary carbohydrate subtypes. OVX-induced obesity may be involved in the induction of insulin resistance from an increased triacylglycerol content, decreased glucose uptake and glycogen synthesis in skeletal muscles, regardless of dietary carbohydrate subtypes.

Increased phosphorylation of skeletal muscle glycogen synthase at NH2-terminal sites during physiological hyperinsulinemia in type 2 diabetes.

In type 2 diabetes, insulin activation of muscle glycogen synthase (GS) is impaired. This defect plays a major role for the development of insulin resistance and hyperglycemia. In animal muscle, insulin activates GS by reducing phosphorylation at both NH(2)- and COOH-terminal sites, but the mechanism involved in human muscle and the defect in type 2 diabetes remain unclear. We studied the effect of insulin at physiological concentrations on glucose metabolism, insulin signaling and phosphorylation of GS in skeletal muscle from type 2 diabetic and well-matched control subjects during euglycemic-hyperinsulinemic clamps. Analysis using phospho-specific antibodies revealed that insulin decreases phosphorylation of sites 3a + 3b in human muscle, and this was accompanied by activation of Akt and inhibition of glycogen synthase kinase-3alpha. In type 2 diabetic subjects these effects of insulin were fully intact. Despite that, insulin-mediated glucose disposal and storage were reduced and activation of GS was virtually absent in type 2 diabetic subjects. Insulin did not decrease phosphorylation of sites 2 + 2a in healthy human muscle, whereas in diabetic muscle insulin infusion in fact caused a marked increase in the phosphorylation of sites 2 + 2a. This phosphorylation abnormality likely caused the impaired GS activation and glucose storage, thereby contributing to skeletal muscle insulin resistance, and may therefore play a pathophysiological role in type 2 diabetes.

Diverse effects of two allosteric inhibitors on the phosphorylation state of glycogen phosphorylase in hepatocytes.

Two distinct allosteric inhibitors of glycogen phosphorylase, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and CP-91149 (an indole-2-carboxamide), were investigated for their effects on the phosphorylation state of the enzyme in hepatocytes in vitro. CP-91149 induced inactivation (dephosphorylation) of phosphorylase in the absence of hormones and partially counteracted the phosphorylation caused by glucagon. Inhibition of glycogenolysis by CP-91149 can be explained by dephosphorylation of phosphorylase a. This was associated with activation of glycogen synthase and stimulation of glycogen synthesis. DAB, in contrast, induced a small degree of phosphorylation of phosphorylase. This was associated with inactivation of glycogen synthase and inhibition of glycogen synthesis. Despite causing phosphorylation (activation) of phosphorylase, DAB is a very potent inhibitor of glycogenolysis in both the absence and presence of glucagon. This is explained by allosteric inhibition of phosphorylase a, which overrides the increase in activation state. In conclusion, two potent phosphorylase inhibitors exert different effects on glycogen metabolism in intact hepatocytes as a result of opposite effects on the phosphorylation state of both phosphorylase and glycogen synthase.

Effect of dietary fish oil on insulin sensitivity and metabolic fate of glucose in the skeletal muscle of normal rats.

The aim of this work was to study the effect of the administration of cod liver oil on the non-oxidative and oxidative fate of glucose metabolism in the skeletal muscle of normal rats. To achieve this goal, the gastrocnemius was examined regarding glucose oxidation, glycogen synthase activity and glycogen storage both at baseline and during euglycemic hyperinsulinemic clamping. The results show that dietary fish oil decreases plasma insulin levels without alteration in glucose homeostasis (at baseline). In addition, the observed enhancement in whole body glucose utilization during clamping suggests an increased peripheral insulin sensitivity. Furthermore, under insulin-stimulated glucose disposal, an enhancement in the glycolytic pathway (increased levels of muscle glucose-6-phosphate and plasma lactate) rather than changes in the oxidation (pyruvate dehydrogenase complex) and storage components of glucose metabolism was observed in the skeletal muscle of rats fed dietary fish oil. These results coupled with the hypolipidemic effects of fish oil may have implications for the prevention and/or management of some pathological states manifested by insulin resistance with or without dyslipidemia.

Signalling pathways and combinatory effects of insulin and amino acids in isolated rat hepatocytes.

Liver metabolism is influenced by hormones and nutrients. Amino acids such as glutamine or leucine induce an anabolic response, which resembles that of insulin in muscle and adipose tissue. In this work, the signalling pathways and the effects of insulin were compared to those of glutamine and leucine in isolated hepatocytes from normal and streptozotocin-diabetic rats. Glutamine increased cell volume and induced an anabolic response characterized by an activation of acetyl-CoA carboxylase (ACC), glycogen synthase (GS) and p70 ribosomal S6 kinase (p70S6K), the key enzymes in fatty acid, glycogen and protein synthesis, respectively. The effects of glutamine were independent of insulin and did not share its signalling components. Leucine, which is poorly metabolized by the liver and does not modify cell volume, activated ACC and p70S6K, and exerted a synergistic effect on the glutamine-induced activation of ACC and p70S6K. These amino acids did not affect insulin signalling. Insulin alone had no anabolic effect in hepatocytes, despite the activation of protein kinase B. Nevertheless, it enhanced the activation of ACC and p70S6K induced by leucine. However, insulin injected intravenously activated rat liver p70S6K. In hepatocytes from streptozotocin-diabetic animals, the metabolic responses to the amino acids and insulin were similar to those in normal hepatocytes. We conclude that glutamine, insulin and leucine exert different effects that are mediated by different signalling pathways, although their effects are combinatory. The anabolic effect of insulin in hepatocytes was strictly dependent on the permissive action of leucine.

Mutations of muscle glycogen synthase that disable activation by glucose 6-phosphate.

Glycogen synthase, an enzyme of historical importance in the field of reversible protein modification, is inactivated by phosphorylation and allosterically activated by glucose 6-phosphate (glucose-6-P). Previous analysis of yeast glycogen synthase had identified a conserved and highly basic 13-amino-acid segment in which mutation of Arg residues resulted in loss of activation by glucose-6-P. The equivalent mutations R578R579R581A (all three of the indicated Arg residues mutated to Ala) and R585R587R590A were introduced into rabbit muscle glycogen synthase. Whether expressed transiently in COS-1 cells or produced in and purified from Escherichia coli, both mutant enzymes were insensitive to activation by glucose-6-P. The effect of phosphorylation was studied in two ways. Purified, recombinant glycogen synthase was directly phosphorylated by casein kinase 2 and glycogen synthase kinase 3, under conditions that inactivate the wild-type enzyme. In addition, phosphorylation sites were converted to Ala by mutagenesis in wild-type and in the glucose-6-P desensitized mutants expressed in COS-1 cells. Phosphorylation inactivated the R578R579R581A mutant but had little effect on the R585R587R590A. This result was surprising since phosphorylation had the opposite effects on the corresponding yeast enzyme mutants. The results confirm that the region of glycogen synthase, Arg-578-Arg-590, is required for activation by glucose-6-P and suggest that it is part of a sensitive and critical switch involved in transitions between different conformational states. However, the role must differ subtly between the mammalian and the yeast enzymes.