Effect of whey protein-derived decapeptide on mood status and blood variables in healthy adults: a randomized, double-blind, placebo-controlled cross-over trial.

PURPOSE: The importance of maintaining good mental health with overall well-being has recently drawn attention from various fields. Functional peptides found from various protein sources reportedly reduce mental health problems. We found a new decapeptide (AJI-801) from whey proteins, which can possibly improve mood status and increase blood acetyl-L-carnitine (ALC) and fibroblast growth factor 21 (FGF21) levels. In this study, we assessed the effects of a single intake of whey protein hydrolysate containing a high amount of AJI-801 (WPH) on blood variables and mood status. METHODS: A randomized, double-blind, placebo-controlled cross-over trial of two doses of WPH (100 and 500 mg) was conducted. Participants, aged between 20 and 59 years with fatigue were allocated to two groups based on the WPH doses received, and set first test food in each study. The blood ALC and FGF21 levels at baseline and after 60, 120, and 180 min of test food intake were analyzed and the responses to the questionnaire items for mood status were obtained at baseline and after 60 and 180 min of test food intake. RESULTS: There were no significant differences in the blood ALC and FGF21 levels between the two groups. As mood status, intake of 500-mg WPH (including 2.5-mg AJI-801) showed significant improvement in Depression/Dejection of the Profile of Mood States Questionnaire second edition and visual analog scale score for depression, as compared to the placebo. CONCLUSIONS: Intake of AJI-801 500-mg WPH (including 2.5-mg AJI-801) contributes to the improvement of feeling down in healthy persons with fatigue. TRIAL REGISTRATION: University Hospital Medical Information Network Clinical Trial Registry (UMIN 000046829).

Chemical Site-Specific Conjugation Platform to Improve the Pharmacokinetics and Therapeutic Index of Antibody-Drug Conjugates.

To overcome a lack of selectivity during the chemical modification of native non-engineered antibodies, we have developed a technology platform termed "AJICAP" for the site-specific chemical conjugation of antibodies through the use of a class of IgG Fc-affinity reagents. To date, a limited number of antibody-drug conjugates (ADCs) have been synthesized via this approach, and no toxicological study was reported. Herein, we describe the compatibility and robustness of AJICAP technology, which enabled the synthesis of a wide variety of ADCs. A stability assessment of a thiol-modified antibody synthesized by AJICAP technology indicated no appreciable increase in aggregation or decomposition upon prolonged storage, indicating that the unexpectedly stable thiol intermediate has a great potential intermediate for payload or linker screening or large-scale manufacturing. Payload conjugation with this stable thiol intermediate generated several AJICAP-ADCs. In vivo xenograft studies indicated that the AJICAP-ADCs displayed significant tumor inhibition comparable to benchmark ADC Kadcyla. Furthermore, a rat pharmacokinetic analysis and toxicology study indicated an increase in the maximum tolerated dose, demonstrating an expansion of the AJICAP-ADC therapeutic index, compared with stochastic conjugation technology. This is the first report of the therapeutic index estimation of site-specific ADCs produced by utilizing Fc affinity reagent conjugation. The described site-specific conjugation technology is a powerful platform to enable next-generation ADCs through reduced heterogeneity and enhanced therapeutic index.

Identification of a vesicular ATP release inhibitor for the treatment of neuropathic and inflammatory pain.

Despite the high incidence of neuropathic and inflammatory pain worldwide, effective drugs with few side effects are currently unavailable for the treatment of chronic pain. Recently, researchers have proposed that inhibitors of purinergic chemical transmission, which plays a key role in the pathological pain response, may allow for targeted treatment of pathological neuropathic and inflammatory pain. However, such therapeutic analgesic agents have yet to be developed. In the present study, we demonstrated that clodronate, a first-generation bisphosphonate with comparatively fewer side effects than traditional treatments, significantly attenuates neuropathic and inflammatory pain unrelated to bone abnormalities via inhibition of vesicular nucleotide transporter (VNUT), a key molecule for the initiation of purinergic chemical transmission. In vitro analyses indicated that clodronate inhibits VNUT at a half-maximal inhibitory concentration of 15.6 nM without affecting other vesicular neurotransmitter transporters, acting as an allosteric modulator through competition with Cl- A low concentration of clodronate impaired vesicular ATP release from neurons, microglia, and immune cells. In vivo analyses revealed that clodronate is more effective than other therapeutic agents in attenuating neuropathic and inflammatory pain, as well as the accompanying inflammation, in wild-type but not VNUT -/- mice, without affecting basal nociception. These findings indicate that clodronate may represent a unique treatment strategy for chronic neuropathic and inflammatory pain via inhibition of vesicular ATP release.

Facile and Efficient Chemoenzymatic Semisynthesis of Fc-Fusion Compounds for Half-Life Extension of Pharmaceutical Components.

The formation of Fc-fusions, in which biologically active molecules and the Fc fragment of antibodies are linked to each other, is one of the most efficient and successful half-life extension technologies to be developed and applied to peptide and protein pharmaceuticals thus far. Fc-fusion compounds are generally produced by recombinant methods. However, these cannot be applied to artificial middle molecules, such as peptides with non-natural amino acids, unnatural cyclic peptides, or pharmaceutical oligonucleotides. Here, we developed a simple, efficient, semisynthetic method for Fc-fusion production involving our previously developed enzymatic N-terminal extension reaction (i.e., NEXT-A reaction) and strain-promoted azide-alkyne cycloaddition, achieving quantitative conversion and high selectivity for the N-terminus of the Fc protein. An Fc-fusion compound prepared by this method showed comparable biological activity to that of the original peptide and a long-circulating plasma half-life. Thus, the proposed method is potentially applicable for the conjugation of a wide range of pharmaceutical components.

Simultaneous quantification of intracellular and secreted active and inactive glucagon-like peptide-1 from cultured cells.

Glucagon-like peptide-1 (GLP-1) is an incretin peptide that regulates islet hormone secretion. During recent years, incretin-based therapies have been widely used for patients with type 2 diabetes. GLP-1 peptides undergo N- and C-terminal processing for gain or loss of functions. We developed a method to quantify picomolar quantities of intact GLP-1 peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By employing this label-free selected reaction monitoring (SRM) method, we were able to analyze secreted GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36 amid from human enteroendocrine NCI-H716 cells after stimulation with nateglinide, glucose, and sucralose. The absolute total concentrations of secreted GLP-1 peptides at baseline and after stimulation with nateglinide, glucose, and sucralose were 167.3, 498.9, 238.3, and 143.1 pM, respectively. Meanwhile, the ratios of GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36 amide) to total GLP-1 peptides were similar (6 ± 3, 26 ± 3, and 78 ± 5%, respectively). The SRM assay can analyze the concentrations of individual GLP-1 peptides and, therefore, is a tool to investigate the physiological roles of GLP-1 peptides. Furthermore, the molecular species secreted from NCI-H716 cells were unknown. Therefore, we performed a secretopeptidome analysis of supernatants collected from cultured NCI-H716 cells. Together with GLP-1 peptides, we detected neuroendocrine convertase 1, which regulates peptide hormones released from intestinal endocrine L-cells.

Activation of the gut calcium-sensing receptor by peptide agonists reduces rapid elevation of plasma glucose in response to oral glucose load in rats.

The calcium-sensing receptor (CaSR) is expressed in various tissues, including the gastrointestinal tract. To investigate the role of gut CaSR on glycemic control, we examined whether single oral administration of CaSR agonist peptides affected the glycemic response in rats. Glucose tolerance tests were performed under oral or duodenal administration of various CaSR agonist peptides (γGlu-Cys, protamine, and poly-d-lysine hydrobromide) in conscious rats. Involvement of CaSR was determined by using a CaSR antagonist. Signaling pathways underlying CaSR agonist-modified glycemia were investigated using gut hormone receptor antagonists. The gastric emptying rate after the administration of CaSR agonist peptides was measured by the phenol red recovery method. Oral and duodenal administration of CaSR agonist peptides attenuated glycemic responses under the oral glucose tolerance test, but the administration of casein did not. The promotive effect on glucose tolerance was weakened by luminal pretreatment with a CaSR antagonist. Treatment with a 5-HT3 receptor antagonist partially diminished the glucose-lowering effect of peptides. Furthermore, the gastric emptying rate was decreased by duodenal administration of CaSR agonist peptides. These results demonstrate that activation of the gut CaSR by peptide agonists promotes glucose tolerance in conscious rats. 5-HT3 receptor and the delayed gastric emptying rate appear to be involved in the glucose-lowering effect of CaSR agonist peptides. Thus, activation of gut CaSR by dietary peptides reduces glycemic responses so that gut CaSR may be a potential target for the improvement of postprandial glycemia.

Effect of imbalance in dietary macronutrients on blood hemoglobin levels: a cross-sectional study in young underweight Japanese women.

Background: Iron deficiency and underweight are common nutritional problems among young Japanese women, many of whom show unhealthy dietary patterns owing to a desire for thinness. We conducted a cross-sectional analysis of the relationship between iron status, nutritional status, and dietary intake among young Japanese women with underweight to identify dietary risk factors for iron deficiency. Methods: Of the 159 young women (18-29 years of age) enrolled, 77 underweight and 37 normal-weight women were included in the study. They were further categorized into four groups based on quartiles of hemoglobin levels among all participants. Dietary nutrient intake was ascertained using a brief self-administered diet history questionnaire. Blood level of hemoglobin and nutritional biomarkers such as total protein, albumin, insulin-like growth factor-1 (IGF-1), and essential amino acids were measured. Results: In underweight, the multiple comparison test showed that dietary intakes of fat, saturated fatty acid, and monosaturated fatty acid were significantly higher and carbohydrate intake was significantly lower in the group with the lowest hemoglobin level, whereas intakes of iron were the same across groups. Multivariate regression coefficients suggested that replacing fat with protein or carbohydrates increased hemoglobin levels under isocaloric conditions. Additionally, significant positive correlations were observed between hemoglobin levels and nutritional biomarkers. Conclusion: Dietary iron intake did not change across different hemoglobin groups among Japanese underweight women. However, our results suggested that an imbalanced dietary macronutrient induces anabolic status and hemoglobin synthesis deterioration among them. Especially, a higher fat intake may be a risk factor for lower hemoglobin.

Biological Evaluation of Maytansinoid-Based Site-Specific Antibody-Drug Conjugate Produced by Fully Chemical Conjugation Approach: AJICAP®.

BACKGROUND: Trastuzumab-emtansine (T-DM1, commercial name: Kadcyla) is well-known antibody-drug conjugate (ADC) and was first approved for human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. This molecular format consisting of trastuzumab and maytansinoid payload (emtansine) is very simple, however, T-DM1 has wide heterogeneity due to non-specific conjugation, lowering its therapeutic index (TI). METHODS: To overcome this issue during the chemical modification of the random conjugation approach to generate T-DM1, we developed a novel chemical conjugation technology termed "AJICAP®" for modification of antibodies in site-specific manner by IgG Fc-affinity peptide based reagents. RESULTS: In this study, we compared site-specific maytansinoid-based ADCs synthesized by AJICAP and T-DM1 in rat safety studies. The results indicated an increase in the maximum tolerated dose, demonstrating an expansion of the AJICAP-ADC therapeutic index compared with that of commercially available T-DM1. Gram scale preparation of this AJICAP-ADC and the initial stability study are also described. CONCLUSIONS: Trastuzumab-AJICAP-maytansinoid produced by this unique chemical conjugation methodology showed higher stability and tolerability than commercially available T-DM1.

Nateglinide stimulates glucagon-like peptide-1 release by human intestinal L cells via a K(ATP) channel-independent mechanism.

A reduced incretin effect is one of the well-known characteristics of patients with type 2 diabetes, and impaired release of glucagon-like peptide-1 (GLP-1) has been reported to be at least partly involved. In this study, we investigated the effect of nateglinide on GLP-1 release in vivo and in vitro. The GLP-1 level in the portal blood at 20 min after oral administration of nateglinide to Wistar rats was about twice that in vehicle-treated rats. To clarify whether this effect of nateglinide was related to direct stimulation of intestinal cells, in vitro studies were performed using human intestinal L cells (NCI-H716). Nateglinide stimulated GLP-1 release in a concentration-dependent manner from 500 µM, along with transient elevation of the intracellular calcium level. However, diazoxide, nitrendipine, and dantrolene did not block this effect of nateglinide. In addition, the major metabolite of nateglinide, tolbutamide, and mitiglinide, all of which augment insulin secretion by the pancreatic islets, had no effect on GLP-1 release by this cell line. On the other hand, capsazepine significantly inhibited the promotion of GLP-1 release by nateglinide in a concentration-dependent manner. These findings indicate that nateglinide directly stimulates GLP-1 release by intestinal L cells in a K(ATP) channel-independent manner. A novel target of nateglinide may be involved in increasing intracellular calcium to stimulate GLP-1 release, e.g., the transient receptor potential channels. Taken together, the present findings indicate that promotion of GLP-1 release from intestinal L cells may be another important mechanism by which nateglinide restores early-phase insulin secretion and regulates postprandial glucose metabolism.

One-step construction of ferritin encapsulation drugs for cancer chemotherapy.

Conventionally, a disassembly and reassembly method has been used for encapsulation of drug molecules in ferritin protein nano-cages. However, clinical applications of ferritin have been greatly restricted by its limited drug-loading capacity and process complexity. Here, we establish a simple high yield process for preparing high drug-loaded ferritin nanomedicine for industrial production. A complex of ferritin and a target drug was obtained by incubating the mixture at an appropriate pH. An electrostatic charge potential and small ferritin cavity facilitates the passage of drug molecules through the pores, traversing the ferritin shell and enabling deposition of the drug in the ferritin cavity. Compared to the disassembly/reassembly method, the loading capacity of a doxorubicin-loaded ferritin heavy chain (DOX-FTH), constructed by our novel method, was over 3-fold higher, while doxorubicin recovery was 10-fold higher. Results of transmission electron microscopy, size exclusion chromatography, dynamic light scattering, and zeta potential indicate that DOX-FTH exhibits the same physicochemical characteristics of natural apo-ferritin. Moreover, DOX-FTH can be taken up and induce apoptosis of cancer cells overexpressing TfR1. Here, we have demonstrated the successful introduction of more than ten drug molecule types into ferritin nano-cages using a novel method. These results demonstrate that this one-step method is a powerful production process to construct a drug-loading ferritin drug delivery system carrier.

Nateglinide prevents fatty liver through up-regulation of lipid oxidation pathway in Goto-Kakizaki rats on a high-fat diet.

Dyslipidemia and fatty liver are important components of the metabolic syndrome and are the factors most commonly associated with the development of nonalcoholic fatty liver disease. Delayed and excessive insulin secretion in response to food intake is a key element in the onset of these risk factors. Nateglinide (NAT) is known to restore early-phase insulin secretion. We assessed the effect of NAT on postprandial hypertriglyceridemia and fatty liver in type 2 diabetic Goto-Kakizaki (GK) rats. The GK rats fed a high-fat diet containing 30% beef tallow twice a day were administered either the vehicle alone or NAT (50 mg/kg) before each meal for 12 weeks. Delayed insulin secretion and an increase of total insulin release were caused by feeding 30% beef tallow to the rats. This diet also induced postprandial hypertriglyceridemia and increased the hepatic triglyceride content. Treatment with NAT restored early-phase insulin secretion without any increase of total insulin release and also reduced postprandial hypertriglyceridemia and the hepatic triglyceride content. There was up-regulation of the hepatic expression of peroxisome proliferators-activated receptor alpha and its downstream enzymes after 12 weeks of NAT treatment, as well as normalization of the plasma total ketone body level. Furthermore, NAT also up-regulated hepatic expression of the adiponectin receptor AdipoR2, although there was no effect on the plasma adiponectin level. These findings indicate that long-term treatment with NAT prevented the development of fatty liver through the up-regulation of hepatic lipid oxidation pathways. Restoration of early-phase insulin secretion and suppression of recurrent postprandial hypertriglyceridemia might be involved in these effects of NAT. The present results may support the use of NAT to prevent the onset and progression of the metabolic syndrome and chronic liver disease.

Repetitive postprandial hypertriglyceridemia induces monocyte adhesion to aortic endothelial cells in Goto-Kakizaki rats.

To compare the effects of postprandial hypertriglyceridemia and postprandial hyperglycemia on monocyte adhesion to endothelial cells, we investigated the effects of twice-daily standard diet (5% fat) and high-fat diet (30% fat) for 3 weeks on monocyte adhesion to endothelial cells and the expression of adhesion molecules in the aortic artery in non-obese type 2 diabetic Goto-Kakizaki rats. Fasting glucose, insulin, non-esterified fatty acid (NEFA), HbA1c, and body weight were comparable between the two diet groups. Postprandial glucose and insulin were higher in the standard diet group, while postprandial NEFA and triglyceride were higher in the high fat diet group, compared with the other group. The number of monocyte adherent to endothelial cells was higher in the high-fat diet group than the standard diet group. Consumption of high-fat diet resulted in overexpression of heme oxygenase-1, intercellular adhesion molecule-1 (ICAM-1), and connecting segment-1 fibronectin on the arterial wall, compared with standard diet. Thus, our data demonstrated that short-term intermittent high-fat diet prevented postprandial hyperglycemia in a model of type 2 diabetes without a significant increase in body weight. However, the resulting postprandial hypertriglyceridemia induces more monocyte adhesion to endothelial cells than postprandial hyperglycemia. This increased monocyte adhesion is associated with the increased aortic expression of adhesion molecules such as ICAM-1, and connecting segment-1 fibronectin.

Repetitive fluctuations in blood glucose enhance monocyte adhesion to the endothelium of rat thoracic aorta.

BACKGROUND: The aim of this study was to elucidate the effect of repetitive fluctuations in blood glucose concentrations on monocyte adhesion to the aortic endothelium. METHODS AND RESULTS: Nonobese type 2 diabetes, Goto-Kakizaki (GK) rats were fed twice daily to induce repetitive postprandial glucose spikes. Then, we compared the number of monocytes adherent to the endothelium of thoracic aorta in these rats with that in rats fed ad libitum. To suppress the glucose spikes, rats were injected with an inhibitor of sodium-glucose transporter, phloridzin, just before each meal for 12 weeks. GK rats fed twice daily showed significantly lower HbA1c than GK rats fed ad libitum. However, the former group showed markedly higher number of monocytes adherent to the endothelium than the latter, together with increased arterial intimal thickening. Phloridzin significantly reduced the number of adherent monocytes in GK rats fed twice daily. CONCLUSIONS: Our data demonstrated that repetitive postprandial fluctuation in glucose concentration evokes monocyte adhesion to endothelial cells that was worse than that induced by stable hyperglycemia in vivo. Suppression of such fluctuations efficiently suppressed monocyte adhesion to the aortic endothelium.

Biliopancreatic limb plays an important role in metabolic improvement after duodenal-jejunal bypass in a rat model of diabetes.

BACKGROUND AND AIMS: Roux-en-Y gastric bypass improves glucose metabolism in clinical practice, and duodenal-jejunal bypass (DJB), an experimental bypass procedure, also improves metabolism in animals. However, the mechanism remains controversial; especially, the role of the biliopancreatic limb (BP-limb) remains unclear. Our aim was to examine the importance of the function of the BP-limb after DJB using a novel operative model. METHODS: Otsuka Long-Evans Tokushima Fatty rats with diabetes were divided into the following groups: DJB with a short alimentary limb (A-limb) and long BP-limb (B-DJB group), DJB with jejunectomy (J-DJB group) in which the entire length of the jejunum used for the BP-limb of the B-DJB group was excised; and a sham operation group. Glucose tolerance, plasma bile acid levels, and the gut microbiota were assessed postoperatively. RESULTS: Glucose tolerance was improved and weight gain was suppressed after surgery in the B-DJB group. In contrast, these effects were cancelled in the J-DJB group. The plasma levels of bile acids in the B-DJB group were greater than those in other groups. The analysis of gut microbiota showed distinct differences between the B-DJB and other groups; especially, the relative abundance of genus Bifidobacterium was much higher in the B-DJB group. CONCLUSION: The BP-limb played an important role in the control of weight gain, glucose tolerance, and increased plasma bile acid levels after DJB in this rat model of type 2 diabetes mellitus. Plasma bile acids and gut microbiota may be involved in these processes.

Decreased blood glucose excursion by nateglinide ameliorated neuropathic changes in Goto-Kakizaki rats, an animal model of non-obese type 2 diabetes.

In the present study, we examined the effect of long-term suppression of postprandial hyperglycemia and glycemic fluctuation in Goto-Kakizaki (GK) rats, a type 2 diabetic animal model, by nateglinide (NG), a fast-acting hypoglycemic agent, on some measures of neuropathy and compared the outcome with the slow-acting effect of glibenclamide (GC). GK rats fed twice daily were given NG (50 mg/kg) or GC (1 mg/kg) orally before each meal for 24 weeks. The dose of NG and GC was determined by the data of their comparable suppressive effects on hyperglycemia as a total sum of glucose values after glucose load. At the end, there was no significant influence of treatment with NG or GC on body weight, fasting blood glucose, and glycated hemoglobin in GK rats. However, NG treatment suppressed postprandial hyperglycemia by 50% throughout the observation period, whereas this effect was not apparent in GC-treated rats. Delayed motor nerve conduction velocity was normalized by NG treatment, while GC had a partial (50%) effect. GK rats showed elevated contents of sorbitol and 3-deoxyglucosone in the sciatic nerve, and these changes were inhibited by NG treatment. Reduced Na(+)/K(+)-adenosine triphosphatase (ATPase) activity in GK rats was not affected by either NG or GC treatment. These results suggest that meticulous control of postprandial hyperglycemia is essential to inhibit the development of neuropathy in type 2 diabetes.

Nateglinide suppresses postprandial hypertriglyceridemia in Zucker fatty rats and Goto-Kakizaki rats: comparison with voglibose and glibenclamide.

Postprandial hypertriglyceridemia, as well as postprandial hyperglycemia, are important factors contributing to the development of cardiovascular disease in patients with type 2 diabetes. Nateglinide is a recently approved antidiabetic that suppresses postprandial hyperglycemia by stimulating the early phase of insulin secretion. In the present study, we investigated the effects of nateglinide on postprandial hypertriglyceridemia in obese Zucker fatty (ZF) rats and non-obese diabetic Goto-Kakizaki (GK) rats. Administration of an oral fat load caused marked hypertriglyceridemia with a peak at 2 h in ZF and GK rats. Nateglinide (50 mg/kg) significantly suppressed the increase of plasma triglycerides after fat loading in both types of rat (delta AUC [0-4 h]: 15+/-69 mg.h/dl for nateglinide vs. 838+/-100 mg.h/dl for vehicle in ZF rats; p<0.01, 81+/-22 mg x h/dl for nateglinide vs. 164+/-17 mg.h/dl for vehicle in GK rats; p<0.01). In contrast, other antidiabetic agents (voglibose and glibenclamide) did not show a significant effect on the increase of triglycerides after fat loading. The triglyceride components suppressed by nateglinide were mainly at the origin and in the pre beta subfraction on agarose gel electrophoresis, suggesting that chylomicrons and very low density lipoproteins were decreased. Plasma insulin levels were significantly increased at 30 min in nateglinide-treated rats, but not in voglibose- or glibenclamide-treated rats. These results suggest that nateglinide not only suppresses postprandial hyperglycemia, but also suppresses postprandial hypertriglyceridemia, by promoting rapid and pulsatile insulin secretion in patients with type 2 diabetes.

Involvement of protein kinase C activation in L-leucine-induced stimulation of protein synthesis in l6 myotubes.

Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [(3)H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, alpha-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A(2) and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C.