Effect of Subcutaneous Tirzepatide vs Placebo Added to Titrated Insulin Glargine on Glycemic Control in Patients With Type 2 Diabetes: The SURPASS-5 Randomized Clinical Trial.

Importance: The effects of tirzepatide, a dual glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptor agonist, as an addition to insulin glargine for treatment of type 2 diabetes have not been described. Objective: To assess the efficacy and safety of tirzepatide added to insulin glargine in patients with type 2 diabetes with inadequate glycemic control. Design, Setting, and Participants: Randomized phase 3 clinical trial conducted at 45 medical research centers and hospitals in 8 countries (enrollment from August 30, 2019, to March 20, 2020; follow-up completed January 13, 2021) in 475 adults with type 2 diabetes and inadequate glycemic control while treated with once-daily insulin glargine with or without metformin. Interventions: Patients were randomized in a 1:1:1:1 ratio to receive once-weekly subcutaneous injections of 5-mg (n = 116), 10-mg (n = 119), or 15-mg (n = 120) tirzepatide or volume-matched placebo (n = 120) over 40 weeks. Tirzepatide was initiated at 2.5 mg/week and escalated by 2.5 mg every 4 weeks until the assigned dose was achieved. Main Outcomes and Measures: The primary end point was mean change from baseline in glycated hemoglobin A1c (HbA1c) at week 40. The 5 key secondary end points included mean change in body weight and percentage of patients achieving prespecified HbA1c levels. Results: Among 475 randomized participants (211 [44%] women; mean [SD] age, 60.6 [9.9] years; mean [SD] HbA1c, 8.31% [0.85%]), 451 (94.9%) completed the trial. Treatment was prematurely discontinued by 10% of participants in the 5-mg tirzepatide group, 12% in the 10-mg tirzepatide group, 18% in the 15-mg tirzepatide group, and 3% in the placebo group. At week 40, mean HbA1c change from baseline was -2.40% with 10-mg tirzepatide and -2.34% with 15-mg tirzepatide vs -0.86% with placebo (10 mg: difference vs placebo, -1.53% [97.5% CI, -1.80% to -1.27%]; 15 mg: difference vs placebo, -1.47% [97.5% CI, -1.75% to -1.20%]; P < .001 for both). Mean HbA1c change from baseline was -2.11% with 5-mg tirzepatide (difference vs placebo, -1.24% [95% CI, -1.48% to -1.01%]; P < .001]). Mean body weight change from baseline was -5.4 kg with 5-mg tirzepatide, -7.5 kg with 10-mg tirzepatide, -8.8 kg with 15-mg tirzepatide and 1.6 kg with placebo (5 mg: difference, -7.1 kg [95% CI, -8.7 to -5.4]; 10 mg: difference, -9.1 kg [95% CI, -10.7 to -7.5]; 15 mg: difference, -10.5 kg [95% CI, -12.1 to -8.8]; P < .001 for all). Higher percentages of patients treated with tirzepatide vs those treated with placebo had HbA1c less than 7% (85%-90% vs 34%; P < .001 for all). The most common treatment-emergent adverse events in the tirzepatide groups vs placebo group were diarrhea (12%-21% vs 10%) and nausea (13%-18% vs 3%). Conclusions and Relevance: Among patients with type 2 diabetes and inadequate glycemic control despite treatment with insulin glargine, the addition of subcutaneous tirzepatide, compared with placebo, to titrated insulin glargine resulted in statistically significant improvements in glycemic control after 40 weeks. Trial Registration: ClinicalTrials.gov Identifier: NCT04039503.

Association of insulin treatment with gastric residue during an esophagogastroduodenoscopy.

The purpose of this study was to investigate the association of glycemic control and diabetes treatment to gastric residue observed during an esophagogastroduodenoscopy. Among 6,592 individuals who had esophagogastroduodenoscopy at our clinic between 2003 and 2019, we retrospectively and longitudinally identified those who had gastric residue during an esophagogastroduodenoscopy. Other data collected were age, sex, diagnosis of diabetes, glycated hemoglobin and diabetes medication. Cox proportional hazards models were used to assess the association of these data with the occurrence of gastric residue. To the best of our knowledge, this is the first retrospective cohort study finding that undergoing insulin treatment is a risk factor for gastric residue independent of age, sex and diabetes or glycated hemoglobin.

Synergistic association of the copper/zinc ratio under inflammatory conditions with diabetic kidney disease in patients with type 2 diabetes: The Asahi Diabetes Complications Study.

AIMS/INTRODUCTION: We aimed to study the relationships among the copper (Cu)/zinc (Zn) ratio, inflammatory biomarkers, and the prevalence of diabetic kidney disease (DKD) in patients with type 2 diabetes. MATERIALS AND METHODS: A cross-sectional study was performed on 651 patients with type 2 diabetes. DKD was defined as a urinary albumin-to-creatinine ratio of ≥30 mg/g creatinine and/or an estimated glomerular filtration rate using cystatin C of < 60 mL/min/1.73 m2 . Areas under the curves (AUCs), cutoff values, and thresholds for detecting DKD were determined for the Cu/Zn ratio, soluble tumor necrosis factor-α receptor 1 (sTNFαR1), and high-sensitivity C-reactive protein (hsCRP). Patients were categorized by each cutoff value of sTNFαR1 and the Cu/Zn ratio. Odds ratios (ORs) and biological interactions for the prevalence of DKD were determined. RESULTS: DKD was identified in 220 patients. AUC/optimal cutoff values were 0.777/1300 pg/mL for sTNFαR1, 0.603/1.1648 for the Cu/Zn ratio, and 0.582/305 ng/mL for hsCRP. The ORs for DKD were higher, but not significantly, in the sTNFαR1 < 1300 and Cu/Zn ≥ 1.1648 group, significantly higher in the sTNFαR1 ≥ 1300 and Cu/Zn < 1.1648 group (P < 0.0001), and further synergistically elevated in the sTNFαR1 ≥ 1300 and Cu/Zn ≥ 1.1648 group (P < 0.0001) compared with the sTNFαR1 < 1300 and Cu/Zn < 1.1648 group after multivariable adjustment. Levels of sTNFαR1 were significantly higher in the sTNFαR1 ≥ 1300 and Cu/Zn ≥ 1.1648 group than in the sTNFαR1 ≥ 1300 and Cu/Zn < 1.1648 group (P = 0.0006). CONCLUSIONS: Under an inflammatory initiation signal of elevated serum sTNFαR1 levels, an increase in the Cu/Zn ratio may further exacerbate inflammation and is synergistically associated with a high prevalence of DKD in patients with type 2 diabetes.

Analysis of efficacy and safety of dulaglutide 0.75 mg stratified by sex in patients with type 2 diabetes in 2 randomized, controlled phase 3 studies in Japan.

We analyzed the efficacy and safety of once weekly dulaglutide 0.75 mg by sex in 2 randomized, controlled phase 3 studies in Japanese patients with type 2 diabetes (a 52-week monotherapy study [comparator liraglutide 0.9 mg] and a 26-week combination therapy study [comparator insulin glargine]). Females comprised 18% of patients in the monotherapy study and 29% of patients in the combination therapy study. Mean reductions from baseline in glycated hemoglobin (HbA1c) were similar between the sexes for dulaglutide- and liraglutide-treated patients (range -1.17% to -1.49%). Females had numerically greater weight loss or less weight gain than males across all treatment groups. The percentages of patients with reductions in both HbA1c and weight from baseline were also greater for females than for males in all treatment groups. In all treatment groups, the incidences of treatment-emergent adverse events tended to be greater among females than among males. No differences in the incidences of total or nocturnal hypoglycemia were observed between the sexes in any treatment group. Overall, in 2 studies in Japan, across all treatment groups it appeared that HbA1c lowering was unaffected by patient sex, while female patients had greater weight loss or less weight gain and greater incidence of adverse events, including nausea, compared to male patients. Incidences of patients discontinuing dulaglutide early due to adverse event were low (<10%) for both sexes, and no new safety concerns related to dulaglutide were identified for either sex. Therefore, the benefit/risk ratio for dulaglutide remains unchanged, positive for both sexes.

Subgroup analysis of phase 3 studies of dulaglutide in Japanese patients with type 2 diabetes.

The efficacy and tolerability of once weekly dulaglutide 0.75 mg in Japanese patients with type 2 diabetes (T2D) were evaluated by subgroups defined by key demographic characteristics. This post hoc analysis included data from patients who received dulaglutide 0.75 mg for up to 26 weeks in three phase 3 trials (one open-label, randomized; one double-blind and open-label, randomized; one open-label, nonrandomized). Patients were classified into subgroups on the basis of sex (male, female), age (<65, ≥65 years), body weight (<70, ≥70 kg), body mass index (BMI; <25, ≥25 kg/m(2)), duration of diabetes (<7, ≥7 years), HbA1c (≤8.5, >8.5%), use of concomitant sulfonylurea (yes, no), and use of concomitant biguanide (yes, no). Efficacy measures analyzed were changes from baseline in HbA1c and body weight and percentages of patients achieving HbA1c <7.0%. Safety measures analyzed were incidence of hypoglycemia and nausea and change from baseline in seated pulse rate. A total of 855 patients were analyzed. Once weekly dulaglutide 0.75 mg improved blood glucose control as measured by HbA1c regardless of patient characteristics; patients with higher baseline HbA1c values had greater improvements compared to patients with lower baseline values. Weight loss was greater in patients with lower baseline HbA1c and in patients taking concomitant biguanides. Concomitant use of sulfonylureas had the greatest effect on the incidence of hypoglycemia. Treatment of T2D with once weekly dulaglutide 0.75 mg for 26 weeks was associated with significant improvement in glycemic control irrespective of age, sex, duration of diabetes, body weight, BMI, or concomitant medication.

Role of elevated serum uric acid levels at the onset of overt nephropathy in the risk for renal function decline in patients with type 2 diabetes.

AIMS/INTRODUCTION: Despite the use of intensive therapies, declining renal function is often observed during the overt nephropathy stage of type 2 diabetes. We aimed at investigating the role of serum uric acid (SUA) levels at the onset of overt nephropathy in the risk of renal function decline in type 2 diabetes patients. MATERIALS AND METHODS: The present cohort study included 290 type 2 diabetes patients who were followed from the onset of overt nephropathy. The relationship between SUA and declining renal function was assessed using Cox regression models after adjusting for known risk factors. RESULTS: Over a median 4.8-year follow-up period, 85 patients (4.9/100 person-years) showed serum creatinine (Cr) doubling with a total cumulative incidence of 71.9% at 20 years of follow up. The highest SUA tertile resulted in significantly a higher incidence (7.7/100 person-years) and cumulative incidence at 20 years (85.7%) than the middle (3.9/100 person-years, 54.2%) and lowest (3.0/100 person-years, 55.5%) tertiles. The univariate Cox hazard model resulted in significant risks for Cr doubling related to female sex, short diabetes duration, smoking and elevated levels of low-density lipoprotein cholesterol (LDL-c), glycated hemoglobin and SUA tertiles. SUA tertiles remained statistically significant in the multivariate model (highest vs lowest hazard ratio 2.68, 95% confidence interval 1.48-5.00, P = 0.0009). CONCLUSIONS: Elevated SUA levels within the normal range (men >6.3 mg/dL, women >5.1) at the onset of overt nephropathy resulted in an increased risk for declining renal function in type 2 diabetes patients.

A few positively charged residues slow movement of a polypeptide chain across the endoplasmic reticulum membrane.

Many polypeptide chains are translocated across and integrated into the endoplasmic reticulum membrane through protein-conducting channels. During the process, amino acid sequences of translocating polypeptide chains are scanned by the channels and classified to be retained in the membrane or translocated into the lumen. We established an experimental system with which the kinetic effect of each amino acid residue on the polypeptide chain movement can be analyzed with a time resolution of tens of seconds. Positive charges greatly slow movement; only two lysine residues caused a remarkable slow down, and their effects were additive. The lysine residue was more effective than arginine. In contrast, clusters comprising three residues of each of the other 18 amino acids had little effect on chain movement. We also demonstrated that a four lysine cluster can exert the effect after being fully exposed from the ribosome. We concluded that as few as two to three residues of positively charged amino acids can slow the movement of the nascent polypeptide chain across the endoplasmic reticulum membrane. This effect provides a fundamental basis of the topogenic function of positively charged amino acids.

Serum butyrylcholinesterase and the risk of future type 2 diabetes: the Kansai Healthcare Study.

OBJECTIVE: Butyrylcholinesterase is synthesized in the liver. The serum butyrylcholinesterase level has been cross-sectionally reported to be higher in patients with diabetes, hyperlipidaemia, obesity and fatty liver than in those without them. It is not known whether serum butyrylcholinesterase is associated with the risk of future type 2 diabetes. DESIGN: A prospective cohort study. PARTICIPANTS: A total of 8470 Japanese men aged 40-55 years without type 2 diabetes at baseline. MEASUREMENTS: Type 2 diabetes was diagnosed if a fasting plasma glucose (FPG) level was ≥7·0 mmol/l, if a HbA1 c level was ≥6·5% or if participants were taking oral hypoglycaemic medication or insulin. RESULTS: During the 42,227 person-years of follow-up, 868 cases had developed type 2 diabetes. Serum butyrylcholinesterase was significantly positively correlated with body mass index (BMI), FPG, alanine aminotransferase (ALT), γ-glutamyltransferase (GGT) and triglycerides (TG), whereas negatively with high-density lipoprotein (HDL) cholesterol. In Cox proportional hazards models, after adjusting for age, BMI, FPG, alcohol consumption, smoking habit, walk to work, regular leisure-time physical activity and family history of diabetes, the highest quartile (398-806 IU/l) of serum butyrylcholinesterase increased the risk of type 2 diabetes compared with the lowest quartile (56-311 IU/l) [hazard ratio (HR) 1·41 (95% confidence interval (CI), 1·14-1·74)]. After further adjusting for ALT and GGT, this association remained [HR 1·40 (95% CI, 1·13-1·73)]. Furthermore, this association was significant independent of TG and HDL cholesterol. CONCLUSIONS: Elevated serum butyrylcholinesterase was independently associated with an increased risk of future type 2 diabetes.

Stop-and-move of a marginally hydrophobic segment translocating across the endoplasmic reticulum membrane.

Many membrane proteins are cotranslationally integrated into the endoplasmic reticulum membrane via the protein-conducting channel, the so-called translocon. The hydrophobic transmembrane segment of the translocating nascent polypeptide chain stops at the translocon and then moves laterally into the membrane. Partitioning of the hydrophobic segment into the membrane is the primary determinant for membrane insertion. Here, we examined the behavior of a marginally hydrophobic segment at the translocon and found that its stop-translocation was greatly affected by the C-terminally attached ribosomes. The marginally hydrophobic segment first stops at the membrane and then moves into the lumen as long as the nascent chain is attached to translating ribosomes. When it is released from the ribosome by the termination codon, the marginally hydrophobic segment does not move. Puromycin or RNase treatment also suppressed movement. The movement was reversibly inhibited by high-salt conditions and irreversibly inhibited by ethylenediaminetetraacetic acid. There is an unstable state prior to the stable membrane insertion of the transmembrane segment. This characteristic state is maintained by the synthesizing ribosome.

Crucial role of cytochrome P450 in hepatotoxicity induced by 2,3-dimethoxy-1,4-naphthoquinone in rats.

Quinone toxicity is induced by two principal mechanisms: arylation/alkylation and a redox cycle. We have previously shown that increases in intracellular levels of superoxide anion and cell death induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a redox cycling quinone, are enhanced by pretreatment of rat primary hepatocytes with cytochrome P450 inhibitors. This indicates a novel interaction of quinones with cytochrome P450, and is thus worthy of further investigation using an in vivo model. The aim of this study was to examine the effects of cytochrome P450 inhibitors on DMNQ-induced hepatotoxicity in rats. When DMNQ was administered intraperitoneally, the activities of serum alanine aminotransferase and aspartate aminotransferase were found to increase in a dose-dependent manner, indicating that hepatotoxicity was induced by treatment with DMNQ. Pretreatment with the cytochrome P450 inhibitors SKF-525A (SKF), cimetidine and ketoconazole potentiated the DMNQ-induced hepatotoxicity. The blood concentration of DMNQ was not affected by administration of SKF. Pretreatment with the antioxidant α-tocopherol almost completely attenuated the hepatotoxicity induced by DMNQ and by the combination of DMNQ with SKF. Levels of reduced glutathione in the liver were decreased and levels of oxidized glutathione were increased by treatment with DMNQ. These effects were potentiated by pretreatment with SKF. DMNQ-induced lipid peroxidation in the liver was also enhanced by pretreatment with SKF. Taken together, these results indicate that DMNQ-induced hepatotoxicity is augmented by inhibition of cytochrome P450 and that this augmentation is due to the enhancement of oxidative stress.

A novel protein kinase B (PKB)/AKT-binding protein enhances PKB kinase activity and regulates DNA synthesis.

Protein kinase B (PKB)/Akt reportedly plays a role in the survival and/or proliferation of cells. We identified a novel protein, which binds to PKB, using a yeast two-hybrid screening system. This association was demonstrated not only in vivo by overexpressing both proteins or by coimmunoprecipitation of the endogenous proteins, but also in vitro using glutathione S-transferase fusion proteins. Importantly, this protein specifically associates with the C terminus of PKB but not with other AGC kinases and enhances PKB phosphorylation and kinase activation without growth factor stimulation. Thus, we termed this Akt-specific binding protein APE (Akt-phosphorylation enhancer). Since APE-induced phosphorylation of PKB did not occur in cells treated with wortmannin or LY294002, APE itself is not a kinase but seems to enhance or prolong the phosphoinositide 3-kinase-dependent phosphorylation of PKB. In cells in which APE was suppressed by small interfering RNA, DNA synthesis was significantly reduced with suppression of PKB phosphorylation, suggesting a synergistic role of APE in PKB-induced proliferation. On the other hand, in cells overexpressing both PKB and APE, despite markedly increased basal phosphorylation of PKB, both DNA rereplication and subsequent Chk2 phosphorylation and apoptosis were seen, suggesting the involvement of APE in the regulation of cell cycling replication licensing. Taking these observations together, APE appears to be a novel regulator of PKB phosphorylation. Furthermore, the interaction between APE and PKB, possibly dependent on the expression levels of both proteins, may be a novel molecular mechanism leading to proliferation and/or apoptosis.

Extremely early onset of ranitidine action on human histamine H2 receptors expressed in HEK293 cells.

BACKGROUND/AIMS: Histamine H2 receptor antagonists are considered to exert their effects on gastric acid secretion more rapidly than proton pump antagonists. However, there are no reports concerning the direct interaction of a histamine H2 receptor antagonist with the human H2 receptor in terms of onset of action. This study aims to characterize how rapidly famotidine and ranitidine, the most widely used histamine H2 receptor antagonists, interact with the human histamine H2 receptor. METHODS: HEK293 cell lines, stably expressing human histamine H2 receptors, were obtained. The dose- and time-dependent effects of famotidine and ranitidine on [3H]-tiotidine binding and histamine-stimulated cAMP production were analyzed. RESULTS: Ranitidine inhibited both [3H]-tiotidine binding and histamine-stimulated cAMP production more promptly than did famotidine. Inhibition of histamine-stimulated cAMP production by Cmax doses of famotidine (20 mg p.o.) and ranitidine (150 mg p.o.) peaked by 15 and 2 min, respectively. [3H]-tiotidine binding was not saturated by 60 min at the famotidine Cmax, while the ranitidine Cmax had produced saturation by 15 min. CONCLUSION: Ranitidine inhibits the human histamine H2 receptor very rapidly.

Hepatic Akt activation induces marked hypoglycemia, hepatomegaly, and hypertriglyceridemia with sterol regulatory element binding protein involvement.

Akt is critical in insulin-induced metabolism of glucose and lipids. To investigate functions induced by hepatic Akt activation, a constitutively active Akt, NH(2)-terminally myristoylation signal-attached Akt (myr-Akt), was overexpressed in the liver by injecting its adenovirus into mice. Hepatic myr-Akt overexpression resulted in a markedly hypoglycemic, hypoinsulinemic, and hypertriglyceridemic phenotype with fatty liver and hepatomegaly. To elucidate the sterol regulatory element binding protein (SREBP)-1c contribution to these phenotypic features, myr-Akt adenovirus was injected into SREBP-1 knockout mice. myr-Akt overexpression induced hypoglycemia and hepatomegaly with triglyceride accumulation in SREBP-1 knockout mice to a degree similar to that in normal mice, whereas myr-Akt-induced hypertriglyceridemia in knockout mice was milder than that in normal mice. The myr-Akt-induced changes in glucokinase, phosphofructokinase, glucose-6-phosphatase, and PEPCK expressions were not affected by knocking out SREBP-1, whereas stearoyl-CoA desaturase 1 induction was completely inhibited in knockout mice. Constitutively active SREBP-1-overexpressing mice had fatty livers without hepatomegaly, hypoglycemia, or hypertriglyceridemia. Hepatic acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, and glucose-6-phosphate dehydrogenase expressions were significantly increased by overexpressing SREBP-1, whereas glucokinase, phospho-fructokinase, glucose-6-phosphatase, and PEPCK expressions were not or only slightly affected. Thus, SREBP-1 is not absolutely necessary for the hepatic Akt-mediated hypoglycemic effect. In contrast, myr-Akt-induced hypertriglyceridemia and hepatic triglyceride accumulation are mediated by both Akt-induced SREBP-1 expression and a mechanism involving fatty acid synthesis independent of SREBP-1.

Differing roles of Akt and serum- and glucocorticoid-regulated kinase in glucose metabolism, DNA synthesis, and oncogenic activity.

Serum- and glucocorticoid-regulated kinase (SGK) is a serine kinase that has a catalytic domain homologous to that of Akt, but lacks the pleckstrin homology domain present in Akt. Akt reportedly plays a key role in various cellular actions, including glucose transport, glycogen synthesis, DNA synthesis, anti-apoptotic activity, and cell proliferation. In this study, we attempted to reveal the different roles of SGK and Akt by overexpressing active mutants of Akt and SGK. We found that adenovirus-mediated overexpression of myristoylated (myr-) forms of Akt resulted in high glucose transport activity in 3T3-L1 adipocytes, phosphorylated glycogen synthase kinase-3 (GSK3) and enhanced glycogen synthase activity in hepatocytes, and the promotion of DNA synthesis in interleukin-3-dependent 32D cells. In addition, stable transfection of myr-Akt in NIH3T3 cells induced an oncogenic transformation in soft agar assays. The active mutant of SGK (D-SGK, substitution of Ser422 with Asp) and myr-SGK were shown to phosphorylate GSK3 and to enhance glycogen synthase activity in hepatocytes in a manner very similar to that observed for myr-Akt. However, despite the comparable degree of GSK3 phosphorylation between myr-Akt and d-SGK or myr-SGK, d-SGK and myr-SGK failed to enhance glucose transport activity in 3T3-L1 cells, DNA synthesis in 32D cells, and oncogenic transformation in NIH3T3 cells. Therefore, the different roles of SGK and Akt cannot be attributed to ability or inability to translocate to the membrane thorough the pleckstrin homology domain, but rather must be attributable to differences in the relatively narrow substrate specificities of these kinases. In addition, our observations strongly suggest that phosphorylation of GSK3 is either not involved in or not sufficient for GLUT4 translocation, DNA synthesis, or oncogenic transformation. Thus, the identification of substrates selectively phosphorylated by Akt, but by not SGK, may provide clues to clarifying the pathway leading from Akt activation to these cellular activities.

Three mitogen-activated protein kinases inhibit insulin signaling by different mechanisms in 3T3-L1 adipocytes.

TNFalpha, which activates three different MAPKs [ERK, p38, and jun amino terminal kinase (JNK)], also induces insulin resistance. To better understand the respective roles of these three MAPK pathways in insulin signaling and their contribution to insulin resistance, constitutively active MAPK/ERK kinase (MEK)1, MAPK kinase (MKK6), and MKK7 mutants were overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated transfection procedure. The MEK1 mutant, which activates ERK, markedly down-regulated expression of the insulin receptor (IR) and its major substrates, IRS-1 and IRS-2, mRNA and protein, and in turn reduced tyrosine phosphorylation of IR as well as IRS-1 and IRS-2 and their associated phosphatidyl inositol 3-kinase (PI3K) activity. The MKK6 mutant, which activates p38, moderately inhibited IRS-1 and IRS-2 expressions and IRS-1-associated PI3K activity without exerting a significant effect on the IR. Finally, the MKK7 mutant, which activates JNK, reduced tyrosine phosphorylation of IRS-1 and IRS-2 and IRS-associated PI3K activity without affecting expression of the IR, IRS-1, or IRS-2. In the context of our earlier report showing down-regulation of glucose transporter 4 by MEK1-ERK and MKK6/3-p38, the present findings suggest that chronic activation of ERK, p38, or JNK can induce insulin resistance by affecting glucose transporter expression and insulin signaling, though via distinctly different mechanisms. The contribution of ERK is, however, the strongest.

Angiotensin II-induced insulin resistance is associated with enhanced insulin signaling.

Angiotensin II (AII) is involved in the pathogenesis of both hypertension and insulin resistance, though few studies have examined the relationship between the two. We therefore investigated the effects of chronic AII infusion on blood pressure and insulin sensitivity in rats fed a normal (0.3% NaCl) or high-salt (8% NaCl) diet. AII infusion for 12 days significantly elevated blood pressure and significant insulin resistance, assessed by a hyperinsulinemic-euglycemic clamp study and glucose uptake into isolated muscle and adipocytes. High-salt loading exacerbated the effects of AII infusion significantly. Despite the insulin resistance, insulin-induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrates, activation of phosphatidylinositol (PI) 3-kinase, and phosphorylation of Akt were all enhanced by AII infusion. Subsequently, to investigate whether oxidative stress induced by AII contributes to insulin resistance, the membrane-permeable superoxide dismutase mimetic, tempol, was administered to AII-infused rats. Chronic AII infusion induced an accumulated plasma cholesterylester hydroperoxide levels, indicating the increased oxidative stress, whereas the treatment with tempol normalized plasma cholesterylester hydroperoxide levels in AII-infused rats. In addition, the treatment with tempol normalized insulin resistance in AII-infused rats, shown as a decreased glucose infusion rate in the hyperinsulinemic euglycemic clamp study and a decreased insulin-induced glucose uptake into isolated skeletal muscle, as well as enhanced insulin-induced PI 3-kinase activation to those in the control rats. These results strongly suggest that AII-induced insulin resistance cannot be attributed to impairment of early insulin-signaling steps and that increased oxidative stress, possibly through impaired insulin signaling located downstream from PI 3-kinase activation, is involved in AII-induced insulin resistance.

Resistin is regulated by C/EBPs, PPARs, and signal-transducing molecules.

Expression of the adipocyte-derived protein resistin, which is thought to play a key role in the development of insulin resistance in vivo, is regulated by a variety of hormones and mediators, including insulin and TNFalpha. Here we describe our use of adenovirus-mediated gene transfer to determine which transcription factors and signaling pathways affect resistin expression in 3T3-L1 adipocytes. We found that resistin expression was enhanced by overexpression of C/EBPalpha and suppressed by C/EBPzeta, a negative regulator of C/EBPalpha. Additionally, C/EBPalpha induced resistin even in L6 myocytes. Overexpression of PPARgamma markedly reduced resistin expression, whereas PPARalpha had no significant effect. Resistin expression was markedly suppressed by overexpression of the PI3-kinase p110alpha catalytic subunit and by Akt. Finally, overexpression of MEK1, MKK6, or MKK7 suppressed resistin expression. These findings indicate that resistin expression is regulated by C/EBPalpha and PPARgamma, partly via modulation of signal transduction in the PI3-kinase and MAP kinase pathways.

Humoral regulation of resistin expression in 3T3-L1 and mouse adipose cells.

Resistin is a hormone secreted by adipocytes that acts on skeletal muscle myocytes, hepatocytes, and adipocytes themselves, reducing their sensitivity to insulin. In the present study, we investigated how the expression of resistin is affected by glucose and by mediators known to affect insulin sensitivity, including insulin, dexamethasone, tumor necrosis factor-alpha (TNF-alpha), epinephrine, and somatropin. We found that resistin expression in 3T3-L1 adipocytes was significantly upregulated by high glucose concentrations and was suppressed by insulin. Dexamethasone increased expression of both resistin mRNA and protein 2.5- to 3.5-fold in 3T3-L1 adipocytes and by approximately 70% in white adipose tissue from mice. In contrast, treatment with troglitazone, a thiazolidinedione antihyperglycemic agent, or TNF-alpha suppressed resistin expression by approximately 80%. Epinephrine and somatropin were both moderately inhibitory, reducing expression of both the transcript and the protein by 30-50% in 3T3-L1 adipocytes. Taken together, these data make it clear that resistin expression is regulated by a variety of hormones and that cytokines are related to glucose metabolism. Furthermore, they suggest that these factors affect insulin sensitivity and fat tissue mass in part by altering the expression and eventual secretion of resistin from adipose cells.

Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes.

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) reportedly activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake by skeletal muscle cells. In this study, we investigated the role of AMPK in AICAR-induced glucose uptake by 3T3-L1 adipocytes and rat soleus muscle cells by overexpressing wild-type and dominant negative forms of the AMPKalpha2 subunit by use of adenovirus-mediated gene transfer. Overexpression of the dominant negative mutant had no effect on AICAR-induced glucose transport in adipocytes, although AMPK activation was almost completely abolished. This suggests that AICAR-induced glucose uptake by 3T3-L1 adipocytes is independent of AMPK activation. By contrast, overexpression of the dominant negative AMPKalpha2 mutant in muscle markedly suppressed both AICAR-induced glucose uptake and AMPK activation, although insulin-induced uptake was unaffected. Overexpression of the wild-type AMPKalpha2 subunit significantly increased AMPK activity in muscle but did not enhance glucose uptake. Thus, although AMPK activation may not, by itself, be sufficient to increase glucose transport, it appears essential for AICAR-induced glucose uptake in muscle.