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Medication is a major determinant of human gut microbiome structure, and its overuse increases the risks of morbidity and mortality. However, effects of certain commonly prescribed drugs and multiple medications on the gut microbiome are still underinvestigated.We performed shotgun metagenomic analysis of fecal samples from 4198 individuals in the Japanese 4D (Disease, Drug, Diet, Daily life) microbiome project. A total of 759 drugs were profiled, and other metadata, such as anthropometrics, lifestyles, diets, physical activities, and diseases, were prospectively collected. Second fecal samples were collected from 243 individuals to assess the effects of drug initiation and discontinuation on the microbiome.We found that numerous drugs across different treatment categories influence the microbiome; more than 70% of the drugs we profiled had not been examined before. Individuals exposed to multiple drugs, polypharmacy, showed distinct gut microbiome structures harboring significantly more abundant upper gastrointestinal species and several nosocomial pathobionts due to additive drug effects. Polypharmacy was also associated with microbial functions, including the reduction of short-chain fatty acid metabolism and increased bacterial stress responses. Even nonantibiotic drugs were significantly correlated with an increased antimicrobial resistance potential through polypharmacy. Notably, a 2-time points dataset revealed the alteration and recovery of the microbiome in response to drug initiation and cessation, corroborating the observed drug-microbe associations in the cross-sectional cohort.Our large-scale metagenomics unravels extensive and disruptive impacts of individual and multiple drug exposures on the human gut microbiome, providing a drug-microbe catalog as a basis for a deeper understanding of the role of the microbiome in drug efficacy and toxicity.

To identify gut and oral metagenomic signatures that accurately predict pancreatic ductal carcinoma (PDAC) and to validate these signatures in independent cohorts.We conducted a multinational study and performed shotgun metagenomic analysis of fecal and salivary samples collected from patients with treatment-naïve PDAC and non-PDAC controls in Japan, Spain, and Germany. Taxonomic and functional profiles of the microbiomes were characterized, and metagenomic classifiers to predict PDAC were constructed and validated in external datasets.Comparative metagenomics revealed dysbiosis of both the gut and oral microbiomes and identified 30 gut and 18 oral species significantly associated with PDAC in the Japanese cohort. These microbial signatures achieved high area under the curve values of 0.78 to 0.82. The prediction model trained on the Japanese gut microbiome also had high predictive ability in Spanish and German cohorts, with respective area under the curve values of 0.74 and 0.83, validating its high confidence and versatility for PDAC prediction. Significant enrichments of Streptococcus and Veillonella spp and a depletion of Faecalibacterium prausnitzii were common gut signatures for PDAC in all the 3 cohorts. Prospective follow-up data revealed that patients with certain gut and oral microbial species were at higher risk of PDAC-related mortality. Finally, 58 bacteriophages that could infect microbial species consistently enriched in patients with PDAC across the 3 countries were identified.Metagenomics targeting the gut and oral microbiomes can provide a powerful source of biomarkers for identifying individuals with PDAC and their prognoses. The identification of shared gut microbial signatures for PDAC in Asian and European cohorts indicates the presence of robust and global gut microbial biomarkers.

To evaluate the effect of multifactorial intervention on the onset and progression of diabetic kidney disease in the patients with type 2 diabetes, we analyzed the effects of intensified multifactorial intervention by step-wise intensification of medications and life-style modifications (intensive therapy treatment targets; HbA1c under 6.2%, blood pressure under 120/75 mmHg, low-density lipoprotein cholesterol under 80 mg/dL) comparing with the guideline-based standard care (conventional therapy treatment targets: HbA1c under 6.9%, blood pressure under 130/80 mmHg, low-density lipoprotein cholesterol under 120 mg/dL) on diabetic kidney disease. A total of 2540 eligible patients in the Japan Diabetes Optimal Integrated Treatment for three major risk factors of cardiovascular diseases (J-DOIT3) cohort were randomly assigned to intensive therapy (1269) and conventional therapy (1271) and treated for a median of 8.5 years. The prespecified kidney outcome measure was a composite of progression from normoalbuminuria to microalbuminuria or progression from normoalbuminuria to macroalbuminuria or progression from microalbuminuria to macroalbuminuria, serum creatinine levels elevated by two-fold or more compared to baseline, or kidney failure. Primary analysis was carried out on the intention-to-treat population. Changes in the estimated glomerular filtration rate and albuminuria were also analyzed. A total of 438 kidney events occurred (181 in the intensive therapy group and 257 in the conventional therapy group). Intensive therapy was associated with a significant 32% reduction in kidney events compared to conventional therapy and was associated with a change in HbA1c at one year from study initiation. Thus, prespecified analysis shows that intensified multifactorial intervention significantly reduced the onset and progression of diabetic kidney disease compared to currently recommended care.

During insulin resistance, lipid uptake by the liver is promoted with peroxisome proliferator activated protein (PPAR) γ-upregulation, leading to hepatic steatosis. Insulin, however, does not directly regulate adipogenic gene expression in liver, and the mechanisms for their upregulation in obesity remains unclear. Here, we show that the Irs2locus, a critical regulator of insulin actions, encodes an antisense transcript ASIrs2, whose expression increased in obesity or after refeeding in liver, reciprocally to that of Irs2. ASIrs2 regulates hepatic Pparg-expression, and its suppression ameliorates steatosis in obese mice. The human orthologue AL162497.1 shows similar genomic organization like ASIrs2, whose expression being correlated with hepatic PPARG and severity of NASH. We also identified HARS2 as a potential binding protein for ASIrs2, functioning as a regulator of Pparg. Collectively, our data revealed a functional duality of Irs2 gene locus, where reciprocal changes of Irs2 and ASIrs2 in obesity cause insulin resistance and steatosis.

During insulin resistance, lipid uptake by the liver is promoted by peroxisome proliferator-activated protein (PPAR) γ upregulation, leading to hepatic steatosis. Insulin, however, does not directly regulate adipogenic gene expression in liver, and the mechanisms for its upregulation in obesity remain unclear. Here, we show that the Irs2 locus, a critical regulator of insulin actions, encodes an antisense transcript, ASIrs2, whose expression increases in obesity or after refeeding in liver, reciprocal to that of Irs2. ASIrs2 regulates hepatic Pparg expression, and its suppression ameliorates steatosis in obese mice. The human ortholog AL162497.1, whose expression is correlated with that of hepatic PPARG and the severity of non-alcoholic steatohepatitis (NASH), shows genomic organization similar to that of ASIrs2. We also identified HARS2 as a potential binding protein for ASIrs2, functioning as a regulator of Pparg. Collectively, our data reveal a functional duality of the Irs2 gene locus, where reciprocal changes of Irs2 and ASIrs2 in obesity cause insulin resistance and steatosis.

Aims To investigate the prevalence of comorbid conditions/complications among Japanese patients with type 2 diabetes mellitus (T2DM) in a real-world setting. Methods We performed retrospective analyses of a large-scale database directly linked to electronic medical records, J-DREAMS (Japan Diabetes compREhensive database project based on an Advanced electronic Medical record System), to determine the prevalence of clinically significant comorbid conditions/complications among Japanese patients with T2DM aged ≥ 20 years with ≥ 1 clinical encounter at a referral center between April 1, 2017 and March 31, 2019. Results Data were available for 10,151 patients (39.2% female). The mean age and T2DM duration were 66.0 years and 16.1 years, respectively. Only 0.5% had isolated T2DM, 6.6% had one comorbid condition/complication, and the remainder had multiple comorbid conditions/complications. Dyslipidemia (84.7%) and hypertension (75.1%) were the most common, followed by chronic kidney disease (35.4%), retinopathy (23.1%), and cardiovascular diseases (22.1%). Overall, 36.0% of patients were overweight/obese (body mass index ≥ 25 kg/m2) and 18.6% had a history of neoplasms. The prevalence of comorbid conditions/complications tended to increase with advancing age and duration of T2DM. Conclusions We revealed a high prevalence of comorbid conditions/complications, including chronic kidney and cardiovascular diseases, in Japanese patients with T2DM.

BACKGROUND: A randomized control trial conducted to assess the effects of intensified multifactorial intervention (intensive therapy) compared with the guideline-based standard care (conventional therapy) on diabetic complications and all-cause mortality, the J-DOIT3 showed a nonsignificant reduction in the primary outcome. The study was intended to determine the effect of intensive therapy on the pre-specified renal outcome as a secondary outcome measure. METHODS: A total of 2540 eligible patients were randomly assigned to intensive therapy (n = 1269) and conventional therapy (n = 1271) and treated for a median of 8.5 years. The renal outcome measure was a composite of the onset or progression of nephropathy defined as progression from normoalbuminuria (urinary albumin < 30 mg/g·creatinine) to microalbuminuria (urinary albumin ≥30 mg/g·creatinine, < 300 mg/g·creatinine); progression from normoalbuminuria to macroalbuminuria (urinary albumin ≥ 300 mg/g·creatinine), progression from microalbuminuria to macroalbuminuria, serum creatinine levels elevated by 2-fold or more compared to baseline, or end-stage renal failure (permanent dialysis initiated or renal transplant performed). The primary analysis was carried out on the intention-to-treat (ITT) population. Changes in the estimated glomerular filtration rate (eGFR) and albuminuria were also analyzed RESULTS: A total of 438 renal events occurred (181 in the intensive therapy group and 257 in the conventional therapy group), and intensive therapy was associated with a significant 32% reduction in renal events compared to conventional therapy (HR, 0.68; 95% CI, 0.56 to 0.82; P < 0.0001) and was associated with a decrease in HbA1c at 1 year from study initiation. CONCLUSIONS: The pre-specified analysis shows that intensified multifactorial intervention significantly reduced the onset and progression of diabetic kidney disease compared to the currently recommended care. TRIAL REGISTRATION: ClinicalTrials.gov, number NCT00300976 FUNDING STATEMENT: This trial was supported by Ministry of Health, Labour and Welfare, Asahi Kasei Pharma, Astellas Pharma, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Daiichi Sankyo, Eli Lilly, GlaxoSmithKline, Kissei Pharmaceutical, Kowa Pharmaceutical, Mitsubishi Tanabe Pharma, Mochida Pharmaceutical, MSD, Novartis Pharma, Novo Nordisk Pharma, Ono Pharmaceutical, Pfizer, Sanwa Kagaku Kenkyusho, Shionogi, Sumitomo Dainippon Pharma, Taisho Toyama Pharmaceutical, and Takeda Pharmaceutical. DECLARATION OF INTERESTS: K. U. reports a funded research department from MSD, Nippon Boehringer Ingelheim, and Novo Nordisk; lecture fees from Astellas, AstraZeneca, Daiichi Sankyo, Eli Lilly, Kissei, Kowa, Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe, MSD, Nippon Boehringer Ingelheim, Novartis, Novo Nordisk, Ono, Sanofi, Sanwa Kagaku, Shionogi, Sumitomo Dainippon, Taisho Toyama Pharmaceutical Co., Ltd., and Takeda; grants and endowments from Astellas, AstraZeneca, Daiichi Sankyo, Eli Lilly, Kowa, Kyowa Hakko Kirin, Mitsubishi Tanabe, MSD, Nippon Boehringer Ingelheim, Novartis, Novo Nordisk, Ono, Sanofi, Sanwa Kagaku, Sumitomo Dainippon, Taisho Toyama, and Takeda. T. S. reports lecture fees from Kissei, Mitsubishi Tanabe, and Takeda. Y. O. reports lecture fees from Nippon Boehringer Ingelheim, and Takeda. S. O. reports lecture fees from AstraZeneca, Eli Lilly, and Mitsubishi Tanabe. H. K. reports lecture fees from Eli Lilly, Mitsubishi Tanabe, Novo Nordisk, and Ono. M. H. reports lecture fees from AstraZeneca, Kissei, and Mitsubishi Tanabe. A. Morita reports lecture fees from Nippon Boehringer Ingelheim. K. O. reports lecture fees from Abbott Japan Co., Ltd., ASKA Pharmaceutical Co. Ltd., Astellas, AstraZeneca, Daiichi Sankyo, Eli Lilly, Johnson & Johnson K. K., Kao Corporation, Kyowa Hakko Kirin, MSD, Nippon Boehringer Ingelheim, Novo Nordisk, Ono, Sanofi K. K., and Takeda. K. H. reports consulting fees from Kissei and Takeda; research support from Novartis. A. Morise reports lecture fees from Nippon Boehringer Ingelheim. Y. O. reports lecture fees from Eli Lilly, Novo Nordisk, Sanofi, and Takeda; consulting fees from Astellas, Chugai Pharmaceutical Co., Ltd., and Kowa; being the chairman of the board of directors, and owning stock in Statcom Co., Ltd. M. N. reports lecture fees from AbbVie G. K., Astellas, Daiichi Sankyo, Eli Lilly, Kissei, Kowa, Kyowa Hakko Kirin, Meiji Seika Pharma Co., Ltd., Mitsubishi Tanabe, MSD, Novo Nordisk, Sanofi, Ono, Taisho Toyama, and Takeda; grants and endowments from AstraZeneca, Daiichi Sankyo, Kowa, Kyowa Hakko Kirin, Mitsubishi Tanabe, Mochida Pharmaceutical Co., Ltd., Sanwa Kagaku, and Takeda. T. K. reports funded research departments from MSD, Nippon Boehringer Ingelheim, Novartis, and Novo Nordisk; lecture fees from Astellas, AstraZeneca, Eli Lilly, Kissei, Kowa, Mitsubishi Tanabe, MSD, Nippon Boehringer Ingelheim, Novo Nordisk, Ono, Sumitomo Dainippon, and Takeda; manuscript fees from Eli Lilly; grants and endowments from Daiichi Sankyo, Mitsubishi Tanabe, Sumitomo Dainippon, and Takeda; funds for contracted research from Daiichi Sankyo, Sanwa Kagaku, and Takeda; funds for collaborative research from Daiichi Sankyo and Novartis. No other potential conflicts of interest relevant to this work are reported. The authors and the participating investigators of the J-DOIT3 Study Group declared duality of interest also to his/her healthcare institution. ETHICS APPROVAL STATEMENT: The study protocol was approved by the ethics committee of each participating institution. All patients provided written informed consent.

Summary The assessment of macrophage function has been a topic of intense discussion due to multiple subtypes. This protocol describes the collection of bone marrow cells from the femur and tibia of mice, differentiation into bone marrow-derived macrophages (BMDM cells), and sampling from cultures. This protocol focuses on the efficient preparation of BMDM cells, providing a way to assess the function of macrophages. For complete details on the use and execution of this protocol, please refer to Toda et al. (2020)., Graphical Abstract, Highlights • Fast and efficient collection of bone marrow from mice • Differentiation into BMDM that are >90% positive for CD11b and F4/80 • Culture of BMDM for in vitro functional analysis of RNA and protein • Cells can be assessed without assuming polarization to M1/M2 subtype, The assessment of macrophage function has been a topic of intense discussion due to multiple subtypes. This protocol describes the collection of bone marrow cells from the femur and tibia of mice, differentiation into bone marrow-derived macrophages (BMDM cells), and sampling from cultures. This protocol focuses on the efficient preparation of BMDM cells, providing a way to assess the function of macrophages.

Objective To explore how patients with type 2 diabetes (T2DM) psychologically and behaviorally respond to internalized stigma through social stigma. Methods A qualitative study with semi-structured interviews was recorded on audiotapes, transcribed verbatim, and analyzed using a grounded theory approach. Participants were adults aged 30–64 years and diagnosed with T2DM. A total of 26 patients participated. Results The qualitative data revealed that participants' responses to social stigma, although varied, could be organized into a four-step process: Encountering Negative Experiences , Reevaluating the Self with Type 2 Diabetes , Reconstructing a Sense of Identity , and Maintaining Balance between Patient and Social Roles. When participants form a negative image of and relationship to their illness, they tend to internalize stigma, which can affect their sense of self-worth, attitude toward social participation, and compliance. Conclusion Participants who internalize stigma tend to have a lower sense of self-worth and their social participation falls somewhere between severely limited ( Social Avoidance ) and highly active ( Role Conflict ). This can hinder devotion to their treatment regimen and affect their degree of compliance with physicians. Practice implications Internalized stigma can be assessed by observing a patient's illness-related negative self-image.

The physiological role of immune cells in the regulation of postprandial glucose metabolism has not been fully elucidated. We have found that adipose tissue macrophages produce interleukin-10 (IL-10) upon feeding, which suppresses hepatic glucose production in cooperation with insulin. Both elevated insulin and gut-microbiome-derived lipopolysaccharide in response to feeding are required for IL-10 production via the Akt/mammalian target of rapamycin (mTOR) pathway. Indeed, myeloid-specific knockout of the insulin receptor or bone marrow transplantation of mutant TLR4 marrow cells results in increased expression of gluconeogenic genes and impaired glucose tolerance. Furthermore, myeloid-specific Akt1 and Akt2 knockout results in similar phenotypes that are rescued by additional knockout of TSC2, an inhibitor of mTOR. In obesity, IL-10 production is impaired due to insulin resistance in macrophages, whereas adenovirus-mediated expression of IL-10 ameliorates postprandial hyperglycemia. Thus, the orchestrated response of the endogenous hormone and gut environment to feeding is a key regulator of postprandial glycemia.

Purpose High plantar pressure is a major risk factor for diabetic foot ulcers. The relationship between plantar pressure and foot mobility has been investigated in some studies. However, when the foot is in motion, foot mobility is only a small feature of the gait. Therefore, we investigated relationship between high plantar pressure and gait and also studied the motion of the trunk. In addition, we investigated the relationship between gait and patient characteristics to identify patients at high-risk of developing diabetic foot ulcers. Methods The relationships between elevated plantar pressure, gait features, and patient characteristics were analyzed. Plantar pressure distribution in the stance phase was divided on the four plantar segments. Elevated plantar pressure was defined as being more than the mean plus one standard deviation of the corresponding segment in non-diabetic subjects. Plantar pressure distribution was measured by an F-scan system, and gait features were measured using wireless motion sensors attached to the sacrum and feet. Patient characteristics were obtained from medical records or by interview. Results Small roll and yaw motions of the body and yaw motion of the foot during the mid-stance phase were related to the elevated plantar pressure in 57 diabetic patients. Furthermore, these gait features were related to sensory neuropathy, diabetes duration, patient weight, toe-gap force, and ankle range of motion. Conclusion Given our findings, it may be possible to prevent diabetic foot ulcers by increasing foot motion during the mid-stance phase. Passive exercise aimed at expanding ankle range of motion in patients with sensory neuropathy or long-standing diabetes may assist in achieving this.

To calculate process quality measures of diabetes care in Japan using nationwide exclusive claims database.Using the National Database of health insurance claims during 2015-2016, the proportions of outpatients who received recommended examinations at least annually among those with regular antidiabetic medication were calculated as quality indicators, reported altogether and by prefecture and institutional certification (from the Japan Diabetes Society). Distributions of institutional-level quality indicators were also reported.Among 4,154,452 outpatients, 96.7% underwent HbA1c or glycated albumin examination. Retinopathy examination was conducted among 46.5% of patients [prefecture (range): 37.5%-51.0%, institutional certification: 44.8% (without) vs. 59.8% (with)]. Urinary qualitative examination was conducted among 67.3% of patients at institutions with200 beds (prefecture: 54.1%-81.9%, institutional certification: 66.8% vs. 92.8%), whereas urinary quantitative albumin or protein examination was conducted among 19.4% of patients (prefecture: 10.8%-31.6%, institutional certification: 18.7% vs. 54.8%). Distributions of institutional-level quality indicators showed that most institutions without institutional certification seldomly order urinary quantitative examination.Although the quality indicator for glycaemic control examination was favourable, some aspects of diabetes care were suboptimal and varied greatly by prefecture and institution; individual and organisational efforts to improve quality of diabetes care would be needed in Japan.

Objective To identify the factors associated with toenail onychomycosis in patients with diabetes. Methods In this cross-sectional, observational study, the presence and severity (area of nail involvement and nail thickness) of toenail onychomycosis and related factors were examined. One hundred and thirteen patients with diabetes were surveyed at the Diabetic Foot Outpatient Clinic at the University hospital. Toenails of all patients enrolled in the survey were examined whether onychomycosis was present or absent by mycological examination. The severity of onychomycosis was assessed by clinical evaluation, using the area of nail involvement and the nail thickness. Results Fifty eight (51.3%) patients had toenail onychomycosis. The presence of onychomycosis was significantly associated with not washing of feet every day (the unadjusted model, OR: 3.45, 95% CI: 1.24–9.65, P =0.018). The median area of nail involvement was 50.0% (range 5.0–100.0%). A larger area of involvement was significantly related to a lower toe brachial index ( β =−67.46, P =0.040). The median nail thickness with onychomycosis was 1.14mm (range 0.68–9.86mm). Increasing thickness was significantly correlated with higher hemoglobin A 1 c levels ( β =0.98, P =0.003). Conclusions This study suggested that daily washing of feet may reduce the risk of onychomycosis in patients with diabetes. This suggested that education regarding the importance of the washing of feet every day and support for continuous self-care might be effective in the prevention of onychomycosis in patients with diabetes. Furthermore, good control of blood glucose might prevent increasing nail thickness. This study may highlight importance of early nursing educational intervention to improve patients' daily life style for prevention of onychomycosis induced diabetic foot ulcers.

Rimonabant has been shown to not only decrease the food intake and body weight but also to increase serum adiponectin levels. This increase of the serum adiponectin levels has been hypothesized to be related to the rimonabant-induced amelioration of insulin resistance linked to obesity, although experimental evidence to support this hypothesis is lacking. To test this hypothesis experimentally, we generated adiponectin knock-out (adipo(-/-))ob/ob mice. After 21 days of 30 mg/kg rimonabant, the body weight and food intake decreased to similar degrees in the ob/ob and adipo(-/-)ob/ob mice. Significant improvement of insulin resistance was observed in the ob/ob mice following rimonabant treatment, associated with significant up-regulation of the plasma adiponectin levels, in particular, of high molecular weight adiponectin. Amelioration of insulin resistance in the ob/ob mice was attributed to the decrease of glucose production and activation of AMP-activated protein kinase (AMPK) in the liver induced by rimonabant but not to increased glucose uptake by the skeletal muscle. Interestingly, the rimonabant-treated adipo(-/-)ob/ob mice also exhibited significant amelioration of insulin resistance, although the degree of improvement was significantly lower as compared with that in the ob/ob mice. The effects of rimonabant on the liver metabolism, namely decrease of glucose production and activation of AMPK, were also less pronounced in the adipo(-/-)ob/ob mice. Thus, it was concluded that rimonabant ameliorates insulin resistance via both adiponectin-dependent and adiponectin-independent pathways.

SummaryInsulin receptor substrate (Irs) mediates metabolic actions of insulin. Here, we show that hepatic Irs1 and Irs2 function in a distinct manner in the regulation of glucose homeostasis. The PI3K activity associated with Irs2 began to increase during fasting, reached its peak immediately after refeeding, and decreased rapidly thereafter. By contrast, the PI3K activity associated with Irs1 began to increase a few hours after refeeding and reached its peak thereafter. The data indicate that Irs2 mainly functions during fasting and immediately after refeeding, and Irs1 functions primarily after refeeding. In fact, liver-specific Irs1-knockout mice failed to exhibit insulin resistance during fasting, but showed insulin resistance after refeeding; conversely, liver-specific Irs2-knockout mice displayed insulin resistance during fasting but not after refeeding. We propose the concept of the existence of a dynamic relay between Irs1 and Irs2 in hepatic insulin signaling during fasting and feeding.

Adipose tissue expression and circulating concentrations of monocyte chemoattractant protein-1 (MCP-1) correlate positively with adiposity. To ascertain the roles of MCP-1 overexpression in adipose, we generated transgenic mice by utilizing the adipocyte P2 (aP2) promoter (aP2-MCP-1 mice). These mice had higher plasma MCP-1 concentrations and increased macrophage accumulation in adipose tissues, as confirmed by immunochemical, flow cytometric, and gene expression analyses. Tumor necrosis factor-alpha and interleukin-6 mRNA levels in white adipose tissue and plasma non-esterified fatty acid levels were increased in transgenic mice. aP2-MCP-1 mice showed insulin resistance, suggesting that inflammatory changes in adipose tissues may be involved in the development of insulin resistance. Insulin resistance in aP2-MCP-1 mice was confirmed by hyperinsulinemic euglycemic clamp studies showing that transgenic mice had lower rates of glucose disappearance and higher endogenous glucose production than wild-type mice. Consistent with this, insulin-induced phosphorylations of Akt were significantly decreased in both skeletal muscles and livers of aP2-MCP-1 mice. MCP-1 pretreatment of isolated skeletal muscle blunted insulin-stimulated glucose uptake, which was partially restored by treatment with the MEK inhibitor U0126, suggesting that circulating MCP-1 may contribute to insulin resistance in aP2-MCP-1 mice. We concluded that both paracrine and endocrine effects of MCP-1 may contribute to the development of insulin resistance in aP2-MCP-1 mice.

The effects of insulin on vascular endothelial growth factor (VEGF) expression in cultured vascular cells and in angiogenesis were characterized. Insulin increased VEGF mRNA levels in mouse aortic smooth muscle cells from 10(-9) to 10(-7) m with an initial peak of 3.7-fold increases at 1 h and a second peak of 2.8-fold after 12 h. The first peak of VEGF expression was inhibited by LY294002, an inhibitor of phosphatidylinositol (PI) 3-kinase, and by the overexpression of dominant negative forms of p85 subunit of PI 3-kinase or Akt. Inhibitors of MEK kinase, PD98059, or overexpression of dominant negative forms of Ras was ineffective. In contrast, the chronic effect of insulin on VEGF expression was partially inhibited by both LY294002 or PD98059 as well as by the overexpression of dominant negatives of PI 3-kinase or Ras. The importance of PI 3-kinase-Akt pathway on VEGF expression was confirmed in mouse aortic smooth muscle cells isolated from insulin receptor substrate -1 knockout (IRS-1-/-) mice that showed parallel reductions of 46-49% in insulin-stimulated VEGF expression and PI 3-kinase-Akt activation. Insulin-induced activation of PI 3-kinase-Akt on hypoxia-induced VEGF expression and neovascularization was reduced by 40% in the retina of neonatal hypoxia model using IRS-1-/- mice. Thus, unlike other cells, insulin can regulate VEGF expression by both IRS-1/PI 3-kinase-Akt cascade and Ras-MAPK pathways in aortic smooth muscle cells. The in vivo results provide direct evidence that insulin can modulate hypoxia-induced angiogenesis via reduction in VEGF expression in vivo.

The p85alpha regulatory subunit of class I(A) phosphoinositide 3-kinases (PI3K) is derived from the Pik3r1 gene, which also yields alternatively spliced variants p50alpha and p55alpha. It has been proposed that excess monomeric p85 competes with functional PI3K p85-p110 heterodimers. We examined embryonic stem (ES) cells with heterozygous and homozygous disruptions in the Pik3r gene and found that wild type ES cells express virtually no monomeric p85alpha. Although, IGF-1-stimulated PI3K activity associated with insulin receptor substrates was unaltered in all cell lines, p85alpha-null ES cells showed diminished protein kinase B activation despite increased PI3K activity associated with the p85beta subunit. Furthermore, p85alpha-null cells demonstrated growth retardation, increased frequency of apoptosis, and altered cell cycle regulation with a G(0)/G(1) cell cycle arrest and up-regulation of p27(KIP), whereas signaling through CREB and MAPK was enhanced. These phenotypes were reversed by re-expression of p85alpha via adenoviral gene transfer. Surprisingly, all ES cell lines could be differentiated into adipocytes. In these differentiated ES cells, however, compensatory p85beta signaling was lost in p85alpha-null cells while increased signaling by CREB and MAPK was still observed. Thus, loss of p85alpha in ES cells induced alterations in IGF-1 signaling and regulation of apoptosis and cell cycle but no defects in differentiation. However, differentiated ES cells partially lost their ability for compensatory signaling at the level of PI3K, which may explain some of the defects observed in mice with homozygous deletion of the Pik3r1 gene.

Insulin-like growth factor-1 (IGF-1) and insulin are known to prevent apoptosis. The signaling network of IGF-1 and insulin occurs via multiple pathways involving different insulin receptor substrates (IRSs). To define their roles in the anti-apoptotic function of IGF-1 and insulin, we established brown pre-adipocyte cell lines from wild-type and IRS knockout (KO) animals. In response to 16 h of serum deprivation, IRS-1-deficient cells showed a significant decrease in response to IGF-1 protection from apoptosis, whereas no changes were observed in the IRS-2, IRS-3, or IRS-4 KO cells. Five hours after serum withdrawal, cells already began to undergo apoptosis. At this early time point, IGF-1 and insulin were able to protect both wild-type and IRS-1 KO cells from death by 85-90%. After a longer period of serum deprivation, the protective ability of insulin and IGF-1 was decreased, and this was especially reduced in the IRS-1 KO cells. Reconstitution of these cells with IRS-1, IRS-2, IRS-3, or IRS-1/IRS-2 chimeras restored the anti-apoptotic effects of IGF-1, whereas overexpression of IRS-4 had no effect at long time points and actually reduced the effect of IGF-1 at the short time point. The biochemical basis of the defect in anti-apoptosis was not dependent on phosphorylation of mitogen-activated protein kinase; whereas phosphoinositide 3-kinase activity was decreased by 30% in IRS-1 KO cells. Akt phosphorylation was slightly reduced in these cells. Phosphorylation of the transcription factors cAMP response element-binding protein and FKHR by IGF-1 and insulin was markedly reduced in IRS-1 KO cells. In addition, both IGF-1 and insulin prevented caspase-3 cleavage in the wild-type cells, and this effect was greatly reduced in the IRS-1-deficient cells. These findings suggest that the IRS proteins may play differential roles in the anti-apoptotic effects of IGF-1 and insulin in brown pre-adipocytes, with IRS-1 being predominant, possibly acting through caspase-3-, CREB-, and FKHR-dependent mechanisms.

Class IA phosphoinositide (PI) 3-kinase is composed of a p110 catalytic subunit and a p85 regulatory subunit and plays a pivotal role in insulin signaling. To explore the physiological roles of two major regulatory isoforms, p85 alpha and p85 beta, we have established brown adipose cell lines with disruption of the Pik3r1 or Pik3r2 gene. Pik3r1-/- (p85 alpha-/-) cells show a 70% reduction of p85 protein and a parallel reduction of p110. These cells have a 50% decrease in PI 3-kinase activity and a 30% decrease in Akt activity, leading to decreased insulin-induced glucose uptake and anti-apoptosis. Pik3r2-/- (p85 beta-/-) cells show a 25% reduction of p85 protein but normal levels of p85-p110 and PI 3-kinase activity, supporting the fact that p85 is more abundant than p110 in wild type. p85 beta-/- cells, however, exhibit significantly increased insulin-induced Akt activation, leading to increased anti-apoptosis. Reconstitution experiments suggest that the discrepancy between PI 3-kinase activity and Akt activity is at least in part due to the p85-dependent negative regulation of downstream signaling of PI 3-kinase. Indeed, both p85 alpha-/- cells and p85 beta-/- cells exhibit significantly increased insulin-induced glycogen synthase activation. p85 alpha-/- cells show decreased insulin-stimulated Jun N-terminal kinase activity, which is restored by expression of p85 alpha, p85 beta, or a p85 mutant that does not bind to p110, indicating the existence of p85-dependent, but PI 3-kinase-independent, signaling pathway. Furthermore, a reduction of p85 beta specifically increases insulin receptor substrate-2 phosphorylation. Thus, p85 alpha and p85 beta modulate PI 3-kinase-dependent signaling by multiple mechanisms and transmit signals independent of PI 3-kinase activation.

Fibroblastic proliferation accompanies many angiogenesis-related retinal and systemic diseases. Since connective tissue growth factor (CTGF) is a potent mitogen for fibrosis, extracellular matrix production, and angiogenesis, we have studied the effects and mechanism by which vascular endothelial growth factor (VEGF) regulates CTGF gene expression in retinal capillary cells. In our study, VEGF increased CTGF mRNA levels in a time- and concentration-dependent manner in bovine retinal endothelial cells and pericytes, without the need of new protein synthesis and without altering mRNA stability. VEGF activated the tyrosine receptor phosphorylation of KDR and Flt1 and increased the binding of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit to KDR and Flt1, both of which could mediate CTGF gene induction. VEGF-induced CTGF expression was mediated primarily by PI3-kinase activation, whereas PKC and ERK pathways made only minimal contributions. Furthermore, overexpression of constitutive active Akt was sufficient to induce CTGF gene expression, and inhibition of Akt activation by overexpressing dominant negative mutant of Akt abolished the VEGF-induced CTGF expression. These data suggest that VEGF can increase CTGF gene expression in bovine retinal capillary cells via KDR or Flt receptors and the activation of PI3-kinase-Akt pathway independently of PKC or Ras-ERK pathway, possibly inducing the fibrosis observed in retinal neovascular diseases.

Insulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on multiple tyrosine residues in response to insulin and binds several Src homology 2 domain-containing proteins, thereby initiating downstream signaling. To assess the tyrosine phosphorylation sites that mediate relevant downstream signaling and biological effects, we created site-directed mutants of IRS-1 and overexpressed them in the Xenopus laevis oocyte. In oocytes overexpressing IRS-1 or IRS-1-895F (Tyr-895 replaced with phenylalanine), insulin activated phosphatidylinositol (PI) 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and induced oocyte maturation. In contrast, in oocytes overexpressing IRS-1-4F (Tyr-460, Tyr-608, Tyr-939, and Tyr-987 of IRS-1 replaced with phenylalanine), insulin did not activate PI 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and failed to induce oocyte maturation. These observations indicate that in X. laevis oocytes overexpressing IRS-1, the association of PI 3-kinase rather than Grb2 (growth factor-bound protein 2) with IRS-1 plays a major role in insulin-induced oocyte maturation. Activation of PI 3-kinase may lie upstream of mitogen-activated protein kinase activation and p70 S6 kinase activation in response to insulin.

This study was undertaken to define intracellular signaling pathways upstream to glycogen synthase activation. First, we examined the role of the two pathways of insulin signaling, Ras-dependent and wortmannin/LY294002-sensitive, in glycogen synthase activation. Although negative dominant Ras (Ras17N) induction in PC12 cells markedly decreased activities of mitogen-activated protein kinase (MAP) and pp90 S6 kinase in response to insulin or insulin-like growth factor I (IGF-I), activation of glycogen synthase by these agents was unaffected by negative dominant Ras induction. In contrast, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), inhibitors of phosphatidylinositol 3-kinase, antagonized glycogen synthase activation in response to insulin or IGF-I. Next, we examined the contribution of pp70 S6 kinase, one of the wortmannin/LY294002-sensitive signaling molecules on glycogen synthase activation. Immunosuppressant rapamycin completely blocked activation of pp70 S6 kinase by insulin or IGF-I, but rapamycin alone or in combination with induction of negative dominant Ras failed to antagonize glycogen synthase activation by these hormones. These data suggest that 1) activation of Ras-MAP kinase is not necessary for stimulation of glycogen synthase and 2) activation of wortmannin/LY294002sensitive pathway, independent of pp70 S6 kinase, plays a key role in glycogen synthase regulation in PC12 cells.

A 59-year-old female with type 1 diabetes and RS3PE had HLA types known to be associated with both diseases. Type 1 diabetes patients suffering from polyarthritis and pitting edema should be examined for possible RS3PE and glucocorticoid therapy may be indicated despite the diabetes.